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About the cover: Mitotic PtK2 cellsin late anaphasestainedblue for DNA and greenfor tubulin. Courtesyof Torsten\gittman.
Library of CongressCataloging-in-PublicationData
Molecular cell biology I Harvey Lodish . . . [et al.]. -6th ed.
p. cm.
Includesbibliographicalreferencesand index.
1. Cytology. 2. Molecular biology. I. Lodish, Harvey F.
QH581.2.M6552007
57L5-dc22 2007006188
ISBN-13: 97 8-0-7167-7601-7
ISBN-10:0-7167-7 601-4
@ 1986,1.990,1995,2000,2004,2008 by tJf.H. Freemanand Company
All rights reserved.

Printed in the United Statesof America

Secondprinting

W. H. Freemanand Company
41 Madison Avenue,New York, NY 10010
Houndmills, Basingstoke
RG21 6XS, England

www.whfreeman.com
To our studentsand to our teachers,
from whom we continueto learn,
and to our families,for their support,
encouragement, and love
 PREFACE

I n writing the sixth edition of Molecular Cell Biology
I we have incorporated many of the spectacularadvances
I made over the past four years in biomedical science,driven
in part by new experimental technologiesthat have revolu-
tionized many fields. High-velocity techniquesfor sequenc-
ing DNA, for example,have generatedthe completesequence
of dozens of eukaryotic genomes;these in turn have led to
important discoveriesabout the organization of the human
genome and regulation of gene expression,as well as novel
insights into the evolution of life-forms and the functions of
individual members of multiprotein families. New imaging
techniqueshave generated profound revelations about cell
organization and movement, and new molecular structures
have greatly increased our understanding of life processes
such as cell-cell signaling, photosynthesis, gene transcrip-
tion, and chromatin structure.
what we know. A number of experimental organisms,from
yeaststo worms to mice, are used throughout so the student
can seehow discoveriesmade with a "lower organism" can
Iead directly to insights even about human biology and dis-
ease.This experimental approach, evident in the text itself,
has also been thoroughly integrated into the pedagogical
framework. For example:
r Experimental Figures lead students through important
experimental results.
r Classic Experiments essaysfocus on historically impor-
tant and Nobel Prize-winningexperiments.
r New and revised Analyze the Data problems at the end
of each chapter require the student to synthesizereal ex-
perimental data to answer a seriesof questions.

r Updated Perspectives for the Future essays explore

New Author Team potential applications of future discoveriesand unanswered
Two new authors have been instrumental in refocusing this questions that lie ahead in research.
book toward these exciting new developments. Anthony
Bretscher of Cornell University is known for identifying and
characterizing new components of the actin cytoskeleton and
elucidating their biological functions in relation to cell polarity
and membrane traffic. Hidde Ploegh, of the Massachusetts
Institute of Technologg has made major contributions to our
understanding of immune system behavior, particularly in
regard to the various tactics that viruses employ to evade our
immune responsesand the ways our immune systemresponds.
Both authors are widely recognized for their researchas well
as their classroomteachingabilities.
'We
are grateful to Paul Matsudaira, Jim Darnell, Larry
ZipurskS and David Baltimore for their exceptional contri-
butions to the previous editions of Molecular Cell Biology.
Much of their vision and insight is apparent at many places
in this book.

Experimental
Emphasis Fluorescence showsthe locationof DNAand multiple
microscopy
The hallmark of Molecular Cell Biology has always been the proteins BNG Giepmans
withinthe samecell.lFrom et al, 2006,Science
use of experiments to teach students how we have learned 3'12:217
|

vtl
 Coiled-coilstalk New Discoveries,
New Methodologies
Methodological advances continue to expand and enrich
our knowledge of molecular cell biology and lead to new
understanding. Following are just a selection of the new
Motor head
experimental methodologiesand cutting-edgescienceintro-
Necklinkers Microtubule duced in this edition:
(+)

r Expanded coverage of proteomics, including organelle

proteome profiling and advances in mass spectroscopy
(Chapter 3)
I ForwardmotorbindsB-tubulin,
^ releasing
ADP r Expanded coverageof RNAi, including the use of shRNAs
to inhibit any gene of interest in a cultured cell or organism
(Chapters5, 8)

r Updated discussionsof chromatin, including structure and

condensation(Chapter 6), control of geneexpressionby chro-
matin remodeling (Chapter 71, and chromatin-remodeling
p ForwardheadbindsATP proteins and tumor development(Chapter 25)
? r Evolution of chromosomes and the mitochondrion
(Chapter 6)

r New molecular models, including pre-initiation complex

p
Conformationalchangein
necklinkercausesrear
head to swing forward
and mediator complex (Chapter 7); annular phospholipids
(Chapter 1,0);Caz* ATPase (Chapter 11); rhodopsin, trans-
ducin, and protein kinase A (Chapter 15); and myosin
ATPase (Chapter 17)

r Latest advancesin light and electron microscopy, includ-

ing cryoelectron tomography (Chapter 9)

r Reactive oxygen species(ROS) (Chapter 12)

[ rue* forwardheadreleases
ADB
^ trailingheadhydrolyzesATP r Role of supercomplexesin electron transport (Chapter 12)
V and releasesP.
r Human epidermal growth factor receptors (HERs) and
treatment of cancer (Chapter 16)

r Myosin ATPasecycle (Chapter 17)

r Kinesin-1MPase cycle (Chapter 18)

r Use of retrovirus infection for tracing cell lineage

(Chapter 21)

r Axon guidance molecules (Chapter 23)

r Somaticgenerearrangementin immune cells (Chapter 24)

r Cancer stem cells (Chapter25)

r Use of DNA microarray analysis in tumor typing

(Chapter25)

Figure18-22 Kinesin-1
usesATPto "walk" downa microtubule

vill PREFACE
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MEDIA AND SUPPLEMENTS

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17 ,7 ,L Cell Mlgra:1jon Coordlnates Fore Gen.radon wlth Cell

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)a:99 -, Aott dol d6. t..t d

PREFACE '
 ACKNOWLEDGMENTS
In updating, revising and rewriting this book, we were given
'We
invaluable help by many colleagues. thank the following
people who generouslygave of their time and expertiseby making
contributions to specific chapters in their areas of interest, pro-
viding us with detailed information about their courses, or by
reading and commenting on one or more chapters:
Steven Ackerman, (Jniuersity of Massacbusetts,Boston
Richard AdIe4 IJniuersity of Michigan, Dearborn
Karen Aguirre, Coastal Carolina lJniuersity
Jeff Bachant, Uniuersity of California, Riuerside
Kenneth Balazovich, Uniuersity of Michigan
Ben A. Barres, Stanford Uniuersity
Karen K. Bernd, Dauidson College
Sanford Bernstein, San Diego State (Jniuersity
Doug Black, Howard Hughes Medical Institute and (Jniuersity
of California, Los Angeles
Richard L. Blanton, North Carolina State (Jniuersny
Justin Blau, New York [Jniuersity
Steven Block, Stanford IJniuersity
Jonathan E. Boyson, Uniuersity of Vermont
Janet Braam. Rice Uniuersity
Roger Bradleg Montana State Uniuersity
IilTilliam S. Bradshaq Brigham Young (Jniuersity
Gregory G. Brown, McGill (Jniuersity
\Tilliam J. Brown, Cornell IJniuersity
Max M. Burger, Friedrich Miescher Institute for Biomedical
Research, Basel, Switzerland
David Burgess,Boston College
Robin K. Cameron, McMaster [Jniuersity
\7. Zacheus Cande, (Jniuersity of California, Berkeley
Steven A. Carr, Broad lnstitute of Haruard (Jniuersity and
Massacbusetts Institute of Technology
Alice Y. Cheung, IJniuersity of Massacbusetts, Amberst
Dennis O. Clegg, Uniuersity of California, Santa Barbara
Paul Clifton, Utah State IJniuersity
Randy \7. Cohen, California State [Jniuersity, Northridge
Richard Dickerson, (Jniuersity of California, Los Angeles
Patrick J. DiMario, Louisiana State Uniuersity
Santosh R. D'Mello, [Jniuersity of Texas, Dallas
Chris Doe, HHMI and [Jniuersity of Oregon
Robert S. Dotson, Tulane [Jniuersity
'Sfilliam
Dowhan, IJniuersity of Texas-Houston
Medical School
Gerald B. Downes, (Jniuersity of Massachusetts,Amherst
Erastus C. Dudley, Huntingdon College
Susan Dutcher, V/ashington (Jniuersity School of Medicine
Matt Elrod-Erickson, Middle TennesseeState Uniuersity
Susan Ely, Cornell Uniuersity
Charles P. Emerson Jr., Boston Biomedical ResearchInstitute
Irene M. Evans, Rochester Institute of Technology
James G. Evans,.Whitehead Institute Bio Imaging Center,
Massachusetts Institute of Technology
Marilyn Gist Farquhar, Uniuersity of California, San Diego
X PREFACE
Xavier Fernandez-Busquets, Bioengineering Instituteof
Catalonia, Uniuersitatde Barcelona,Spain
TerrenceG. Frey,San Diego State Uniuersity
Margaret T. Fuller,Stanford UniuersitySchoolof Medicine
KendraJ. Golden, WbitmanCollege
David S. Goldfarb,RochesterUniuersity
Martha J. Grossel,ConnecticutCollege
LawrenceI. Grossman,WayneStateUniuersitySchoolof Medicine
Michael Grunstein, Uniuersityof California, Los Angeles,
Schoolof Medicine
Barry M. Gumbiner, Uniuersityof Virginia
'Wei
Guo, Uniuersityof Pennsyluarua
Leah Haimo, Uniuersityof California, Riuerside
Heidi E. Hamm, Vanderbilt(JniuersityMedicalSchool
Craig M. Hart, Louisiana State Uniuersity
Merill B. Hille, Uniuersityof Washington
Jerry E. Honts, Drake Uniuersity
H. Robert Horvitz, Massachusetts Institute of Technology
Richard Hynes, Massachusetts lnstitute of Technologyand
Howard HughesMedical Inshtute
Harry Itagaki,Kenyon College
ElizabethR. Jamieson,Smitb College
Marie A. Janicke,State Uniuersityof New York, Buffalo
Bradley'W.Jones,Uniuersityof Mississippi
Mark Kainz, ColgateUniuersity
Naohiro Kato, Louisiana State Uniuersity
Amy E. Keating, Massachusetts lnstitute of Technology
CharlesH. Keith, Uniuersityof Georgia
Thomas C. S. Keller lll, Florida State Uniuersity
'V/estern
Greg M. Kelly, Uniuersityof Ontario
StephenKendall, California State Uniuersity,Fullerton
FelipeKierszenbaum, MichiganStateUniuersity
Cindy Klevickis,JamesMadison lJniuersity
Brian Kobilka, StanfordUniuersityMedicalSchool.
Martina Koniger, WellesleyUniuersfiy
CatherineKoo, Caldwell College
Keith G. Kozminski, Uniuersityof Virginia
StevenI(. IJHernault, Emory Uruuerstty
Douglas Lauffenburger, Massacbusetts Institute of Tecbnology
RobertJ. Lefkowitz, HHMI and Duke UniuersityMedical School
R. L. Levine,McGill Uniuersity
FangJu Ltn, CoastalCarolina [Jniuersity
ElizabethLord, Uniuersityof California, Riuerside
Liqun Luo, StanfordUniuersity
Grant MacGregor, Uniuersityof California, Iruine
Jennifer O. Manilay, IJniuersityof California, Merced
Barry Margul ies,Towson Uniuer stty
C. William McCurdy, Uniuersityof California, Dauis,
and LawrenceBerkeleyNational Laboratory
Dennis \il/. McGee, State Uniuersityof New York, Binghamton
JamesMcGrath, RochesterSchoolof Medicine
David D. McKemy, Uniuersityof SouthernCalifornia
Roderick MacKinnon, Rockefeller(Jniuersity
JamesA. McNew, Rice Uniuersity
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CONTENTS IN BRIEF

Part I Chemical
and MolecularFoundations1
1. L i f e B e g i n sw i th C e l l s 1
2. C h e mi caFl o u n d a ti o n s3 1
3. ProteinStructureand Function 63

Part ll Genetics
and MolecularBiology 111
4. B a s i cM o l e c u l aG
r e n e t i cM e c h a n i s m1s 1 1
5. M o l e cu l a Ge
r n e ti cT e ch n i ques1G5
6. G e n e s,Ge n o mi cs, a n d C h rom osomes 215
7. Transcriptional Controlof Gene Expression269
8. Post-transcriptional GeneControl 323

Part lll CeflStructureand Function 371

9. V i su a l i zi n gF, ra cti o n a ti n g, and Cultur ingCells 371
10. BiomembraneStructure 409
11. Transmembrane Transportof lonsand small Molecures437
12. C e ll u l a rE n e rg e ti cs4 7 9
13. M o vi n gP ro te i n si n to Me mbr anes and Or ganelles533
14. VesicularTraffic,Secretion,and Endocytosis579
15. C e llS i g n a l i n gl : S i g n a Tl ra nsduction and Shor t- Ter m
CellularResponses 62 3
16. c e l l si g n a l i n gl l : si g n a l i n gPathwaysThat contr ol GeneActivity 665
17. C e l lOrg a n i za ti o n a n d Mo v ementl: M icr ofilaments713
18. C e l lOrg a n i za ti o n a n d Mo v ementll: Micr otubules and Inter m ediate
Filaments157
19. I n t e g ra ti n gC e l l si n to T i ssues801

Part lV CellGrowth and Development847

20. Regulatingthe EukaryoticCellCycle 847
21. C e l lB i rth ,L i n e a g ea, n d D e ath 905
22. T h e Mo l e cu l a C r e l lB i o l o g yof Developm entg4g
23. N e rveC e l l s 1 0 0 1
24. l m m u n o l o g y1 0 5 5
25. Cancer 1107
 CONTENTS

and MolecularFoundations
PartI Chemical

C e l l sG r o w a n d D i v i d e 18
1 L I F EB E G I N S
WITHCELLS CellsDie from AggravatedAssaultor an Internal Program 19

E The Diversityand Commonality InvestigatingCellsand Their Parts 20

of Cells 1 !!|
Cell BiologyRevealsthe Size,Shape,Location,
All CellsAre Prokaryoticor Eukaryotic 1 and Movementsof Cell Components 20
r r g a n i s m sH e l p a n d H u r t U s
U n i c e l l u l aO 4 Biochemistryand BiophysicsRevealthe Molecular
VirusesAre the Ultimate Parasites 6 Structureand Chemistryof PurifiedCell Constituents 21
C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n 6 of DamagedGenes 22
GeneticsRevealsthe Consequences
EvenSingleCellsCan HaveSex 7 GenomicsRevealsDifferencesin the Structure
of EntireGenomes
and Expression 23
We Developfrom a SingleCell 8
DevelopmentalBiologyRevealsChangesin the
Stem Cells,Fundamentalto FormingTissues 23
Propertiesof Cellsas They Specialize
and Organs,Offer MedicalOpportunities
Choosingthe Right ExperimentalOrganismfor the Job 25
BiologicalStudiesUse Multiple
The Most Successful
@The M o l e c u l eos f a c e l l Approaches 27

Small MoleculesCarryEnergy,TransmitSignals,
a n d A r e L i n k e di n t o M a c r o m o l e c u l e s ' on Evolution 28
A GenomePerspective
s|
ProteinsGive CellsStructureand Perform
Most CellularTasks 10 Metabolic Proteins,the GeneticCode,and Organelle
StructuresAre NearlYUniversal 28
NucleicAcidsCarryCoded Information for Making
't1 Darwin'sldeasAbout the Evolutionof Whole Animals
Proteinsat the Right Time and Place
Are Relevantto Genes
The Genome ls Packagedinto Chromosomesand
R e p l i c a t e dD u r i n gC e l lD i v i s i o n 12 Many GenesControllingDevelopmentAre Remarkably
S i m i l a ri n H u m a n sa n d O t h e r A n i m a l s 28
Mutations May Be Good, Bad, or lndifferent 13
Human Medicinels Informed by Researchon Other
Organisms 29

E Thework of cells 14

C e l l sB u i l da n d D e g r a d eN u m e r o u sM o l e c u l e s
and Structures 15 FOUNDATIONS
2 CHEMICAL 31
Animal CellsProduceTheir Own External
E n v i r o n m e nat n d G l u e s 16
CellsChangeShapeand Move 16 ![ covalent Bondsand Noncovalent
lnteractions 32
CellsSenseand Send Information 16
CellsRegulateTheir Gene Expression to Structureof an Atom Determines
TheElectronic the
M e e t C h a n g i n gN e e d s 17 Numberand Geometry of CovalentBondslt CanMake 33

CONTENTS .
 E l e c t r o n sM a y B e S h a r e dE q u a l l yo r U n e q u a l l y L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l eC h e m i c a l
i n C o v a l e n tB o n d s 34 Reactionswith EnergeticallyFavorableReactions 57
CovalentBondsAre Much Strongerand More Hydrolysisof ATPReleases
SubstantialFreeEnergy
StableThan NoncovalentInteractions and DrivesMany CellularProcesses 57
lonic InteractionsAre Attractionsbetween Oppositely ATPls GeneratedDuring Photosynthesis
and Respiration 59
C h a r g e dl o n s 36 N A D - a n d F A DC o u p l eM a n y B i o l o g i c aO
l xidation
HydrogenBondsDeterminethe Water Solubility and ReductionReactions
o f U n c h a r g e dM o l e c u l e s 37
Van der Waals InteractionsAre Causedby
T r a n s i e nD
t iooles 37
The HydrophobicEffectCausesNonpolar
3 P R O T E ISNT R U C T U R
ANED
Moleculesto Adhere to One Another 38 FUNCTION 63
M o l e c u l a rC o m p l e m e n t a r i tM y e d i a t e dv i a
NoncovalentInteractionspermitsTight,
H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s 3e ![ Structureof proteins
Hierarchical 64
The PrimaryStructureof a Protein ls lts Linear
Arrangementof Amino Acids 65
@ C h e m i c alB u i l d i n gB l o ckso f C e i l s 40 SecondaryStructuresAre the Core Elements
A m i n o A c i d sD i f f e r i n gO n l y i n T h e i rS i d eC h a i n s of ProteinArchitecture 66
ComposeProteins 41 Overall Foldingof a PolypeptideChainYields
FiveDifferent NucleotidesAre Usedto Build Its TertiaryStructure 67
N u c l e i cA c i d s Different Waysof Depictingthe Conformationof
MonosaccharidesJoined by GlycosidicBonds ProteinsConveyDifferent Typesof Information 68
Form Linearand Branchedpolysaccharides 44 StructuralMotifs Are RegularCombinationsof
PhospholipidsAssociateNoncovalentlyto Form Secondaryand TertiaryStructures 68
the BasicBilayerStructureof Biomembranes S t r u c t u r aal n d F u n c t i o n aD
l o m a i n sA r e M o d u l e s
of TertiaryStructure 70
ProteinsAssociateinto Multimeric Structuresand
![ C h e m i c aEl q u i l i b ri u m 49 M a c r o m o l e c u l aAr s s e m b l i e s 72
EquilibriumConstantsReflectthe Extentof a Membersof Protein FamiliesHavea Common
ChemicalReaction 50 EvolutionaryAncestor 72
ChemicalReactionsin CellsAre at SteadyState 50
DissociationConstantsof Binding ReactionsReflect
the Affinity of InteractingMolecules ![ ProteinFolding 74
50
BiologicalFluidsHaveCharacteristic
pH Values 51 P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o
Which ProteinsCan Fold 74
Hydrogenlons Are Releasedby Acidsand
Taken Up by Bases 52 I n f o r m a t i o nD i r e c t i n ga P r o t e i n ' sF o l d i n gl s
E n c o d e di n l t s A m i n o A c i d S e q u e n c e
74
B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l aar n d
E x t r a c e l l u l aFrl u i d s 52 Foldingof Proteinsin Vivo ls Promotedby Chaperones 7 5
AlternativelyFoldedProteinsAre lmplicatedin Diseases 77

@ B i o c h e m i caEl n e rg e ti cs 54
SeveralFormsof EnergyAre lmportant in !f, ProteinFunction 78
BiologicalSystems 54 S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e
CellsCan TransformOne Typeof Energyinto Another 55 Functionsof Most Proteins 7g
The Changein Free EnergyDeterminesthe Direction EnzymesAre Highly Efficientand SpecificCatalysts 79
of a ChemicalReaction 55 An Enzyme'sActive Site BindsSubstratesand
The AG'' of a ReactionCan Be Calculated CarriesOut Catalysis g0
from lts K"o 56 SerineProteasesDemonstrateHow an
The Rateof a ReactionDependson the Activation Enzyme'sActive Site Works g1
EnergyNecessary to Energizethe Reactantsinto Enzymesin a Common PathwayAre Often physically
a TransitionState 56 Associatedwith One Another g4

xvi . coNTENTs
EnzymesCalledMolecularMotors ConvertEnergy Purifying,Detecting,and
into Motion 85 CharacterizingProteins 92
CentrifugationCan SeparateParticlesand
!E- r.r
RegulatingProteinFunctionl: MoleculesThat Differ in Massor Density 92
!l|
ProteinDegradation 86 ElectrophoresisSeparatesMoleculeson the Basis
Ratio
of Their Charge-to-Mass 94
RegulatedSynthesisand Degradationof Proteins
is a FundamentalPropertyof Cells 86 Liquid ChromatographyResolvesProteinsby
Mass,Charge,or BindingAffinitY 96
The Proteasomels a ComplexMolecularMachine
Usedto DegradeProteins Highly SpecificEnzymeand Antibody AssaysCan
Detect IndividualProteins 98
Ubiquitin Marks CytosolicProteinsfor Degradation
88 Radioisotopes Are IndispensableToolsfor Detecting
in Proteasomes
BiologicalM o l e c u l e s 99

MassSpectrometryCan Determinethe Mass

RegulatingProteinFunctionll: and Sequenceof Proteins 101
Noncovalentand Covalent Protein PrimaryStructureCan Be Determinedby
Modifications 88 ChemicalMethods and from Gene Sequences 103

NoncovalentBinding PermitsAllosteric, Protein Conformationls Determinedby Sophisticated

PhysicalMethods 103
or Cooperative,Regulationof Proteins 89
NoncovalentBindingof Calciumand GTPAre Widely
UsedAs AllostericSwitchesto Control ProteinActivity 90 105
g Proteomics
Phosphorylationand DephosphorylationCovalently
RegulateProteinActivitY 91 Proteomicsls the Studyof All or a LargeSubset
of Proteinsin a BiologicalSYstem 105
ProteolyticCleavagelrreversiblyActivatesor
lnactivatesSome Proteins 91 AdvancedTechniquesin MassSpectrometryAre
Criticalto ProteomicAnalYsis 106
Higher-OrderRegulationIncludesControl of Protein
Locationand Concentration 92

Partll Geneticsand MolecularBiologY

Organizationof GenesDiffersin Prokaryoticand

4 B A S I CM O L E C U L AGRE N E T I C EukaryoticDNA 122
MECHANISMS 111 EukaryoticPrecursormRNAsAre Processed to
F o r mF u n c t i o n am
l RNAs 123
AlternativeRNA Splicinglncreases the Number of
structureof NucleicAcids 113 ProteinsExpressed from a SingleEukaryoticGene 125
@
A NucleicAcid Strand ls a LinearPolymerwith
End-to-EndDirectionality 113 @ The Decodingof mRNAbY tRNAs 127
N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y MessengerRNA CarriesInformation from DNA in a
A n t i p a r a l l eS
l trands 114 Three-LetterGeneticCode 127
DNA Can Undergo ReversibleStrandSeparation 115 The FoldedStructureof IRNA Promoteslts Decoding
117 Functions 129
TorsionalStressin DNA ls Relievedby Enzymes
Different Typesof RNA ExhibitVarious NonstandardBasePairingOften OccursBetween
118 C o d o n sa n d A n t i c o d o n s 130
ConformationsRelatedto Their Functions
Amino Acids BecomeActivatedWhen Covalently
L i n k e dt o t R N A s 131
@ T r a n s c r i p t i o n o f P ro te i n -C o d i ng
Genesand Formationof Functional
MRNA 120 @ StepwiseSynthesisof Proteins
on Rabosomes 132
A TemplateDNA Strand ls Transcribedinto a
120 Machines
RibosomesAre Protein-Synthesizing 132
ComplementaryRNA Chain by RNA Polymerase

CONTENTS XVII
 Methionyl-tRNA,tttRecognizes the AUG StartCodon 133
T r a n s l a t i o nI n i t i a t i o nU s u a l l yO c c u r sa t t h e F i r s t 5 M O L E C U L AGRE N E T T C
E C H N T Q U1E6Ss
A U G f r o m t h e 5 ' E n do f a n m R N A 133
D u r i n gC h a i nE l o n g a t i o nE a c hl n c o m i n g
Aminoacyl-tRNAMovesThrough Three E! GeneticAnalysisof Mutationsto
RibosomalSites ldentifyand StudyGenes 166
135
Translationls Terminatedby ReleaseFactors R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e sG e n e r a l l y
When a Stop Codon ls Reached 137 HaveOppositeEffectson Gene Function 166
Polysomesand RapidRibosomeRecyclingIncrease Segregationof Mutations in BreedingExperiments
the Efficiencyof Translation 138 RevealsTheir Dominanceor Recessivity 167
ConditionalMutations Can Be Usedto Study
EssentialGenesin Yeast EO

!f| DNAReptication 139 Recessive Lethal Mutations in DiploidsCan Be

l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e di n
s e q u i r ea p r i m e r t o I n i t i a t e
D N A P o l y m e r a s eR Heterozygotes 171
Replication 140 ComplementationTestsDetermineWhether Different
Duplex DNA ls Unwound and Daughter StrandsAre RecessiveMutations Are in the SameGene 't7
1
Formedat the DNA ReplicationFork 't41
Double Mutants Are Usefulin Assessingthe Order
SeveralProteinsParticipatein DNA Replication 141 in Which ProteinsFunction 171
D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l lfyr o m GeneticSuppressionand SyntheticLethalityCan
E a c hO r i g i n 143 RevealInteractingor Redundantproteins 173
GenesCan Be ldentified by Their Map position
on the Chromosome 174
!!| D N A R e p a i ra n d R e co mb i n a ti o n 145
DNA Polymerases IntroduceCopyingErrors p| DNACloningand Char acter i z ati on176
and Also CorrectThem 145
RestrictionEnzymesand DNA LigasesAllow
C h e m i c aal n d R a d i a t i o nD a m a g et o D N A C a n
Insertionof DNA Fragmentsinto CloningVectors 176
Leadto Mutations 145
E. coli PlasmidVectorsAre Suitablefor Cloning
H i g h - F i d e l i tD
y N A E x c i s i o n - R e p aSiyr s t e m s
lsolatedDNA Fragments 178
Recognize a n d R e p a i rD a m a g e 147
cDNA LibrariesRepresentthe Sequences
of
B a s eE x c i s i o n R e p a i r sT . G M i s m a t c h eas n d Protein-CodingGenes 179
D a m a g e dB a s e s 147
cDNAsPreparedby ReverseTranscriptionof Cellular
MismatchExcisionRepairsOther Mismatchesand
mRNAsCan Be Clonedto GeneratecDNA Libraries 1 8 1
S m a l lI n s e r t i o n a
s nd Deletions 147
DNA LibrariesCan Be Screenedby Hybridization
NucleotideExcisionRepairsChemicalAdducts
to an Oligonucleotidperobe 181
That Distort Normal DNA Shape 148
YeastGenomicLibrariesCan Be Constructedwith
Two SystemsUtilize Recombinationto Repair
ShuttleVectorsand Screenedby Functionar
D o u b l e - S t r a nB
d r e a k si n D N A 149 Complementation 182
H o m o l o g o u sR e c o m b i n a t i oC
n a n R e o a i rD N A Gel Electrophoresis
Allows Separationof Vector
D a m a g ea n d G e n e r a t eG e n e t i cD i v e r s i t y 150 DNA from Cloned Fragments 184
Cloned DNA MoleculesAre SequencedRapidly
by the DideoxyChain-TerminationMethoc 187
@ V i r u s e s : pa ra si te o
s f th e C e l l u l a r The PolymeraseChain ReactionAmplifiesa Specific
GeneticSystem 154 DNA Sequencefrom a ComplexMixture 188
Most Viral Host RangesAre Narrow 154
Viral CapsidsAre RegularArraysof One or a UsingClonedDNA Fragments
Few Typesof protein
ff|
154 to Study GeneExpression 191
VirusesCan Be Clonedand Counted in plaqueAssays 1 5 5
HybridizationTechniquespermit Detection
LyticViral Growth CyclesLeadto the Death of Host Cells 1 5 5 't91
of SpecificDNA Fragmentsand mRNAs
Viral DNA ls Integrated into the Host-CellGenome
DNA MicroarraysCan Be Usedto Evaluatethe
in SomeNonlyticViral Growth Cycles 158 Expression
of Many Genesat One Time 192

XVIII ' CONTENTS

ClusterAnalysisof Multiple ExpressionExperiments g e n e sE n c o d eF u n c t i o n a l
N o n p r o t e i n - C o d i nG
ldentifies Co-regulatedGenes 193 RNAs 222
SystemsCan ProduceLarge
E. coli Expression
Quantitiesof Proteinsfrom ClonedGenes
Or ganization
Chr om osomal
PlasmidExpression VectorsCan Be Designedfor 223
U s ei n A n i m a l C e l l s 196
of Genesand NoncodingDNA
s o n t a i nM u c h
G e n o m e so f M a n y O r g a n i s m C
N o n f u n c t i o n aD
l NA
f[ l d e n t i f y in ga n d L o ca ti n gH u ma n Most Simple-SequenceDNAsAre Concentrated
Disease Ge n e s 198 in SpecificChromosomalLocations 224

Many InheritedDiseasesShow One of Three Major DNA FingerprintingDependson Differences

199 DNAs
in Length of SimPle-Sequence 225
Patternsof Inheritance
D N A P o l y m o r p h i s mAsr e U s e di n L i n k a g e - M a p p i n g SpacerDNA Occupiesa Significant
Unclassified
200 Portion of the Genome 225
H u m a nM u t a t i o n s
LinkageStudiesCan Map DiseaseGeneswith a
Resolutionof About 1 Centimorgan 201 (Mobile)DNA
]f| Transposable
FurtherAnalysisls Neededto Locatea DiseaseGene Elem ents 226
i n C l o n e dD N A 202
M a n y I n h e r i t e dD i s e a s eRs e s u l ft r o m M u l t i p l eG e n e t i c Movement of Mobile ElementsInvolvesa
D N A o r a n R N AI n t e r m e d i a t e 226
Defects 203
DNA TransposonsAre Presentin Prokaryotes
and Eukaryotes 227

ffl Inactivatingthe Functionof LTRRetrotransposons BehaveLike Intracellular

S p e c i f i cGe n e si n E u ka ryo te s 204 Retroviruses 229
Non-LTRRetrotransposons Transposeby a Distinct
Normal YeastGenesCan Be Replacedwith Mutant 230
Mechanism
A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n 205
Other Retrotransposed RNAsAre Found in Genomic
Transcriptionof GenesLigatedto a Regulated 234
DNA
PromoterCan Be ControlledExperimentally
M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l Iyn f l u e n c e d
SpecificGenesCan Be PermanentlyInactivatedin 234
Evolution
t h e G e r m L i n eo f M i c e 207
SomaticCell RecombinationCan InactivateGenes
in SpecificTissues 208 or ganelleDNAs 236
@
D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y
M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N A M o l e c u l e s 237
l n h i b i t S o m eG e n e s
mtDNA ls InheritedCytoplasmically 237
RNA InterferenceCausesGene Inactivationby
D e s t r o y i n gt h e C o r r e s p o n d i nm
g RNA 210 The Size,Structure,and Coding Capacityof mtDNA
Vary ConsiderablyBetweenOrganisms 238

Productsof MitochondrialGenesAre Not Exported 240

M i t o c h o n d r i aE v o l v e df r o m a S i n g l eE n d o s y m b i o t i c
, E N O M I C SA, N D
6 G E N E SG EventInvolvinga Rickettsia-like Bacterium 240
CHROMOSOMES 215 MitochondrialGeneticCodesDiffer from the
StandardNuclearCode 240
Mutations in MitochondrialDNA CauseSeveral
EukaryoticGeneStructure 217
][ G e n e t i cD i s e a s eisn H u m a n s
Most EukaryoticGenesContain Intronsand Produce ChloroplastsContain LargeDNAsOften Encoding
m R N A sE n c o d i n gS i n g l eP r o t e i n s 217 M o r e T h a n a H u n d r e dP r o t e i n s 242
S i m p l ea n d C o m p l e xT r a n s c r i p t i oU
n nits
A r e F o u n di n E u k a r y o t i cG e n o m e s 217
Genome- wide
Genom ics: Anal Ys i s
g e n e sM a y B e S o l i t a r yo r B e l o n g
P r o t e i n - C o d i nG
219
of GeneStructureand Expression 243
t o a G e n eF a m i l y
HeavilyUsedGene ProductsAre Encodedby Multiple StoredSequences SuggestFunctionsof Newly
221 ldentified Genesand Proteins 243
Copiesof Genes

CONTENTS xtx
 Comparisonof RelatedSequences from Different S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i oonf M a n y
SpeciesCan Give Cluesto Evolutionary BacterialGenesvia DNA-BindingRepressors
Among proteins
Relationships 244 and Activators 273
G e n e sC a n B e l d e n t i f i e dW i t h i n G e n o m i cD N A TranscriptionInitiation from Some Promoters
Sequences 244 RequiresAlternativeSigmaFactors 273
T h e N u m b e ro f P r o t e i n - C o d i nG
g e n e si n a n Transcriptionby osa-RNAPolymerasels Controlled
Organism'G s e n o m el s N o t D i r e c t l yR e l a t e d by ActivatorsThat Bind Farfrom the Promoter 274
to lts BiologicalComplexity
Many BacterialResponses
Are Controlledby
S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n eC o p y - Two-ComponentRegulatorySystems 275
Number VariationAre lmportant Determinants
of DifferencesBetween Individualsof a Species 246

@ overview of EukaryoticGene
St r u c t u r alOrg a n i za ti o n Controland RNAPolymerases 276
][
of EukaryoticChromosomes 247 RegulatoryElementsin EukaryoticDNA Are Found
Both Closeto and Many KilobasesAway from
ChromatinExistsin Extendedand CondensedForms 248 TranscriptionStart Sites 276
Modificationsof HistoneTailsControl Chromatin
Three EukaryoticPolymerases
CatalyzeFormation
Condensationand Function 250 of Different RNAs 278
NonhistoneProteinsProvidea StructuralScaffold
The LargestSubunit in RNA Polymerasell Hasan
for Long ChromatinLoops 254 EssentialCarboxyl-TerminalRepeat 279
A d d i t i o n a lN o n h i s t o n ep r o t e i n sR e g u l a t e
RNA Polymerasell InitiatesTranscriptionat DNA
Transcriptionand Replication 2s6 SequencesCorrespondingto the 5' Cap of mRNAs 280

@ M o r p h o l og ya n d F u n cti o n aEl l e m ents

o f E u k a r y o ti C
c h ro mo so me s E in pr ote i n-
Regulator ysequences
257 CodingGenes 282
ChromosomeNumber;Size,and Shapeat Metaphase
Are Species-Specific The TATABox, Initiators,and CpG lslandsFunction
257 as Promotersin EukaryoticDNA 282
During Metaphase,ChromosomesCan Be
D i s t i n g u i s h ebdy B a n d i n gp a t t e r n sa n d Promoter-Proximal
ElementsHelp Regulate
EukaryoticGenes 282
C h r o m o s o m eP a i n t i n g 25g
C h r o m o s o m eP a i n t i n ga n d D N A S e q u e n c i n g Distant EnhancersOften Stimu late Transcription
Reveal
the Evolutionof Chromosomes by RNA Polymerasell 284
259
InterphasePolyteneChromosomesArise by DNA Most EukaryoticGenesAre Regulatedby Multiple
A m p l i fi c a t i o n Transcription-Control
Elements
260
Three FunctionalElementsAre Requiredfor Replication
a n d S t a b l eI n h e r i t a n c eo f C h r o m o s o m e s 261
CentromereSequences Vary Greatlyin Length 263 @Activator s and Repr essorof
s
Transcription 286
Addition of TelomericSequencesby Telomerase
PreventsShorteningof Chromosomes 263 Footprintingand Gel-ShiftAssaysDetect protein-DNA
Interactions 286
ActivatorsAre Modular ProteinsComposed
of DistinctFunctionalDomainsand promote
7 TRANSCRIPTIONALCONTROL Transcription 288
O F G E N EE X P R E S S I O N 269 Repressors
Inhibit Transcriptionand Are the
FunctionalConverseof Activators 290
D N A - B i n d i n gD o m a i n sC a n B e C l a s s i f i eidn t o
W Controlof GeneExpression
in NumerousStructuralTypes 290
Bacteria 271 StructurallyDiverseActivation and Repression
TranscriptionInitiation by BacterialRNA polymerase DomainsRegulateTranscription 293
RequiresAssociationwith a SigmaFactor 271 TranscriptionFactorInteractionsIncrease
Initiation of /ac Operon TranscriptionCan Be Gene-ControlOptions 294
Repressed and Activated 271 M u l t i p r o t e i nC o m p l e x e F
s o r mo n E n h a n c e r s 295

XX O CONTENTS
 TranscriptionInitiation by RNA Mitochondrialand ChloroplastDNAsAre
RNA
Transcribedby Organelle-Specific
Polymerasell 296
Polymerases 317
GeneralTranscriptionFactorsPositionRNA
Polymerasell at Start Sitesand Assistin Initiation 296
SequentialAssemblyof ProteinsFormsthe Pol ll
TranscriptionPreinitiationComplexin Vitro GENE
8 POST- TRANSCRIPTIONAL
In Vivo TranscriptionInitiation by Pol ll Requires CONTROL 323
Additional Proteins 298

f[ of EukaryoticPre-mRNA325
Processing
@ M o l e c u l a Me o f T ra n sc r iption
r ch a n i sms
and Activation 299 The 5' Cap ls Added to NascentRNAsShortlyAfter
Repression 325
TranscriptionInitiation
Formationof HeterochromatinSilencesGene A DiverseSet of Proteinswith ConservedRNA-B|nding
Expressionat Telomeres,Near Centromeres, DomainsAssociatewith Pre-mRNAs 326
and in Other Regions 299
SplicingOccursat Short,ConservedSequencesin
Can Direct HistoneDeacetylationand
Repressors Pre-mRNAsvia Two TransesterificationReactions 329
Methylation at SpecificGenes 303
with Pre-mRNA
During Splicing,snRNAsBase-Pair 330
ActivatorsCan Direct HistoneAcetylationand
305 Spliceosomes,Assembledfrom snRNPsand a
Methylation at SpecificGenes 330
Pre-mRNA,CarryOut SPlicing
Chromatin-Remodeling FactorsHelp Activate or
RepressTranscription 306 C h a i nE l o n g a t i o nb y R N AP o l y m e r a slel l s C o u p l e d
to the Presenceof RNA-Processing Factors 333
HistoneModificationsVary Greatlyin Their Stabilities 307
SRProteinsContributeto Exon Definition in Long
The Mediator ComplexFormsa MolecularBridge Pre-mRNAs
BetweenActivation Domainsand Pol ll 307
Group ll Introns ProvideCluesto the
Self-Splicing
Transcriptionof Many GenesRequiresOrdered Binding Evolutionof snRNAs 334
and Functionof Activatorsand Co-activators 308
3' Cleavageand Polyadenylationof Pre-mRNAs
The YeastTwo-HybridSystemExploitsActivator Flexibility Are Tightly CouPled 335
to DetectcDNAsThat EncodeInteractingProteins 310
NuclearExonucleases DegradeRNAThat ls Processed
Out of Pre-mRNAs 336

E Regulationof Transcription-Factor
Activity 311
Regulation of Pre-mRNA
All NuclearReceptorsSharea Common Domain Processing 337
Structure 312
AlternativeSplicingls the PrimaryMechanismfor
Nuclear-ReceptorResponseElementsContain Inverted 337
RegulatingmRNA Processing
or Direct Repeats 313
A Cascadeof RegulatedRNA SplicingControls
Hormone Bindingto a NuclearReceptorRegulateslts 338
DrosophilaSexualDifferentiation
Activity as a TranscriptionFactor 313
SplicingRepressorsand ActivatorsControl Splicing
at AlternativeSites 339

![ RegulatedElongationand of
RNA Editing Alters the Sequences
Terminationof Transcription 314 SomePre-mRNAs 340

Transcriptionof the HIV Genome ls Regulatedby

an Antitermination Mechanism 315
]f| Transportof mRNAAcrossthe
Pausingof RNA Polymerasell
Promoter-Proximal NuclearEnveloPe 341
Occursin Some RapidlyInducedGenes 316
NuclearPore ComplexesControl lmport and Export
from the Nucleus 342
@ other EukaryoticTranscription Pre-mRNAs in SpliceosomesAre Not Exportedfrom
Systems 315 the Nucleus 345
TranscriptionInitiation by Pol I and Pol lll ls HIV Rev Protein Regulatesthe Transportof Unspliced
Analogousto That by Pol ll 316 Viral mRNAs 346

CONTENTS ' xxi

 f!| CytoplasmicMechanisms of post- Localizationof mRNAsPermitsProductionof
transcriptionalControl Proteinsat SpecificRegionsWithin the
347
Cytoplasm 357
Micro RNAsRepressTranslationof SpecificmRNAs 347
RNA InterferenceInducesDegradationof precisely
ComplementarymRNAs 349 f[ Processing
of rRNAand IRNA 358
CytoplasmicPolyadenylationpromotesTranslation Pre-rRNAGenesFunctionas NucleolarOrganizers
of SomemRNAs 351 a n d A r e S i m i l a ri n A l l E u k a r y o t e s 359
Degradationof mRNAsin the CytoplasmOccurs Small NucleolarRNAsAssistin Processing
Pre-rRNAs 360
by SeveralMechanisms 352 Self-Splicing
Group I IntronsWere the First
ProteinSynthesisCan Be GloballyRegulated 353 Examplesof CatalyticRNA 363
RNA-BindingproteinsControl
Sequence-Specific Pre-tRNAsUndergo ExtensiveModification in the
SpecificmRNATranslation 356 Nucleus 363
SurveillanceMechanismspreventTranslationof N u c l e a rB o d i e sA r e F u n c t i o n a l lS
y p e c i a l i z eN
d uclear
lmproperlyProcessedmRNAs 357 Domains 3G4

Partlll CellStructureand Function

Phase-Contrast and DifferentiallnterferenceContrast

9 V I S U A L I Z IN G,
F R A C T ION A T IN G, MicroscopyVisualizeUnstainedLivingCells 381
A N D C U L T U R I NC GE L L S 371 Fluorescence MicroscopyCan Localizeand euantify
S p e c i f i cM o l e c u l e si n L i v eC e l l s 382
l m a g i n gS u b c e l l u l aDr e t a i l sO f t e n R e q u i r e tsh a t t h e
lll Organellesof the EukaryoticCell 372 SamplesBe Fixed,Sectioned,and Stained
T h e P l a s m aM e m b r a n eH a sM a n y C o m m o nF u n c t i o n s lmmunofluorescence MicroscopyCan Detect Specific
in All Cells 372 Proteinsin FixedCells 38s
EndosomesTake Up 5oluble Macromolecules from Confocaland DeconvolutionMicroscopyEnable
the Cell Exterior 372 Visualizationof Three-Dimensional
Objects 386
LysosomesAre Acidic OrganellesThat Contain a Graphicsand lnformaticsHaveTransformed
Batteryof DegradativeEnzymes 373 Modern Microscopy
Peroxisomes
DegradeFattyAcidsand ToxicCompounds 374
The EndoplasmicReticulumls a Network of
InterconnectedInternal Membranes 375 Eil ElectronMicroscopy:
Methods
The Golgi ComplexProcesses and SortsSecreted and Applications 388
a n d M e m b r a n ep r o t e i n s 376 Resolutionof Transmission
ElectronMicroscopyis
P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e sa n d E n a b l ea VastlyGreaterThan That of Light Microscopy 3gg
C e l lt o E l o n g a t eR a p i d l y 377 CryoelectronMicroscopyAllows Visualizationof
T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A ParticlesWithout Fixationor Staining 399
SyntheticApparatus,and a FibrousMatrix 378 ElectronMicroscopyof Metal-CoatedSpecimens
MitochondriaAre the PrincipalSitesof ATp Can RevealSurfaceFeaturesof Cellsand Their
Productionin Aerobic NonphotosyntheticCells 378 Components 390
ChloroplastsContain Internal Compartmentsin
Which Photosynthesis Takesplace 379
!!| Purificationof Cell Organelles 391
g Disruptionof CellsReleases
Their Organelles
L i g h t M i cro sco p y:
vi su a l i zi n gC ell and Other Contents 391
Structureand Localizingproteins
W i t h i n C el l s CentrifugationCan SeparateMany Typesof
380 Organelles 392
The Resolutionof the Light Microscopels About Organelle-Specific AntibodiesAre Usefulin
0.2 pm 381 P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s 393

xxii . coNTENrs
 Lipid-Binding M o t i f s H e l pT a r g e tP e r i p h e r a l
l s o l a t i o nC , n d Differentiation
, u l tu re a 427
Proteinsto the Membrane
of MetazoanCells 394
ProteinsCan Be Removedfrom Membranes
Flow CytometrySeparatesDifferent CellTypes 394 by Detergentsor High-SaltSolutions 427
C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h
M e d i a a n d S p e c i aSl o l i dS u r f a c e s 395
P r i m a r yC e l lC u l t u r e sC a n B e U s e dt o S t u d yC e l l $f, sphingolipids,
Phospholipids,
Differentiation 396 andCholesterol:SYnthesis
P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v ea F i n i t e Movement
and Intracellular 429
L i f eS p a n 396
Fatty AcidsSynthesisls Mediated by Several
TransformedCellsCan Grow Indefinitelyin Culture 397 430
lmportant Enzymes
SomeCell LinesUndergo Differentiationin Culture 398
SmallCytosolicProteinsFacilitateMovement of
H y b r i dC e l l sC a l l e dH y b r i d o m a sP r o d u c eA b u n d a n t FattyAcids 430
M o n o c l o n aA l ntibodies 400
Incorporationof Fatty Acids into Membrane Lipids
HATMedium ls CommonlvUsedto lsolate TakesPlaceon OrganelleMembranes 431
HybridCells 402
s o v e P h o s p h o l i p i df sr o m O n e M e m b r a n e
F l i p p a s eM
Leafletto the OppositeLeaflet 431
organelles 407
Cnsstc ExprRturrur9.1 separating Cholesterolls Synthesizedby Enzymesin the Cytosol
and ERMembrane 432
Cholesterola nd Phospholi pids Are Transported
BetweenOrganellesby SeveralMechanisms 433
1 O B I O M E M B R A NS
ET R U C T U R E 409

s[ B i o m e mb ra n e L s:i p i dC o mp o sition
a n d S t r u ctu raOrg
l a n i za ti o n 411 1 1 T R A N S M E M B R A NTER A N S P O R T
P h o s p h o l i p i dSsp o n t a n e o u s lFyo r mB i l a y e r s 411 OF IONSAND SMALLM OLECU LES437
Phospholipid B i l a y e r sF o r ma S e a l e dC o m p a r t m e n t
S u r r o u n d i n ga n I n t e r n a lA q u e o u sS p a c e 411
s o n t a i nT h r e eP r i n c i p aC
B i o m e m b r a n eC l lasses E overview of Membrane
of Lipids 415 Transport 438
M o s t L i p i d sa n d M a n y P r o t e i n sA r e L a t e r a l l yM o b i l e O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s sM e m b r a n e s
in Biomembranes 416 b y S i m p l eD i f f u s i o n 438
L i p i dC o m p o s i t i o nI n f l u e n c etsh e P h y s i c a l Membrane ProteinsMediate Transportof
Propertiesof Membranes 418 Most Moleculesand All lons Across
Biomembranes 439
Lipid Compositionls Different in the Exoplasmic
and CytosolicLeaflets 419
s l u s t e rw i t h S p e c i f i c
C h o l e s t e r oal n d S p h i n g o l i p i dC
P r o t e i n si n M e m b r a n eM i c r o d o m a i n s 420
@ uniport Transportof Glucose
and Water 441

$[| Biomembranes:ProteinComponents SeveralFeaturesDistinguishUniport Transportfrom

and BasicFunctions 421 S i m p l eD i f f u s i o n 44'l

ProteinsInteractwith Membranesin Three GLUT1UniporterTransportsGlucoseinto Most

421 M a m m a l i a nC e l l s 442
Different Ways
Most TransmembraneProteinsHave s F a m i l yo f S u g a r -
T h e H u m a nG e n o m eE n c o d e a
M e m b r a n e - S p a n n i nagH e l i c e s TransportingGLUTProtetns 443

M u l t i p l e B S t r a n d si n P o r i n sF o r m TransportProteinsCan Be EnrichedWithin Artificial

424 M e m b r a n e sa n d C e l l s 443
M e m b r a n e - S p a n n i n"gB a r r e l s "
CovalentlyAttached HydrocarbonChainsAnchor OsmoticPressureCausesWater to Move Across
424 Membranes 4M
Some Proteinsto Membranes
A l l T r a n s m e m b r a nPer o t e i n sa n d G l y c o l i p i dA
s re AquaporinsIncreasethe Water Permeabilityof Cell
Membranes 4M
y r i e n t e di n t h e B i l a y e r
A s y m m e t r i c a l lO

CONTENTS O xxiii
 ATP-powered pumpsand the BacterialSymporterStructureRevealsthe
@
I n t r a c el l u l al ro n i cE n vi ro n me nt 447 Mechanismof SubstrateBinding 467
Na*-LinkedCa2*Antiporter ExportsCa2* from
Different Classes
of PumpsExhibitCharacteristic C a r d i a cM u s c l eC e l l s
Structuraland Functionalproperties 447
SeveralCotransportersRegulate
ATP-Poweredlon PumpsGenerateand
CytosolicpH 468
M a i n t a i nl o n i cG r a d i e n t sA c r o s sC e l l u l a r
Membranes A PutativeCation ExchangeProtein Playsa
448
K e y R o l ei n E v o l u t i o no f H u m a nS k i n
MuscleRelaxationDependson Ca2*ATpases That Pigmentation 469
Pump Ca" from the Cytosolinto the
S a r c o p l a s mR
i ce t i c u i u m NumerousTransportProteinsEnablePlant
449
Vacuolesto AccumulateMetabolitesand lons 469
CalmodulinRegulates the plasmaMembrane
Ca'* PumpsThat Control CytosolicCa2+
Concentrations 451
N a - / K - A T P a s eM a i n t a i n st h e I n t r a c e l l u l aN ft| Transeprrnerar
Transporr 470
r a*
a n d K * C o n c e n t r a t i o nisn A n i m a l C e l l s 452 Multiple TransportProteinsAre Neededto
V-ClassH* ATPases Maintain the Acidity of Move Glucoseand Amino AcidsAcross
Lysosomes and Vacuoles Epithelia 471
453
BacterialPermeases Are ABC proteinsThat SimpleRehydrationTherapyDependson the
lmport a Variety of Nutrientsfrom the OsmoticGradientCreatedby Absorption
Environment o f G l u c o s ea n d N a + 411
454
The Approximately50 MammalianABCTransporters ParietalCellsAcidify the StomachContentsWhile
PlayDiverseand lmportant Rolesin Cell and M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H 472
O r g a nP h y s i o l o g y 455
Cusslc ExprntueruT
11.1 stumbting
Upon
C e r t a i nA B CP r o t e i n s" F l i p ' ,p h o s p h o l i p i d s
ActiveTransport 477
and Other Lipid-SolubleSubstratesfrom
One Membrane Leafletto the Opposite
Leaflet 456

12 CELLULAR
ENERGETICS 479
El N o n g a te dto n C h a n n e l a
s n d th e
RestingMembrane potential 458
@ FirstStepsof Glucoseand Fatty
SelectiveMovement of lons Createsa
TransmembraneElectricpotential
Acid CatabolismGlycolysis
:
Difference 458
and the CitricAcid €ycle 480
T h e M e m b r a n eP o t e n t i a li n A n i m a l C e l l sD e p e n d s (Stagel), CytosolicEnzymes
During Glycolysis
Largelyon Potassiumlon MovementsThrough ConvertGlucoseto Pyruvate 481
O p e n R e s t i n gK + C h a n n e l s 460 The Rate of Glycolysis
ls Adjustedto Meet
lon ChannelsContain a SelectivityFilter Formed the Cell'sNeed for ATP 483
from ConservedTransmembraneSegments 461 Glucosels FermentedUnder AnaerobicConditions 485
PatchClampsPermit Measurementof lon
U n d e r A e r o b i cC o n d i t i o n s ,M i t o c h o n d r i a
M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s 463 E f fi c i e n t l yO x i d i z eP y r u v a t ea n d G e n e r a t e
Novel lon ChannelsCan Be Characterizedby a ATP (Stagesll-lV) 485
Combinationof Oocyte Expressionand MitochondriaAre DynamicOrganelleswith Two
P a t c hC l a m p i n g 464 Structurallyand FunctionallyDistinctMembranes 485
N a - E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e
In Stagell, Pyruvatels Oxidizedto CO2and High-
Changein FreeEnergy(AG) 464 EnergyElectronsStored in ReducedCoenzymes 487
Transportersin the Inner MitochondrialMembrane
H e l p M a i n t a i nA p p r o p r i a t eC y t o s o l i a
c nd Matrix
@| Cotransportby symportersand Concentrationsof NAD* and NADH
Antiporters 465 MitochondrialOxidation of Fatty AcidsGenerates
N a * - L i n k e dS y m p o r t e r lsm p o r t A m i n o A c i d sa n d ATP
G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h PeroxisomalOxidation of Fatty AcidsGenerates
ConcentrationGradients 466 No ATP 491

xxiv . coNTENTs
 El e ctro nT ra n sp o rtch a i n a n d PhotoelectronTransportfrom EnergizedReaction-
@ Th e CenterChlorophylla Producesa Charge
Generationof the Proton-Motive Separation 514
Force 493
I n t e r n a lA n t e n n a a n d L i g h t - H a r v e s t i n g
StepwiseElectronTransportEfficientlyReleases
the C o m p l e x e sI n c r e a s et h e E f f i c i e n c yo f
EnergyStored in NADH and FADH2 493 Photosynthesis 515
ElectronTransportin Mitochondria ls Coupledto
P r o t o nP u m p i n g 493
ElectronsFlow from FADH2and NADHto 02
MolecularAnalysisof
Through Four Multiprotein Complexes 494 Photosystems 517
ReductionPotentialsof ElectronCarriersFavor The SinglePhotosystemof PurpleBacteria
ElectronFlow from NADH to 02 499 Generatesa Proton-MotiveForcebut No 02 517
ExperimentsUsing PurifiedComplexesEstablished LinearElectronFlow Through Both Plant Photosystems,
the Stoichiometryof Proton Pumping 499 PSlland PSl,Generatesa Proton-MotiveForce,
02, and NADPH 519
The Q CycleIncreases the Numberof Protons
Translocated as ElectronsFlow Through g o m p l e xl s L o c a t e do n
A n O x y g e n - E v o l v i nC
C o m p l e xl l l 500 t h e L u m i n a lS u r f a c eo f t h e P S l lR e a c t i o n
Center 520
The Proton-MotiveForcein Mitochondria ls Due
Largelyto a Voltage GradientAcrossthe Inner CellsUse Multiple Mechanismsto ProtectAgainst
Membrane 502 Damagefrom ReactiveOxygenSpeciesDuring
PhotoelectronTransPort 521
ToxicBy-productsof ElectronTransportCan
D a m a g eC e l l s 502 CyclicElectronFlow Through PSIGeneratesa
Proton-MotiveForcebut No NADPHor 02 522
I and ll Are
RelativeActivitiesof Photosystems
[[ Harnessingthe Proton-Motive Regulated 523
F o r c ef o r E n e rg y-R e q u i ri n g
Processes 503
[[l During
co2 Metabolism
The Mechanismof ATPSynthesisls Shared
Photosynthesis 524
Among Bacteria,Mitochondria,and Chloroplasts 505
ATPSynthaseComprisesTwo Multiprotein RubiscoFixesCO2in the ChloroplastStroma 525
ComplexesTermedFeand F1 505 Synthesisof SucroseUsing FixedCO2ls Completed
Rotation of the Ft 1 Subunit,Driven by in the Cytosol 525
Proton Movement Through F6,Powers Light and RubiscoActivaseStimulateCO2
ATPSynthesis 506 Fixation 525
ATP-ADPExchangeAcrossthe Inner Mitochondrial Which Competeswith
Photorespiration,
Membrane ls Poweredby the Proton-Motive Photosynthesis,ls Reducedin PlantsThat Fix
Force s09 CO2by the C4PathwaY 527
l x i d a t i o nN o r m a l l yD e p e n d s
R a t eo f M i t o c h o n d r i aO
on ADP Levels 510
Brown-FatMitochondria Usethe Proton-Motive
Forceto GenerateHeat 510 1 3 M O V I N GP R O T E I NISN T O
MEMBRANES A N D O R G A N E L L E Ss33

@ Ph o t o s yn th e siasn d L i g h t-A b sor bing

Pigments 511
$[ Translocationof secretoryProteins
Acr ossthe ERMembr ane 535
ThylakoidMembranesin ChloroplastsAre the Sites
in Plants
of Photosynthesis 511 A HydrophobicN-TerminalSignalSequenceTargets
Three of the Four Stagesin Photosynthesis Occur NascentSecretoryProteinsto the ER 536
O n l y D u r i n gl l l u m i n a t i o n 511 CotranslationalTranslocationls lnitiated by Two
EachPhoton of Light Hasa DefinedAmount GTP-Hydrolyzing Proteins 537
of Energy 513 Passageof Growing PolypeptidesThrough the
PhotosystemsComprisea ReactionCenterand Transloconls Driven by EnergyReleasedDuring
AssociatedLight-Harvesting
Complexes 514 Translation 539

CONTENTS . XXV
 ATPHydrolysisPowersPost-translational
Translocationof SomeSecretoryProteinsin yeast s[ Sortingof Peroxisomal
Proteins sG7
540
CytosolicReceptorTargetsProteinswith an SKL
Sequenceat the C-Terminusinto the Peroxisomal
$[ Insertionof proteinsinto the Matrix
E RM e mb ra n e 542 P e r o x i s o m aM
l e m b r a n ea n d M a t r i x P r o t e i n sA r e
Incorporated by Different Pathways s68
SeveralTopologicalClasses
of Integral Membrane
ProteinsAre Synthesizedon the ER 543
InternalStop-Transfer and Signal-AnchorSequences
proteins s[ Transportinto and out of the
DetermineTopologyof Single-Pass 544
Nucleus 569
M u l t i p a s sP r o t e i n sH a v eM u l t i p l e I n t e r n a l
TopogenicSequences 546 Largeand Small MoleculesEnter and Leavethe
A P h o s p h o l i p iA d n c h o rT e t h e r sS o m eC e l l - S u r f a c e Nucleusvia NuclearPoreComplexes 570
Proteinsto the Membrane 547 lmportinsTransportProteinsContainingNuclear-
The Topologyof a Membrane ProteinOften Can L o c a l i z a t i oS
n i g n a l si n t o t h e N u c l e u s 571
Be Deducedfrom lts Sequence 547 ExportinsTransportProteinsContainingNuclear-Export
S i g n a l so u t o f t h e N u c l e u s 573
Most mRNAsAre Exportedfrom the Nucleusby a
Pr o t e i nMo d i fi ca ti o n s,
F o l d i n g, R a n - l n d e p e n d e nMt e c h a n i s m 573
s[
and Quality Control in the ER 549
A Preformed/V-LinkedOligosaccharide ls Added to
M a n y P r o t e i n si n t h e R o u g hE R 550
O l i g o s a c c h a r i dSei d eC h a i n sM a y p r o m o t eF o l d i n g 1 4 V E S I C U L ATRR A F F I CS, E C R E T I O N ,
and Stabilityof Glycoproteins ss2 AND ENDOCYTOSIS 579
D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e db y
P r o t e i n si n t h e E RL u m e n 552
C h a p e r o n eas n d O t h e r E Rp r o t e i n sF a c i l i t a t eF o l d i n g ![ Techniquesfor Studyingthe
and Assemblyof Proteins ss2 SecretoryPathway s80
lmproperlyFoldedProteinsin the ERlnduce Transportof a protein Through the Secretory
Expressionof Protein-FoldingCatalysts 555 pathway Can Be Assayedin tiving Cells 5g2
U n a s s e m b l eodr M i s f o l d e dP r o t e i n si n t h e E RA r e YeastMutants Define Major Stagesand Many
Often Transportedto the Cytosolfor Componentsin VesicularTransport 584
Degradation 556
Cell-FreeTransportAssaysAllow Dissectionof
I n d i v i d u aS
l t e p si n V e s i c u l aTr r a n s p o r t 585

E!| Sortingof Proteinsto Mitochondria

and Chloroplasts 557 M olecularMechanisms
of
@
A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c eD sirect Vesicular Traffic 585
Proteinsto the MitochondrialMatrix 558 Assembryof a protein coat DrivesVesicle
M i t o c h o n d r i aP
l r o t e i nl m p o r t R e q u i r e sO u t e r - M e m b r a n e F o r m a t i o na n d S e l e c t i o no f C a r g o
R e c e p t o ras n d T r a n s l o c o ni sn B o t h M e m b r a n e s 559 Molecules 5g6
Studieswith ChimericProteinsDemonstratelmportant A ConservedSet of GTPaseSwitch proteinsControls
Featuresof Mitochondriallmport 550 Assemblyof Different VesicleCoats 5g7
T h r e eE n e r g yI n p u t sA r e N e e d e dt o l m p o r t P r o t e i n s T a r g e t i n gS e q u e n c e os n C a r g o p r o t e i n sM a k e
into Mitochondria 501 S p e c i f i cM o l e c u l a rC o n t a c t sw i t h C o a t
Multiple Signalsand PathwaysTarget Proteinsto Proteins 588
SubmitochondrialCompartments 561 Rab GTPases Control Dockingof Vesicleson Target
T a r g e t i n go f C h l o r o p l a sSt t r o m a lP r o t e i n sl s S i m i l a rt o Membranes 589
lmport of MitochondrialMatrix Proteins 565 PairedSetsof SNAREproteinsMediate Fusionof
ProteinsAre Targetedto Thylakoidsby Mechanisms Vesicleswith Target Membranes 591
Relatedto TranslocationAcrossthe Bacterial Dissociationof SNAREComplexesAfter Membrane
I n n e rM e m b r a n e 565 F u s i o nl s D r i v e nb y A T PH y d r o l y s i s 5g1

xxvi . coNTENTs
@ EarlyStagesof the Secretory l: SIGNAL
1 5 C E L LS I G N A L I N G
Pathway 592
TRANSDUCTION AND SHORT- T ER M
COPIIVesiclesMediate Transportfrom the ER CELLULAR RESPONSES 623
to the Golgi
COPIVesiclesMediate RetrogradeTransportwithin
t h e G o l g ia n d f r o m t h e G o l g i t o t h e E R ss4 Signalto cellular
FromExtracellular
[[
AnterogradeTransportThrough the Golgi Occurs Response 625
by CisternalMaturation 595
S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e
Signaling
Molecules 625
Later stages of the secretory SignalingMoleculesCan Act Locallyor at a Distance 625
@
Pathway 597 B i n d i n go f S i g n a l i n gM o l e c u l e sA c t i v a t e s
Receptorson Target Cells 626
VesiclesCoatedwith Clathrinand/or Adapter Proteins
Mediate SeveralTransportSteps 598
D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f o f C l a t h r i n studying cell-Surface
Vesicles 599 $[
Receptors 627
Mannose6-PhosphateResiduesTargetSoluble
Proteinsto Lysosomes 600 ReceptorProteinsBind LigandsSpecifically 627

Study of LysosomalStorageDiseases
RevealedKey The DissociationConstantls a Measureof the
Componentsof the LysosomalSorting Pathway 602 Affinity of a Receptorfor lts Ligand 628

ProteinAggregation in the trans-GolgiMay Function BindingAssaysAre Usedto Detect Receptorsand

in Sorting Proteinsto RegulatedSecretoryVesicles 602 DetermineTheir Affinities for Ligands 628

Some ProteinsUndergo ProteolyticProcessing to a SignalingMolecule

MaximalCellularResponse
After Leavingthe trans-Golgi 603 UsuallyDoesNot RequireActivationof All
Receptors 629
SeveralPathwaysSort Membrane Proteinsto the
Apical or BasolateralRegionof PolarizedCells 604 Sensitivityof a Cellto ExternalSignalsls Determined
by the Number of SurfaceReceptorsand Their
Affinity for Ligand 631

!f| Receptor-Mediated
Endocytosis 606 ReceptorsCan Be Purifiedby Affinity Techniques 631

CellsTake Up Lipidsfrom the Blood in the Form of ReceptorsAre FrequentlyExpressedfrom Cloned Genes 6 3 1
Large,Well-Defined LipoproteinComplexes 606
Receptorsfor Low-DensityLipoproteinand Other
LigandsContain Sorting SignalsThat Target
Highly ConservedComPonents
Them for Endocytosis 608 Signal-Transduction
of Intracellular
Pathways 632
The Acidic pH of Late EndosomesCausesMost
Receptor-Ligand Complexesto Dissociate 610 GTP-BindingProteinsAre FrequentlyUsedAs On/Off
The EndocyticPathwayDeliverslron to Cellswithout Switches 633
Dissociationof the Receptor-Transferrin
Complex Protein Kinasesand Phosphatases are Employedin
in Endosomes 611 Virtually All SignalingPathways 634
SecondMessengers Carryand Amplify Signalsfrom
Many Receptors 634
[!| DirectingMembraneProteins
and CytosolicMaterialsto the
Lysosome 612 of G Protein-
GeneralElements
s!|
MultivesicularEndosomesSegregateMembrane Coupled SYstems
RecePtor 535
ProteinsDestinedfor the Lysosomal
Membranefrom ProteinsDestinedfor G Protein-CoupledReceptorsAre a Largeand Diverse
Familywith a Common Structureand Function 635
LysosomalDegradation 612
RetrovirusesBud from the PlasmaMembrane by a Process G Protein-CoupledReceptorsActivate Exchangeof
Endosomes 614
Similarto Formationof Multivesicular G T Pf o r G D Po n t h e a S u b u n i to f a T r i m e r i cG
Protein 637
14.1 Following
Cnsstc ExprRturruT a Protein Different G ProteinsAre Activated by Different GPCRs
out of thecell 621 and ln Turn RegulateDifferent EffectorProteins 539

CONTENTS . xxvii
 [f| G Protein-Coupled Receptors I n s u l i na n d G l u c a g o nW o r k T o g e t h e rt o M a i n t a i na
T h a t R eg u l a tel o n C h a n n e l s 640 StableBlood GlucoseLevel

AcetylcholineReceptorsin the Heart Muscle Clnsstc ExprnlveruT

15.1 TheInfancy
of signal
Activate a G ProteinThat OpensK+ Channels 641 Transduction-GTPStimulationof cAMP Synthesis 663
Light ActivatesG..-CoupledRhodopsins 641
Activationof RhodopsinInducesClosingof
cGMP-GatedCation Channels 642
Rod CellsAdapt to Varying Levelsof Ambient Light 15 CELL-SIGNALIN
l lG
: SIGNALING
Becauseof Opsin Phosphorylationand Binding PATHWAYSTHAT C O N T R O L
GENE
of Arrestin 644
ACTIVITY 66s

$!| G Protein-GoupledReceptorsThat TGFFReceptors

sl| and the Direct
Activateor Inhibit Adenylyl Activation of Smads 668
Cyclase 646
A TGFBSignalingMoleculels Formedby Cleavage
Adenylyl Cyclasels Stimulatedand Inhibited of an InactivePrecursor 668
by Different Receptor-LigandComplexes 646
RadioactiveTaggingWas Usedto ldentify TGFp
StructuralStudiesEstablishedHow G,,.GTpBinds Receptors 669
to and ActivatesAdenylyl Cyclase 646
ActivatedTGFBReceptorsPhosphorylateSmad
cAMP ActivatesProtein KinaseA by Releasing TranscriptionFactors 670
CatalyticSubunits 547
NegativeFeedbackLoopsRegulateTGFB/Smad
GlycogenMetabolismls Regulatedby Signaling 671
Hormone-lnducedActivation of Protein KinaseA 648
Lossof TGFBSignalingPlaysa Key Role
cAMP-MediatedActivation of Protein KinaseA in Cancer 671
ProducesDiverseResponses in Different Cell Types 649
S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y
SignalingPathways 550
S e v e r aM
l e c h a n i s mD
s o w n - R e g u l a tS
e ignaling cytokineReceptors
andthe
from G Protein-CoupledReceptors 651
sf|
JAK/STATPathway 672
Anchoring ProteinsLocalizeEffectsof cAMp to
SpecificRegionsof the Cell CytokinesInfluenceDevelopmentof Many Cell
Types 672
CytokineReceptorsHaveSimilarStructuresand
sfl G Protein-coupled
Receptors
That Activate SimilarSignalingPathways 673
ActivatePhospholipase
C 553 JAK KinasesActivate STATTranscriptionFactors 674
PhosphorylatedDerivativesof InositolAre ComplementationGeneticsRevealedThat
lmportant SecondMessengers 654 JAK and STATProteinsTransduceCytokine
C a l c i u ml o n R e l e a s e
from the Endoplasmic Signals 677
Reticulumis Triggeredby lp3 654 Signalingfrom CytokineReceptorsls Regulated
The Ca2*/CalmodulinComplexMediatesMany by NegativeSignals 678
CellularResponsesto ExternalSignals 655 Mutant ErythropoietinReceptorThat Cannot Be
Diacylglycerol(DAG)Activatesprotein KinaseC, TurnedOff Leadsto IncreasedNumbersof
Which RegulatesMany Other proteins Erythrocytes 679
6s6
Signal-lnducedRelaxationof VascularSmooth
Musclels Mediated by cGMp-Activated
Protein KinaseG 656
s[ ReceprorTyrosrneKrnases 679
IntegratingResponses of cells L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i oann d
$f| Activation of IntrinsicKinasein RTKs
t o En v i ro n me n taInl fl u e n ce s 680
657
Overexpression
of HER2,a Receptor
Integrationof Multiple SecondMessengersRegulates TyrosineKinase,Occursin Some Breast
Glycogenolysis 557 Cancers 680

XXVIII ' CONTENTS

 D o m a i n sA r e l m p o r t a n tf o r B i n d i n gS i g n a l -
Conserved HedgehogSignalingRelievesRepressionof Target
TransductionProteinsto ActivatedReceptors Genes 700
Down-regulationof RTKSignalingOccursby
Endocytosis
and LysosomalDegradation 683
@ PathwaysThat InvolveSignal - l nduc ed
ProteinCleavage 703
@ Act i v a t i o n o f R a sa n d MA P K i n ase Degradationof an Inhibitor ProteinActivatesthe
Pathways 584 NF-rBTranscriptionFactors 703
Ras,a GTPaseSwitch Protein,CyclesBetweenActive Ligand-ActivatedNotch ls CleavedTwice,Releasing
and InactiveStates 68s a TranscriptionFactor 705

ReceptorTyrosineKinasesAre Linkedto Rasby Matrix Metalloproteases CatalyzeCleavageof Many

Adapter Proteins 685 S i g n a l i n gP r o t e i n sf r o m t h e C e l lS u r f a c e 706

GeneticStudiesin Drosophilaldentified Key InappropriateCleavageof Amyloid PrecursorProtein

Signal-TransducingProteinsin the Ras/MAP Can Leadto Alzheimer'sDisease
KinasePathway 685 RegulatedIntramembraneProteolysisof SREBP
Binding of SosProteinto InactiveRasCausesa Releasesa TranscriptionFactorThat Acts to
ConformationalChangeThat ActivatesRas 587 Maintain Phospholipidand CholesterolLevels
SignalsPassfrom Activated Rasto a Cascadeof
Protein Kinases 688
MAP KinaseRegulatesthe Activity of Many Transcription
FactorsControlling Early-ResponseGenes 690 1 7 C E L LO R G A N I Z A T I OANN D
G Protein-CoupledReceptorsTransmitSignalsto 713
M O V E M E N Tl : M I C R O F I L A M E N T S
MAP Kinasein YeastMating Pathways 691
ScaffoldProteinsSeparateMultiple MAP Kinase
Pathwaysin EukaryoticCells 692 @ and Actin
Microfilaments
Structures 715
The Ras/MAPKinasePathwayCan InduceDiverse
C e l l u l a rR e s p o n s e s 693 717
Actin ls Ancient,Abundant, and Highly Conserved
G-ActinMonomersAssembleinto Long, Helical
F-ActinPolymers 717
sf| Phosphoinositides
as signal 718
F-ActinHasStructuraland FunctionalPolarity
Transducers 694
C" ls Activatedby SomeRTKsand
Phospholipase
CytokineReceptors 6e4 Dynamicsof Actin Filaments 718
@
Recruitmentof Pl-3 Kinaseto Hormone-Stimulated
ReceptorsLeadsto Synthesisof Phosphorylated Actin Polymerizationin Vitro Proceedsin Three Steps 7 1 9
Phosphatidylinositols Actin FilamentsGrow Fasterat (+) EndsThan at
(-) Ends 720
in the Plasma
Accumulationof Pl 3-Phosphates
Membrane Leadsto Activation of SeveralKinases 695 Actin FilamentTreadmillingls Acceleratedby
P r o f i l i na n d C o f i l i n 721
Activated Protein KinaseB InducesMany
CellularResponses 696 Thymosin-B4 Providesa Reservoirof Actin for
Polymerization 722
The Pl-3KinasePathwayls NegativelyRegulated
by PTENPhosphatase CappingProteinsBlockAssemblyand Disassembly
a t A c t i n F i l a m e n tE n d s 722

s!| Activationof GeneTranscription

by Seven-spanningCell-Surface @ of Actin Filament
Mechanisms
Receptors 697 AssemblY 723
CREBLinkscAMP and Protein KinaseA to Activation F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s 723
of GeneTranscription 698 The Arp2/3 ComplexNucleatesBranchedFilament
Arrestin ActivatesSeveralKinaseCascades 698
GPCR-Bound Assembly 724
Wnt SignalsTrigger Releaseof a Transcription lntracellularMovementsCan Be Poweredby Actin
Factorfrom CytosolicProteinComplex Polymerization 726

CONTENTS xxix
 ToxinsThat Perturbthe Pool of Actin Monomers ChemotacticGradientsInduceAltered Phosphoinositide
Are Usefulfor StudyingActin Dynamics 726 LevelsBetweenthe Front and Backof a Cell 750

Cnsstc ExpenlvlrruT
17.1 Looking
at Muscle
@ o r g a n i z a ti o no f A cti n -B a secellular
d Contraction 755
Structures 728
C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t si n t o
Bundlesor Networks 728 1 8 C E L LO R G A N I Z A T I OANN D
Adaptor ProteinsLink Actin Filamentsto M O V E M E N Tl l : M I C R O T U B U L E S
Membranes 728 AND INTERMEDIATE FILAM EN T S 757

fifl Myosins:Actin-Based
Motor [[ Micr otubuleStr uctur eand
Proteins 731 Or ganization 758
M y o s i n sH a v eH e a d ,N e c k ,a n d T a i l D o m a i n sw i t h MicrotubuleWalls Are PolarizedStructuresBuilt
DistinctFunctions 732 f r o m a B - T u b u l i nD i m e r s 758
M y o s i n sM a k e U p a L a r g eF a m i l yo f MicrotubulesAre Assembledfrom MTOCsto
MechanochemicalMotor proteins 733 GenerateDiverseOrganizations 760
C o n f o r m a t i o n aCl h a n g e si n t h e M y o s i nH e a d
CoupleATPHydrolysisto Movement 736
Myosin HeadsTake DiscreteStepsAlong ftf| Microtubule
Dynamics 762
Actin Filaments 736 MicrotubulesAre DynamicStructuresDue to
MyosinV Walks Hand Over Hand Down an Actin Kinetic Differencesat Their Ends 763
Filament 737 I n d i v i d u aM
l i c r o t u b u l e sE x h i b i tD y n a m i cI n s t a bIi t y 763
LocaIized A s s e m b l ya n d " S e a r c h - a n d - C a p t u r e "
H e l p O r g a n i z eM i c r o t u b u l e s 766
@ Myosin-poweredMovements 738 DrugsAffecting Tubulin PolymerizationAre Useful
M y o s i nT h i c kF i l a m e n t sa n d A c t i n T h i n F i l a m e n t s Experimentallyand to Treat Diseases 766
i n S k e l e t aM
l u s c l eS l i d eP a s tO n e A n o t h e r
D u r i n gC o n t r a c t i o n 738
S k e l e t aM
l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d [f! Regulationof MicrotubuleStructure
ScaffoldingProteins 740 and Dynam ics 767
Contractionof SkeletalMusclels Regulatedby Ca2* MicrotubulesAre Stabilizedby Side-and
a n d A c t i n - B i n d i n gP r o t e i n s 740 End-Binding Proteins 767
Actin and Myosin ll Form ContractileBundlesin M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g
N o n m u s c l eC e l l s 741 and SeveringProteins 768
Myosin-DependentMechanismsRegulate
C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s 742
Myosin-V-Bound VesiclesAre CarriedAlong @ Kinesinsand Dyneins:Micr otubul e-
Actin Filaments 743 BasedMotor Proteins 769
Organellesin AxonsAre TransportedAlong
Microtubulesin Both Directions 769
@ c e l l M i g r a t i o ns: i g n a l i n ga n d
Kinesin-1PowersAnterogradeTransportof
Chemotaxis 745 VesiclesDown Axons Toward the (+) End of
C e l lM i g r a t i o nC o o r d i n a t e F
s o r c eG e n e r a t i o nw i t h Microtubules 770
C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g 745 KinesinsForm a LargeProtein Familywith Diverse
The SmallGTP-BindingProteinsCdc42,Rac,and Rho Functions 771
Control Actin Organization 747 Kinesin-l ls a Highly Processive
Motor 772
C e l lM i g r a t i o nI n v o l v e st h e C o o r d i n a t eR e g u l a t i o n Dynein Motors TransportOrganellesTowardthe (-)
of Cdc42,Rac,and Rho 748 E n do f M i c r o t u b u l e s 774
Migrating CellsAre Steeredby Chemotactic Kinesinsand DyneinsCooperatein the Transport
Molecules 750 o f O r g a n e l l eT
s hroughout he Cell 715

XXX . CONTENTS
 Ciliaand Flagella:
Microtubule- Microfilamentsand MicrotubulesCooperateto
fifl TransportMelanosomes
BasedSurfaceStructures 777
Cdc42Coordi nates M icrotubules and M icrofi laments
E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e t D u r i n g C e l lM i g r a t i o n 797
MicrotubulesBridged by Dynein Motors 777
C i l i a r ya n d F l a g e l l a Br e a t i n gA r e P r o d u c e db y
C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t
Microtubules 778 1 9 I N T E G R A T I NCGE L L SI N T O
IntraflagellarTransportMoves Material Up and
TISSUES 801
D o w n C i l i aa n d F l a g e l l a 779
Defectsin IntraflagellarTransportCauseDisease
by Affecting SensoryPrimaryCilia 780 $[ cell- celland cell- Matr ixAdhes i on:
An Overview 803
C e l l - A d h e s i oMn o l e c u l e sB i n dt o O n e A n o t h e r a n d
$!| Mitosis 781 t o I n t r a c e l l u l aPr r o t e i n s 803

MitosisCan Be Dividedinto Six Phases 782 The ExtracellularMatrix Participatesin Adhesion,

S i g n a l i n ga, n d O t h e r F u n c t i o n s 805
s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n
C e n t r o s o m eD
Preparationfor Mitosis 783 The Evolutionof MultifacetedAdhesionMolecules
Enabledthe Evolutionof DiverseAnimal Tissues 807
of
The Mitotic SpindleContainsThree Classes
Microtubules 784
M i c r o t u b u l eD y n a m i c sI n c r e a s eDs r a m a t i c a l l y Junctio ns
cell- celland celI- ECM
in Mitosis 784 @
and TheirAdhesionMolecules 808
M i c r o t u b u l e sT r e a d m i l D
l u r i n gM i t o s i s 785
t p i c a l ,L a t e r a l a
l e l l sH a v eD i s t i n c A
E p i t h e l i aC , nd
The KinetochoreCapturesand HelpsTransport 808
BasalSurfaces
Chromosomes 786
ThreeTypesof JunctionsMediate Many Cell-Cell
DuplicatedChromosomesAre Aligned by Motors 809
and Cell-ECMInteractions
a n d T r e a d m i l l i n gM i c r o t u b u l e s 788
l l d h e s i o n si n A d h e r e n s
C a d h e r i n sM e d i a t eC e l l - C e A
AnaphaseA MovesChromosomes
to Polesby 810
J u n c t i o n sa n d D e s m o s o m e s
Microtubule Shortening 789
Tight JunctionsSealOff Body Cavitiesand Restrict
AnaphaseB SeparatesPolesby the Combined 814
D i f f u s i o no f M e m b r a n eC o m p o n e n t s
A c t i o n o f K i n e s i n sa n d D y n e i n 789
Adhesionsin EpithelialCells 816
IntegrinsMediateCelI-ECM
A d d i t i o n a lM e c h a n i s mC
s o n t r i b u t et o S D i n d l e
Formation 789 G a p J u n c t i o n sC o m p o s e do f C o n n e x i n A
s llow Small
Moleculesto PassDirectlyBetweenAdjacent Cells 817
CytokinesisSplitsthe DuplicatedCell in Two 789
P l a n tC e l l sR e o r g a n i z T
e h e i r M i c r o t u b u l e sa n d
B u i l da N e w C e l l W a l li n M i t o s i s 790
M atr ix l:
The Extr acellular
The BasalLamina 820
IntermediateFilaments 791 l a m i n aP r o v i d e sa F o u n d a t i o nf o r
The BasaL
$fl Assemblyof Cellsinto Tissues 820
IntermediateFilamentsAre Assembledfrom M a t r i x P r o t e i n ,H e l p s
L a m i n i n ,a M u l t i a d h e s i v e
S u b u n i tD i m e r s 792 821
Cross-linkComponentsof the BasalLamina
Intermediate FilamentsProteins Are Expressed
Sheet-Forming Type lV Collagenls a Major Structural
in a Tissue-Specific
Manner 792 821
C o m p o n e n to f t h e B a s a L
l amina
I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c 795
Components
Perlecan,a Proteoglycan,Cross-links
Defectsin Laminsand KeratinsCauseManv Diseases 795 of the BasalLaminaand Cell-SurfaceReceptors 824

coordinationand cooperation @The ExtracellulaMra t r i xl l :

[[
between CytoskeletalElements 796 Connectiveand Other Tissues 825

lntermediateFilament-Associated Proteins s r e t h e M a j o r F i b r o u sP r o t e i n s
F i b r i l l a rC o l l a g e n A
C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n 796 in the ECMof ConnectiveTissues 825

CONTENTS XXXI
F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e di n t o ConnectionsBetweenthe ECMand Cytoskeleton
FibrilsOutsideof the Cell 926 Are Defectivein MuscularDystrophy 835
T y p eI a n d l l C o l l a g e n A
s ssociate
with Nonfibrillar l g C A M sM e d i a t eC e l l - C e l l A d h e s i oi n N e u r o n a l
Collagensto Form DiverseStructures 826 and Other Tissues 836
Proteoglycans
and Their ConstituentGAGsPlay LeukocyteMovement into Tissuesls Orchestrated
DiverseRolesin the ECM 827 by a Precisely
Timed Sequenceof Adhesive
HyaluronanResistsCompression, Facilitates
Cell Migration, Interactions 837
and GivesCartilagelts Gel-likeProperties 829
FibronectinsInterconnectCellsand Matrix, Influencing
Cell Shape,Differentiation,and Movement 830
p[ PtantTissues 839
T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s eF i b r i l s
in a Matrix of Glycoproteins
[[ AdhesiveInteractionsin Motile Looseningof the Cell Wall PermitsPlant Cell
a n d N o n mo ti l eC e l l s 833 Growth 840
I n t e g r i n sR e l a yS i g n a l sB e t w e e nC e l l sa n d T h e i r Plasmodesmata DirectlyConnectthe Cytosolsof
T h r e e - D i m e n s i o nE an l vironment 833 A d j a c e n tC e l l si n H i g h e rP l a n t s
Regulationof Integrin-MediatedAdhesionand Only a Few AdhesiveMoleculesHave Been
S i g n a l i n gC o n t r o l sC e l l M o v e m e n t ldentified in Plants 841

PartlV CellGrowth and Development

20 R E G U L AT IN TGH E E U K A R Y OT IC ![fl Cyclin-Dependent

KinaseRegulation
C E L LC Y C L E Dur ingMitosis 8s9
847
MPFComponentsAre ConservedBetween Lower
and Higher Eukaryotes 860
E[ Overviewof the CellCycle Phosphorylationof the CDKSubunit Regulatesthe
and lts Control 849 KinaseActivity of MPF 851
The Cell Cyclels an Ordered Seriesof Events ConformationalChangesInducedby Cyclin
L e a d i n gt o C e l lR e p l i c a t i o n Binding and PhosphorylationIncrease
849
MPFActivity 862
RegulatedProtein Phosphorylationand
DegradationControl PassageThrough the
Cell Cycle 849
DiverseExperimentalSystemsHave Been Usedto @ MolecularMechanisms
for
Control proteins
ldentify and lsolateCell-Cycle 851 Regulating Mitotic Events 864
Phosphorylationof NuclearLaminsand Other
ProteinsPromotesEarlyMitotic Events 864
@ Controlof Mitosisby Cyclinsand
U n l i n k i n go f S i s t e rC h r o m a t i d sI n i t i a t e sA n a p h a s e 867
MPF Activity 853
ChromosomeDecondensationand Reassembly
of the
Maturation-PromotingFactor(MpF)Stimulates NuclearEnvelopeDependon Dephosphorylation
Meiotic Maturation in Oocytesand Mitosis of MPFSubstrates 870
i n S o m a t i cC e l l s 854
Mitotic CyclinWas Firstldentified in EarlvSea
U r c h i nE m b r y o s 856
!!f| Cyclin-cDK
and Ubiquitin-protein
CyclinB Levelsand KinaseActivity of Mitosis-
Promoting Factor(MPF)ChangeTogetherin
Ligase Control of S phase 872
CyclingXenopusEgg Extracts 8s6 A Cyclin-Dependent Kinase(CDK)ls Criticalfor
Anaphase-PromotingComplex(APC/C)Controls S-PhaseEntry in S. cerevisiae 872
Degradationof Mitotic Cyclinsand Exit from Three G1CyclinsAssociatewith 5. cerevrsrae
CDK
Mitosis 8s8 to Form S-Phase-Promoting Factors 874

. CONTENTS
Degradationof the S-Phase
Inhibitor TriggersDNA
Replication 2 1 C E L LB I R T H L, I N E A G EA, N D
Multiple CyclinsRegulatethe KinaseActivity of DEATH 90s
5. cerevisiaeCDK During Different Cell-CyclePhases 877
Replicationa t E a c hO r i g i n l s I n i t i a t e dO n l y O n c e
During the Cell Cycle 877 |ffirhe Birthof cells:Stemcells,
N i c h e s ,a n d L i n e a g e 905

cell-cyclecontrolin Mammalian Stem CellsGive Riseto Both Stem Cellsand

!fil DifferentiatingCells 906
Cells 879
RestrictedDuring
Cell FatesAre Progressively
M a m m a l i a nR e s t r i c t i o n
P o i n t l s A n a l o g o u st o Development 907
STARTin Yeast Cells 880 908
The CompleteCell Lineageof C. elegansls Known
Multiple CDKsand CyclinsRegulatePassage of
HeterochronicMutants ProvideCluesAbout Control
M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e 881 909
of Cell Lineage
RegulatedExpression of Two Classes of Genes
Cultured EmbryonicStem CellsCan Differentiateinto
R e t u r n sG eM a m m a l i a nC e l l st o t h e C e l lC y c l e 881 911
V a r i o u sC e l lT y p e s
Through the RestrictionPoint Dependson
Passage
Adult Stem Cellsfor Different Animal TissuesOccupy
Phosphorylationof the Tumor-Suppressor
Rb Protein 882 912
S u s t a i n i n gN i c h e s
C y c l i nA l s R e q u i r e df o r D N A S y n t h e s iasn d C D K 1
MeristemsAre Nichesfor Stem Cellsin Postnatal
for Entry into Mitosis 883 92O
Plants
Two Typesof Cyclin-CDKInhibitorsContributeto
Control in Mammals
Cell-Cycle 883

@ cell-Typespecificationin Yeast 921

!![ in cell-Cycle
Checkpoints Mati ng-TypeTranscription FactorsSpecify
Regulation 884 CellTypes 922

The Presenceof UnreplicatedDNA PreventsEntry MCMl and a1-MCM1ComplexesActivate Gene

Transcription 923
into Mitosis 888
lmproper Assemblyof the Mitotic SpindlePrevents a2-MCMI and cr2-a1ComplexesRepressTranscription 923
the Initiation of Anaphase 888 PheromonesInduceMating of ct and a Cellsto
G e n e r a t ea T h i r d C e l l T Y P e 923
ProperSegregationof Daughter Chromosomesls
Monitored by the Mitotic Exit Network 889
Arrest of Cellswith DamagedDNA Depends
Cell-Cycle
on Tumor Suppressors 891 !!| and Differentiation
Specification
of Muscle 924
EmbryonicSomitesGive Riseto Myoblasts 925
!![ Meiosis:A specialTypeof cell
Division 892 MyogenicGenesWere Firstldentified in Studieswith
Cultured Fibroblasts 925
Key FeaturesDistinguishMeiosisfrom Mitosis 892 Two Classesof Regulatory FactorsAct in Concert to
Protein
of G1Cyclinsand a Meiosis-Specific
Repression Guide Productionof MuscleCells 926
KinasePromote Premeiotic5 Phase 895 Differentiationof Myoblastsls Under Positiveand
Recombinationand a Meiosis-SpecificCohesinSubunit NegativeControl 927
Are Necessaryfor the SpecializedChromosome Cell-CellSignalsAre Crucialfor Determinationand
Segregationin Meiosis| 895 Migration of Myoblasts 928
SpecialPropertiesof Rec8Regulatelts Cleavagein bHLH RegulatoryProteinsFunctionin Creationof
M e i o s i sI a n d l l 896 Other Tissues 929
The Monopolin ComplexCo-orientsSister
Kinetochoresin Meiosis| 898
Tensionon SpindleMicrotubulesContributesto
@ Regulationof Asymmetric
ProperSpindleAttachment 898 CellDivision 930
20.1 cellBiology
Cnsstc ExpenlueruT Emerging YeastMating-TypeSwitchingDependsupon
from the Sea:The Discoveryof Cyclins 903 AsymmetricCell Division 930

CONTENTS ' xxxiii

ProteinsThat RegulateAsymmetryAre Localizedat
Controlof Body Segmentation:
OppositeEndsof Dividing Neuroblastsin Drosophila 9 3 1
Themesand Variationsin Insects
and Vertebrates 969

!@ cell Deathand tts Regulation 936 Early Drosophila Development ls an Exercisein Speed 970

ProgrammedCell Death OccursThrough Apoptosis TranscriptionalControl Specifiesthe Embryo's

937
Anterior and Posterior 971
Neurotrophinspromote Survivalof Neurons 937
TranslationInhibitorsReinforceAnterior-Posterior
A Cascadeof CaspaseProteinsFunctionsin One Patterning 973
Apoptotic Pathway 938
InsectSegmentationls Controlledby a Cascade
Pro-ApoptoticRegulatorsPermit CaspaseActivation of Transcription Factors 974
in the Absenceof TrophicFactors 941
VertebrateSegmentationls Controlledby Cyclical
SomeTrophicFactorsInduceInactivationof Expressionof RegulatoryGenes 977
a Pro-ApoptoticRegulator 942
DifferencesBetweenSegmentsAre Controlledby
Tumor NecrosisFactorand RelatedDeath Signals Hox Genes 978
PromoteCell Murder by Activating Caspases 943
Hox-GeneExpressionls Maintained by a Variety
of Mechanisms 982
Flower DevelopmentRequiresSpatiallyRegulated
2 2 T H EM O L E C U L ACRE L LB I O L O G Y Productionof TranscriptionFactors 983
O FD E V E L O P M E N T 949
!![ cell-TypeSpecification
in Early
Neural Development 985
@ H i g h l i g h tso f D e ve l o p me n t 950
N e u r u l a t i o nB e g i n sF o r m a t i o no f t h e B r a i na n d
DevelopmentProgresses
from Egg and Sperm SpinalCord 986
to an EarlyEmbryo 9s0 SignalGradientsand TranscriptionFactorsSpecifyCell
As the EmbryoDevelops,Cell LayersBecomeTissues Typesin the NeuralTube and Somites 987
and Organs 951
Most Neuronsin the BrainArise in the Innermost
GenesThat RegulateDevelopmentAre at the NeuralTube and Migrate Outward 988
Heart of Evolution 952
LateralInhibition Mediated by Notch Signaling
CausesEarlyNeural Cellsto BecomeDifferent 988

@ and Fertilization g53

Gametogenesis
!![ Growth and Patterningof Limbs 990
G e r m - l i n eC e l l sA r e A l l T h a t W e I n h e r i t 953
FertilizationUnifiesthe Genome Hox GenesDeterminethe Right Placesfor
955
Limbsto Grow 990
Genomiclmprinting ControlsGene Activation
Accordingto Maternal or PaternalChromosome Limb DevelopmentDependson Integration
Origin 958 o f M u l t i p l e E x t r a c e l l u l aSri g n a lG r a d i e n t s 991
Too Much of a Good Thing: The X Chromosomels Hox GenesAlso Control Fine Patterning
Regulatedby DosageCompensation 958 of Limb Structures 992
So Fa[ So Good 994

!f, cell Diversityand patterning Cussrc ExprRlueruT

22.1 UsingLethal
in EarlyVertebrateEmbryos Mutationsto Study Development 999
959
CleavageLeadsto the FirstDifferentiationEvents 960
The Genomesof Most SomaticCellsAre Complete 961 23 NERVE
CELLS 1001
GastrulationCreatesMultiple TissueLayers,Which
BecomePolarized 961
SignalGradientsMay InduceDifferent Cell Fates 963 !fl Neurons andGlia:Building
SignalAntagonistsInfluenceCell Fatesand Tissue Blocksof the NervousSystem lOOz
Induction 965 Information FlowsThrough Neuronsfrom Dendrites
A Cascadeof SignalsDistinguishes
Left from Right 956 to Axons 1003

xxxiv . coNTENTs
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tgot Iyunr.uur;anrldepy pue aleuul qlog sesseduo:u3 EZol uotpeJluo) al)snlAol speel
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ol Jelrursieuuel4 e ur pa6uerreagaJV saueg U)I
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selqueseu.roldaraulla)-I aql lo alnpnrls aql arnpnJls paruasuo)e e^eH sut;nqo;6ounuu.t1
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' sr ol da) au a r n lr n r ls :su lln q o l6 0 u nuul
l la) -I's lto) L @
SpecificMutations TransformCulturedCells MutationsCausingLossof
into Tumor Cells 1113 and
Gr owth- lnhibiting
A Multi-hit Model of CancerInduction ls Supported Cell-CycleControls 1134
by SeveralLinesof Evidence 1114
from
MutationsThat Promote UnregulatedPassage
OncogenicMutations Can Be Traced
Successive 1134
Gr to 5 PhaseAre Oncogenic
in Colon Cancers 1116
MutationsAffecting Chromatin-
Loss-of-Function
DNA MicroarrayAnalysisof Expression
PatternsCan 1135
RemodelingProteinsContributeto Tumors
RevealSubtle DifferencesBetweenTumor Cells 1116
Lossof p53 Abolishesthe DNA-DamageCheckpoint 1136
Apoptotic GenesCan Functionas Proto-oncogenes
G e ne ti cB a si so f ca n ce r 1119 or Tumor-SuppressorGenes 1137
@ Th e
Failureof Cell-Cycle CheckpointsOften Leadsto
Gai n-of-FunctionM utationsConvertProto-oncogenes i n T u m o rC e l l s 1138
Aneuploidy
into Oncogenes 1119
Cancer-Causing VirusesContainOncogenesor
Activate CellularProto-oncogenes 1121
and CaretakerGenes
Carcinogens
Mutations in Tumor-Suppressor
Loss-of-Function in Cancer 11 3 9
GenesAre Oncogenic 1123
CarcinogensInduceCancerby DamagingDNA 11 3 9
Genes
Inherited Mutations in Tumor-Suppressor
lncreaseCancerRisk 1123 SomeCarcinogensHave Been Linkedto
Specific Cancers 1139
Aberrationsin SignalingPathwaysThat Control
DevelopmentAre Associatedwith Many Cancers 1124 Lossof DNA-RepairSystemsCan Leadto Cancer 1141
TelomeraseExpressionContributesto
lmmortalizationof CancerCells 1143
!fl oncogenicMutationsin Growth-
Promoting Proteins 1127 GLOSSARY G-1
OncogenicReceptorsCan Promote Proliferationin
the Absenceof External Growth Factors 1127 INDEX l-1
Viral Activators of Growth-Factor ReceptorsAct
as Oncoproteins 1128
Many OncogenesEncodeConstitutivelyActive
Sig nal-Transduction
Proteins 1129
InappropriateProductionof NuclearTranscription
FactorsCan InduceTransformation 11 3 0
M o l e c u l a rC e l lB i o l o g yl s C h a n g i n gH o w C a n c e r
ls Treated 1132

CONTENTS . xxxvtl
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e 'sllo)+oremole1r1-ra6urJ q)el 'a.loquMoqsaurlsalur q6norqlarr;s ol pasnereq)tqM 's1)elsrn)QoDe7alPasaqf
aql urse'leaqsler;eqlrda ue urol ue)slla)(6) uro[oqeql ]e qlnq alou:euol)eqnl(e) satlt^t])e )tloqelaupue'(sllallr1ofue1na snslan
'o^oLll ltaqtul le]]lp
o] ^lt1lqe
eq1sr{poq llo)oql lurolord luerseronl; e }o uorl)npo.r}ur{q a;qrsrn rrlo,telord)uotleztue6lo lPulalul
apeurseMllo)aq1'salrrpuap ]o )roMlaupaq)uerq aq1qbnorqls1lar sllar,{6oloqdroLu o} uol}lppe u1'sqdelooloqd asaqlul pa}el}snlll
raqloqllMsuot])auuo) ooo'00 t ueqlarourulro]uel leql ;1at e6re1 srsllo)]o ,ileuen;e:roo;otldloul oq] jo auos 'sazlspue sadeqs
,{;qrperrurue 'urnllaqara)aLl}}o uornauelurlrn4el6urs y (+)slla) lo luauruosse6ulpunolseue ul auo) slla) l-l lunglJ V
( a ) P r o k a r y o t icce l l (b) Eukaryoticcell

P e r i p l a s m iscp a c e
a n d c e l lw a l l
Golgi vesicles

Lysosome

O u t e rm e m b r a n e I n n e r( p l a s m a ) Nucleoid
membrane lu'btrm I

Nucleoid N u c l e a rm e m b r a n e
P l a s m am e m b r a n e

Golgi vesicles
Mitochondrion
Peroxisome

Lysosome

I n n e r( p l a s m a )m e m b r a n e

Periplasmicspace

Outer membrane
R o u g he n d o p l a s m i c
reticulum

A FIGURE 1-2 Prokaryotic cellshavea simplerinternal of thecellular

segregation DNAwithina definednucleus, whichis
organizationthan eukaryoticcells.(a)Electron micrograph of a bounded by a doublemembrane Theouternuclear membrane is
thin sectionof Escherichia
coli a commonintestinalbacteriumThe continuouswiththe roughendoplasmic reticulum, a factory for
nucleoid, of the bacterial
consisting DNA,is notenclosed withina assemblingproteinsGolgivesicles process andmodifyproteins,
membraneE coli andsomeotherbacterra aresurrounded by two mitochondriagenerate energy, lysosomes digestcellmaterials to
membranes separatedbythe periplasmicspaceThethincellwallis them,peroxisomes
recycle process molecules usingoxygen, and
adjacent to the innermembrane (b)Electron
mrcrograph of a plasma secretory carrycellmaterials
vesicles to the surfaceto release them
cell,a typeof whitebloodcellthatsecretes antibodiesOnlya single D
lPart(a)courtesyoflJ B u r d e t t a n G
d RE M u r r a y . P a r t ( b ) fromPC
membrane (theplasma membrane) surroundsthecell,butthe andK L Mercer,
Cross 1993,CellandTissue lJltrastructure: A Functional
interiorcontains manymembrane-limited compartments, or W H Freeman
Perspective, andCompanyl
organelles.Thedefining cellsis
of eukaryotic
characteristic

and 40 miles up in the atmosphere;they are quite adaptable!
The carbon stored in bacteria is nearly as much as the car-
bon stored in plants.
Eukaryotic cells, unlike prokaryotic cells, contain a de-
fined membrane-bound nucleus and extensive internal mem-
branes that enclose other compartments called organelles
(Figure 1-2b). The region of the cell lying betweenthe plasma
membrane and the nucleus is the cytoplasm, comprising the
cytosol (aqueousphase)and the organelles.Eukaryotescom-
priseall membersof the plant and animal kingdoms,including
the fungi, which exist in both multicellular forms (molds) and
unicellular forms (yeasts),and the protozoans (proto, primi-
tive zoan, animal), which are exclusively unicellular. Eukary-
otic cells are commonly about 10-100 pm across' generally
much larger than bacteria. A typical human fibroblast, a con-
nective tissuecell, might be about 15 p,m acrosswith a volume
and dry weight some thousandsof times those of an E. coli
bacterial cell. An ameba, a single-celledprotozoan, can be
more than 0.5 mm long. An ostrich egg beginsas a singlecell
that is evenlarger and is easilyvisible to the naked eye.

Y N D C O M M O N A L I T YO F C E L L S
THE DIVERSITA
 All cells are thought to have evolved from a common archaeacell membraneshave chemical properties that differ
progenitor becausethe structures and moleculesin all cells dramatically from those of bacteria and eukaryotes.
have so many similarities. In recent years, detailed analysis Many archaeansgrow in unusual,often extreme,environ-
of the DNA sequencesfrom a variety of prokaryotic organ- ments that may resemblethe ancient conditions that existed
isms has revealed two distinct types: the bacteria and the when life first appeared on earth. For instance, halophiles
archaea. Working on rhe assumption that organisms with ("salt loving") require high concentrationsof salt to survive,
more similar genesevolvedfrom a common progenitor more and thermoacidophiles("heat and acid loving") grow in hot
recently than those with more dissimilar genes,researchers (80' C) sulfur springs,where a pH of lessthan 2 is common.
have developed the evolutionary lineage tree shown in Still other archaeanslive in oxygen-free milieus and generate
Figure 1-3. According to this tree, the archaeaand the eu- methane(CH+) by combining water with carbon dioxide.
karyotes diverged from bacteria billions of years ago before
they diverged from each other. In addition to DNA sequence
distinctions that define the three groups of organisms, U n i c e l l u l aO
r r g a n i s m sH e l p a n d H u r t U s
Bacteria and archaea, the most abundant single-celledor-
ganisms,are commonly 1-2 pm in size. Despite their small
Animals Plants size and simple architecture,they are remarkable biochemi-
Fungi cal factories,converting simple chemicalsinto complex bio-
Ciliates logical molecules.Bacteria are critical to the earth's ecology,
Euglena
but some cause major diseases:bubonic plague (Black
Microsporidia EUKARYOTA
S I i m em o l d s Death) from Yersiniapestis,strep throat from Streptomyces,
Diplomonads tuberculosis from Mycobacterium tubercwlosis, anthrax
(Giardia lamblia) from Bacillus anthracis, cholera from Vibrio cholerae, and
food poisoning from certain types of E. coli and Salmonella.
EUBACTERIA Humans are walking repositories of bacteria, as are all
E. coli Sulfolobus plants and animals.'We provide food and shelter for a stag-
ARCHAEA
gering number of "bugs," with the greatestconcentration in
B. subtilus Thermococcus our intestines.In return for the food and shelter that allow
Thermotoga Methanobacteriu m them to reproduce, bacteria help us digest our food. One
common gut bacterium,E. coli, is also a favorite experimen-
Halococcus
Flavobacteria tal organism. In responseto signalsfrom bacteria such as E.
coli, the intestinal cells form appropriate shapesto provide a
G r e e ns u l f u r Halobacterium niche where bacteria can live, thus facilitating proper diges-
bacteria
Methanococcus tion by the combined efforts of the bacterial and the intes-
Borrelia
jannaschii tinal cells. Conversely,exposure to intestinal cells changes
burgdorferi
the properties of the bacteria so that they participare more
effectivelyin human digestion. Such communication and re-
sponseis a common feature of cells.
P r e s u m e dc o m m o n p r o g e n i t o r
The normal, peacefulmutualism of humans and bacteria is
of all extant organisms
sometimesviolated by one or both parties. When bacteria be-
P r e s u m e dc o m m o n p r o g e n i t o r gin to grow where they are dangerousto us (e.g.,in the blood-
of archaebacteria and eukaryotes stream or in a wound), the cells of our immune system fight
A FIGURE 1-3 All organismsfrom simplebacteriato complex back, neutralizingor devouring the intruders. Powerful antibi-
mammalsprobablyevolvedfrom a common,single-celled otic medicines,which selectivelypoison prokaryotic cells, pro-
progenitor.Thisfamilytreedepicts theevolutionary relationsamong vide rapid assistanceto our relatively slow-developingimmune
thethreemajorlineages of organisms.Thestructure of thetreewas response.Understanding the molecular biology of bacterial
initially
ascertained frommorphological criteria:creaturesthat look cells leads to an understanding of how bacteria are normally
alikewereput closetogetherMorerecently the sequences of DNA poisonedby antibiotics,how they becomeresistantto antibi-
andproteins havebeenexamined asa moreinformation-rich otics, and what processesor structurespresent in bacterial but
criterionfor assigning relationshipsThegreater thesimilaritiesin not human cells might be usefully targeted by new drugs.
thesemacromolecular sequences, the moreclosely relatedorganisms
arethoughtto be.Thetreesbasedon morphological Like bacteria) protozoa are usually beneficial members of
comparisons
andthefossilrecordgenerally the food chain. They play key roles in the fertility of soil, con-
agreewellwith thosebasedon
molecular data.Althoughallorganisms in the eubacterial trolling bacterial populations and excreting nitrogenous and
and
archaean lineagesareprokaryotes, archaea aremoresimilar to phosphatecompounds,and are key playersin wastetreatment
eukaryotes thanto eubacteria ("true"bacteria) in somerespects For systems-both natural and man-made.These unicellular eu-
instance, archaean andeukaryotic genomes encodehomologous karyotesare also critical parts of marine ecosystems, consum-
histone proteins,whichassociate with DNA;in contrast, bacteria lack ing large quantities of phytoplankton and harboring photo-
histonesLikewise, the RNAandproteincomponents of archaean synthetic algae, which use sunlight to produce biologically
ribosomes aremorelikethosein eukaryotes thanthosein bacteria. usefulenergyforms and small fuel molecules.

C H A P T E RI I L I F EB E G I N SW I T H C E L L S
S' vid"o: Plasmodium Enteringand Exitinga LiverCell
Sporozoite
(a) (b)

Redbloodcel
E ,I
SPorulation
,, *

"F1 \
Merozoites-
,11
,H;,", )A
Gametocytes
|,Ervuy
uo, rEo

Huum
maann
\ -\ / . / - \ - /
F=>^
/\
l,r' I
Mosquito +/L +
( 0;r"""Y"!

FfGURE 1-4 Plasmodiumorganisms,the parasitesthat cause burstforth in synchrony from largenumbersof infectedcells4. lt
malaria,are single-celledprotozoanswith a remarkablelife isthiseventthatbringson thefeversandshaking chillsthatare
cycle.ManyPlasmodium speciesareknown,andtheycaninfecta thewell-known symptoms of malariaSomeof the released
varrety of animals, cyclingbetweeninsect andvertebrate hosts. The merozoites infectadditional RBCs, creating a cycleof production
fourspecies thatcausemalaria in humansundergo severaldramatic andinfection. Eventually,somemerozoites develop intomaleand
transformations withintheirhumanandmosquito hosts.(a)Diagram femalegametocytes 5, anothermetamorphosis. These cells,
of the lifecycleSporozoites entera humanhostwhenan infected whichcontainhalftheusualnumberof chromosomes, cannot
Anopheles mosquitobitesa personfl Theymigrateto the liver, survivefor longunless theyaretransferred in bloodIo an Anopheles
wheretheydevelop intomerozoites, whicharereleased intothe mosquito. In the mosquito s stomach, the gametocytes are
bloodE. Merozortes fromsporozoites,
differsubstantially sothis transformed intospermor eggs(gametes), yetanother
transformation isa metamorphosis (Greek,"to transform" or "many metamorphosis markedby development of longhairlike flagellaon
shapes")Circulating merozoites invaderedbloodcells(RBCs) and the sperm6 Fusion of spermandeggsgenerates zygotesZ,
reproduce withinthem B Proteins producedby somePlasmodium whichimplant intothecellsof thestomach wallandgrowinto
species moveto the surface of infectedRBCs, causing the cellsto oocysts,essentiallyfactories for producing sporozoites Rupture of
adhere to thewallsof bloodvessels. Thrsprevents infectedRBCs an oocystreleases thousands of sporozoites E; thesemigrateto the
fromcirculating to thespleen,wherecellsof the immunesystem salivaryglands, settingthe stagefor infectionof anotherhuman
woulddestroy the RBCs andthe Plasmodium organisms theyharbor host.(b)Scanning electron micrograph of matureoocysts and
Aftergrowingandreproducing in RBCsfor a periodof time emerging sporozoites. Oocysts abut the external sudace of stomach
characteristic of eachPlasmodium the merozoites
species, suddenly wallcellsandareencased withina membrane that protects them
fromthe hostimmunesystem. (b)courtesy
lPart of R E Sinden ]

However, some protozoa do give us grief: Entamoeba
histolytica causesdysentery;Trichomonas uaginalis,vagini-
tis; and Trypanosoma brucei, sleeping sickness.Each year
the deadliest of the protozoa, Plasmodium falciparum and
related species,is the cause of more than 300 million new
casesof malaria, a diseasethat kills 1.5 to 3 million people
annually. These protozoans inhabit mammals and mosqui-
toes alternately,changing their morphology and behavior in
responseto signalsin each of theseenvironments.They also
recognize receptors on the surfacesof the cells they infect.
The complex life cycle of Plasmodium dramatically
illustrates how a singlecell can adapt to each new challenge
it encounters(Figure 1-4). All of the transformations in cell
type that occur during the Plasmodium Iife cycle are gov-
erned by instructions encodedin the geneticmaterial of this
parasite and triggered by environmental inputs.
The other group of single-celledeukaryotes, the yeasts,
also have their good and bad points with respect to hu-
mans. as do their multicellular cousins, the molds. Yeasts
and molds, which collectively constitute the fungi, have an
important ecological role in breaking down plant and ani-
mal remains for reuse.They also make numerous antibi-
otics and are used in the manufacture of bread, beer,wine,
and cheese.Not so pleasant are fungal diseases'which
range from relatively innocuous skin infections such as
jock itch and athlete's foot to life-threatening Pneumocys-
tis carinii pneumonia, a common cause of death among
AIDS patients.

A N D C O M M O N A L I T YO F C E L L S
THE DIVERSITY
VirusesAre the Ultimate Parasites
Not all microscopic pathogensare cells. The other most fa-
miliar disease-causingorganisms are the viruses, which
make use of the machinery inside the cellsthey infect to copy
themselves.Virus-causeddiseasesare numerous and all too
familiar: chickenpox, influenza, some types of pneumonia,
polio, measles,rabies,hepatitis,the common cold, and many
others. Smallpox, once a worldwide scourge,was eradicated
by a decade-longglobal immunization effort beginning in
the mid-1960s. Mral infections in plants (e.g.,dwarf mosaic
virus in corn) have a major economic impact on crop pro-
duction. Planting of virus-resistantvarieties, developed by
traditional breeding methods and more recently by genetic-
engineeringtechniques,can reduce crop lossessignificantly.
Most viruses have a rather limited host range, infecting cer-
tain bacteria,plants, or animals(Figure1-5).
Becausevirusescannot grow or reproduce on their own,
they are in this sensenot consideredto be alive. To survive.
a virus must infect a host cell and take over its internal ma-
chinery to synthesizeviral proteins and in some casesrepli-
cate the viral geneticmaterial. When newly made virusesare
releasedby budding from the cell membrane or when the in-
fected cell bursts, the cycle starts anew. Viruses are much
smaller than cells,on the order of 100 nanometer (nm) in di-
ameter; in comparison, bacterial cells are usually >1000 nm
(1 nm : 10-e meters).A virus is typically composedof a
protein coat that enclosesa core containing the genetic ma-
terial, which carries the information for producing more
viruses (Chapter 4). The coat protects a virus from the envi-
ronment and allows it to stick to, or enter,specifichost cells.
In some viruses, the protein coat is surrounded by an outer
membrane-likeenvelope.
The ability of viruses to rransport genetic material into
cells and tissuesrepresentsa medical menaceand a medical
(a)T4 bacteriophage
A FIGURE 1-5 Virusesmust infect a host cell to grow and
reproduce.Theseelectronmicrographs illustrate
someof the
structural
varietyexhibited
byviruses. (a)T4 bacteriophage (bracket)
attaches
to a bacterial
cellviaa tailstructureViruses that infect
bacteria
arecalledbacteriophages, or simplyphages. (b)Tobacco
mosarcviruscausesa mottlingof the leaves of infected
tobacco
C H A P T E R1 I L I F EB E G I N SW I T H C E L L S
opportunity. Viral infections can be devastatingly destruc-
tive, causing cells to break open and tissuesto fall apart.
However, many methods for manipulating cells depend on
using virusesto convey geneticmaterial into cells.To do this,
the portion of the viral genetic material that is potentially
harmful is replaced with other genetic material, including
human genes.The altered viruses, or vectors, still can enter
cellstoting the introduced geneswith them (Chapter 9). One
day, diseasescausedby defectivegenesmay be treated by us-
ing viral vectors to introduce a normal copy of a defective
gene into patients. Current research is dedicated to over-
coming the considerableobstaclesto this approach, such as
getting the introduced genesto work at the right placesand
trmes.
C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n
The most remarkable feature of organismsis their ability to
reproduce.Biological reproduction, combined with continu-
ing evolutionary selectionfor a highly functional body plan,
is why today's horseshoecrabs look much as they did 300
million years ago, a time span during which entire mountain
ranges have risen or fallen. The Teton Mountains in
S7yoming,now about 14,000 feet high and still growing, did
not exist a mere 10 million years ago. Yet horseshoecrabs,
with a life span of about 19 years,have faithfully reproduced
their ancient selvesmore than half a million times during
that period. The common impression that biological struc-
ture is transient and geologicalstructure is stableis the exact
opposite of the truth. Despite the limited duration of our
individual lives, reproduction gives us a potential for im-
mortality that a mountain or a rock does not have.
Whereas some species have changed little over great
periods of time, other organismshave changeddramatically
(b)Tobaccomosaicvirus
50nm
, ,
(c) Adenovirus
plantsandstuntstherrgrowth.(c)Adenovirus
causeseyeand
respiratory
tractrnfections
in humans.
Thisvirushasan outer
membranous envelope fromwhichlongglycoprotein
spikesprotrude
[Part(a) from A Levine,1991, Viruses,ScientificAmericanLibrary,p 20
Part(b) courtesyof R C Valentine Part(c) courtesyof RobleyC Williams,
Universityof Californial
during the same period. The changescame in responseto
pressuresfrom the environment that causedincreasedsur-
vival of variant individuals. Both stasis and change are
possible becausethe machinery of cells does an amazingly
precisejob of copying geneticmaterial, yet its rare errors
introduce some variation. If environmental conditions
continue to select more or less the existing form, as in the
caseof horseshoecrabs, the specieswill changelittle. If a
new variant has a survival advantage, perhaps because
conditions have changed, it may persist and replace the
old form.
Populations of bacteria exposedto antibiotics, for exam-
ple, changetheir properties dramatically to escapeand live.
They do this becauserare mutations, changesin the genetic
material that allow antibiotic resistance,keep some cells
alive while the cells without those mutations die. Most pop-
ulations of any single specieshave a large repertoire of ge-
netic alterations becausethere is a low but significant error
rate in copying the genes.That error rate increasesin the
presenceof radiation such as sunlight or certain chemical
poisons. Current genomeproiects are exploring geneticvari-
ation among humans. "The" human genome sequencethat
has been determined already is just one version among bil-
lions. Understanding variation is essentialto learn how we
respond differently to certain infections or drugs and to ex-
ploring how our geneticheritage combines with our experi-
enceand learning to make each of us unique.
Underlying the reproduction of organismsis the copying
of cells, something that must be precise in order to control Budding lS. cerevisi ael
the size, shape, and organization of animals and to prevent
unwanted growth, such as cancer.The cell is a machine that A FfGURE 1-6 The yeast Saccharomycescerevisraereproduces
can copy itself, unlike viruses,which cannot do so on their sexuallyand asexually'(a)Twocellsthat differin matingtype'
own. As we will seein Chapters 20 and 21',the cell cycle- calleda andcr,canmateto forman a/a cellIl. Thea anda cellsare
from a single cell copying its own contents through division haploid,meaning theycontaina singlecopyof eachyeast
into two cells-is controlled by a seriesof elegant switches chromosome, halfthe usualnumber'Matingyieldsa diploida/ctcell
and cross-checking mechanisms. Reproducing cells accu- containing copiesof eachchromosome.
two Duringvegetative
growth,diploidcellsmultiply by mitoticbudding,an asexualprocess
rately is a matter of life and death.
E Understarvation conditions, a
diploidcellsundergomeiosis,
specialtypeof celldivision, to formhaploidascospores B. Rupture
EvenSingleCellsCan Have Sex of an ascusreleases fourhaploidspores, whichcangerminate into
haploid cells4. These alsocanmultiply asexuallyE. (b)Scanning
If genetic material was never shared or exchanged,each of buddingyeastcells.Aftereachbudbreaks
electron micrograph
individual would be the beginning of a new clone of indi-
free,a scaris left at the buddingsite,so the numberof previous buds
viduals, and the members of a clone would share most of canbe counted.Theorange cellsarebacteria (b)
[Part M AbbeyA/isuals
the same genetic strengths and weaknesses.Sex is a lncl
Unlimited,
processof mingling genetic variation from two individu-
als, creating new individuals with a combination of prop-
erties unlike either parent and that may be beneficial for book becauseit has proven to be a great experimental
survival and reproduction. Each chromosome except the organism. Like many other unicellular organisms' yeasts
sex chromosomes is representedtwice, one copy from the h"u. t*o mating types that are conceptually like the male
father and one from the mother. Since each pair of chro- and female gametes(eggsand sperm) of higher organisms'
mosomes trades piecesduring the formation of eggs and
sperm, new combinations of genesare created and inher-
ited together in the next generation-variation is acceler-
ated. The other benefit of having two copiesof each chro-
mosome is that a poorly functioning gene is backed up by
the other copy.
The common yeast used to make bread and beer,
Saccharomycescereuisiae,appearsfairly frequently in this ubiquitous.

A N D C O M M O N A L I T YO F C E L L S . 7
THE DIVERSITY
We Developfrom a SingleCell
In 1827, German physician Karl von Baer discovered that
mammals grow from eggs that come from the mother's
ovary. Fertilization of an egg by a sperm cell yields a
zygote, a visually unimpressive cell 200 pm in diameter.
Every human being begins as a zygote,which housesall the
necessaryinstructions for building a human body contain-
ing about 100 trillion (1014)cells, an amazing feat. Devel-
opment begins with the fertrlizedegg cell dividing into two,
four, then eight cells, forming the very early embryo (Figure
1-7). Continued cell proliferation and then differentiation
Chapters 16 and 22.
Making different kinds of cells-muscle, skin, bone, neu-
ron, blood cells-is not enough to produce the human body.
The cells must be properly arranged and organized into tis-
sues,organs, and appendages.Our two hands have the same
kinds of cells, yet their different arrangements-in a mirror
image-are critical for function. In addition, many cells ex-
hibit distinct functional and/or structural asymmetries, a
property often called polarity. From such polarized cells
arise asymmetric, polarized tissuessuch as the lining of the
intestinesand structures such as hands and hearts. The fea-
tures that make some cellspolarized and how they arise also
are coveredin later chapters,including Chapter 21.
Video:EarlyEmbryonicDevelopment
< FIGURE 1-7 The first few
cell divisionsof a fertilized
egg set the stagefor all
subsequentdevelopment.A
developing mouseembryois
shownat (a)the two-cell,(b)
and(c)eight-cell
four-cell,
Theembryoissurrounded
supporting membranes. The
corresponding stepsin human
development occurduringthe
firstfew daysafterfertilization.
IClaudeEdelmann/Photo
Researchers.
I n cl
C H A P T E R1 I L I F EB E G I N SW I T H C E L L S
Stem Cells,Fundamentalto FormingTissues
and Organs,Offer MedicalOpportunities
The biology of stem cells, cells that can give rise to specific
cell types and tissues,has generated, great interest. 'We can
'Sfhen
contrast stem cells to the simpler types of bacteria. a
bacterial E. coli cell divides, both daughter cells are pretty
much equivalent in content, size, and shape. In some other
bacteria and in many casesof eukaryotic cell division, the
two daughters differ in important ways. Although both will
have the same genetic material, the cells may differ in size,
shape, and contents. The cells may have different fates, that
is, they may become different types of differentiated cell. A
division that produces two different daughter cells is some-
times described as an asymmetric cell division.
Stem-celldivisions are a specialcaseof asymmetric divi-
sion. One of the two daughter cells is identical to the parent
cell; the other follows a path of differentiation, such as be-
coming a blood cell. The parent cell, called a stem cell, can
go on reproducing itself at every division, at each division
also producing anorher blood cell. Mosr tissuesin our bod-
ies form from stem cells. Blood, for example, is produced
from stem cells that residein the bone marrow and continue
to produce new blood cellsfor our entire lives. This is the ba-
sis of the often successfulbone marrow transplants that are
used to treat cancer patients who have had their blood stem
cells damaged by cancer treatments: what is being trans-
planted is stem cells.However, blood stem cells produce onlv
more of themselvesand blood cells, not othir cell types.
Thus eachtissuemust have its own stem cells,at leastduring
the period of development when the tissue is formed. Stem
cells for each tissue arise from even more capable stem cells
that have the ability to form multiple stem cell types. The
first stem cells are found in early embryos,where all the cells
are capable of producing all cell types.
In mammals the ultimate stem cell is the fertilized egg,
which produces early embryo cells capable of forming all
the tissuesof the body. This capability is illustrated by the
formation of identical twins, which occur naturally when
stages. the mass of cells composing an early embryo divides into
by two parts, each of which develops and grows into an indi-
vidual animal. This means that the cells cannor have di-
vided up their embryo-forming duties prior to the time of
embryo division. Each cell in an eight-cell-stagemouse em-
bryo has the potential to give rise to any part of the entire
animal. Cells with this capability are referred to as embry-
onic stem (ES)cells. As we will learn in Chapter 22, ES cells
can be grown in the laboratory (cultured) and will develop
into various types of differentiated cells under appropriate
conditions.
The ability to make and manipulate mammalian em-
bryos in the laboratory has led to new medical opportunities
as well as various social and ethical concerns.In vitro fertil-
ization, for instance, has allowed many otherwise infertile
couples to have children. One technique involves extractron
of nuclei from defective sperm incapable of normally fertiliz-
ing an egg,injection of the nuclei into eggs,and implantation
of the resulting fertilized eggsinto the mother.
 In recent years, nuclei taken from cells of adult animals critical shortage of twins and triplets). Of much gteater
have been used to produce new animals. In this procedure' scientific and medical interest is the ability to generatespe-
the nucleus is removed from a body cell (e.g., skin or blood cific cell types starting from embryonic or adult stem cells'
cell) of a donor animal and introduced into an unfertilized This procedure, somatic cell nuclear transfer (SCNT)' pro-
mammalian egg that has beendeprived of its own nucleus.In duces cells that are grown in culture and never turned into
a step that has now been done with mice, cows, sheep,
mules, and some other animals, the egg with its donor nu-
cleus is implanted into a foster mother. The ability of such a
donor nucleusto direct the developmentof an entire animal
shows that all the information required for life is retained in
the nuclei of some adult cells. Sinceall the cells in an animal
produced in this way have the genesof the single original
donor cell, the new animal is a genetic clone of the donor If the cells are produced using a donor nucleus from a pa-
(Figure 1-8), though the animals may differ anyway due to tient, the properties of the cells may allow them to escapere-
their distinct environments and experiences.Repeating the jection by the patient's immune system,opening new possi-
processcan give rise to many clones. Nuclei taken from ES bilities for cell-transplanttherapies.
cellswork especiallywell, whereasnuclei from other parts of
the body at later times in life work far lesswell. The major-
ity of embryos produced by this technique do not survive
E The Moleculesof a Cell
due to birth defects,so the donor nuclei may not have all the
needed information or the nuclei may be damaged by the Molecular cell biologists explore how all the remarkable
cloning process.Even those animals that are born alive have
abnormalities,including acceleratedaging. The "rooting" of
plants, in contrast, is a type of cloning that is readily accom-
plished by gardeners,farmers, and laboratory technicians.
Scientific interest in the cloning of humans is very lim-
ited. Virtually all scientistsoppose it becauseof its high risk
to the embryo (also, most people don't believe there is a ture and function.

Small MoleculesCarryEnergy,TransmitSignals,
and Are Linkedinto Macromolecules
Much of the cell's content is a watery soup flavored with
small molecules (e.g., simple sugars' amino acids, vitamins)
and ions (e.g.,sodium, chloride, calcium ions)' The locations
conce.tirations of small molecules and ions within the
"nd controlled by numerous proteins inserted in cellular
cell are
membranes. These pumps' transporters, and ion channels
move nearly all small moleculesand ions into or out of the
cell and its organelles(Chapter 11).
One of the best-known small molecules is adenosine
triphosphate (ATP), which storesreadily available chemical
'When
..r"rgy itt two of its chemical bonds (seeFigure 2-31)'
cells"splitapart theseenergy-richbonds in AIP' the released
..r.rgy ."n b. harnessed to power an energy-requiring
pro..tt such as muscle contraction or protein biosynthesis'
a FIGURE 1-8 Fivegeneticallyidenticalclonedsheep.An early To obtain energy for making ATR cells break down food
sheepembryowasdivided intofivegroupsof cellsandeachwas molecules.For instance, when sugar is degraded to carbon
separately implanted intoa surrogate mother, much likethe natural dioxide and water, the energy stored in the original chemical
process of twinningAt an earlystagethecellsareableto adjustand bonds is releasedand much of it can be "captured" in ATP
forman entireanimal; laterin development the cellsbecome (Chapter 72).Bacterial,plant, and animal cells can all make
progressively andcanno longerdo so.An alternative
restricted way
ATP ty this process.In addition, plants and a few other or-
to cloneanimals isto replace the nucleiof multiplesingle-celled
ganisms can harvest energy from sunlight to form ATP in
embryos with donornucleifromcellsof an adultsheep'Eachembryo
was photosynthesis.
'
will be geneticallyidenticalto the adultfromwhichthe nucleus
theseprocedures to Other small moleculesact as signalsboth within and be-
obtained.Lowpercentages of embryos survive
givehealthy animals, andthefull impactof the techniques on the tween cells; such signals direct numerous cellular activities
effect on our bodies of
animalsis not yet known.lGeoff Photo
Tompkinson/Science Library/Photo (Chapters iS uttd f e ;. fne powerful
a frightening event comes from the instantaneous flooding of
Incl
Researchers,

S F A CELL
T H E M O L E C U L EO O 9
 the body with epinephrine, a small-moleculehormone that
mobilizes the "fight-or-flight" response.The movements
neededto fight or flee are triggered by nerve impulses that
flow from the brain to our muscleswith the aid of neuro-
transmitters, another type of small-moleculesignal that we
discussin Chapter23.
acids. Sugars,for example, are the monomers used to form
polysaccharides. These macromolecules are critical struc-
tural components of plant cell walls and insect skeletons.A
typical polysaccharideis a linear or branched chain of re-
peating identical sugar units. Such a chain carries informa-
tion: the number of units. However, if the units arenot iden-
tical, then the order and type of units carry additional
information. As we will seein Chapter 6, some polysaccha-
rides exhibit the greaterinformational complexity associated
with a linear code made up of different units assembledin a
particular order. But this property is most typical of the two
other types of biological macromolecules-proteins and
nucleic acids.
ProteinsGive CellsStructureand perform Most
CellularTasks
The varied, intricate structures of proteins enable them to
carry out numerous functions. Cells string together Z0 dif_
ferent amino acids in a linear chain to foim a protein (see
Figure 2-14).Proteins commonly range in length from 100
Insulin
Lrturamtne
Glutaminesynthetase
synthetase Hemoglobin
FIGURE 1-9 Proteinsvary greatly in size,shape,and
function.Thesemodels of thewater-accessiblesurface of some
representative
proteinsaredrawnto a commonscaleandreveal the
numerous projections
andcreviceson thesurface. Eachprotern hasa
definedthree-dimensionalshape(conformation) thatisstabilizedby
numerous chemrcalinteractions
discussed in Chapters2 and3. The
illustrated
proteinsincludeenzymes (glutamine synthetase and
10 C H A P T E R1 | L | F EB E G T N W
S |TH CELLS
to 1000 amino acids, but some are much shorter and others
longer. We obtain amino acids either by synthesizingthem
from other moleculesor by breaking down proteins that we
eat. The "essential" amino acids, from a dietary standpoint,
are the eight that we cannot synthesizeand must obtain from
food. Beans and corn together have all eight, making their
combination particularly nutritious. Once a chain of amino
acids is formed, it folds into a complex shape,conferring a
distinctive three-dimensionalstructure and function on each
protein (Figure1-9).
Some proteins are similar to one another and therefore
can be consideredmembers of a protein family. A few hun-
dred such families have been identified. Most proreins are
designedto work in particular places within a cell or to be
releasedinto the extracellular (extra, "outside") space.Elab-
orate cellular pathways ensurethat proteins are transported
to their proper intracellular (intra, "within") locations or se-
creted (Chapters 13 and14).
Proteins can serveas structural componentsof a cell. for
example,by forming an internal skeleton(Chapters tO, tZ,
and 18). They can be sensorsthat changeshapeas rempera-
ture, ion concentrations, or other properties of the cell
change. They can import and export substancesacross the
plasmamembrane(Chapter11). They can be enzymes,caus-
ing chemicalreactionsto occur much more rapidly than they
would without the aid of theseprotein catalysts(Chapter 3).
They can bind to a specificgene,turning it on or off (Chap-
ter 7). They can be extracellular signals,releasedfrom one
cell to communicatewith other cells, or intracellular signals,
carrying information within the cell (Chapters 15 and 16).
They can be motors that move other moleculesaround, burn-
ing chemicalenergy (ATP) to do so (Chapters17 and 1g).
DNA molecule
lmmunoglobulin Adenylate L i p i db i l a y e r
K tn a s e
adenylatekinase),an antibody(immunoglobuiln),a hormone
(insulin),
and the bloods oxygencarrier(hemoglobin)Modelsof a
segmentof the nucleicacid DNA and a smallregionof the lipid
bilayerthat formscellularmembranes(seeSection1.3)demonstrate
the relativewidth of thesestructures
comparedwith that of tvpical
proteins.[Courtesyof GarethWhite]
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 can be "painted" for easy
FIGURE 1-12 Chromosomes
A normalhumanhas23 pairsof morphologically
identification.
fromthe
onememberof eachpairis inherited
chromosomes;
distinct
motherandthe othermemberfromthefather(Left)A chromosome
whenthe
spreadfroma humanbodycellmidwaythroughmitosis,
chromosomes Thispreparation
arefullycondensed wastreatedwith
thatalloweachof the 22 pairs
reaqents
staininq
fluorescent-labeled
Chapter 4. The molecular design of DNA and the remark-
able properties of the replisome ensure rapid, highly accu-
rate copying. Many DNA polymerasemoleculeswork in
concert, each one copying part of a chromosome.The entire
genome of fruit flies, about 1.2 x 10o nucleotideslong, can
be copied in three minutes! Becauseof the accuracyof DNA
replication, nearly all the cells in our bodies carry the same
genetic instructions, and we can inherit Mom's brown hair
and Dad's blue eyes.
A rather dramatic example of genecontrol involves inac-
'Women
tivation of an entire chromosomein human females.
have two X chromosomes,whereas men have one X chro-
mosome and one Y chromosome, which has different genes
than the X chromosome. Yet the genes on the X chromo-
some must, for the most part, be equally active in female
cells(XX) and male cells(XY). To achievethis balance'one
of the X chromosomesin female cells is chemically modified
and condensedinto a very small mass called a Barr body'
which is inactive and never transcribed.
Surprisingly,we inherit a small amount of geneticmate-
rial entirely and uniquely from our mothers. This is the cir-
cular DNA present in mitochondria' the organelles in eu-
karyotic cells that synthesizeAIP using the energy released
by the breakdown of nutrients. Mitochondria contain multi-
ple copies of their own DNA genomes,which code for some
of the mitochondrialproteins(Chapter6). Becauseeachhu-
man inherits mitochondrial DNA only from his or her
mother (it comes with the egg but not the sperm), the
andtheX andY chromosomes to appearin a differentcolorwhen
viewedin a fluorescence microscope Thistechnique of multiplex
fluorescence (M-FISH)
in situhybridization sometimes iscalled
chromosome painting(Chapter 6) (RiSht)Chromosomes fromthe
preparationon the leftarranged in pairsrndescending orderof size,
an arraycalleda karyotype.Thepresence of X andY chromosomes
identifies asmale [Courtesy
the sexof the individual of M R Speicher]
distinctive features of a particular mitochondrial DNA can
be used to trace maternal history. Chloroplasts, the or-
ganelles that carry out photosynthesisin plants' also have
Ih.i, o*tt circular genomes'Both mitochondria and chloro-
olasts are believedto be derived from endosymbionts,bacte-
ii" th"t took up residenceinside eukaryotic cells in a mutu-
ally beneficialpartnership.The mitochondrial and chloroplast
circular DNAs appeatto have originated as bacterial genomes'
which also are usually circular,though the organellegenomes
have lost most of the bacterialgenes.
Mutations May Be Good, Bad,or Indifferent
Mistakes occasionallydo occur spontaneouslyduring DNA
replication, causing changesin the sequenceof nucleotides'
Soch ch"ng.s, or mutations, also can arise from radiation
that causesdamageto the nucleotidechain or from chemical
poisons, such as those in cigarette smoke, that lead to errors
iuring the DNA-copying process (Chapter 25)' Mutations
.o-.1., various forms: a simple swap of one nucleotide for
another; the deletion, insertion, or inversion of one to mil-
lions of nucleotides in the DNA of one chromosome; and
translocation of a stretch of DNA from one chromosome to
another.
In sexually reproducing animals such as ourselves,muta-
tions can be inherited only if they are presentin cellsthat po-
tentially contribute to the formation of offspring' Such
germJine cells include eggs'sperm' and their precursor cells'
T H E M O L E C U L EOSF A C E L L O 13
 Body cells that do not contribute to offspring are called so-
matic cells. Mutations that occur in these cells never are in-
herited, although they may contribute to the onset of cancer.
Plants have a less distinct division between somatic and
germ-line cells, since many plant cells can function in both
capacltres.
Mutated genesthat encode altered proteins or that can-
not be controlled properly ."u.. .rurn..ous inherited dis-
eases.For example, sickle-celldiseaseis atributable to a sin-
gle nucleotide substitution in the hemoglobin gene, which
encodesthe protein that carries oxygen in red blood cells.
The single amino acid changecausedby the sickle cell muta-
Sequencingof the human genome has shown that a
transcription factors typically are only 70-12 nucleotides
long, a few single-nucleotidemutations might convert a non-
functional bit of DNA into a functional protein-binding
regulatory site.
Much of the nonessentialDNA in both eukaryotesand
prokaryotes consistsof highly repeatedsequencesthat can
move from one place in the genome to another. These mo-
bile DNA elementscan jump (transpose)inro genes,most
commonly damaging but sometimes activating them.
Jumping generallyoccursrarely enoughto avoid endanger-
ing the host organism. Mobile elements,which *... di.-
coveredfirst in plants, are responsiblefor leaf color varie-
gation and the diverse beautiful color patterns of Indian
corn kernels.By jumping in and out of ge.resthat control
prgmentationas plant developmentprogresses,the mobile
elemenrsgive rise to elaboratecolorid putt...rr. Mobile el-
ements were later found in bacteria, in which they often
carry and, unfortunatelS disseminategenesfor antibiotic
resrstance.
Now we understand that mobile elements have multi_
plied and slowly accumulatedin genomesover evolutionary
time, becoming a universal property of genomesin preseni_
day_organisms. They account for an astounding45 percent
of the human genome. Some of our own mobile DNA ele_
ments are copies-often highly mutated and damaged_of
genomesfrom viruses that spend part of their life iycle as
DNA segmentsinserted into host-cell DNA. Thus we carry
in our chromosomes the genetic residues of infections ac_
quired by our ancestors.Once viewedonly as molecularpar_
asites,mobile DNA elementsare now thoueht to have ion_
tnbuted significantly to the evolution of higher organisms
(Chapter6).
'14 . cHAprE1
R | L | F EB E G t Nwsl r H c E L L s
IE TheWorkof Cetts
In essence,any cell is simply a compartment with a watery
interior that is separatedfrom the external environment by a
surfacemembrane (the plasma membrane) that preventsthe
free flow of moleculesin and out. In addition, as we've
noted, eukaryotic cells have extensive internal membranes
that further subdivide the cell into various comparrmenrs,
the organelles.Each compartment has contents and proper-
ties, such as specializedproteins or a certain pH, suited to its
job. The plasma membrane and other cellular membranes
are composedprimarily of two layers of phospholipid mole-
cules. These bipartite moleculeshave a "water-loving" (hy-
drophilic) end and a "water-hating" (hydrophobic) end. The
two phospholipid layersof a membrane are orienred with all
the hydrophilic ends directed toward the inner and outer
surfacesand the hydrophobic ends buried within the interior
(Figure1-13). Smalleramounts of other lipids, such as cho-
lesterol, and many kinds of proteins are inserted into the
phospholipid framework. The lipid moleculesand somepro-
teins can float sidewisein the plane of the membrane,giving
membranes a fluid character. This fluidity allows cells to
change shape and even move. However, the attachment of
some membrane proteins to other molecules inside or out-
side the cell restricts their lateral movement. We will learn
more about membranes and how molecules cross them in
Chapters10 and 11.
The cytosol and the internal spacesof organellesdiffer
from each other and from the cell exterior in terms of acid-
itg ionic composition, and protein contents. For example,
the composition of saltsinside the cell is often drastically dif-
ferent from what is outside. Becauseof these different ,,mi-
croclimates," each cell compartment has its own assigned
Water-seeking
Cholesterol h e a dg r o u p
Water
FIGURE 1-13Thewatery interiorof cellsis surroundedby
the plasmamembrane,a two-layeredshell of phospholipids.
Thephospholipid moleculesareorientedwith theirfattyacylchains
(blacksquiggly lines)
facinginwardandtheirwater-seeking head
groups(whitespheres) facingoutward.Thusbothsidesof the
membrane arelinedby headgroups, mainlycharged phosphates,
adjacentto thewateryspaces tnsideandoutside thecell.All
biologicalmembranes havethesamebasicphospholipid bilayer
structureCholesterol (red)andvariousproteins(notshown)are
embedded in the bilayer.
Theinteriorspaceisactuallymuchlarger
relative
to thevolumeof the plasma membrane depicted here.
tasks in the overall work of the cell (Chapters 10, L2, and
13). The unique functions and microclimates of the various
cell compartm€nts are due largely to the proteins that reside
in their membranesor interior.
'We
can think of the entire cell compartment as a factory
dedicatedto sustainingthe well-being of the cell. Much cellu-
lar work is performed by molecular machines, some housed
in the cytosol, some attached to the cytoskeleton' and some in
various organelles.Here we quickly review the major tasks
that cells carry out in their pursuit of the good life.
Cells needto break down worn-out or obsoleteparts into
small molecules that can be discarded or recycled' This
housekeepingtask is assignedlargely to lysosomes,organelles
crammedwith degradativeenzymes.The interior of a lyso-
some has a pH of about 5.0' roughly 100 times more acidic
than that of the surrounding cytosol. This aids in the break-
down of materials by lysosomal enzymes' which are specially

CellsBuild and DegradeNumerous

Moleculesand Structures
As chemical factories,cells produce an enormous number of
complex molecules from simple chemical building blocks.
All of this syntheticwork is powered by chemical energy ex-
tracted primarily from sugarsand fats or sunlight, in the case
of plant cells, and stored primarily in ATP, the universal
"currency" of chemical energy (Figure 1-14). In animal and
plant cells, most ATP is produced by large molecular ma-
chines located in two organelles,mitochondria and chloro-
plasts. Similar machines for generating ATP are located in
the plasma membrane of bacterial cells. Both mitochondria
and chloroplasts are thought to have originated as bacteria
that took up residenceinside eukaryotic cells and then be-
came welcome collaborators (Chapter 12). Directly or indi-
rectlS all of our food is created by plant cells using sunlight
to build complex macromolecules during photosynthesis.
Even underground oil suppliesare derived from the decay of
plant material.
Most of the structural and functional properties of cells de-
cell and most membrane proteins, however' are made on ribo-
somes associatedwith the endoplasmic reticulum (ER)' This
organelleproduces,processes'and ships out both proteins and
lipids. Prolein chains produced on the ER move to the Golgi
comple*, where they are further modified before being for-
*ari.d to their final destinations. Proteins that travel in this
way contain short sequencesof amino acids or attached sugar
ch"itts (oligosaccharides)that serve as addressesfor directing
them to their correct destinations. These addresseswork be-
causethey arerecognizedand bound by other proteins that do
the sorting and shipping in various cell compartments'

llll+ overviewAnimation:BiologicalE lnterconversions

Light (photosynthesis)
or
compoundswith high
potentialenergY(resPiration
)

Transportof Generationof an Heat

Synthesisof Synthesisof other Cellularmovements,
cellularconstituents includingmusclecon- moleculesagainst electricpotential
cellularmacro-
traction,crawling move- a concentration acrossa memDrane
molecules(DNA, (suchas membrane
phospholipidsand ments of entire cells, gradient for nerve
RNA, proteins,
polysaccharides) certainrequired and movement of 1fi..:.,;t;,",
metabolites) chromosomesduring
mitosis
breakdown of sugars by
andfatsin mostcells.Theenergyreleased
A FIGURE1-14 ATP is the most common molecule used by
of PifromATPdrivesmanycellular
(hydrolysis)
thesplitting processes
cells to capture and transfer energy. ATPis formed from ADP and
inorganicphosphate(Pi)by photosynthesis in plantsand by the

THEWORK OF CELLS 15
 lntermediate f ilaments
FIGURE 1-15 The three types of cytoskeletalfilamentshave
characteristic distributionswithin cells.Threeviewsof the same
cell.A culturedfibroblastwastreatedwith threedifferentantibodv
preparations. Eachantibody bindsspecifically
to the protein
monomers formingonetypeof filamentandischemically linkedto a
differently
colored fluorescentdye(green,blue,or red).Visualization
A n i m a l C e l l sP r o d u c eT h e i r O w n E x t e r n a l
E n v i r o n m e nat n d G l u e s
The simplest multicellular animals are sinsle cells embedded
in a jelly of proteins and polysaccharides."ll.d th. extracel-
lular matrix. Cells themselvesproduce and secretethesema-
terials, thus creating their own immediate environment
(Chapter 19). Collagen, the single most abundant protein in
the animal kingdom, is a major component of the extracel-
lular matrix in most tissues.In animals, the extracellularma-
trix cushions and lubricates cells. A specialized,especially
tough matrix, the basal lamina, forms a supporting layer un_
derlying sheetlikecell layers and helps pr.u"rrt the cells from
rlppmg apart.
The cells in animal tissuesare ..glued" togerher by cell_
adhesionmolecules(CAMs) embeddedin their surfacemem_
C e l l sC h a n g eS h a p ea n d M o v e
Cells changeshapeand move becausetheir internal skeleton.
the cytoskeleton,exerts forces on the rest of the cell and its
attachments- Three types of protein filaments, organized
into networks and bundles, form the cytoskeleton within
16 . c H A p r E1R | L I F E
B E G t Nwst r H c E L L s
Microtubules Microfilaments
of the stainedcellin a fluorescence microscope
reveals
the location
of filamentsboundto a particular dye-antibodypreparation In this
case,intermediate filaments arestainedgreen;microtubules, blue;
and microfilaments, red.All threefibersystemscontributeto the
shapeandmovements of cells.lcourtesy
of V Small
]
animal cells (Figure 1-15). The cytoskeleton prevents the
plasma membrane of animal cells from relaxing into a
sphere(Chapter 10); it also functions in cell locomotion and
the intracellular transport of vesicles,chromosomes, and
macromolecules(Chapters 17 and 18). The cytoskeletoncan
be linked through the cell surfaceto the extracellular matrix
or to the cytoskeletonof other cells,thus helping to form tis-
sues(Chapter 19).
All cytoskeletal filaments are long polymers of protein
subunits. Elaborate systemsregulatethe assembly dirur-
sembly of the cytoskeleton,thereby controlling cell".rd
shape.In
some cells the cytoskeletonis relatively stable, but in others
it changesshapecontinuously. Shrinkageof the cytoskeleton
in some parts of the cell and its growrh in other parts can
produce coordinated changesin shape that result in cell lo-
comotion. For instance,a cell can send out an extensionthat
attachesto a surfaceor to other cellsand then retract the cell
body from the other end. As this processcontinues due to
coordinated changesin the cytoskeleton,the cell moves for-
ward. Cells can move at rates on the order of 20 pm./second.
largement but not movement of cells from one position to
another.
CellsSenseand SendInformation
A living cell continuously monitors its surroundings and ad-
justs its own activities and composition accordingly. Cells
also communicate by deliberatelysendingsignalsthat can be
receivedand interpreted by other cells. Suchsignalsare com_
mon not only within an individual organism but also be_
tween organisms.For instance,the odor of a pear signals a
food source to us and other animals; consumption of the
pear by an animal aids in distributing the pear'sseeds.Every- (a)
Surfacereceptors B o u n ds i g n a l
one benefits! The signals employed by cells include simple
small chemicals, gases, proteins, light, and mechanical )?'..
movements. Cells possessnumerous receptor proteins for
detecting signals and elaborate pathways for transmitting
OO
them within the cell to evoke a response.At any time, a cell
o
may be able to senseonly some of the signalsaround it, and
o 6
how a cell responds to a signal may change with time. In s
some cases,receivingone signal primes a cell to respond to a
I
subsequentdifferent signal in a particular way. enzyme Active enzyme
Both changesin the environment (e.g.,an increaseor de-
creasein a particular nutrient or the light level) and signals (b)
receivedfrom other cells representexternal information that
Cytosolic Receptor-hormone
cells must process.The most rapid responsesto such signals
generally involve changesin the location or activity of pre-
m R N A+ o
existing proteins. For instance, soon after you eat a carbo- + oo
j----\ Protein
hydrate-rich meal, glucosepours into your bloodstream.The tm # ot
Oc ^

rise in blood glucose is sensedby B cells in the pancreas, I

which respond by releasingtheir stored supply of the protein mRNA
hormone insulin. The circulating insulin signal causesglu-
lncreasedtranscription
cose transporters in the cytoplasm of fat and muscle cells to of specificgenes
Nucleus
move to the cell surface,where they begin importing glucose.
Meanwhile, liver cellsalso are furiously taking in glucosevia FIGURE 1-16 Externalsignalscommonlycausea changein
a different glucosetransporter.In both liver and musclecells, the activity of preexistingproteinsor in the amountsand
an intracellular signalingpathway triggeredby binding of in- typesof proteinsthat cellsproduce.(a)Binding of a hormone or
othersignaling molecule receptors
to itsspecific cantriggeran
sulin to cell-surfacereceptorsactivatesa key enzymeneeded the activityof a
pathway
intracellular that increasesor decreases
to make glycogen,a large glucosepolymer (Figure 1-16a). protein. bindingof insulin to receptors in
preexisting Forexample,
The net result of thesecell responsesis that your blood glu- of liverandmuscle cellsleadsto activation of
the plasma membrane
coselevel falls and extra glucoseis stored as glycogen,which glycogen synthase, a keyenzyme in thesynthesis of glycogen from
your cells can use as a glucosesource when you skip a meal glucose(b)Thereceptors for steroidhormones arelocated within
to cram for a test. cells,noton the surface
cell The hormone-receptor complexes
The ability of cells to send and respond to signalsis cru- activatetranscription of specifictarqetgenes,leading to increased
cial to development. Many developmentallyimportant sig- production of the encoded proteins.Manysignals that bindto
nals are secretedproteins produced by specific cells at spe- receptors on the cellsurface alsoact,by morecomplex pathways, to
cific times and places in a developing organism. Often a modulate geneexPression
receiving cell integratesmultiple signals in deciding how to
behave,for example, to differentiate into a particular tissue
type, to extend a process,to die, to send back a confirming controlled at the level of transcription, the first step in the
s i g n a l( y e s ,I ' m h e r e ! ) ,o r t o m i g r a t e . production of proteins. In this way cells can produce a par-
The functions of about half the proteins in humans, iicular mRNA only when the encoded protein is needed'
roundworms, yeast, and severalother eukaryotic organisms thus minimizing wasted energy. Producing an mRNA is,
have beenpredicted basedon analysesof genomic sequences however,only the first in a chain of regulatedeventsthat to-
(Chapter6). Suchanalyseshave revealedthat at least10-15 gether determine whether an active protein product is pro-
percent of the proteins in eukaryotesfunction as secretedex- duced from a particular gene.
tracellular signals, signal receptors, or intracellular signal- Transcriptional control of geneexpressionwas first deci-
transduction proteins, which pass along a signal through a sively demonstratedin the responseof the gut bacterium
seriesof steps culminating in a particular cellular response E. coli to different sugar sources.E. coli cells prefer glucose
(e.g., increased glycogen synthesis).Clearly, signaling and as a sugar source,but they can survive on lactosein a pinch'
signal transduction are maior activities of cells. These bacteria use both a DNA-binding repressor protein
and a DNA-binding actiuator protein to change the rate of
transcription of three genesneededto metabolize lactosede-
CellsRegulateTheir Gene Expressionto Meet pending on the relative amounts of glucoseand lactosepres-
C h a n g i n gN e e d s int (Chapter 4). Such dual positiveinegativecontrol of gene
In addition to modulating the activities of existing proteins, exoression fine-tunes the bacterial cell's enzymatic equip-
cells often respond to changing circumstancesand to signals -..rt fo. the job at hand.
from other cells by altering the amount or types of proteins Like bacterial cells, unicellular eukaryotes may be sub-
they contain. Gene expression,the overall processof selec- jected to widely varying environmental conditions that
tively reading and using genetic information, is commonly require extensivechangesin cellular structuresand function'

T H EW O R K O F C E L L S 17
 For instance,in starvation conditions yeastcellsstop growing
and form dormant spores (seeFigure 1-6). In multicellular
organisms, however, the environment around most cells is
relatively constant. The major purpose of gene control in us
and in other complex organismsis to tailor the properties of
various cell types to the benefit of the entire animal or plant.
Control of gene activity in eukaryotic cells usually in-
volves a balance between the actions of transcriptional acti-
vators and repressors.Binding of activators to specificDNA
regulatory sequencescalled enhancers turns on transcrip-
tion, and binding of repressorsto other regulatory sequences
called silencersturns off transcription. In Chapters 7 and 8,
we take a closelook at transcriptional activators and repres-
sors and how they operate, as well as other mechanismsfor
controlling gene expression.In an extreme case,expression
of a particular gene could occur only in part of the brain,
only during eveninghours, only during a certain stageof de-
velopment, only after a large meal, and so forth.
Many external signals modify the activity of transcrip-
tional activators and repressorsthat control specific genes.
For example, lipid-soluble steroid hormones, such as estro-
gen and testosterone,can diffuse across the plasma mem-
brane and bind to their specific receptors located in the cy-
toplasm or nucleus(Figure 1-16b).Hormone binding changes
Overview Animation: Life Cycle of a Cell {lltt
Nondividing
cells
Resting
the shape of the receptor so that it can bind to specific en-
hancer sequencesin the DNA, thus turning the receptor into
a transcriptional activator. By this rather simple signal-
transduction pathway, steroid hormones cause cells to
change which genes they transcribe (Chapter 7). Since
steroid hormones can circulate in the bloodstream, they can
affect the properties of many or all cells in a temporally co-
ordinated manner. Binding of many other hormones and of
growth factors to receptors on the cell surface triggers dif-
ferent signal-transduction pathways that also lead to
changesin the transcription of specific genes (Chapters 15
and 16). Although thesepathways involve multiple compo-
nents and are more complicated than those transducing
steroid hormone signals,the generalidea is the same.
CellsGrow and Divide
As we have discussed,reproduction is at the heart of biol-
ogy; rocks don't do it. The reproduction of organisms de-
pends on the reproduction of cells. The simplest type of re-
production entails the division of a "parent" cell into two
"daughter" cells.This occurs as part of the cell cycle,a series
of eventsthat preparesa cell to divide followed by the actual
division process, called mitosis. The eukaryotic cell cycle
commonly is representedas four stages(Figure 1-17). The
chromosomes and the DNA they carry are copied during
the S (synthesis)phase.The replicatedchromosomesseparare
during the M (mitotic) phase,with each daughter cell getting
a copy of each chromosome during cell division. The M and
S phasesare separatedby two gap stages,the G1 phase and

o
R N Aa n d
protein
synthesis
G2
A FIGURE 1-17 Duringgrowth, eukaryoticcellscontinually
progressthrough the four stagesof the cell cycle,
generatingnew daughtercells.In mostproliferating cells,the
four phases
of the cellcycleproceedsuccessively, takingfrom
10-20 hoursdepending on celltypeanddevelopmental state
Duringinterphase, whichconsists of the G,,,S,andG2phases,
cellroughly
doubles itsmassReplication
of DNAdurinqthe S
phaseleavesthe cellwith four copiesof eachtypeof
chromosome Inthe mitotic(M)phase, thechromosomes
are
evenlypartitionedto two daughter cells,andthecytoplasm
dividesroughlyin halfin mostcases. Undercertainconditions
suchasstarvation or whena tissuehasreached itsfinalsize,cells
willstopcyclingandremainin a waitingstatecalledGs Mostcells
in Gscanreenter thecycleif conditions chanqe.
c el l s
G2 phase, during which mRNAs and proteins are made. In
single-celledorganisms,both daughtercellsoften (though not
always) resemblethe parent cell. In multicellular organisms,
stem cells can give rise to two different cells, one that resem-
bles the parent cell and one that does not. Such asymmetric
cell division is critical to the generationof different cell types
in the body (Chapter21).
During growth the cell cycle operatescontinuously with
newly formed daughter cells immediately embarking on
their own path ro mitosis. Under optimal conditions bacteria
can divide to form two daughter cells once every 30 minutes.
At this rate, in an hour one cell becomesfour: in a day one
cell becomesmore than 101a, which if dried would weigh
about 25 grams. Under normal circumstances, however,
growth cannot continue at this rate becausethe food supply
becomeslimiting.
Most eukaryotic cells take considerably longer than bacte-
rial cells to grow and divide. Moreover, the cell cycle in adult
plants and animalsnormally is highly regulated(Chapter20).
the
This tight control prevents imbalanced, excessivegrowth of
tissueswhile ensuring that worn-out or damaged cells are re-
placed and that additional cells are formed in responseto new
circumstancesor developmental needs.For instance, the pro-
liferation of red blood cellsincreasessubstantiallywhen a per-
son ascendsto a higher altitude and needs more capacity to
capture oxygen. Somehighly specializedcells in adult animals,
such as nervecellsand striatedmusclecells,rarely divide, if at
all. The fundamental defect in cancer is loss of the ability to

18 C H A P T E R1 | L t F EB E G T N W
S |TH CELLS
control the growth and division of cells.In Chapter 25, we ex-
amine the molecular and cellular eventsthat lead to inappro-
priate, uncontrolled proliferation of cells. Diploid (2n)
Mitosis is an asexual process since the daughter cells
carry the exact samegeneticinformation as the parental cell.
ln sexwalreproduction, fusion of two cells produces a third
cell that contains genetic information from each parental
cell. Sincesuch fusions would causean ever-increasingnum-
ber of chromosomes, sexual reproductive cycles employ a Haploid(1nl
(",,o )
special type of cell division, called meiosis,that reducesthe
number of chromosomesin preparation for fusion (seeFig-
ure 5-3). Cells with a full set of chromosomes are called
diploid cells. During meiosis, a diploid cell replicates its
chromosomes as usual for mitosis but then divides twice One type Two types
of female of male
without copying the chromosomesin between. Each of the gamete gamete
resulting four daughter cells, which have only half the full
number of chromosomes,is said to be haploid.
Diploid (2n)
Sexual reproduction occurs in animals and plants and
even in unicellular organismssuch as yeasts(seeFigure 1-5).
Animals spendconsiderabletime and energygeneratingeggs Female Male
zygote zygote
and sperm, the haploid cells, called gametes,which are used
for sexualreproduction. A human femalewill produce about FIGURE 1-18 Dad madeyou a boy or girl. In animals,meiosis
half a million eggsin a lifetime, all of thesecells forming be- cellsformseggsandsperm(gametes).
of diploidprecursor Themale
fore she is born; a young human male produces about 100 parentproduces two typesof spermanddetermines the sexof the
million sperm each day. Gametes are formed from diploid zygoteIn humans, asshown here,
X andY arethe sex chromosomes;
precursor germ-line cells,which in humans contain 46 chro- the zygotemustreceivea Y chromosome fromthe maleparentto
mosomes.In humans the X and Y chromosomesare called developintoa male A : autosomes (non-sex
chromosomes).
sex chromosomes becausethey determine whether an indi-
vidual is male or female. In human diploid cells, the 44 re- bing" between the fingers; the cells in the webbing subse-
maining chromosomes,called autosomes,occur as pairs of quently die in an orderly and precisepattern that leavesthe
22 different kinds. Through meiosis,a man produces sperm fingers and thumb free to play the piano. Nerve cells in the
that have 22 chromosomes plus either an X or a Y, and a brain soon die if they do not make proper or useful electrical
woman produces ova (unfertilized eggs) with 22 chromo-
somesplus an X. Fusion of an egg and sperm (fertilization)
yields a fertilized egg,the zygote,with46 chromosomes,one
pair of each of the 22 kinds and a pair of Xs in femalesor an
X and a Y in males(Figure1-18). Errors during meiosiscan
lead to disorders resulting from an abnormal number of
chromosomes.These include Down's syndrome, caused by
an extra chromosome 21, and Klinefelter's syndrome,
causedby an extra X chromosome.

CellsDie from AggravatedAssaultor an

lnternal Program
'lfhen
cells in multicellular organismsare badly damagedor
infectedwith a virus, they die. Cell death resulting from such
a traumatic event is messy and often releasespotentially
toxic cell constituents that can damage surrounding cells.
FIGURE 1-19ApoPtoticcellsbreakapartwithout spewing
Cells also may die when they fail to receivea life-maintaining
forth cell constituentsthat might harm neighboringcells.
signal or when they receive a death signal. In this type of looklikethe cellon the left Cells
Whitebloodcellsnormally
programmed cell death, called apoptosis, a dying cell actu- programmed celldeath(apoptosis), likethe cellon the
undergoing
ally produces proteins necessaryfor self-destruction.Death right,formnumerous surface blebsthateventually arereleased The
by apoptosis avoids the releaseof potentially toxic cell con- cellisdyingbecause it lackscertaingrowth Apoptosis
signals. is
stituents(Figure1-19). important to eliminate virus-infectedcells,to remove cellswherethey
Programmed cell death is critical to the proper develop- arenot needed (likethewebbingthatdisappears asfingersdevelop),
ment and functioning of our bodies (Chapter 21). During andto destroy immunesystem cellsthatwouldreactwith ourown
fetal life, for instance,our hands initially developwith "web- bodies[Gopal Murtil/isuals Inc]
Unlimited,

THE WORK OF CELLS 19

connectionswith other cells. Some developinglymphocytes,
the immune-systemcells intended to recognizeforeign pro-
teins and polysaccharides,have the ability to reac against
our own tissues.Such self-reactivelymphocytes becomepro-
grammed to die before they fully marure. If these cells are
not weededout before reaching maturity, they can causeau-
toimmune diseases,in which our immune system destroys
the very tissuesit is meant to protect.
IE InvestigatingCellsand TheirParts
To build an integrared understanding of how the various
molecular components that underlie cellular functions work
together in a living cell, we must draw on various perspec-
tives. Here, we look at how five disciplines-cell biology,
biochemistry and biophysics,genetics,genomics,and devel-
opmental biology-can conrribure to our knowledge of cell
structure and function. The experimental approachesof
each field probe the cell's inner workings in different ways,
allowing us to ask different types of questions about cells
and what they do. Cell division provides a good example to
illustrate the role of different perspectivesin analyzing a
complex cellular process.Although we discussthe different
disciplinesseparatelyfor clarity, in practice most biologists
use multiple approachesin concert. This is part of the fun of
cell biologg putting together genetics with microscopy or
enzymology with development.
The realm of biology rangesin scalemore than a billion-
fold (Figure 1-20). Beyond that, it's ecology and earth sci-
ence at the "macro" end, chemistry and physics at the "mi-
cro" end. The visible plants and animals that surround us
are measuredin meters (100-102 m). By looking closely,we
can seea biologicalworld of millimeters(1 mm : 10-3 m)
and even tenths of millimeters (10-a m). Setting aside oddi-
ties like chicken eggs, most cells are 1-100 micrometers
(1 pm : 10-5 m) long and thus clearlyvisibleonly when mag-
nified. To seethe structureswithin cells, we must go farther
down the sizescaleto 10-100 nanometers(1 nm : 10 v m).
Cell Biology Revealsthe Size,Shape,Location,
and Movementsof Cell Components
The goal of cell biologists is to understandhow a cell is able
to control its own shape and surface properties, transport
materials to the right locations, copy itself, and receiveand
send signals.Cell biologists use severaltypes of microscopy
to observecells, while at the same time labeling specificcell
components and altering them to seewhat happens.Analy-
sesare generally done at the micrometer scale.
Actual observation of cells awaited development of the
first, crude microscopes in the early 1600s. A compound

Nanometers Micrometers Millimeters Meters

Small Assemblies
molecules
Macro-
Atoms molecules Cells M u l t i c e l l u l aor r g a n i s m s
Glucose Ribosome Bacterium C. elegans Newborn human

C-C bond Hemoglobin Mitochondrion Bumblebee

I
J '1
1010m 10sm 1 0 ' 8m 0-7m 10'6m 1 0 - 5m 1 0 - am 1 0 - 3m 1 0 2m 1 0 ' 1m 1 0 0m
0.1nm 1 nm 10nm 1 0 0n m 1 pm 10pm 1 0 0u m 1 mm 1 0m m 100mm 1m
FIGURE 1-20 Biologistsare interestedin objectsrangingin about15 mm across(d)Emperor
spider, penguins
areabout1 m tall.
sizefrom smallmolecules to the tallesttrees.A sampling
of [Part(a)Will andDeniMclntyrePart(b)YorgasNikas/photoResearchers,
Inc
biological
objects
alignedon a logarithmicscale(a)TheDNAdouble Part(c)GaryGauglerl/isuals
Unlimited,Inc Part(d)HughS Rosefuisuals
helixhasa diameter
of about2 nm (b)Eight-cell-stagenuman Unlimited.
lncl
embryothreedaysafterfertilization,
about200 rr"macross(c)A wolf

20 CHAPTER
1 I L I F EB E G I N S
WITHCELLS
microscope,the most useful type of light microscope,has
two lenses.The total magnifying power is the product of the
magnificationby each lens. As better lenseswere invented,
the magnifying power and the ability to distinguish closely
spacedobjects, the resolution, increasedgreatly. Modern
cclmpoundmicroscopesmagnify the view about a thousand-
fold, so that a bacterium 1 micrometer (1 p-) long looks
like it's a millimeterlong. Objectsabout 0.2 pm apart can be
discernedin theseinstruments.
Microscopy is most powerful when particular compo-
nents of the cell are stained or labeled specifically,enabling
them to be easilyseenand locatedwithin the cell. A simple
exampleis stainingwith dyesthat bind specificallyto DNA
to visualize the chromosomes. Specific proteins can be de-
tected by harnessingthe binding specificityof antibodies,the
proteins whose normal task is to help defend animals against
infection and foreign substances.In general,eachtype of an-
tibody binds to one protein or large polysaccharideand no
other (Chapter 3). Purified antibodies can be chemically
linked to a fluorescent molecule, which permits their detec-
tion in a specialfluorescencemicroscope(Chapter 3). lf a
cell or tissueis treated with a detergentthat partially dis-
solvescell membranes,fluorescentantibodiescan drift in
and bind to the specific protein they recognize. When the
sample is viewed in the microscope,the bound fluorescent
antibodies identify the location of the target protein (see
F i g u r e1 - 1 5 ) .
Betrer still is pinpointing proteins in living cells with in-
tact membranes. One way of doing this is to introduce an
engineeredgenethat codesfor a hybrid protein: part of the
hybrid protein is the cellular protein of interest;the other
part is a protein that fluoresceswhen struck by ultraviolet
light. A common fluorescentprotein used for this purpose is
green fluorescent protein (GFP), a natural protein that
makes some iellyfish colorful and fluorescent. GFP
"tagging" could reveal,for instance,that a particular pro-
tein is first made on the endoplasmic reticulum and then is
moved by the cell into the lysosomes.In this case,first the
endoplasmicreticulum and later the lysosomeswould glow
in the dark.
Chromosomesare visible in the light microscopeonly
during mitosis,when they becomehighly condensed.The ex-
traordinary behavior of chromosomesduring mitosis first
was discoveredusing the improved compound microscopes
of the late 1800s.About halfway through mitosis,the repli-
catedchromosomesbeginto move apart. Microtubules,one
of the three types of cytoskeletalfilaments, participate in this
movementof chromosomesduring mitosis.Fluorescenttag-
ging of tubulin, the protein subunitthat polymerizesto form
microtubules, reveals structural details of cell division that
otherwisecould not be seenand allows observationof chro-
mosomemovement(Figure1-21).
Electron miqroscopesuse a focusedbeam of electronsin-
stead of a beam of light. In transmission electron mi-
croscopy, specimensare cut into very thin sections and
placedunder a high vacuum,precludingexaminationof liv-
ing cells. The resolution of transmission electron micro-
llll| 5e6nsAnimation:Mitosis
A FIGURE1-21 During the later stages of mitosis,
microtubules (red) pull the replicated chromosomes(black)
toward the ends of a dividing cell. Thisplantcell is stained
with a DNA-bindingdye (ethidium)to revealchromosomes and
antibodiesspecificfor tubulinto reveal
with f luorescent-tagged
microtubulesAt this stagein mitosis,the two copiesof each
replicatedchromosome(calledchromatids)haveseparatedand are
movinqawavf rom eachother [Courtesy of AndrewBajer ]
scopes,about 0.1 nm, permits fine structural details to be
distinguished,and their powerful magnification would make
a 1-pm-long bacterialcell look like a soccerball. Most of the
organelles in eukaryotic cells and the double-layered
structure of the plasma membrane were first observedwith
electron microscopes(Chapter 9). \fith new specializedelec-
tron microscopy techniques,three-dimensionalmodels of
organellesand large protein complexes can be constructed
from multiple images.But to obtain a more detailed look at
the individual macromoleculeswithin cells, we must turn to
techniqueswithin the purview of biochemistry and bio-
physics.
s e v e a tl h e
B i o c h e m i s t r ay n d B i o p h y s i c R
M o l e c u l a rS t r u c t u r ea n d C h e m i s t r yo f P u r i f i e d
Cell Constituents
Biochemistsextract the contentsof cellsand separatethe con-
stituents based on differencesin their chemical or physical
properties,a processcalledfractionation. The attention to in-
dividual moleculesmeans operating at the nanometer scale.
Of particular interest are proteins, the workhorses of many
A typical fractionation schemeinvolvesuse
cellular processes.
of various separationtechniquesin a sequentialfashion.These
separationtechniquescommonly are basedon differencesin
the size of moleculesor the electric charge on their surface
(Chapter 3). To purify a particular protein of interest, a
purification scheme is designed so that each step yields a
GE L L SA N D T H E I RP A R T S
I N V E S T I G A T I NC 2'l
 tive is to engineera gene that encodesa protein of interest
*o ^c with a small attached protein "tag," which can be used to
;s'-

J 1
4"8"
2
-s'

3 - 4 - 5
pull out the protein from whole cell extracts.
Purification of a protein is a necessaryprelude to studies
on how it catalyzesa chemical reaction or carries out other
functions and how its activiry is regulated. Some enzymesare
made of multiple protein chains (subunits) with one chain cat-
alyzing a chemical reaction and other chains regulating when
and where that reaction occurs. The molecular machines that
perform many critical cell processesconstitute even larger as-
sembliesof proteins. By separating the individual proteins
composing such assemblies,their individual catalytic or other
activitiescan be assessed. For example,purification and study
- - of the activity of the individual proteins composing the DNA
replication machine provided clues about how they work to-
gether to replicate DNA during cell division (Chapter 4).
I
The folded, three-dimensionalstructure, or conformation,
of a protein is vital to its function. To understand the relation
between the function of a protein and its form, we need to
know both what it does and its detailed structure. The most
widely used method for determining the complex structures of
proteins, DNA, and RNA is x-ray crystallography, one of the
powerful tools of biophysics.Computer-assistedanalysisof the
data often permits the location of every atom in a large, com-
FIGURE 1-22 Biochemicalpurificationof a protein from a plex molecule to be determined. The double-helix structure of
cell extract often requiresseveralseparationtechniques.The
DNA, which is key to its role in herediry was first proposed
purificationcanbe followedby gelelectrophoresis of thestarting
basedon x-ray crystallographic studies.Throughout this book
proteinmixture andthefractions obtainedfromeachpurification
you will encounter numerous examplesof protein structuresas
step Inthisprocedure, a sample isappliedto wellsin thetop of a
gelatin-likeslabandan electric fieldisappliedInthe presence we zero in on how proteins work.
of
appropriate saltanddetergent concentrations, the proteins move
throughthefibersof the geltowardtheanode,with largerproteins GeneticsRevealsthe Consequences
movingmoreslowlythroughthe gelthansmaller ones(seeFigure
3-35)Whenthegelisstained, separated proteins
of DamagedGenes
arevisible as
distinctbandswhoseintensities areroughlyproportional to the Biochemical and crystallographic studies can tell us much
proteinconcentration Shownhereareschematic depictions of oels about an individual protein, but they cannot prove that it is
for thestarting mixtureof proteins (lane1)andsamples takenaiter required for cell division or any other cell process.The im-
eachof several purificationstepsIn the firststep,saltf ractionation, portance of a protein is demonstratedmost firmly if a muta-
proteinsthat precipitated with a certainamountof saltwerere- tion that prevents its synthesis or makes it nonfunctional
dissolved; electrophoresisof thissample (lane2) showsthatit adverselyaffects the processunder study.
contains fewerproteins thantheoriginal mixtureThesample then !(/e define the genotype of an organism as its composi-
wassubjected in successionto threetypesof column
tion of genes;the term also is commonly used in referenceto
chromatography thatseparate proteins by electrical
charge, size,or
different versionsof a singlegeneor a small number of genes
bindingaffinityfor a particularsmallmolecule (seeFigure 3-37).The
finalpreparation isquitepure,ascanbe seenfromtheappearance of interest in an individual organism. A diploid organism
of justoneproteinbandin lane5 [AfterJBergetal,2o02,Biochemistry generally carries two versions (alleles)of each gene, one de-
W H Freeman andCompany, p 87l rived from each parent. There are important exceptions,
such as the geneson the X and Y chromosomesin males of
some species,including our own. The phenotype is the visi-
preparation with fewer and fewer contaminating proteins, un- ble outcome of a gene's action, such as blue eyes versus
til finally only the protein of interestremains (Figure 1-22). brown eyesor the shapesof peas.In the early days of genet-
The initial purification of a protein of interest from a cell ics, the location and chemical identity of genes were un-
extract often is a tedious, time-consumingtask. Once a small known; only the observablecharacteristics,the phenotypes,
amount of purified protein is obtained, antibodies to it can could be followed. The concept that genesare like "beads"
be produced by methods discussedin Chapter 19. For a bio- on a long "string," the chromosome,was p,roposedearly in
chemist, antibodies are near-perfecttools for isolating larger the 1900s basedon geneticwork with the fruit fly Drosophila.
amounts of a protein of interest for further analysis.In ef- In the classicalgeneticsapproach, mutants are isolated that
fect, antibodies can "pluck out" the protein they specifically lack the ability to do something a normal organism can do.
recognizeand bind from a semipure sample containing nu- Often large genetic "screens" are done to look for many dif-
merous different proteins. An increasinglycommon alterna- ferent mutant individuals (e.g., fruit flies, yeast cells) that are

22 C H A P T E R1 I L I F EB E G I N SW I T H C E L L S
unable to complete a certain process,such as cell division or
muscle formation. In experimental organismsor cultured cells,
mutations usually are produced by treatment with a mutagen,
a chemical or physical agent that promotes mutations in a
largely random fashion.But how can we isolateand maintain
mutant organismsor cellsthat are defectivein some process,
such as cell division, that is necessaryfor survival?One way is
to look for organisms with a temperature-sensitivemutation.
Thesemutants are able to grow at one temperature,the permis-
siue temperature, but not at anothe5 usually higher tempera-
ture, the nonpermissiuetemperature.Normal cells can grow at
either temperature. In most cases,a temperature-sensitivemu-
tant produces an altered protein that works at the permissive
temperaturebut unfolds and is nonfunctional at the nonpermis-
sive temperature. Temperature-sensitive screensare readily
done with viruses,bacteria, yeast,roundworms, and fruit flies.
By analyzing the effectsof numerous different temperature-
sensitivemutations that altered cell division, geneticistsdis-
coveredall the genesnecessaryfor cell division without know-
ing anything, initially, about which proteins they encode or
how theseproteinsparticipatein the process.The greatpower
of geneticsis to reveal the existenceand relevanceof proteins
without prior knowledge of their biochemicalidentity or mo-
lecular function. Eventually these "mutation-defined" genes
were isolatedand replicated(cloned)with recombinantDNA
techniquesdiscussedin Chapter 5. \fith the isolatedgenesin
hand, the encodedproteins could be produced in the test tube
or in engineeredbacteria or cultured cells. Then the bio-
chemistscould investigatewhether the proteins associatewith
other proteins or DNA or catalyze particular chemical reac-
tions during cell division (Chapter20).
The analysis of genome sequencesfrom various organ-
isms during the past decadehas identified many previously
unknown DNA regions that are likely to encode proteins
(i.e., protein-coding genes).The generalfunction of the pro-
tein encoded by a sequence-identifiedgene may be deduced
by analogy with known proteins of similar sequence.Rather
than randomly isolating mutations in novel genes, several
techniquesare now available for inactivating specific genes
by engineering mutations into them or destroying their
mRNA with interfering RNA molecules(Chapter 5). The ef-
fects of such deliberategene-specificinactivation procedures
provide information about the role of the encoded proteins
in living organisms. This application of genetic techniques
starts with a gene/proteinsequenceand ends up with a mu-
tant phenotype; traditional genetics starts with a mutant
phenotype and ends up with a gene/proteinsequence.
GenomicsRevealsDifferencesin the Structure
and Expressionof EntireGenomes
Biochemistry and geneticsgenerally focus on one gene and
'$flhile
its encoded protein at a time. powerful, these tradi-
tional approachesdo not give a comprehensiveview of the
structure and activity of an organism'sgenome,its entire set
of genes.The field of genomicsdoes just that, encompassing
the molecular characterizationof whole genomesand the de-
termination of global patterns of geneexpression.The recent
completion of the genome sequencesfor more than 100
speciesof bacteria and severaleukaryotesnow permits com-
parisons of entire genomes from different species.The re-
sults provide overwhelming evidenceof the molecular unity
of life and the evolutionary processesthat made us what we
are (seeSection 1.5). Genomics-basedmethods for compar-
ing thousands of piecesof DNA from different individuals
all at the same time are proving useful in tracing the history
and migrations of plants and animals and in following the
inheritance of diseasesin human families.
DNA microarrays can simultaneously detect all the
mRNAs presentin a cell, thereby indicating which genesare
being transcribed. Such global patterns of gene expression
clearly show that liver cells transcribe a quite different set of
genesthan do white blood cellsor skin cells. Changesin gene
expressionalso can be monitored during a diseaseprocess'
in responseto drugs or other external signals'and during de-
velopment. For instance,the identification of all the mRNAs
presentin cultured fibroblasts before, during' and after they
divide has given us an overall view of transcriptional
changesthat occur during cell division (Figure 1-23). Cancet
diagnosis is being transformed becausepreviously indistin-
guishablecancer cells have distinct gene expressionpatterns
and prognoses (Chapter 25). Similar studies with different
organisms and cell types are revealing what is universal
about the genesinvolved in cell division and what is specific
to particular organisms.To find out which genesare directly
regulatedby a transcription factor, chromatin containing the
protein of interest can be purified with an antibody and the
associated DNA analyzed on microarrays, a procedure
called chromatin immunopreclpltatlon.
The entire complement of proteins in a cell, its proteome'
is controlled in part by changesin genetranscription.The reg-
ulated synthesis,processing,localization' and degradationof
specific proteins also play roles in determining the proteome
of a particular cell. Learning how proteins bind to other pro-
teins, often in large, multiprotein complexes, is providing a
comprehensiveview of the molecular machines important for
cell functioning. The field of proteomics will advance dramat-
ically once high-throughput x-ray crystallography, currently
under development,permits researchersto rapidly determine
the structuresof hundredsor thousandsof proteins.
DevelopmentalBiology RevealsChangesin
the Propertiesof Cellsas They Specialize
Another approach to viewing cells comes from studying how
they changeduring development of a complex organism. Bac-
teria, algae, and unicellular eukaryotes (protozoans, yeasts)
often, but by no means always, can work solo. The concerted
actions of the trillions of cells that compose our bodies require
an enormous amount of communication and division of labor.
During the development of multicellular organisms, differen-
tiation processesform hundreds of cell types, each specialized
for a particular task: transmission of electric signals by neu-
rons, transport of oxygen by red blood cells, destruction of in-
fecting bacteria by macrophages' contraction by muscle cells,
chemicalprocessingby liver cells,and so on.
GE L L SA N D T H E I RP A R T S
I N V E S T I G A T I NC 23
 expression is readily apparent in an early fly embryo, in
which all the cells look alike until they are stained to detect
the proteins encoded by particular genes (Figure 1-24).
Transcription can changewithin one cell type in responseto
an external signal or in accordancewith a biological clock;
some genes,for instance,undergo a dalIy cycle between low
and high transcription rates.
Producing different kinds of cells is not enough to make
an organism, any more than collecting all the parts of a truck
in one pile gives you a truck. The various cell types must be
organized and assembled into all the tissues and organs.
Even more remarkable, these body parts must work almost
immediately after their formation and continue working
during the growth process. For instance, the human heart
begins to beat when it is lessthan 3 mm long, when we are
mere 23-day-old embryos, and continues beating as it grows
into a fist-sizemuscle. From a few hundred cells to billions,
and still ticking.
In the developing organism, cells grow and divide at
some times and not others, they assembleand communicate,
they prevent or repair errors in the developmentalprocess,
and they coordinate each tissuewith others. In the adult or-
EXPERIMENTAL FIGURE 1-23 Microarrayanalysisof normal ganism,cell division largely stops in most organs.If part of
growing braincellsand braintumor cells.An experiment likethis an organ such as the liver is damaged or removed, cell divi-
isa startingpointfor learning howtumorcellsdifferfromnormal sion resumesuntil the organ is regenerated.The legend goes
cellsRNAwasextracted fromnormalgrowingmousebraincells that Zets punished Prometheus for giving humans fire by
fromthecerebellum andfroma tumorof thecerebellum TheRNA
fromthe tumorwaslabeled with a reddye,andRNAfromthe
normal,non-tumorous cerebellum waslabeled with a greendye The
two RNApreparations weremixedandhybridized to a microarray
containing thousands of spotsof DNA Eachspotcontains the DNA
sequence of onegene Unbound RNAwaswashedawayandthe
microarray wasexposed to UVlight,whichcauses the dyesto
fluoresce Spotsthataregreenhaveboundmostlynormalcerebellum
RNA,spotsthatareredhaveboundmostlytumorRNA,andspots
thatareyellowhaveboundroughly equalamounts of each.The
faintlystained spotsrepresent genesfor whichthereislittleRNAin
eithersampleThedataindicate whichgeneshavebeentranscribed
in tumors,normalcerebellum, or both Onlypartof the datais
shownhereTheentiredatasetrequires analyzing the colorsof more
than25,000spots,allof whichcanbefittedontoonemicroscope
slide.Precisemeasurements of colorintensityareactuallymadeby a
spectrophotometer, but lookingby eyeshowsthat manygenesare
morehighlyexpressed in normalor tumorcells.Someof these FIGURE 1-24 Differentialgene expressioncan be detectedin
differences arethe consequence of the changeintotumorcells,but early fly embryosbefore cellsare morphologicallydifferent.
somemayreveal geneexpression changes thatcausethetumorsto An earlyDrosophila embryohasabout6000cellscoveringitssurface,
form In addition, proteins madeexclusively in tumors,andperhaps mostof whichareindistinguishable by simplelightmicroscopy. lf the
necessary for uncontrolled growth,maybe candidate targetsfor embryois madepermeable to antibodies with a detergentthat
discovering anti-cancer drugs [Courtesyof TalRavehandl\y'atthewScott, partiallydissolves
membranes, theantibodies canfindandbindto
StanfordUniversitySchoolof Medicinel
the proteinstheyrecognize Inthisembryowe seeantibodies tagged
with a fluorescentlabelboundto proteins thatarein the nuclei;each
smallsphere corresponds to onenucleus. Threedifferentantibodies
wereused,eachspecific for a differentproteinandeachgivinga
Many of the differences among differentiated cells are
distinctcolor(yellow,green,or blue)in a fluorescence microscope.
due to production of specificsetsof proteins neededto carry
Theredcolorisaddedto highlight overlaps between theyellowand
out the unique functions of each cell type; that is, only a
bluestainsThelocations of thedifferent proteinsshowthatthe cells
subsetof an organism'sgenesis transcribedat any given time arein factdifferentat thisearlystage,with particular genesturned
or in any given cell. Such differential gene expressionat dif- on in specific
stripesof cells.Thesegenescontrolthesubdivision of
ferent times or in different cell types occurs in bacteria, the bodyintorepeating segments, likethe blackandyellowstripes of
fungi, plants, animals, and even viruses. Differential gene a hornet.[Courtesyof Sean Carroll,
University
of Wisconsin
]

24 CHAPTER
1 I L I F EB E G I N S
WITHCELLS
chaining him to a rock and having an eagleeat his liver. The
punishment was eternal because,as the Greeks evidently
knew, the liver regenerates.
Developmental studies involve watching where, when,
and how different kinds of cells form, discovering which
signals trigger and coordinate developmentalevents,and
understandingthe differential gene action that underliesdif-
ferentiation (Chapters 16 and 21). During development we
can see cells change in their normal context of other cells.
Cell biology, biochemistry, cell biology, genetics, and ge-
nomics approachesare all employed in studying cells during
development.
C h o o s i n gt h e R i g h t E x p e r i m e n t aOl r g a n i s m
for the Job
Our current understanding of the molecular functioning of
cells rests on studieswith viruses,bacteria, yeast, protozoa,
slime molds, plants, frogs, sea urchins, worms, insects,fish,
chickens, mice, and humans. For various reasons, some
organisms are more appropriate than others for answering
particular questions.Becauseof the evolutionary conserva-
tion of genes,proteins, organelles,cell types, and so forth,
discoveriesabout biological structuresand functions obtained
with one experimental organism often apply to others. Thus
researchersgenerallyconduct studieswith the organism that
is most suitable for rapidly and completely answering the
question being posed, knowing that the results obtained in
one organismare likely to be broadly applicable.Figure1-25
summarizesthe typical experimental usesof various organ-
isms whose genomeshave been sequencedcompletely or
nearly so. The availability of the genomesequencesfor these
organisms makes them particularly useful for geneticsand
genomicsstudies.
Bacteria have several advantagesas experimental or-
ganisms: they grow rapidlS possesselegant mechanisms
for controlling gene activity, and have powerful genetics.
This latter property relates to the small size of bacterial
genomes,the easeof obtaining mutants, the availability of
techniquesfor transferring genes into bacteria, an enor-
mous wealth of knowledge about bacterial gene control
and protein functions, and the relative simplicity of map-
ping genesrelative to one another in the genome. Single-
celledyeastsnot only have some of the sameadvantagesas
bacteria but also possessthe cell organization,marked by
the presenceof a nucleusand organelles,that is character-
istic of all eukaryotes.
Studiesof cellsin specializedtissuesmake use of animal
and plant "models," that is, experimentalorganismswith
attributes typical of many others. Nerve cells and muscle
cells, for instance,traditionally were studied in mammals
or in creatureswith especiallylarge or accessible cells,such
as the giant neural cells of the squid and sea hare or the
flight musclesof birds. More recently,muscle and nerve de-
velopment have been extensively studied in fruit flies
(Drosophila melanogaster),roundworms (Caenorhabditis
elegans),and zebrafish(Danio rerio), in which mutants can
be readily isolated. Organisms with large-celledembryos
that develop outside the mother (e.g.' frogs, sea urchins,
fish, and chickens) are extremely useful for tracing the fates
of cells as they form different tissues and for making ex-
tracts for biochemicalstudies.For instance,a key protein in
regulating mitosis was first identified in studies with frog
and sea urchin embryos and subsequentlypurified from ex-
tracts (Chapter 20lt.
Using recombinant DNA techniques,researcherscan en-
gineer specificgenesto contain mutations that inactivate or
increaseproduction of their encodedproteins. Suchgenescan
be introduced into the embryos of worms, flies, frogs, sea
urchins, chickens,mice, a variety of plants, and other organ-
isms,permitting the effectsof activating a geneabnormally or
inhibiting a normal gene function to be assessed.This ap-
proach is being used extensivelyto produce mouse versions
of human genetic diseases.Inactivating particular genes by
introducing short piecesof interfering RNA is allowing quick
tests of gene functions possiblein many organisms.The ex-
pansion of genomeproiectsto critically important diseaseor-
ganisms,such as malaria, and to creaturesthat span the evo-
lutionary tree is bringing new options for medicine and new
insights into how living organisms have diversified to take
advantageof every possibleecologicalniche.
Mice have one enormous advantage over other experi-
mental organisms:they are the closestto humans of any an-
imal for which powerful geneticapproachesare feasible.En-
gineered mouse genes carrying mutations similar to those
associatedwith a particular inherited diseasein humans can
be introduced into mouse embryonic stem (ES) cells. These
cells can be injected into an early embryo, which is then im-
planted into a pseudopregnantfemale mouse (a mouse
treated with hormones to trigger physiological changes
neededfor pregnancy) (Chapter 5). If the mice that develop
from the injected ES cells exhibit diseasessimilar to the hu-
man disease,then the link between the diseaseand muta-
tions in a particular geneor genesis supported. Once mouse
models of a human diseaseare available, further studies on
the molecular defectscausing the diseasecan be done and
new treatmentscan be tested,thereby minimizing human ex-
posure to untested treatments. Large-scalegenetic screens
are being done that take advantageof newly designedmuta-
genic transposons.The transposons allow efficient genera-
tion of mouse mutants and rapid identification of the gene
that has been hit in each one.
A continuous unplanned genetic screen has been per-
\What we mean
formed on human populations for millennia.
is that all sorts of human variations have arisen and have
been noticed, since they affect visible or noticeable human
characteristics.Thousandsof inherited traits have beeniden-
tified and, more recently,mapped to locations on the chro-
mosomes.Some of these traits are inherited propensitiesto
get a disease;others are eye color or other minor character-
istics. Geneticvariations in virtually every aspectof cell biol-
ogy can be found in human populations, allowing studiesof
normal and diseasestatesand of variant cells in culture.
Less-commonexperimental organisms offer possibilities
for exploring unique or exotic properties of cells and for
studying standard properties of cells that are exaggeratedin
GE L L SA N D T H E I RP A R T S
I N V E S T I G A T I NC 25
 Podcast:Common ExperimentalOrganisms

Viruses Bacteria

Proteinsinvolvedin DNA, RNA, Proteinsinvolvedin DNA, RNA,

protein synthesis protein synthesis,
Gene regulation metabolism
Cancerand control of cell Gene regulation
oroliferation Targetsfor new antibiotics
Transportof proteinsand Cell cycle
organellesinsidecells Signaling
Infectionand immunity
Possiblegene therapy approaches

Yeast (Saccharo myce s cerev is i ael Roundworm I Caenorh abd itis

elegansl
Controlof cell cycle and cell division
Proteinsecretionand membrane Developmentof the body plan
biogenesis C e l ll i n e a g e
Functionof the cytoskeleton Formationand function of the
Cell differentiation nervous system
Aging Controlof programmedcell death
Gene regulationand chromosome Cell proliferationand cancergenes
structure Aging
Behavior
Gene regulationand chromosome
structure

Fruit ffy (Drosophi Ia mel anoga sterl Zebrafish

Developmentof the body plan Developmentof vertebratebody

Generationof differentiatedcell tissues
lineages Formationand function of brain and
Formationof the nervoussystem, nervous syslem
heart,and musculature Birth defects
Programmedcell death Cancer
Geneticcontrol of behavior
Cancergenesand control of cell
proliferation
Controlof cell polarization
Effectsof drugs, alcohol,pesticides

Mice, includingculturedcells Plant (A ra b i d ops is th aIi anal

Developmentof body tissues Developmentand patterningof

F u n c t i o no f m a m m a l i a ni m m u n e tissues
system Geneticsof cell biology
Formationand function of brain Agricultural applications
and nervoussystem Physiology
Models of cancersand other Gene regulation
human diseases lmmunity
Gene regulationand inheritance Infectiousdisease
Infectiousdisease

26 C H A P T E R1 I L I F EB E G I N SW I T H C E L L S
< FIGURE 1-25 Eachexperimentalorganismusedin cell Cell division was viewed, and indeed discovered,by some
biology has advantagesfor certaintypes of studies.Viruses (a) of the earliest usersof microscopes.More recently a variety
andbacteria (b)havesmallgenomes amenable to genetic dissection of kinds of microscopy, including confocal and electron
Manyinsights intogenecontrolinitially camefromstudies with these microscopy and time-lapseimaging (Chapter 9), have been
organismsTheyeastSaccharomyces cerevisiae (c)hasthe cellular
used to characterizethe stepsof the cell cycle. Most biology
organization of a eukaryote but isa relatively simplesingle-celled begins with this sort of observation, defining the mysteries
organism thatiseasyto growandto manipulate genetically.Inthe
that must be tackled experimentally.Then manipulations be-
nematode worm Caenorhabditis elegans (d),whichhasa small
gin. Antibodies were made againstproteins that play critical
numberof cellsarranged in a nearlyidentical wayin everyworm,the
formation of eachindividual roles in cell division both to detect proteins and in some
cellcanbe tracedThefruitfly
Drosophila melanogaster (e),firstusedto discover the properties of casesto interfere with the functions of those proteins. Key
chromosomes, hasbeenespecially valuable in identifying genesthat proteins were fused to fluorescentproteins, starting with the
controlembryonic development Manyof thesegenesare iellyfish greenfluorescentprotein (GFP),so that key proteins
evolutionarily conserved in humansThezebraf ishDaniorerio(f)is could be followed in living cells. Questions about when and
usedfor rapidgenetic screens to identify genesthatcontrol where proteins work could be addressedand their functions
development andorganogenesis. Of theexperimental animal defined to an extent.
systems, mice(Musmusculus) (g)areevolutionarily the closestto The apparatusof cell division, such as the mitotic spindle
humans andhaveprovided models for studying numerous human and other protein complexes,was purified and analyzed us-
genetic andinfectious diseases. Themustard-family weed ing the approaches of biochemistry and biophysics. Each
Arabidopsis thaliana,sometimes described asthe Drosophila of the protein had to be purified to find out whether it is part of a
plantkingdom, hasbeenusedfor genetic screens to identifygenes complex of proteins bound together as a machine, and the
involved in nearlyeveryaspect of plantlife Genome sequencing is structuresof key proteins were determined using x-ray crys-
completed for manyviruses andbacterial species, theyeast
tallography and other methods (Chapter 3). Previously un-
Saccharomyces cerevisiae, the roundwormC. elegans,the f ruit fly
known enzymatic activities were detected in extracts using
D. melanogaste4 humans,andthe plantArabidopsis thalianallis
mostlycompleted for miceandin progress for zebrafish assays such as measuring the attachment of phosphate
Other
organisms, particularly frogs,seaurchins, chickens, andslimemolds, groups to cell division regulatory proteins by kinases, and
continue to be immensely valuable for cellbiologyresearch then the relevant kinasescould be purified.
Increasingly, a widevariety of otherspecies areused,especially for Finding a novel protein in a complex of proteins involved
studies of evolution of cellsandmechanisms [Part (a)VisualsUnlimited, in cell division makes it a good bet that the protein does
Inc Part(b)KariLountmaa/Science PhotoLibrary/Photo Researchers,Inc Part something important, a sort of "guilt by association," but it
(c)ScimavPhoto Researchers,Inc Part(d)Photo Researchers, IncPart(e) doesnot provide proof that the protein matters. For that one
Darwin Dale/Photo Researchers,Inc,Part(f)IngeSpencel'/isuals Unlimited, Inc must turn to genetics. Genetics can be used to identify
Part(g)J M Labavjancanatuisuals Unlimited,Inc Part(h)Darwin Dale/Photo mutants in the newly found protein. If cell division fails in a
Researchers, Inc] living organism when the protein is not working, you know
the protein matters. Geneticsis also a way to identify previ-
ously unknown genesand proteins sincescreenscan be done
a useful fashion in a particular animal. For example, the (especiallyin bacteria and yeast, but also in more complex
ends of chromosomes,the telomeres,are extremely dilute in Iab organisms)to look for all the genesthat are neededfor
most cells.Human cells typically contain 92 telomeres(46 cell division. The newly discoveredproteins can be incorpo-
chromosomesX 2 endsper chromosome).In contrast,some rated into a complete picture of the mechanicsof cell divi-
protozoawith unusual "fragmented" chromosomescontain sion machinery.
millions of telomeres per cell. Taking advantage of the Genomics provides another way to look for working
unique properties of this well-chosen experimental organ- parts of the cell-division machine. Since it is often true that
ism has led to important recent discoveriesabout telomere mRNAs and proteins are produced only when they are
structure. needed, using microarrays to look for all geneswhose ex-
pressionvaries with the cell cycle is a powerful approach to
identify candidatesfor cell division regulators.
The Most SuccessfulBiologicalStudiesUse
Having identified new genesthat are required for cell di-
M u l t i p l eA p p r o a c h e s vision, one must find out how thesegenes'protein products
We have discussedfive classesof approachesto biological work. Simply knowing that a protein matters to cell division
problems: cell biology biochemistry and biophysics, genet- is not enough to understand the mechanism.Thus it is nec-
ics, genomics,and developmentalbiology. Each has its own essaryto return to biochemical and biophysical approaches
types of experiments,and most biological problems require to work out the molecular biology and to cell biology to
more than one approach in order to reach a satisfying un- monitor protein locations and movements.
derstanding of mechanism. Now we will survey how these Finally, cell division does not happen in a vacuum; it
approacheshave been applied to the study of cell division happens in the context of the life cycle of the organism. To
to emphasizehow important it is to use multiple types of fully appreciatehow the regulation works and is used, it is
experlments. important to use the approachesof developmental biology

GE L L SA N D T H E I RP A R T s
I N V E S T I G A T I NC 27
to revealwhen and where cell division normally happensand
why. In theseexperimentsthe times and placesof cell division
during the developmentof the organism are monitored, and
then the signals that stimulate or suppresscell division are
identified and studied. Errors in developmental control of
cell division, revealedby studying mutants, can causeorgans
and tissuesto be the wrong sizeor cancer.
The samekinds of approachesthat work for cell division
can be applied to many other biological challenges,such as
Iearning how muscles form or how they work or how the
brain functions. Often it's good to use every tool in the
toolkit.
tf,| A GenomePerspective
on Evolution
Comprehensivestudies of genesand proteins from many
organismsare giving us an extraordinary documentationof
the history of life. Nature is a laboratory that has been
conducting experimentsfor billions of years, and some of
the most successfulgenomesthat emergedare still with us.
'We
share with other eukaryotes thousands of individual
proteins, hundredsof macromolecularmachines,and most
of our organelles,all as a result of our sharedevolutionary
history. New insights into molecular cell biology arising
from genomicsare leadingto a fuller appreciationof rhe el-
egant molecular machines that arose during billions of
yearsof genetictinkering and evolutionaryselectionfor the
most efficient, precise designs. Due ro alternative RNA
splicing,the number of proteins vastly exceedsthe number
of genes,and the functions of many variant proteins and
assembliesof proteins remain to be discovered. Once a
more complete description of cells is in hand, we will be
ready to fully investigate the rippling, flowing dynamics of
living systems.
MetabolicProteins,the GeneticCode,
a n d O r g a n e l l eS t r u c t u r e sA r e N e a r l yU n i v e r s a l
Even organisms that look incredibly different share many
biochemical properties. For instance, the enzymesthat cat-
alyze degradation of sugars and many other simple chemi-
cal reactions in cells have similar structures and mecha-
nisms in most living things. The genetic code whereby the
nucleotide sequencesof mRNA specifiesthe amino acid se-
quencesof proteins can be read equally well by a bacterial
cell and a human cell. Becauseof the universalnature of the
genetic code, bacterial "factories" can be designedto man-
ufacture growth factors, insulin, clotting factors, and other
human proteins with therapeutic uses. The biochemical
similarities among organisms also extend to the organelles
found in eukaryotic cells.The basic structuresand functions
of these subcellular components are largely conservedin all
eukaryotes.
28 CHAPTER
1 I L I F EB E G I N S
WITHCELLS
Computer analysisof DNA sequencedata, now avail-
able for numerousbacterialspeciesand severaleukaryotes,
can locate protein-coding geneswithin genomes.\fith the
aid of the geneticcode, the amino acid sequencesof pro-
teins can be deduced from the corresponding gene se-
quences.Although simple conceptually, "finding" genes
and deducing the amino acid sequencesof their encoded
proteins is complicated in practice becauseof the many
noncodingregionsin eukaryoticDNA (Chapter5). Despite
the difficulties and occasional ambiguities in analyzing
DNA sequences, comparisonsof the genomesfrom a wide
range of organisms provide stunning, compelling evidence
for the conservation of the molecular mechanisms that
build and change organisms and for the common evolu-
t i o n a r y h i s t o r yo f a l l s p e c i e s .
Darwin'sldeasAbout the Evolutionof Whole
A n i m a l sA r e R e l e v a n t o G e n e s
Darwin did not know that genesexist or how they change,
but we do: the DNA replication machine makes an error, or
a mutagen causesreplacement of one nucleotide with an-
other or breakage of a chromosome. Some changesin the
genomeare innocuous, some mildly harmful, some deadly; a
very few are beneficial. Mutations can change the sequence
of a gene in a way that modifies the activity of the encoded
protein or alters when, where, and in what amounts the pro-
tein is produced in the body.
Gene-sequencechangesthat are harmful will be lost
from a population of organisms becausethe affected indi-
viduals cannot survive as well as their relatives. This selec-
tion process is exactly what Darwin described without
knowing the underlying mechanismsthat cause organisms
to vary. Thus the selectionof whole organisms for survival
is really a selectionof genes,or more accurately sets of
genes.A population of organismsoften contains many vari-
ants that are all roughly equally well-suited to the prevailing
conditions. When conditions change-a fire, a flood, loss of
preferred food supply, climate shift-variants that are better
able to adapt will survive, and those less suited to the new
conditions will begin to die out. In this way, rhe genetic
composition of a population of organisms can change over
tlme.
M a n y G e n e sC o n t r o l l i n gD e v e l o p m e n t
A r e R e m a r k a b l yS i m i l a ri n H u m a n s
a n d O t h e rA n i m a l s
As humans, we probably have a biasedand somewhat exag-
gerated view of our status in the animal kingdom. Pride in
our swollen forebrain and its associatedmental capabilities
may blind us to the remarkably sophisticated abilities of
other species:navigation by birds, the sonar systemof bats,
homing by salmon, or the flight of a fIy.
Despite all the evidencefor evolutionary unity at the cel-
lular and physiological levels,everyoneexpectedthat genes
(a) < FIGURE 1-26 Similargenes,conservedduringevolution,
regulatemanydevelopmental processes in diverseanimals.
Insects andmammals areestimated to havehada commonancestor
Genes abouthalfa billionyearsago.Theysharegenesthatcontrolsimilar
processes, suchasgrowthof heartandeyesandorganization of the
bodyplan,indicating conservation of functionfromancient times.
(a)Hoxgenesarefoundin clusters on thechromosomes of mostor
allanimalsHoxgenesencoderelated proteinsthatcontrolthe
activities of othergenesHoxgenesdirectthedevelopment of
different segments alongthe head-to-tail axisof manyanimals, as
Flv Mammal indicated by corresponding colors. Eachgeneisactivated
(transcriptionally) in a specificregionalongthe head-to-tail axisand
controls the growthof tissues there Forexample, in micethe Hox
genesareresponsible for thedistinctive shapes of vertebrae
Mutations affecting Hoxgenesin fliescausebodypartsto formin
thewronglocations, suchaslegsin lieuof antennae on the head.
These genesprovide a head-to-tail address andserveto direct
formation of the rightstructures in the rightplaces(b)Development
of the largecompound eyesin fruitfliesrequires a genecalled
eyeless (namedfor the mutantphenotype). (c)Flieswith inactivated
eyeless geneslackeyes(d)Normalhumaneyesrequire the human
gene,calledPax6,that corresponds (e)Peoplelacking
to eyeiess.
adequatePax6functionhavethe geneticdisease anrrdia,a lackof
irisesin the eyesPax6andeyeless encodehighlyrelatedproteins
that regulate theactivities of othergenesandaredescended from
the sameancestral gene lParts (b)and(c)Andreas Hefti,
Interdepartmental
Electron Microscopy (lEM)Biocenter of BaselPart(d)
of theUniversity
(d) (e)
@Simon Fraser/Photo Researchers.Inc Part(e)VisualsUnlimitedl

that, as far as we can tell, are utterly absentfrom certain lin-

eagesof animals. Plants, not surprisingly,exhibit many such
differencesfrom animals after a billion-year separation in
their evolution. Yet certain DNA-binding proteins differ be-
tween peasand cows at only two amino acids out of 1'021

regulating animal development would differ greatly from
one phylum to the next. After all, insectsand seaurchins and
mammals look so different. \(e must have many unique
proteins to createa brain like ours . . . or must we? The fruits
of research in developmental genetics during the past two
decadesrevealthat insectsand mammals, which have a com-
mon ancestor about half a billion years ago, possessmany
similar development-regulating genes(Figure 1-26). Indeed,
a large number of these genes appear to be conserved in
many and perhaps all animals. Remarkably, the develop-
mental functions of the proteins encoded by thesegenesare
also often preserved.For instance,certain proteins involved
in eye development in insects are related to protein regula-
tors of eye developmentin mammals. Samefor development
of the heart, gut, lungs, and capillaries and for placementof
body parts along the head-to-tail and back-to-front body
axes (Chapter19).
This is not to say that all genesor proteins are evolution-
arily conserved. Many striking examples exist of proteins
H u m a nM e d i c i n el s I n f o r m e db y R e s e a r c h
on OtherOrganisms
Mutations that occur in certain genesduring the course of
our lives contribute to formation of various human cancers.
The normal, wild-type forms of such "cancer-causing"genes
generallyencodeproteins that help regulatecell proliferation
or death (Chapter 21').'Wealso can inherit from our parents
mutant copies of genesthat causeall manner of genetic dis-
eases,such as cystic fibrosis, muscular dystrophy' sickle cell
anemia, and Huntington's disease.Happily we can also in-
herit genesthat make us robustly resist disease.A remark-
able number of genesassociatedwith cancer and other hu-
man diseasesare present in evolutionarily distant animals.
For example, a recent study shows that more than three-
quarters of the known human diseasegenes are related to
genesfound in the fruit fIy Drosophila.
Vith the identification of human diseasegenesin other
organisms,experimental studies in experimentally tractable
organisms should lead to rapid progress in understanding

OEN E V O L U T I O N
A G E N O M EP E R S P E C T I V 29
the normal functions of the disease-relatedgenes and what
occurs when things go awry. ConverselS the disease states
themselvesconstitute a genetic analysis with well-studied
phenotypes. All the genesthat can be altered to cause a cer-
tain diseasemay encode a group of functionally related pro-
teins. Thus clues about the normal cellular functions of pro-
30 . cHAPTERI I L I F EB E G I N SW I T H C E L L S
teins come from human diseasesand can be used to guide
initial research into mechanism. For instance, genesinitially
identified becauseof their link to cancer in humans can be
studied in the context of normal development in various
model organisms, providing further insight about the func-
tions of their protein products.
 CHAPTER

CHEMICAL
FOUNDATIONS
Polarized
light microscopic
imageof crystalsof ATBwhose
isa primarysourceof energythat drivesmanycellular
hydrolysis
chemicalreactions[Dr.ArthurM SiegelmaMr'isuals
Unlimited
]

he life of a cell dependson thousands of chemical in-
teractions and reactions exquisitely coordinated with
one another in time and sDaceand under the influence
of the cell's genetic instructions and its environment. By un-
derstandingat a molecular level theseinteractions and reac-
tions, we can begin to answer fundamental questionsabout
cellular life: How doesa cell extract critical nutrients and in-
formation from its environment? How does a cell convert
the energy stored in nutrients into work (movement,synthe-
sis of critical components)?How does a cell transform nutri-
ents into the fundamental structuresrequired for its survival
(cell wall, nucleus, nucleic acids, proteins, cytoskeleton)?
How does a cell link itself to other cells to form a tissue?
How do cells communicate with one another so that a com-
plex, efficiently functioning organism can develop and
thrive? One of the goals of Molecular Cell Biology is to pro-
vide answers to these and other questions about the struc-
ture and function of cells and organisms in terms of the
properties of individual moleculesand ions.
For example, the properties of one such molecule,water,
have controlled and continue to control the evolution,
structure, and function of cells. You cannot understand bi-
ology without appreciating how the properties of water
control the chemistry of life. Life first arose in a watery en-
vironment. Constituting 70-80 percent by weight of most
cells, water is the most abundant molecule in biological sys-
tems. It is within this aqueous milieu that small molecules
and ions, which make up about 7 percent of the weight of
living matter, assembleinto the larger macromoleculesand
macromolecular aggregatesthat make up a cell's machinery
and architecture and so the remaining mass of organisms.
These small molecules include amino acids (the building
blocks of proteins), nucleotides (the building blocks of
DNA and RNA), lipids (the building blocks of biomem-
branes), and sugars (the building blocks of starchesand
cellulose).
Many biomolecules(e.g.,sugars)readily dissolvein wa-
ter; these molecules are called hydrophilic (water liking).
Others (e.g., cholesterol)are oilS fatlike substancesthat
shun water; these are said to be hydrophobic (water fear-
ing). Still other biomolecules(e.g.,phospholipids)are a bit
schizophrenic, containing both hydrophilic and hydropho-
bic regions; these molecule are said to be amphipathic.
Phospholipids are used to build the flexible membranes
that form the wall-like boundaries of cells and their inter-
nal organelles.The smooth functioning of cells, tissues'
and organisms depends on all these molecules, from the
smallest to the largest. Indeed, the chemistry of the simple
proton (H*) can be as important to the survival of a hu-
man cell as that of each gigantic, genetic-code-carrying
DNA molecule (the mass of the DNA molecule in human
OUTLINE
2.1 CovalentBondsand NoncovalentInteractions 32
40
2.2 BuildingBlocks
Chemical of Cells
2.3 Equilibrium
Chemical 49
2.4 Energetics
Biochemical 54

31
 (a) Molecularcomplementarity (bl Chemicalbuilding blocks
ProteinA

Polymerization
p&.
d'

",llrill

ProteinB 1
S m a l lm o l e c u l e Macromolecule
subunits

{c} Chemicalequilibrium (dl Chemicalbond energy

"High-energy"
phosphoan h y dr i d e

e#
bonds
kr

Kl

K1
K^^_ Adenosine
k,
triphosphate

A FIGURE2-1 Chemistry of life: four key concepts.(a) Molecular (nght)depends

reactions on the rateconstants of the forward(k1,
complementarity liesat the heartof all biomolecular interactions,as upperarrow)andreverse (k,,lowerarrow)reactions Theratioof
when two proteinswith complementary shapesand chemical these,K"o,providesan informative measureof the relativeamounts
propertiescometogetherto form a tightlybound complex (b) Small of products
andreactants thatwill be present
at equilibrium (d)In
moleculesserveas buildingblocksfor IargerstructuresForexample, manycases, the source of energyfor chemical reactionsin cellsisthe
to generatethe information-carrying macromolecule DNA,four small hydrolysis
of the molecule ATPThisenergyisreleased whena high-
nucleotidebuildingblocksare covalentlylinkedinto long strings energyphosphoanhydride bondlinkingthe B andy phosphates in
(polymers), which then wrap aroundeachother to form the double theATPmolecule (red)isbrokenby the addition of a watermolecule,
helix (c) Chemicalreactionsare reversible, and the distributionof the TOrmlnOAUI'311O l',.
chemicalsbetweenstartingreagents(/eft)and the productsof the

c h r o m o s o m e1 i s 8 . 6 x 1 0 r 0 t i m e s t h a t o f a p r o t o n ! ) . T h e chemical energetics,including the central role of ATP

chemical interactions of all of these molecules,large and
small, with water and with one anothet define the nature
of life.
Luckily, although many types of biomolecules interacr
and react in numerous and complex pathways to form func-
tional cells and organisms, a relatively small number of
chemical principles are necessaryto understand cellular
processes at the molecularlevel (Figure 2-l).ln this chapter
we review these key principles, some of which you already
know well. Sfe begin with the covalent bonds that connect
atoms into a molecule and the noncovalent forcesthat stabi-
lize groups of atoms into functional structures within and
between molecules. We then consider the key properties of
the basic chemical building blocks of macromoleculesand
macromolecular assemblies.After reviewing those aspectsof
chemical equilibrium that are most relevant to biological
systems,we end the chapter with basic principles of bio-
(adenosinetriphosphate) in capturing and transferring en-
ergy in cellular metabolism.
A CovalentBondsand
NoncovalentInteractions
Strong and weak attractive forces between atoms are the
"glue" that holds them together in individual moleculesand
permits interactions between different biomolecules.Strong
forces form a covalent bond when two atoms share one pair
of electrons("single" bond) or multiple pairs of electrons
("double" bond, "triple" bond, etc.). The weak attractive
forces of noncovalent interactions are equally important in
determining the properties and functions of biomolecules
such as proteins, nucleic acids, carbohydrates,and lipids.
.We
will first review covalent bonds and then discussthe four

32 cHAPTER2 | CHEMICALFOUNDATTONS
 Electrons Covalentbond try defines the structures of many biomolecules.A carbon
H (or any other) atom bonded to four dissimilar atoms or
groups in a nonplanar configuration is said to be asymmet-
ric. The tetrahedral orientation of bonds formed by an asym-
H metric carbon atom can be arranged in three-dimensional
space in two different ways, producing molecules that are
mirror imagesof eachother, a property calledchirality (from
H
the Greek word cheir,meaning "hand") (Figure2-4). Such
moleculesare called optical isomers,or stereoisomers.Many
molecules in cells contain at least one asymmetric carbon
H
atom, often called a chiral carbon atom. The different
stereoisomersof a molecule usually have completely differ-
A FIGURE 2-2 Covalentbondsform by the sharingof electrons. ent biological activities becausethe arrangement of atoms
Covalentbonds,the strongforcesthat holdatomstogetherinto within their structures differs, yielding their unique abilities
molecules,
formwhenatomsshareelectrons fromtherroutermost to interact and chemically react with other molecules.
electron Eachatomformsa definednumberandqeometrv
orbitals.
of covalent
bonds
Some drugs are mixtures of the stereoisomersof small
moleculesin which only one stereoisomerhas the bio-
logical activity of interest.The use of a pure single stereoiso-
major types of noncovalent interactions:ionic bonds, hydro-
mer of the chemical in place of the mixture can result in a
gen bonds, van der Waals interactions,and the hydrophobic
more potent drug with reduced sideeffects.For example,one
effect.
stereoisomerof the antidepressantdrug citalopram (Celexa) is

The ElectronicStructureof an Atom

D e t e r m i n e st h e N u m b e ra n d G e o m e t r y (al Formaldehyde
of CovalentBondslt Can Make
Hydrogen, oxygen, carbon, nitrogen,phosphorus,and sul- H
fur are the most abundant elementsin biological molecules. c:o
Theseatoms, which rarely exist as isolatedentities,readily H
form covalent bonds, using electronsin the outermost elec-
tron orbitals surrounding their nuclei (Figure 2-2\. As a
rule, each type of atom forms a characteristicnumber of co-
valent bonds with other atoms, with a well-defined geome- (b) Methane
try determined by the atom's size and by both the distribu-
tion of electrons around the nucleus and the number of H
electronsthat it can share.In some cases(e.g.,carbon), the I
H-C-H
number of stable covalent bonds formed is fixed; in other I
H
cases (e.g., sulfur), different numbers of stable covalent
bonds are possible.
All the biological building blocks are organizedaround Chemical Ball-and-stick Space-filling
structure model model
the carbon atom, which normally forms four covalent bonds
with three or four other atoms. As illustrated in Figure 2-3a A FIGURE 2-3 Geometryof bondswhen carbonis covalently
for formaldehyde, carbon can bond to three atoms, all in a linked to three or four other atoms.(a)A carbonatomcanbe
common plane. The carbon atom forms two typical single bondedto threeatoms,asin formaldehyde (CHzO). Inthiscase,the
bonds with two atoms and a double bond (two shared elec- carbon-bonding electrons participate in two singlebondsandone
tron pairs) with the third atom. In the absenceof other con- , h i c ha l ll i ei n t h es a m ep l a n eU. n l i k a
d o u b l eb o n dw etoms
straints, atoms joined by a single bond generally can rotate connected by a singlebond,whichusually canrotatef reelyaboutthe
bondaxis,thoseconnected by a doublebondcannot(b)Whena
freely about the bond axis, whereas those connected by a
carbonatomformsfoursinglebonds,asin methane (CH+), the
double bond cannot. The rigid planarity imposed by double
bonded atoms (all H in this case) are oriented in space in the formof
bonds has enormous significancefor the shapesand flexibil- indicates
a tetrahedron. Theletterrepresentation on the leftclearly
ity of biomoleculessuch as phospholipids, proteins, and nu- of the molecule andthe bondingpattern
theatomiccomposition
cleic acids. Theball-and-stick modelin the centerillustrates the geometric
Carbon can also bond to four rather than three atoms. arrangement of the atoms and bonds, but the diameters of the balls
As illustrated by the methane (CHa) molecule,when carbon representing the atomsandtheirnonbonding electrons are
is bonded to four other atoms, the angle between any two unrealistically smallcompared with the bondlengthsThesizesof the
bonds is 109.5' and the positions of bonded atoms define electron cloudsin the space-filling modelon the rightmore
the four points of a tetrahedron(Figure2-3b). This geome- accuratelv represent thestructure in threedimensions

C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S 33
 that can participate in noncovalent interactions. Sulfur
forms two covalent bonds in hydrogen sulfide (H2S)but also
can accommodate six covalent bonds, as in sulfuric acid
(H2SO4)and its sulfate derivatives.Nitrogen and phospho-
rus each have five electronsto share.In ammonia (NH3), the
nitrogen atom forms three covalent bonds; the pair of elec-
trons around the atom not involved in a covalent bond can
take part in noncovalent interactions. In the ammonium ion
(NH+*), nitrogen forms four covalent bonds, which have a
tetrahedral geometry. Phosphoruscommonly forms five co-
valent bonds, as in phosphoric acid (H3POa) and its phos-
phate derivatives,which form the backbone of nucleic acids.
Phosphategroups covalently attachedto proteins play a key
role in regulating the activity of many proteins, and the cen-
tral moleculein cellular energetics,ATP, contains three phos-
FIGURE 2-4 Stereoisomers. Manymolecules in cellscontainat phate groups (seeSection 2.4). A summary of common co-
leastoneasymmetric carbonatom Thetetrahedral orientationof valent linkagesand functional groups (portions of molecules
bondsformedbyan asymmetric carbonatomcanbearranged in
that confer distinctive chemical properties) is provided in
three-dimensional spacein two different
ways,producing molecules
Table 2-2.
that aremirrorimages, or stereoisomers,
of eachother.Shownhereis
thecommonstructure of an aminoacid,with itscentral asymmetric
carbonandfourattached groups,includingthe Rgroup,discussed in ElectronsMay Be SharedEqually
Section 2 2. Aminoacidscanexistin two mirror-image forms,
designated r ando. Although thechemical properties of such o r U n e q u a l l yi n C o v a l e n tB o n d s
stereoisomers areidentical,
theirbiological
activities
aredistinctOnly The extent of an atom's ability to attract an electron is called
r aminoacidsarefoundin oroterns its electronegatiuity. ln a bond between atoms with identical
or similar electronegativities,the bonding electrons are es-
170 times more potent than the other. Somestereoisomershave sentially sharedequally betweenthe two atoms, as is the case
very different activities. Darvon is a pain reliever,whereas its for most C-C and C-H bonds. Such bonds are called non-
stereoisomer,Novrad (Daruon spelled backward), is a cough polar. In many molecules,the bonded atoms have different
suppressant. One stereoisomer of ketamine is an anesthetic, electronegativities,resulting in unequal sharing of the elec-
whereas the other causeshallucinations. I trons. The bond betweenthem is said to be polar.
One end of a polar bond has a partial negativecharge
The number of covalent bonds formed by other common (6-), and the other end has a partial positive charge ( 6+).
atoms is shown in Table 2-1,.A hydrogen atom forms only In an O-H bond, for example, the greater electronegativ-
one covalent bond. An atom of oxygen usually forms only ity of the oxygen atom relative to hydrogen results in the
two covalent bonds but has two additional pairs of electrons electrons spending more time around the oxygen atom
than the hydrogen. Thus the O-H bond possessesan elec-
tric dipole, a positive charge separated from an equal but
opposite negative charge. The amount of 6- charge on the
oxygen atom of a O-H dipole is approximately 25 per-
cent of that of an electron, with an equivalent 6+ charge
AT()MAIIO USUAI.
I,IUMBTB TYPICAL on the H atom. Becauseof its two O-H bonds that are nor
I)UTERTI.ICIROI'IS {]FCl)VATElrlT BI]NDS GIOMETBY on exact opposite sides of the O atom, water molecules
Bt]I'IIl
(HzO) are dipoles (Figure 2-5) that form electrostatic,non-
H I H covalent interactions with one another and with other mol-
ecules. These interactions play a critical role in almost
o z every biochemical interaction and so are fundamental to
,ror
cell biology.
S 214,or5 St The polarity of the O:P double bond in H3PO4 results
,'
in a resonancehybrid, a structure between the two forms
N 3or4 -T- shown below in which nonbonding electrons are shown as
pairs of dots:
5
P
-'r-
P H
I
H
I
o :o
I I l*
4 -?- H-O-P-O-H
il"
e H-O-P-O-H
I
o. o

34 CHAPTER2 I CHEMICALFOUNDATIONS
 FUNCTIONAL GROUPS
LINKAGES
o o
til
-c-o-c-
I I
In the resonancehybrid on the right, one of the electrons
from the P:O double bond has accumulatedaround the O
atom, giving it a negativecharge and leaving the P atom with
a positive charge. These chargesare important in noncova-
lent interactions.
C o v a l e n tB o n d sA r e M u c h S t r o n g e r
and More StableThan NoncovalentInteractions
Covalentbonds are very stable(i.e.,consideredto be strong)
becausethe energies required to break them are much
greater than the thermal energy available at room tempera-
ture (25 "C) or body temperature(37'C). For example,the
ol H though these interactions are weak and have a transient
A FIGURE 2-5 The dipolenatureof a water molecule.The
symbolE representsa partialcharge(aweakerchargethantheone
on an electron
or a proton) Because of thedifferencein the
of H andO, eachof the polarH-O bondsin
electronegativities
waterisa dipoleThesizes anddirections of the dipoles
of eachof
the bondsdetermine the netdrstanceandamountof charqe
or dipolemoment,of the molecule
separation,
o o
-c- -c-o-
Carbonyl Carboxyl
(ketone) ( c a r b o x y l i ca c i d )
o
oo
-o-P-o- iltl
I -o-P-o-P-
o-
Phosphate
o- o-
Pyrophosphate
(phosphorylated
molecule) (diphosphate)
ll
-N-C-
Amide
thermal energy at25 "C is approximately 0.6 kilocalorie per
mole (kcal/mol), whereas the energy required to break the
carbon-carbonsingle bond (C-C) in ethane is about 140
times larger (Figure 2-6). Consequently' at room tempera-
ture (25 "C), fewer than f. in 1012ethanemoleculesis broken
into a pair of 'CH3 molecules,each containing an unpaired,
nonbondingelectron(calleda radical).
Covalent single bonds in biological moleculeshave ener-
gies similar to the energy of the C-C bond in ethane. Be-
cause more electrons are shared between atoms in double
bonds, they require more energy to break than single bonds.
For instance, it takes 84 kcal/mol to break a single C-O
bond but 170 kcal/mol to break a C:O double bond' The
most common double bonds in biological molecules are
C:O, C:N, C:C, and P:O.
In contrast, the energy required to break noncovalent
interactions is only 1-5 kcalimol, much less than the bond
energiesof covalent bonds (seeFigure 2-5). Indeed' nonco-
valent interactions are weak enough that they are con-
stantly being formed and broken at room temperature' Al-
existenceat physiologicaltemperatures(25-37 "C)' multi-
ple noncovalent interactions can, as we will see' act to-
gether to produce highly stable and specific associations
between different parts of alatge molecule or between dif-
ferent macromolecules. Below, we review the four main
types of noncovalent interactions and then consider their
roles in the binding of biomoleculesto one another and to
other molecules.
C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S 35
> FIGURE 2-6 Relativeenergiesof covalent Noncovalent interactions Covalentbonds
bondsand noncovalentinteractions. Bond
energies aredefinedastheenergyrequired to lonic
breaka particular typeof linkageCovalent Hydrogen
h o s ef o r s i n g l (eC - C ) a n d
b o n d si ,n c l u d i nt g bonds
double(C:C) carbon-carbon bonds.areoneto Thermal Hydrolysisof ATP
two powersof 10stronger thannoncovalent energy p h o s p h o a n h y d r i dbeo n d c-c C=C
interactions Thelatteraresomewhat greater
thanthethermalenergyof theenvironment at
normalroomtemperature (25"C) Many
biological processes arecoupled to the energy 0.24 240
released duringhydrolysis of a phosphoanhydride kcal/mol
bondin ATP
Increasingbond strength

lonic InteractionsAre Attractions

between OppositelyChargedlons
Ionic interactions result from the attraction of a positively
charged ion-a cation-for a negatively charged ion-an
anion. In sodium chloride (NaCl), for example, the bonding
electron contributed by the sodium atom is completely trans-
ferred to the chlorine arom. (Figure 2-7a). Unlike covalent
bonds, ionic interactionsdo not have fixed or specificgeometric
orientations becausethe electrostatic field around an ion-its
attraction for an opposite charge-is uniform in all directions.
In solid NaCl, many ions pack tightly together in an alternating
pattern to permit opposite charges to align and thus form a
highly orderedcrystallinearray (saltcrystals)(Figure2-7b).
tVhen solid saltsdissolvein water, the ions separatefrom
one another and are stabilized by their interactionswith wa-
ter molecules.In aqueoussolutions,simpleions of biological
significance,suchas Na*, K*, Ca2*,Mg2* rand Cl-, are hy-
drated, surrounded by a stable shell of water moleculesheld
in place by ionic interactions betweenthe central ion and the
oppositely charged end of the water dipole (Figure 2-7c).
Most ionic compounds dissolvereadily in water becausethe
energy of hydration, the energy releasedwhen ions tightly
bind water molecules,is greater than the lattice energy that
stabilizesthe crystal structure. Parts or all of the aqueousby-
dration shell must be removed from ions when they directly
interact with proteins. For example, water of hydration is
lost when ions pass through protein pores in the cell mem-
brane during nerve conduction.
The relative strength of the interaction betweentwo ions,
A- and C-, dependson the concentration of other ions in a
solution. The higher the concentration of other ions (e.g.,
Na* and Cl-), the more opportunities A and C* have to

(al (b)

1,'
+ Hro
-> dissolving
cl
.-# -d
---.+ Cl
s q{-
+--
Crystallizing

Donationof electron -d

A FIGURE
U
2-7 Electrostaticinteractionsof oppositelycharged dissolved
in water,the ionsseparateandtheircharges, no longer
ionsof salt(NaCl)in crystalsand in aqueoussolution.(a)In balancedby immediately adjacentionsof opposite charge,are
crystalline
tablesalt,sodiumatomsarepositively charged ions(Na+) stabilized
by interactions
with polarwaterWatermolecules andthe
dueto the lossof oneelectron each,whereas chloride
atomsare ionsareheldtogetherby electrostatic
interactionsbetween the
correspondingly negatively
charged (Ct I 5Ugainingoneelectron chargeson the ionandthe partialcharges on thewater'soxygenand
each(b)Insolidform,ioniccompounds formneatlyorderedarrays, hydrogen atomsIn aqueous solutions,
all ionsaresurrounded by a
or crystals,
of tightlypackedionsin whichthe positive
andnegatively hydrationshellof watermolecules
charged ionscounterbalance eachother.(c)Whenthe crvstalsare

36 CHAPTER2 I CHEMICALFOUNDATIONS
 (a) (b)
iuH H o-H
tll
O-H :O-H :O-H :O-H
HH HH
ll :O- CHs
H-O: H-O : O-H
Water-water Alcohol-water
A FIGURE 2-8 Hydrogenbondingof water with itself and with
other compounds. Eachpairof nonbonding
outerelectronsin an
oxygen or a nitrogen
atomcanaccepta hydrogen atomin a hydrogen
bond Thehydroxyl andtheaminogroupscanalsoformhydrogen
bondswithwater.(a)In liquidwater,eachwatermoleculeforms
hydrogen
transient bondswithseveralothers,
creating
a dynamic
interact ionically with these other ions and thus the lower
the energyrequired to break the interaction betweenA- and
C*. As a result, increasingthe concentrationsof saltssuch as
NaCl in a solution of biological moleculescan weaken and
even disrupt the ionic interactions holding the biomolecules
together.
HydrogenBondsDeterminethe Water
S o l u b i l i t yo f U n c h a r g e dM o l e c u l e s
A hydrogen bond is the interaction of a partially positively
charged hydrogen atom in a molecular dipole (e.9., water)
with unpaired electronsfrom another atom, either in the same
(intramolecular\or different Untermolecular)molecule.Nor-
mall5 a hydrogen atom forms a covalent bond with only one
other atom. However, a hydrogen atom covalentlybonded to
an electronegativedonor atom D may form an additional
weak association,the hydrogen bond, with an acceptoratom
A, which must have a nonbonding pair of electronsavailable
for the interaction:
D6--H6+ + : 46- i- D6--H6*:.....:: 46-
uyarolln uona
The length of the covalent D-H bond is a bit longer than it
would be if there were no hydrogen bond becausethe accep-
tor "pulls" the hydrogen away from the donor. An impor-
tant feature of all hydrogen bonds is directionality. In the
strongest hydrogen bonds, the donor atom, the hydrogen
atom, and the acceptor atom all lie in a straight line. Non-
linear hydrogen bonds are weaker than linear ones; still,
multiple nonlinear hydrogen bonds help to stabilize the
three-dimensionalstructuresof many proteins.
Hydrogen bonds are both longer and weaker than co-
valent bonds betweenthe same atoms. In water, for exam-
ple, the distance between the nuclei of the hydrogen and
oxygen atoms of adjacent, hydrogen-bondedmoleculesis
about 0.27 nm, about twice the length of the covalent
(c) :O-H
I
H
H
I :o
-i-rtr- H-O:
I I
I
H H
H H
I I
:O-H :N-CHs .H-O: o
I
-i - o-
H H
Amine-water Peptide group-water Ester group-water
networkof hydrogen-bonded (b)Wateralsocanform
molecules
hydrogen andamines,
bondswith alcohols accountingfor the high
of thesecompounds
solubility (c)Thepeptidegroupandestergroup,
whicharepresent commonly
in manybiomolecules, participatein
hydrogen bondswithwateror polargroupsin othermolecules.
O-H bonds within a singlewater molecule (Figure2-8a).
The strengthof a hydrogen bond betweenwater molecules
(approximately 5 kcal/mol) is much weaker than a cova-
lent O-H bond (roughly 110 kcal/mol), although it is
greater than that for many other hydrogen bonds in bio-
logical molecules(t-2 kcall mol). The extensivehydrogen
bonding between water molecules accounts for many of
the key properties of this compound' including its unusu-
ally high melting and boiling points and its ability to in-
teract with (e.g.,dissolve)many other molecules.
The solubility of unchargedsubstancesin an aqueousen-
vironment dependslargely on their ability to form hydrogen
bondswith water.For instance,the hydroxyl group (-OH) in
an alcohol (XCH2OH) and the amino group (-NH2) in
amines (XCH2NH2) can form severalhydrogen bonds with
water, enabling these moleculesto dissolvein water to high
concentrations(Figure2-8b). In general,moleculeswith polar
bonds that easilyform hydrogen bonds with water' as well as
charged moleculesand ions that interact with the dipole in
water, can readily dissolve in water; that is' they are
hydrophilic (water liking). Many biological moleculescon-
tain, in addition to hydroxyl and amino groups' peptide and
ester groups, which form hydrogen bonds with water via oth-
erwisenonbondedelectronson their carbonyl oxygens(Figure
2-8c). X-ray crystallography combined with computational
analysispermits an accuratedepiction of the distribution of
the outermostunbondedelectronsof atoms as well as the elec-
trons in covalent bonds, as illustrated in Figure 2-9.
Van der WaalsInteractions
Are Causedby TransientDiPoles
When any two atoms approach each other closel5 they cre-
ate a weak, nonspecific attractive force called a van der
Waals interaction. Thesenonspecificinteractionsresult from
the momentary random fluctuations in the distribution of
the electrons of any atom' which give rise to a transtent
C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S 37
 clouds, the atoms are said to be in van der $7aalscontact. The
strength of the van der rWaalsinteraction is about 1 kcal/mol.
weaker than typical hydrogen bonds and only slightly higher
than the averagethermal energy of moleculesat 25 "C. Thus
multiple van der Waals interactions,a van der'Waalsinterac-
tion in conjunction with other noncovalent interactions, or
both are required to significantly influencethe stability of in-
ter- and intramolecular conracrs.

The HydrophobicEffectCausesNonpolar
Moleculesto Adhere to One Another
Becausenonpolar molecules do not contain charged groups,
possessa dipole moment, or becomehydrated,they are insoluble
or almost insolublein water; that is, they are hydrophobic (water
fearing).The covalentbonds betweentwo carbon atoms and be-
tween carbon and hydrogen atoms are the most common non-
polar bonds in biological systems.Hydrocarbons-molecules
made up only of carbon and hydrogen-are virtually insoluble
in water. Large triacylglycerols(or triglycerides),which make up
A FIGURE 2-9 Distributionof bondingand outer nonbonding
animal fats and vegetableoils, also are insolublein water. As we
electronsin the peptidegroup.Shownhereisa peptidebond
will see later, the major portion of these moleculesconsistsof
linkingtwo aminoacidswithina proteincalledcrambinTheblack
long hydrocarbon chains. After being shaken in water, triacyl-
linesrepresent thecovalent bondsbetween atomsThered(negative)
andblue(positive) glycerols form a separatephase.A familiar example is the sepa-
linesrepresentcontoursof chargedetermined
usingx-raycrystallography andcomputational ration of oil from the water-basedvinegar in an oil-and-vinegar
methods. Thegreater
the numberof contourlines,the higherthe charge. Thehighdensity salad dressing.
of redcontourlinesbetweenatomsrepresents the covalentbonds Nonpolar moleculesor nonpolar portions of molecules
(shared electron pairs).
Thetwo setsof redcontourlinesemanating tend to aggregatein water owing to a phenomenoncalled
fromtheoxygen(O)andnotfallingon a covalent bond (blackline) the hydrophobic effect. Becausewater molecules cannot
represent thetwo pairsof nonbonded electronson the oxygen that form hydrogen bonds with nonpolar substances,they tend
areavailable to participate
in hydrogen bondingThehighdensity of to form "cages" of relatiuely rigid hydrogen-bonded
bluecontourlinesnearthe hydrogen (H)bondedto nitrogen (N) pentagons and hexagons around nonpolar molecules
represents a partialpositive
charge,indicatingthatthisH canactasa
donorin hydrogen bonding. [From etal, 2000,procNat,t.
C Jelsch Acad
SciUSA97.3171 CourtesyofM M Teeterl

unequal distribution of electrons. If two noncovalently

bonded atoms are close enough together, electrons of one
atom will perturb the electronsof the other. This perturba-
tion generatesa transient dipole in the secondatom, and the
two dipoles will attract each other weakly (Figure 2-10).
Similarly, a polar covalent bond in one molecule will attract i<---->i k----t
an oppositely oriented dipole in another. Covalent van derWaals
Van der'Waals interactions, involving either transiently radius radius
( 0 . 0 6n
2m ) ( 0 . 1 4n m )
induced or permanent electric dipoles, occur in all types of
molecules, both polar and nonpolar. In particular, van der A FIGURE 2-10 Two oxygenmoleculesin van der Waals
\Waalsinteractions are responsible contact.ln thismodel,redindicates negative chargeandblue
for the cohesion between
nonpolar moleculessuch as heptane,CH3-(CH2)s-CH:, indicatespositivechargeTransientdipolesin theelectron clouds of all
that cannot form hydrogen bonds or ionic interactionswith atomsgiveriseto weakattractive forces,calledvander Waals
'Waals interactions.Eachtypeof atomhasa characteristic
vanderWaals
other molecules.The strength of van der interactions
decreasesrapidly with increasingdistance;thus thesenonco- radiusat whichvanderWaalsinteractions withotheratomsare
optimal.Because atomsrepeloneanotherif theyarecloseenough
valent bonds can form only when atoms are quite closeto one
together for theirouterelectrons
to overlapwithoutbeingshared in a
another. However, if atoms get too close together,they be-
covalentbond,thevanderWaalsradiusisa measure of thesizeof
come repelled by the negative charges of their electrons.
theelectron cloudsurrounding an atom Thecovalent radrus indicated
\fhen the van der Waals attraction between rwo aroms ex- hereisfor thedoublebondof O:O; thesinqle-bond covalent radius
actly balances the repulsion between their two electron of oxygenisslightlylonger.

38 CHAPTER2 | cHEM|CALFOUNDAT|ONS
 Watersreleasedinto bulk the higher their concentration)' the more likely they are to
Nonpolar H i g h l yo r d e r e d solution encounter one another.Vhen two moleculesencounter each
substance w a t e rm o l e c u l e s other, they most likely will simply bounce apart becausethe
\\ o
\.t

noncovalent interactions that would bind them together are

o'o o weak and have a transient existenceat physiological temper-
atures. However, moleculesthat exhibit molecular comple-
o mentarity, a lock-and-key kind of fit between their shapes,
charges,or other physical properties,can form multiple non-
Hydrophobic '$7hen
aggregation covalent interactions at close range. two such struc-
turally complementarymoleculesbump into each other' they
can bind (stick) together.
Figure 2-12 tllustrates how multiple, different weak
bonds can bind two proteins together. Almost any other
arrangement of the same groups on the two surfaceswould
@o not allow the molecules to bind so tightly. Such multiple,
Lowerentropy Higherentropy specificinteractions betweencomplementary regions within
a orotein molecule allow it to fold into a unique three-
FIGURE 2-11 Schematic depictionof the hydrophobiceffect' dimensionalshape(Chapter 3) and hold the two chains of
Cagesof watermolecules thatformaroundnonpolar molecules in DNA togetherin a double helix (Chapter 4)' Similar inter-
solution aremoreordered thanwatermolecules in thesurrounding actionsunderliethe associationof groups of more than two
b u l kl i q u i dA g g r e g a t i o nf n o n p o l amr o l e c u l e s en u m b e r
r esd u c et h
molecules into multimolecular complexes, leading to for-
of watermolecules involved in highlyordered cages, resulting in a
mation of muscle fibers' to the gluelike associationsbe-
higher-entropy, moreenergetical lyf avorable state(nErht) compared
tween cells in solid tissues,and to numerous other cellular
with the unaggregated state(/eft)
structures.
Depending on the number and strength of the noncova-
lent interactions betweenthe two moleculesand on their en-
(Figure 2-1I, Ieft). This state is energeticallyunfavorable vironment, their binding may be tight (strong) or loose
becauseit decreasesthe randomness(entropy) of the pop- (weak) and, as a consequence,either long lasting or tran-
ulation of water molecules.(The role of entropy in chemi- sient.The higher the affinity of two moleculesfor each other,
c a l s y s t e m si s d i s c u s s e di n a l a t e r s e c t i o n . )I f n o n p o l a r the better the molecular "fit" between them, the more non-
moleculesin an aqueousenvironment aggregatewith their covalent interactionscan form, and the tighter they can bind
hydrophobic surfacesfacing each other, the hydrophobic
s u r f a c e a r e a e x p o s e dt o w a t e r i s r e d u c e d ( F i g u r e 2 - 1 1 ,
right). As a consequence,lesswater is neededto form the
cages surrounding the nonpolar molecules, and entropy
increases(an energeticallymore favorable state)relative to
the unaggregatedstate. In a sense,then, water squeezes
the nonpolar moleculesinto spontaneouslyforming aggre-
gates.Rather than constituting an attractive force such as
-cH3 |
in hydrogen bonds, the hydrophobic effect resultsfrom an
a v o i d a n c e o f a n u n s t a b l e s t a t e ( e x t e n s i v ew a t e r c a g e s -CH3 H3(

a r o u n d i n d i v i d u a ln o n p o l a r m o l e c u l e s ) . , _cH3 H3C_

Nonpolar moleculescan also associate,albeit weakly'

through van der Vaals interactions.The net result of the
hydrophobic and van der Waals interactionsis a very pow-
erful tendencyfor hydrophobic moleculesto interact with
one another,not with water. Simply put, like dissolueslike' ProteinA ProteinB ProteinA ProteinC
Polar moleculesdissolve in polar solvents such as water; Stable complex Less stable comPlex
nonpolar moleculesdissolvein nonpolar solventssuch as complementarity andthe bindingof
FIGURE 2-12 Molecular
hexane. proteinsvia multiplenoncovalentinteractions. Thecomplementary
shapes, andhydrophobicity
polarity,
charges, of two proteinsurfaces
M o l e c u l a rC o m p l e m e n t a r i t My ediated permrt weakinteractions,
multiple whichin combination produce a
stronginteractionandtight binding.Because deviations frommolecular
via Noncovalent Interactions PermitsTight,
complementarity weaken
substantially binding,a parlicularsurface
H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s region of anygivenbiomolecule usuallycanbindtightlyto onlyoneor a
Both inside and outside cells, ions and molecuiesare con- verylimitednumber of othermolecules Thecomplementarity of the
stantly bumping into one another.The greaterthe number of two proteinmolecules on the leftpermitsthem to bindmuch more
copiesof any two types of moleculesper unit volume (i'e., tightlythanthetwo noncomplementary proteins on therighi

INTERACTIONS T
C O V A L E N TB O N D SA N D N O N C O V A L E N T 39
together.An important quantitative measureof affinity is the
binding dissociationconstant K6, describedlater.
As we discussin Chapter 3, nearly all the chemicalre-
actionsthat occur in cellsalso dependon the binding prop-
ertiesof enzymes.Theseproteins not only speedup, or cat-
aIyze, reactions but also do so with a high degree of
specificity, a reflection of their ability to bind tightly to
only one or a few related molecules. The specificity of in-
termolecularinteractionsand reactions,which dependson
molecular complementarity,is essentialfor many processes
critical to life.
Covalent Bonds and Noncovalent Interactions
Covalent bonds, which bind the atoms composing a
olecule in a fixed orientation, consist of pairs of elec-
trons shared by two atoms. They are stable in biological
systems because the relatively high energies required to
break them (50-200 kcal/mol) are much larger than the
thermal kinetic energyavailableat room (25 .C) or body
(37 "C) temperatures.
r Many moleculesin cells contain at least one asymmet-
ric carbon atom, which is bonded to four dissimilar
atoms. Such moleculescan exist as optical isomers (mir-
ror images),designatedn and r (seeFigure 2-4), which
have different biological activities.In biological systems,
nearly all sugarsare D isomers,whereasnearly all amino
acids are L isomers.
trons may be sharedequally or unequally in covalent
Atoms that differ in electronegativityform polar co-
bonds in which the bonding electronsare distributed
unequally. One end of a polar bond has a partial positive
charge and the other end has a partial negativechaige (see
Figure2-5).
r Noncovalent interactions between atoms are consider-
ably weaker than covalent bonds, with bond energlesrang-
ing from about 1-5 kcal/mol (seeFigure 2-6).
r Four main types of noncovalent interactions occur in bi-
ological systems:ionic bonds, hydrogen bonds, van der
\faals interactions, and interactions due to the hvdrooho-
bic effect.
r Ionic bonds result from the electrostatic attraction be-
tween the positive and negativechargesof ions. In aqueous
solutions, all cations and anions are surrounded by a shell
of bound water molecules(seeFigure Z-7c).Increasingthe
salt (e.g.,NaCl) concentrarionof a solutioncan weakenthe
relative strength of and even break the ionic bonds between
biomolecules.
hydrogen bond, a hydrogen atom covalently bonded
electronegativeatom associateswith an accepror
whose nonbonding electrons attract the hydrogen
gure 2-8).
40 CHAPTER2 | cHEMTCALFOUNDATTONS
r Weak and relatively nonspecificvan der'Sfaalsinterac-
tions are createdwheneverany two atoms approach each
other closely. They result from the attraction between
transient dipoles associatedwith all molecules (seeFig-
ure2-10).
r In an aqueousenvironment, nonpolar moleculesor non-
polar portions of larger molecules are driven together by
the hydrophobic effect, thereby reducing the exrent of their
direct contactwith water molecules(seeFigure2-11).
r Molecular complementarity is the lock-and-key fit be-
tween moleculeswhose shapes,charges,and other physical
properties are complementary.Multiple noncovalent inter-
actions can form betweencomplementary molecules,caus-
ing them to bind tightly (seeFigure2-12),but not between
moleculesthat are not complementary.
r The high degreeof binding specificity that results from
molecular complementarity is one of the features that un-
derlies intermolecular interactions and thus is essentialfor
many processescritical to life.
E Chemical
BuildingBlocksof Cells
A common theme in biology is the construction of large mol-
ecules (macromolecules)and structures by the covalent or
noncovalent associationof many similar or identical smaller
molecules.The three most abundant classesof the critically
important biological macromolecules-proteins, nucleic
acids, and polysaccharides-are all polymers composed of
multiple covalently linked building block small molecules,or
monomers (Figure 2-13). Proteins are linear polymers con-
taining 10 to severalthousand amino acids linked by peptide
bonds. Nucleic acids are linear polymers containing hun-
dreds to millions of nucleotides linked by phosphodiester
bonds. Polysaccharidesare linear or branched polymers of
monosaccharides(sugars)such as glucose linked by glyco-
sidic bonds. Although the actual mechanismsby which co-
valent bonds betweenmonomers form are complex and will
be discussedlater, the formation of a covalent bond between
two monomer molecules usually involves the net loss of a
hydrogen (H) from one monomer and a hydroxyl (OH) from
the other monomer-or the net loss of one water-and can
therefore be thought of as a debydration reaction. These
bonds are stable under normal biological conditions (e.g.,
37"C, neutral pH), and so thesebiopolymers are stable and
can perform a wide variety of jobs in cells (store informa-
tion, catalyze chemical reactions, serve as structural ele-
ments in defining cell shapeand movement, etc.).
Macromolecular structures can also be assembledusing
noncovalent interactions. The macromolecular two-layered
(bilayer) structure of cellular membranesis built uo bv the
noncovalent assemblyof many thousandsof small molecules
called phospholipids (seeFigure 2-13).In this chapter, we
will focus on the characteristicsof the monomeric chemical
 MONOMERS POLYMERS

HO HO HHOHHOHHOHHO
ttl ttl | | ll | | ll I l ll,l L ll
H2N-C -C-OH + H-N-C-C- oH H-N-C-ciN-C-C N-C-CrN-C-C-OH
I I I lt | | I I
R R R, l" R2 R3 I R*
I
Amino acid peptidebond
PolYPePtide

B
o Base L
tl
HO-P-O
I ?1,
HO-P-OJs, .OH

6-
Nucleotide Nucleic acid

glYcosidicbond

Monosaccharide Polysaccharide

I Hyoropr'irt.
h e a dg r o u p
J

Phospholipid

(Chapter4), andpolysaccharides from monosaccharides (sugars)Each

FIGURE2-13 Overviewof the cell'sprincipalchemical
monomer iscovalentlylinkedintothe polymer by a whose
reaction
buifdingblocks.(Top)The
threemajortypesof biological
(Boftom)
net resultis lossof a watermolecule (dehydration) In
are
macromolecules eachassembledbythe polymerization
of
phospholipid
contrast, monomers noncovalentlyassemble intoa bilayer
(monomers)
smallmolecules
multiple type:proteins
of a particular
whichformsthebasis
structure, membranes
of allcellular (chapter10)
fromaminoacids(Chapter acidsfromnucleotides
3),nucleic

building blocks-amino acids,nucleotides,sugars'and phos-
pholipids. The structure, function, and assemblyof proteins,
nucleic acids, polysaccharides,and biomembranes are dis-
cussedin subsequentchapters.
A m i n o A c i d sD i f f e r i n gO n l y i n T h e i r
SideChainsComposeProteins
The monomeric building blocks of proteins ate 20 amino
acids, which when incorporated into a protein polymer are
sometimescalled residues.All amino acidshave a characteris-
tic structureconsistingof a central alpha (cr)carbon atom (C')
bonded to four different chemical groups: an amino (NHz)
group, a carboxylic acid or carboxyl (COOH) group (hence
Ih. *-. amino acid),a hydrogen (H) atom, and one variable
group, called a side chain or R group' Becausethe ct carbon
i.-nali amino acids except glycine is asymmetric,thesemole-
cules can exist in two mirror-image forms called by conven-
tion the I (dextro) and the r- (levo) isomers (seeFigure 2-4)'
The two isomerscannot be interconverted(one made identi-
cal with the other) without breaking and then re-forming a
chemical bond in one of them. !7ith rare exceptions, only
the I- forms of amino acids are found in proteins'
To understand the three-dimensional structures and
functions of proteins' discussedin detail in Chapter 3, you
must be familiar with some of the distinctive properties of

B U I L D I N GB L O C K SO F C E L L S
CHEMICAL 41
 H Y D R O P H O BA
I CM I N OA C I D S

coo- coo- coo- coo- coo- coo- coo- coo-

*H3N-C-H
I + H . N - c I- H I I I I I I
+H3N-c-H +H3N-c-H 'H"N-C-H *H.N-C-H
I
"l -l -l
cHs CH H-C - CH. CH, CH,
HsC CHs t" t- t-
CH, CH C:CH
l- HaC CHs
cHs
Lr*t
OH
\/
Alanine Valine lsoleucine Leucine Methionine Phenylalanine
( A l ao r A l (Val orVl
Tyrosine Tryptophan
( l l eo r l l (Leu or Ll (Met or M) (Phe or Fl (TyrorYl (TrporW)

H Y D R O P H I LA
I CM I N OA C I D S
Acidic amino acids Polar amino acids with uncharged R groups

coo- coo- coo-

+H.N-c-H
I tl
*H3N-C-H +H3N-C-H
coo- coo- -l
I I r"l
+H3N-c-H 'H"N-C-H CH, CH, H-C-OH
-l t-i
I
CHt CH, coo- oH cHs
l- t- Aspartate Serine Threonine
CH, CH, (Asp or Dl (Ser or Sl (Thr orT)
t- l-
CH, CH,
t- l- coo-
CH, NH 'H"N-C-H
I
l- i -l
NHs* C: NHr+
l- CH,
NHz I-
cH,
Lysine Arginine )'
(Lys or Kl (Argor Rl coo-

SPECIALAMINO ACIDS Glutamate

( G l uo r E )

FIGURE 2-14 The 20 commonaminoacidsusedto build

proteins.Thesidechain(Rgroup;red)determines thecharacteristic
propertiesof eachaminoacidandisthe basisfor groupingamino
acidsintothreemaincategories:hydrophobic,hydrophilic,
and
special.
Shownarethe ionized formsthatexistat the pH(=7)of the
Cysteine Glycine proline cytosolIn parentheses
(Cys or C)
arethethree-letter
andone-letter
(Gty or G) (pro or p)
abbreviationsfor eachaminoacid.

amino acids, which are determined in part by their side
chains. You need not memorize the detailed structure of each
type of side chain to understandhow proteins work because
amino acids can be classified into several broad categories
basedon the size,shape,charge,hydrophobicity (a measure
of water solubility), and chemical reactivity of the side
chains (Figure 2-74). However, you should be familiar with
the generalproperties of each category.
Amino acids with nonpolar side chains are hydrophobic
and so poorly soluble in water. The larger the nonpolar side
chain, the more hydrophobic-less water soluble-the amino
acid. The noncyclic side chains of alanine, ualine, leucine, and.
isoleucine(calledaliphatic),as well as methionine,consisren_
tirely of hydrocarbons, except for the one sulfur arom in me_
thionine, and all are nonpolar. phenylalanine, tyrosine, and.
tryptophan have large, bulky aromatic side chains. In later
chapters, we will see in detail how these hydrophobic side
chains under the influence of the hydrophobic effeci often pack
in the interior of proteins or line the surfacesof proteins that
are embeddedwithin hydrophobic regions of biomembranes.
Amino acids with polar side chains are hydrophilic; the
most hydrophilic of theseamino acids is the subsetwith side
chains that are charged (ionized) at the pH typical of biolog-
ical fluids (=7)-both inside and outside the cell (seeSectio-n
2.3). Arginine and lysine have positively charged side chains
and are called basic amino acids;aspartic acid and glutamic
acid have negatively charged side chains due to the car-
boxylic acid groups in their side chains (their charged forms
are called aspartate and glutamate) and are called acidic. A
fifth amino acid, histidine, has a side chain containing a ring

42 . c H A p r E R 2| c H E M t c A L F o u N D A T t o N s
with two nitrogens, called imidazole, which can shift from
I
being positively charged to uncharged depending on small A c e t y ll y s i n e C H 3 - C - N - C H 2 - C H 2 - C H 2 - C H " - C H - C-Ol O -
changesin the acidity of its environment: NH.-
ll o
- o - Ptl o - c H 2 - c H - c o o -
Phosphoserine
ll
O NH"-

OH
I
pH 5.8 PH 7.8 3-Hydroxyproline H,9-9H
II
H2C\
The activities of many proteins are modulated by shifts
* N //CH-COO-
H,
in environmental acidity through protonation or deprotona-
tion of histidine side chains. Asparagine and glutamine are
HC:C-CH2-CH-COO-
uncharged but have polar side chains containing amide trl
3-Methylhistidine
groups with extensivehydrogen-bondingcapacities'Similarl5 H3C-N-C-.N NH.-
serine and threonine are unchargedbut have polar hydroxyl H
groups, which also participate in hydrogen bonds with other -ooc
polar molecules.
Lastl5 cysteine,glycine, and proline exhibit specialroles
y-Carboxyglutamate c H - c H' "l - c H - c o o -
-OOC
in proteins becauseof the unique properties of their side ilH.*
chains. The side chain of cysteine contains a reactive of aminoacidside
A FIGURE 2-15 €ommonmodifications
sulfhydryl group (-SH), which can oxidize to form a cova- andnumerous
residues others
chainsin proteins.Thesemodified
lent disulfide bond (-S-S-/ to a secondcysteine:
areformedby additionof various groups(red)to theamino
chemical
of a polypeptide
acidsidechainsduringor aftersynthesis chain
I I
N-H N-H
I I- H
H-C-CH2- SH+ HS-CH2q reveals that they contain upward of 100 different amino
ll acids.Chemical modifications of the amino acids account for
C:O C:O
ll
tl
r''l N-H
H-N
ll
H-C- C H 2- S - S - C H 2- c - H
ll
O:C C:O
ll

Regionswithin a singleprotein chain (intramolecular) or

and plants but less studied-perhaps becauseof the relative
in separatechains (intermolecular) sometimesare cross-
insta-bilityof phosphorylatedhistidine-and apparently rare
linked through disulfide bonds. Disulfide bonds stabilizethe
in mammals.ltt. iia. chains of asparagine,serine,and thre-
folded structure of some proteins. The smallestamino acid,
onine are sitesfor glycosylation,the attachmentof linear and
glycine, has a singlehydrogen atom as its R group. Its small
branched carbohydrate chains. Many secretedproteins and
sizeallows it to fit into tight spaces.Unlike the other common
membrane proteins contain glycosylated residues' Other
amino acids, the side chain of proline bends around to form
amino acid modifications found in selectedproteins include
a ring by covalently bonding to the nitrogen atom (amino
the hydroxylation of proline and lysine residuesin collagen
group) attached to the Co. As a result' proline is very rigid
(Chapter 19), the methylation of histidine residuesin mem-
and createsa fixed kink in a protein chain, limiting how a
b."ne rec.ptors' and the 7 carboxylation of glutamate in
protein can fold in the region of proline residues.
blood-clotting factors such as prothrombin'
Some amino acids are more abundant in proteins than
Acetylation, addition of an acetyl group' to the amino
others. Cysteine,tryptophan, and methionine are rare amino
gro,rp oi the N-terminal residue, is the most common form
acids: together they constitute approximately 5 percent of
6f acid chemical modification, affecting an estimated
the amino acids in a protein. Four amino acids-leucine, ser- "-ino
80 percentof all proteins:
ine, lysine, and glutamic acid-are the most abundant amino
acids, totaling 32 percent of all the amino acid residuesin a o RO
typical protein. However, the amino acid composition of lllll
cH3-c- r y- g- c-
proteins can vary widely from thesevalues. ll
HH
Although cellsusethe 20 amino acidsshown in Figure2-14
Acetylated N-terminus
inthe initial synthesisof proteins' analysisof cellular proteins

OF CELLS
B U I L D I N GB L O C K S O 43
CHEMICAL
 This modification may play an important role in controlling PURINES
the life span of proteins within cells becausenonacetylateJ
NH, O
proteins are rapidly degraded. ltl

Five Different Nucleotides

A r e U s e dt o B u i l d N u c l e i cA c i d s
Two types of chemically similar nucleic acids, DNA (de- HH
oxyribonucleic acid) and RNA (ribonucleic acid). are the Adenine (A) (Gl
Guanine
principal genetic-information-carryingmoleculesof the cell.
The monomers from which DNA and RNA polymers are PYRIMIDINES
built, called nucleotides,all have a common structure: a NH,
il t-
phosphategroup linked by a phosphoesterbond to a pentose
(a five-carbon sugar molecule) that in turn is linked io a ni- ru/?--cH
trogen- and carbon-containing ring structure commonly re- r"^tl
ferred to as a base (Figure 2-16a).In RNA, the pentoseis .r'"'ii"
vl
ribose; in DNA, it is deoxyribose that at posirion 2, has a
H
proton rather than the hydroxyl group at that site in ribose
Cytosine (C)

A FIGURE 2-17 Chemical structuresof the principalbasesin

nucleicacids.ln nucleic acidsandnucleotides, nitrogen 9 of purines
andnitrogen1 of pyrimidines (red)arebondedto the 1, carbonof
riboseor deoxyribose U isonlyin RNA,andT isonlyin DNA Both
R N Aa n dD N Ac o n t a iA
n , G ,a n dC .

which form ionic interactions with the negatively charged

phosphates.
Cells and extracellular fluids in organismscontain small
concentrationsof nucleosides,combinations of a baseand a
sugar without a phosphate.Nucleotides are nucleosidesthat
position 1 of a pyrimidine (N1). The acidic character of
have one, two, or three phosphate groups esterified at the
5' hydroxyl. Nucleoside monophosphateshave a single
esterified phosphate (seeFigure 2-16a); nucleoside diphos-
phates contain a pyrophosphategroup:

oo
(a)
-o-l-o-A-o-
Nhz (b)
Adenine l
'-c' 5', o- o-
N ; , 6 ; -c \ HOCH2 Pyrophosphate
daH
HC.1: 1c s,/
-'N' and nucleosidetriphosphateshave a third phosphate.Table 2-3
N
lists the names of the nucleosidesand nucleotidesin nucleic
o acids and the various forms of nucleosidephosphates.The
- o - P - O - c H5', nucleosidetriphosphatesare used in the synthesisof nucleic
acids,which we cover in Chapter 4. Among their other func-
o- 5',
tions in the cell, GTP participares in intracellular signaling
Phosphate HOCH2
2'
a_ndacts as an energy reservoir,particularly in protein syn-
OH thesis, and ATP, discussedlater in this chapter, is the most
H
Ribose widely usedbiologicalenergycarrier.
Adenosine 5'-monophosphate
(AMP} 2-Deoxyribose
M o n o s a c c h a r i d eJso i n e db y G l y c o s i d i B
c onds
FIGURE 2-15 Commonstructureof nucleotides. (a)Adenosine
5' -monophosphate (AMp),a nucleotide
Form Linearand Branchedpolysaccharides
presentin RNAByconvention,
thecarbonatomsof the pentose sugarin nucleotides arenumbered The building blocks of the polysaccharidesare rhe simple sug-
with primes. the 1, carbonisjoinedby a B
In naturalnucleotides, ars, or monosaccharides.Monosaccharides are carbohy_
linkageto the base(inthiscaseadenine); boththe base(blue)and drates,which are literally covalently bonded combinationsof
the phosphate on the 5, hydroxyl
(red)extendabovethe planeof the carbon and water in a one-to-one ratio (CH2O),, where n
sugarring.(b)Ribose anddeoxyribose, the pentoses in RNAand e q u a l s3 , 4 , 5 , 6 , o r 7 . H e x o s e s( n : 6 ) a n d p e n t o s e (sn : 5 )
DNA,respectively are the most common monosaccharides. All monosaccharides

44 . c H A p r E R 2| c H E M t c A L F o u N D A T t o N S
 uBACrr(lJ)
ADENINE(A) GUANINT(G} cYT0srNt(q THYMINE(I)

nNe Adenosine Guanosine Cytidine Uridine

Jin
t,,'o^o Deoxyadenosine Deoxyguanosine Deoxycytidine DeoxYthYmidine

innNe Adenylate Guanylate Cytidylate UridYlate

f
Iino*e Deoxyadenylate DeoxyguanYlate Deoxycytidylate Deoxythymidylate

Nucleoside monophosphates AMP GMP CMP UMP

Nucieosidediphosphates ADP GDP CDP UDP

Nucleoside triphosphates ATP GTP CTP UTP

Deoxynucleoside mono-,
di-, and triphosphates dAMP,etc. dGMP,etc' dCMP, etc dTMP' etc.

contain hydroxyl (-OH) groups and either an aldehyde or

Htt, 6
a keto group: (-rQ
oo H n-J'-oH
- c t- icl - H - c -Lcf - c - l 1
HO-J.-H
lll <_ lo
Aldehyde Keto H-C-OH
HOH HOH
Many biologically important sugarsare hexoses,includ- H- lLoH o-Glucopyranose
o-Glucofuranose ol
-cH20H (common)
ing glucose,mannose,and galactose(Figure2-18). Mannose (rarel

is identical with glucose except that the orientation of the o-Glucose

groups bonded to carbon 2 is reversed.SimilarlS galactose,
another hexose,differs from glucoseonly in the orientation (b) H,r, Ht,ro
rO
(^. (-
of the groups attached to carbon 4. Interconversion of glu-
cose and mannose or galactose requires the breaking and Ho-l'?-H H-l'-oH
making of covalent bonds; such reactions are carried out by
enzymescaIIedepimerases. HO-ILH HO-ILH
l-Glucose (C6H12O6)is the principal externalsourceof H- lLos HO-JLH
energy for most cells in higher organisms and can exist in
H-lu-os H-CLoH
three different forms: a linear structure and two different ulr,,o,-, ul*ro*
hemiacetalring structures(Figure 2-18a).lf the aldehyde
group on carbon 1 reactswith the hydroxyl group on carbon o-Mannose D-Galactose

5, the resulting hemiacetal,o-glucopyranose'contains a six- 2-18 Chemical structuresof hexoses'All hexoses

FIGURE
member ring. In the cr anomer of o-glucopyranose,the hy- havethe samechemical formula(C6H1206) andcontainan aldehyde
droxyl group attachedto carbon 1 points "downward" from or a ketogroup.(a)Theringformsof o-glucose aregenerated from
the ring as shown in Figure 2-18a; in the B anomer, this hy- the linearmolecule by reaction of the aldehydeat carbon 1 with the
droxyl points "upward." In aqueoussolution the ct and B hydroxylon carbon 5 or carbon 4. The threeforms arereadily
anomers readily interconvert spontaneously;at equilibrium although
interconvertible, the pyranose form(right)predominates in
there is about one-third ct anomer and two-thirds B, with biologicalsystems.(b)In o-mannose ando-galactose, the
very little of the open-chain form. Becauseenzymescan dis- configurationof the H (green) andOH(blue)boundto onecarbon
tinguish between the ct and B anomers of n-glucose, these atomdiffersfrom that in glucose. Thesesugars, likeglucose,exist
forms have distinct bioloeical roles. Condensationof the primarilyasPYranoses.

B U I L D I N GB L O C K SO F C E L L S . 45
CHEMICAL
 hydroxyl group on carbon 4 of the linear glucose with its
aldehydegroup results in the formation of o-glucofuranose.
a hemiacetalcontaining a five-memberring. Although ali
three forms of n-glucose exist in biological systems,the
pyranose form is by far the most abundant.
The pyranosering in Figure 2-18a is depictedas planar. In
fact, because of the tetrahedral geometry carbon
"rorrrd
atoms, the most stableconformation of a pyranose ring has a
nonplanar, chairlike shape.In this conformation, each bond
from a ring carbon to a nonring atom (e.g.,H or O) is either
nearly perpendicular to the ring, referred to as axial (a), or
nearly in the plane ofthe ring, referredto as equatorial (e):
bacteria, and molds produce cellulose-degradingenzymes.
Cows and termites can break down cellulose becausethey
harbor cellulose-degradingbacteriain their gut.
The enzymes that make the glycosidic bonds linking
monosaccharidesinto polysaccharidesare specific for the a
or B anomer of one sugar and a particular hydroxyl group
on the other. In principle, any two sugar molecules can be
linked in a variety of ways becauseeachmonosaccharidehas
multiple hydroxyl groups that can participate in the forma-
tion of glycosidic bonds. Furthermore, any one monosaccha-
ride has the potential of being linked to more than two other
monosaccharides,thus generating a branch point and non-
linear polymers. Glycosidic bonds are usually formed be-
tween the growing polysaccharide chain and a covalently
modified form of a monosaccharide.Such modifications in-
clude a phosphate(e.g.,glucose6-phosphate)or a nucleotide
(e.g.,UDP-galactose) :

yranoses
tPyranoses c_o-Glucopyranose

into common table sugar (Figure2-19). Glucose 6-phosphate UDp-galactose

Larger polysaccharides,containing dozensto hundredsof
The epimeraseenzymesthat interconvert differenr monosac-
charides often do so using the nucleotide sugarsrather than
the unsubstitutedsugars.
Many complex polysaccharidescontain modified sugars
that are covalently linked to various small groups, particu-
larly amino, sulfate, and acetyl groups. Such modifications
are abundant in glycosaminoglycans,major polysaccharide
components of the extracellular matrix that we describein
Chapter 19.

PhospholipidsAssociateNoncovalentlyto Form
polymer of the B anomer of glucose. Human digestiveen- the BasicBilayerStructureof Biomembranes
zymescan hydrolyze the c glycosidic bonds in starch but nor Biomembranes are large flexible sheets that serve as the
the B glycosidic bonds in cellulose.Many speciesof plants, boundaries of cells and their intracellular organelles and

cHroH
):o
*oi
H
il \?' H

HO

> FIGURE 2-19 Formationof HOH HOH

the disaccharides lactoseand Galactose Glucose
sucrose.In anyglycosidic Iinkage,
theanomeric carbonof onesugar
molecule (ineithertheo or B CH,OH cH2oH
conformation) islinkedto a ,f.o.
hydroxyloxygenon anothersugar \T
1)a
moleculeThelinkages arenamed H O
OH
accordingly;thuslactose contains
a B(1--+4) bond,andsucrose HOH OHH
containsan ct(1-+ 2) bond Glucose Fructose Sucrose

46 CHAPTER2 | cHEM|CALFOUNDAT|ONS
 Fatty acid chains

Hydrophobictail

PHOSPHATIDYI.

FIGURE2-20 phosphatidylcholine,a typical phosphoglyceride.
All phosphoglycerides are amphipathicphospholipids,
havinga
hydrophobictail (yellow)and a hydrophilichead(blue)in which
glycerolis linkedvia a phosphategroup to an alcohol.Eitheror both
of the fatty acylsidechainsin a phosphoglyceride may be saturated
or unsaturatedIn phosphatidic acid(red),the simplestphospholipid,
the phosphateis not linkedto an alcohol

form the outer surfacesof some viruses.Membranes literally
define what is a cell (the outer membrane and the contents
within the membrane) and what is not (the extracellular
space outside the membrane). Unlike the proteins, nucleic
acids, and polysaccharides,membranesare assembledby the
noncoualentassociationof their componentbuilding blocks.
The primary building blocks of all biomembranesare phos-
pholipids, whose physical properties are responsiblefor the
formation of the sheetlike structure of membranes.The
structuresand functions of membranes,which include in ad-
dition to phospholipidsa variety of other molecules(e.g''
cholesterol,glycolipids, proteins), will be describedin detail
in Chapter 10.
Phospholipidsconsistof two long-chain,nonpolar fatty
acid groups linked (usually by an ester bond) to small,
highly polar groups, including a phosphateand a short or-
ganic molecule, such as glycerol (trihydroxy propanol)
( F i g u r e2 - 2 0 ) .
Fatty acids consist of a hydrocarbon chain attachedto
a carboxyl group (-COOH)' Like glucose,fatty acids are
an important energy source for many cells (Chapter 12)'
They differ in length, although the predominant fatty acids
in cells have an even number of carbon atoms' usually 14,
16. 18. or 20. The maior fatty acids in phospholipids are
listed in Table 2-4. Fatty acids often are designatedby the
abbreviationCx:y, where x is the number of carbonsin the
chain and y is the number of double bonds. Fatty acids
containing 12 or more carbon atoms are nearly insolublein
aqueous solutions becauseof their long hydrophobic hy-
drocarbon chains.
Fatty acids with no carbon-carbon double bonds are
said to be saturated;thosewith at leastone double bond are
unsaturated. Unsaturated fatty acids with more than one
carbon-carbon double bond are referred to as polyunsatu-
rated. Two "essential" polyunsaturated fatty acids, linoleic
acid (C18:2)and linolenicacid (C18:3),cannotbe synthesized

(IONIZEO
OFACII)
NAME
COMMON INPARENTHESES} ABBREVIATI()N
T()RM

SATURATEDFATTY ACIDS

Myristic (myristate) C14:0 cH3(cH2)12cooH

Palmitic (palmitate) C|6:0 cH3(cH2)14cooH

Stearic (stearate) C18:0 cH3(cH2)15cooH

UNSATURMED FATTY ACIDS

Oleic (oleate) C18:1 CH3(CH2)7CH:CH(CHz)7COOH

Linoleic (linoleate) C18:2 CH3(CH2)4CH:CHCHzCH:CH(CHz)7COOH

Arachidonic (arachidonate) C20:4 CH3(CH2)4(CH:CHCHz):CH:CH(CH2)3COOH

OF CELLS
B U I L D I N GB L O C K S
CHEMICAL 47
 by mammals and must be suppliedin their diet. Mammals can
synthesizeother common fatty acids. Two stereoisomeric
configurations, cis and trans, are possible around each
carbon-carbondouble bond:
Trans
A cis double bond introducesa rigid kink in the other-
wise flexible straight chain of afatty acid (Figure2-21).
In general, the unsaturated fatty acids in biological systems
contain only cis double bonds. Saturatedfatty acids without
solid margarine sticks) and other food producrs are not nat-
ural, arising from the catalytic processused for hydrogena-
tion. Saturated and trans fatty acids have similar physical
properties, and their consumption, relative to the consump-
tion of unsaturatedfats, is associatedwith increasedplasma
cholesterollevels.
Fatty acids can be covalently attached to another mole-
cule by a type of dehydration reaction calledesterification,
in which the OH from the carboxyl group of the fatty acid
and a H from a hydroxyl group on the other molecule are
Iost. In the combined molecule formed by this reaction, the
portion derived from the fatty acid is called an acyl group, or
fatty acyl group. This is illustrated by the most common
form of phospholipids, phosphoglycerides,with two acyl
groups attached to rwo of the hydroxyl groups of glycerol
HHHHHHHHHHHHHH
H s -c gt t-l lgl l-l lgl-t ' g
t t-t 'qt z- c, o- _ _ c _ c _ _ _ _ _
c c c c c a c- \. ^ _
.HtH. .Ht H. H H Ht lHt Hl tHLHt iH U T i
t t t
Palmitate
(ionized form of palmitic acid)
FfGURE2-21 The effect of a double bond on the shape of
fatty acids.Shownare chemicalstructuresof the ionizedform of
palmiticacid,a saturatedfatty acidwith 16 C atoms,and oleicacid,
an unsaturated one with 18 C atoms In saturatedfattv acids.the
48 cHAPTER2 | cHEMTCALFOUNDATTONS
(seeFigure 2-20) and triacylglycerols,or triglycerides,which
contain three acyl groups esterfiedto glycerol:
?
H 3 C - ( C H 2 ) , - C- O - C H 2
ol
H3C-(CH2)"-C-O-CH
ol
H3C-(CH2),-C-O-CH'
Triacylglycerol
Fatty acyl groups also can be covalently linked to another
fatty molecule,cholesterol,to form cholesterylesters.
Triglycerides and cholesteryl esters are extremely water-
insoluble compounds in which fatty acids and cholesterol are
either stored or transported. Tiiglycerides are the storageform
of fatty acids in the fat cells of adiposetissue and are the prin-
cipal components of dietary fats. Cholesteryl estersand triglyc-
erides are transported between tissues through the blood-
streamin specializedcarrierscalledlipoproteins(Chapter 14).
In phosphoglycerides,one hydroxyl group of the glycerol
is esterifiedto phosphate while the other two normally are
esterified to fatty acids. The simplest phospholipid, phos-
phatidic acid, contains only these components. In most
phospholipids found in membranes,the phosphate group is
also esterified to a hydroxyl group on another hydrophilic
compound. In phosphatidylcholine, for example, choline is
attached to the phosphate (see Figure 2-20). The negative
charge on the phosphate as well as the charged or polar groups
esterifiedto it can interact strongly with water. The phosphate
and its associatedesterifiedgroup, the "head" group of a phos-
pholipid, is hydrophilic, whereas the fatty acyl chains, the
"tails," are hydrophobic. (Other common phosphoglycerides
and associatedheadgroupsare shown in Table2-5.) Molecules
such as phospholipids that have both hydrophobic and
i U
Oleate
(ionized form of oleic acid)
hydrocarbonchainis often linear;the cis doublebond in oleate
createsa rigid kink in the hydrocarbonchain [AfterL Stryer,
1994,
Biochemistry,4thed, W H Freeman andCompany, p 265l
 sine (C), thymine (T), and uracil (U) (seeFigure Z-17). A,
G, ! and C are in DNA, and A, G' U, and C are in RNA.
r Glucoseand other hexosescan exist in three forms: an
open-chain linear structure, a six-member (pyranose)
PHOSPHOGIYCERIOES HEAD
COMMON GBOUP
ring, and a five-member(furanose)ring (seeFigure 2-18).
In biological systems,the pyranose form of l-glucose
c H g^ , , predominates.
| ,rung
Phosphatidylcholine o&N\cr. r Glycosidic bonds are formed betweeneither the o or the
B anomer of one sugar and a hydroxyl group on another
Gholine sugar, leading to formation of disaccharidesand other
polysaccharides (seeFigure2-L9).
H
l.H r Phospholipids are amphipathic molecules with a hy-
Phosphatidylethanolamine o&N\n drophobic tail (often two fatty acyl chains) connectedby a
small organic molecule (often glycerol) to a hydrophilic
Ethanolamine head (seeFigure 2-20). The long hydrocarbon chain of a
fatty acid may contain no carbon-carbon double bond (sat-
H
lrH urated) or one or more double bonds (unsaturated);a cis
double bond bends the chain'
o---rN H
Phosphatidylserine I
o4o
Serine E C h e m i c aEl q u i l i b r i u m
We now shift our discussionto chemical reactions in which
bonds, primarily covalent bonds in reactant chemicals' are
Phosphatidylinositol broken and new bonds are formed to generatereaction
products. At any one time, severalhundred different kinds of
lnositol chemical reactions are occurring simultaneouslyin every
cell, and many chemicalscan, in principle' undergo multiple
chemical reactions. Both the extent to which reactions can
hydrophilic regionsare calledamphipathic.In Chapter 10, we proceed and the rate at which they take place determine the
will seehow the amphipathic properties of phospholipids are chemical composition of cells.
'$7hen
responsiblefor the assemblyof phospholipidsinto sheetlike reactants first mix together-before any products
bilayer biomembranes in which the fatty acyl tails point into have been formed-their rate of reaction to form products
the center of the sheet and the head groups point outward (forward reaction)is determinedin part by their initial concen-
toward the aqueousenvironment(seeFigure2-13).a trations, which determine the likelihood of reactants bumping
into one another and reacting (Figure 2-22). As the reaction
products accumulate' the concentration of each reactant de-
ir."r.r and so does the forward reaction rate. Meanwhile'
C h e m i c a lB u i l d i n g B l o c k so f C e l l s some of the product molecules begin to participate in the re-
versereaction, which re-forms the reactants (the ability of a re-
r Three major biopolymersformed by polymerizationreac-
action to go "backward" is called microscopicreuersibility).
tions (net dehydration)of basicchemicalbuilding blocks are
This reversereaction is slow at first but speedsup as the con-
presentin cells:proteins,composedof amino acidslinked by
centrationofproduct increases. Eventually,the ratesofthe for-
peptidebonds;nucleicacids,composedof nucleotideslinked
composedof ward and reversereactionsbecomeequal, so that the concen-
by phosphodiesterbonds;and polysaccharides,
(sugars) glycosidic bonds (see trations of reactantsand products stop changing.The systemis
monosaccharides linked by
fourth major chemical then said to be in chemical equilibrium (plural: eqwilibria).
Figure 2-13). Phospholipids, the
At equilibrium, the ratio of products to reactants,
building block, assemble noncovalently into biomembranes.
called the equilibrium constant' is a fixed value that is in-
r Differences in the size, shape, charge, hydrophobicity, dependent of the rate at which the reaction occurs. The
and reactivity of the side chains of the 20 common amino raie of a chemical reaction can be increased by a catalyst,
acids determine the chemical and structural properties of which acceleratesthe chemical transformation (making
proteins (seeFigure2-14). and breaking of covalent bonds) but is not permanently
r The basesin the nucleotidescomposing DNA and RNA changedduring a reaction (seeSection2.4)'ln this section,
are carbon- and nitrogen-containingrings attached to a we discuss several aspectsof chemical equilibria; in the
pentose sugar. They form two groups: the purines- next section, we examine energy changes during reactions
adenine (A) and guanine (G)-and the pyrimidines-cyto- and their relationshipto equilibria.

C H E M I C A LE Q U I L I B R I U M 49
 Rate."r"rr. : 1. By rearranging theseequations,we can express
the equilibrium constant as the ratio of the rate consrants
Rateof forward reaction
(concentrationof reactantsdecreases)
K-^ _T
k1
t) -1\
1
o
C h e m i c ael o u i l i b r i u m ChemicalReactionsin CellsAre at SteadyState
; (forwardand reverseratesare
e q u a l ,n o c h a n g ei n c o n c e n t r a t i o n Under appropriate conditions and given sufficient time, indi-
E of reactantsand products)
o vidual biochemical reactions carried out in a test tube even-
tually will reach equilibrium. Within cells, however, many
Rate of reverse reaction
(concentration of products increases) reactions are linked in pathways in which a product of one
reaction servesas a reactarftin another or is pumoed out of
the cell. In this more complex situation,when the iate of for-
When reactantsare first mixed. mation of a substanceis equal to the rate of its consumption,
initial concentrationof products= 0
the concentration of the substanceremains constant, and the
systemof linked reactionsfor producing and consuming that
T i m e+
substanceis said to be in a steady state (Figure 2-23). One
A FIGURE 2-22 Timedependence of the ratesof a chemical consequenceof such linked reactionsis that they prevent the
reaction.Theforwardandreverse ratesof a reaction dependin oart accumulation of excessintermediates,protecting cells from
on the initialconcentrationsof reactants
andproducts. Thenet the harmful effects of intermediatesthat have the Dotential
forwardreaction rateslowsasthe concentration of reactants of beingtoxic at high concentrations.
decreases, whereas the netreversereaction rateincreases asrne
concentration of productsincreasesAt equilibrium, the ratesof the
forwardandreverse reactions
areequalandthe concentrations of
DissociationConstantsof Binding Reactions
reactantsandproducts remainconstant Reflectthe Affinity of lnteractingMolecules
The concept of equilibrium also appliesto the binding of one
E q u i l i b r i u mC o n s t a n t sR e f l e c t h e
molecule to another. Many important cellular processesde-
Extent of a ChemicalReaction pend on such binding "reactions," which involve the making
The equilibrium constant K.o dependson the nature of the and breaking of various noncovalentinteractionsrather than
reactants and products, the temperature, and the pressure covalentbonds,as discussedabove.A common exampleis the
(particularly in reacrions involving gases).Under standard binding of a ligand (e.g.,the hormone insulin or adrenaline)to
physicalconditions (25'C and 1 atm pressurefor biological its receptor on the surface of a cell forming a multimolecular
systems),the K.o is always the same for a given reaction, assembly,or complex, that triggersa biological response.An-
whether or not a catalystis present. other exampleis the binding of a protein to a specificsequence
For the general reaction with three reactants and three of basepairs in a moleculeof DNA, which frequently causes
products the expressionof a nearby geneto increaseor decrease(Chap-
ter 7). lf the equilibrium constant for a binding reaction is
aA + bB * cC ; . zZ + yY + xX (2_1)
(a)Testtube equilibriumconcentrations
where capital letters representparticular moleculesor atoms
and lowercaseletters representthe number of each in the re- BBB
action formula, the equilibrium constant is given by AAA = -BBB
BBB

(2-2) (b) Intracellularsteady-stateconcentrations

where bracketsdenote the concentrationsof the moleculesat
equilibrium. The rate of the forward reaction (left to right in
Equation (2-1) is
R a r e 6 o , * , ,:6 k r l A l " l B l b [ C | .
where Ai is the rate constant for the forward reaction. Similarly,
the rate of the reversereaction (right to left in Equation 2-1) is
: k,[X]" [y lv
Ratereverse [Z]"
where A, is the rate constant for the reversereaction. At equi-
librium the forward and reverserates are equal, so Rateso*..6/
n n - B B_B - cC
BBB _
CC
A FIGURE2-23 Comparisonof reactionsat equilibrium and
steady state. (a) ln the test tube, a biochemicalreaction(A -+ B)
eventuallywill reachequilibrium,in which the ratesof the fon,rardand
reversereactionsare equal(asindicatedby the reactionarrowsof equal
length).(b) In metabolicpathwayswithin cells,the productB commonly
would be consumed,in this exampleby conversionto C. A pathwayof
linkedreactionsis at steadystatewhen the rateof formationof the
intermediates (e g , B) equalstheir rateof consumption.As indicatedby
the unequallengthof the arrows,the individualreversible reactions
constitutinga metabolicpathwaydo not reachequilibrium Moreover,
the concentrations of the intermedrates at steadystatecan differfrom
what theywould be at eouilibrium.

s0 CHAPTER2 I CHEMICALFOUNDATIONS
 CanBind MultipleLigands
ffi Rodcast:Macromolecules
> FIGURE 2-24 Macromolecules can havedistinctbinding
sitesfor multipleligands.A largemacromolecule (eg , a protein,
bindingsites(A-C)isshown;eachbinding B i n d i n gs i t e B ( K 6 s )
blue)withthreedistinct
siteexhibitsmolecular
complementarityto threedifferentbinding
partners(ligands
A-C)with distinct
dissociation
constants (KdAc)
known, the intracellular stability of the resultingcomplex can ( e . 9 . s, m a l l
be predicted.To illustrate the generalapproach for determin- m o l e c u l e )
ing the concentrationof noncovalentlyassociatedcomplexes,
we will calculatethe extent to which a protein (P) is bound to
DNA (D)forming a protein-DNA complex (PD):
P+D.-PD
Most commonly, binding reactions are describedin terms of
the dissociation constant K6, which is the reciprocal of the
equilibrium constant. For this binding reaction, the dissocia-
tion constant is given by
tPIIDl
ro:
TDi
Typical reactionsin which a protein binds to a specificDNA se-
quencehavea K6 of 10-10 M, where M symbolizesmolariry or
moles per liter (mol/L). To relate the magnitude of this dissocia-
tion constant to the intracellular ratio of bound to unbound
DNA, let's consider the simple example of a bacterial cell hav-
1s L
ing a volume of 1.5 x 10 and containing L moleculeof
DNA and 10 moleculesof the DNA-binding protein P. In this
0
case,givena Ka of 10-1 M, 99 percentof the time this specific
DNA sequencewill have a molecule of protein bound to it and
1 percent of the time it will not, even though the cell contains
only 10 moleculesof the protein! Clearly P and D bind very
tightly (have a high affinity), as reflectedby the low value of the
dissociation constant for their binding reaction. For protein-
' M
protein and protein-DNA binding, K6 values of <10
-10 o M (micromolar)
(nanomolar)are consideredto be tight,
modestlytight, and -10-' M (millimolar) relativelyweak.
The large size of biological macromolecules,such as pro-
teins, can result in the availability of multiple surfacesfor
complementaryintermolecularinteractions (Figure 2-24). As
a consequence,many macromoleculeshave the capacity to
bind severalother moleculessimultaneously.In some cases,
these binding reactionsare independent,with their own dis-
tinct Ka valuesthat are constant.In other cases,binding of a
moleculeat one site on a macromoleculecan changethe three-
dimensionalshapeof a distant site, thus altering the binding
interactionsof that distant sitewith someother molecule.This
is an important mechanismby which one moleculecan alter
(regulate)the activity of a secondmolecule(e.g.,a protein) by
changingits capacityto interact with a third molecule.'Weex-
amine this regulatory mechanismin more detail in Chapter 3.
RoshanKetab 021-6 69 50 639
B i o l o g i c aFl l u i d sH a v eC h a r a c t e r i s t ipcH V a l u e s
The solventinsidecellsand in all extracellularfluids is water. An
important characteristic of any aqueous solution is the
M u l t i l i g a n db i n d i n gm a c r o m o l e c u l (ee . 9 . ,p r o t e i n )
Bindingsite A (K6a)
L i g a n dB
L i g a n dA
( e . 9 . s, m a l l p r o t e i n )
L i g a n dC
(e 9.,polysaccharide)
16:\
s i t eC ( K 6 6 )
Binding
d
(2-4)
concentrationof positivelychargedhydrogenions (H+ ) and neg-
atively chargedhydroxyl ions (OH-). Becausetheseions are the
dissociationproducts of H2O, they are constituentsof all living
systems,and they are liberatedby many reactionsthat take place
between organic moleculeswithin cells.These ions also can be
transported into or out of cells, as when highly acidic gastric
juice is secretedby cellslining the walls of the stomach.
\fhen a water moleculedissociates,one of its polar H-O
bonds breaks.The resultinghydrogenion, often referredto as
a proton, has a short lifetime as a free ion and quickly com-
bines with a water molecule to form a hydronium ion
(HrO*). For convenience,however'we refer to the concentra-
tion of hydrogenions in a solution, [H*], eventhough this re-
ally representsthe concentrationof hydronium ions, [H3O-].
Dissociationof H2O generatesone OH- ion along with each
H*. The dissociationof water is a reversiblereaction:
H2O:H*+OH-
At 25 'C, [H+][OH-]_: 1O-r4 M2, so that in pure water,
l H * l: t o H - l : 1 o - ' M .
The concentration of hydrogen ions in a solution is ex-
pressedconventionally as its pH, defined as the negativelog
of the hydrogen ion concentration. The pH of pure water at
25 'C is 7:
p H : - l o g [ H -:] l " s E L : b e + = - : z
lnj,J
It is important to keep in mind that a 1 unit differencein pH
representsa tenfold difference in the concentration of pro-
tons. On the pH scale,7.0 is consideredneutral: pH values
below 7.0 indicateacidicsolutions(higher[H*]), and values
above 7.0 indicate basic (alkaline) solutions (Figure 2-25)'
For instance, gastric juice, which is rich in hydrochloric acid
EQUILIBRIUM
CHEMICAL 51
 I n c r e a s i n g lbya s i c ric acid (HCl) or the carboxylgroup (-COOH), which tends
( l o w e rH +c o n c e n t r a t i o n )
to dissociateto form the negativelychargedcarboxylate ion
(-COO ). Likewise,a baseis any molecule,ion, or chemical
pH scale group that readily combineswith a H+, such as the hydroxyl
ion (OH-); ammonia (NH:), which forms an ammonium ion
1 4 S o d i u mh y d r o x i d(e1 N ) (NH+-); or the amino group (-NH2).
rWhenacid is addedto an aqueoussolution,the
13 [H+] in-
H o u s e h o lb
dl e a c h creases(the pH goes down). ConverselS when a base is
1)
A m m o n i a( 1 N ) addedto a solution,the [H+] decreases (the pH goesup). Be-
11 cause[H+][OH ] : 10-14M2, any increasein [H*] is cou-
1 0 1^ pled with a commensuratedecrease in [OH-] and vice versa.
sea water Many biologicalmoleculesconrain both acidic and basic
, _.1r
groups.For example,in neutral solutions(pH : 7.0), many
8 I n t e r i o ro f c e l l
amino acidsexist predominantlyin the doubly ionizedform,
j' Fertilizedegg
in which the carboxyl group has lost a proton and the amino
U n f e r t i l i z eedg g
6 Urine group has acceptedone:

I n t e r i o ro f t h e l y s o s o m e NH,*
t-
H-C-COO-
G r a p e f r u ijtu i c e
R

G a s t r i cj u i c ewhere R representsthe uncharged side chain. Such a mole-

cule, containing an equal number of positive and negative
H y d r o c h l o r i ac c i d ( 1 N )
ions, is called a zwitterion. Zwitterions, having no net
charge,are neutral. At extreme pH values,only one of these
two ionizable groups of an amino acid will be charged.
The dissociationreaction for an acid (or acid group in a
I n c r e a s i n gal cy i d i c larger molecule)HA can be wrirten as HA == H+ + A . The
( gr e a t eH r +c o n c e n t r a t i o n )
equilibriumconstantfor this reaction,denotedK. (thesubscript
FfGURE2-25 pH valuesof commonsolutions.ThepHof an
d standsfor "acid"), is definedas K" : [H*][A ]/lHAl. Taking
aqueous solution isthenegative logof thehydrogen ionconcentratron the logarithm
of both sidesand rearrangingthe result yields a
ThepHvalues for mostintracellular andextracellular
biological
fluids
very usefulrelation betweenthe equilibrium constantand pH:
arenear7 andarecarefully regulated to permittheproperfunctioning
of cells,organelles, andcellular secretions
pH: pK"+ loe#l e-s)
(HCl), has a pH of about 1. ks [H+] is roughly a millionfold lr1Al

greaterthan that of cytoplasmwith a pH of abort 7.2.

where pK" equals-log K".
Although the cytosolof cellsnormally has a pH of about
From this expression,commonly known astheHenderson-
7.2, the pH is much lower (about 4.5) in the interior of lyso,
HasselbaLch equation,it can be seenthar the pK. of any acid is
somes,one type of organellein eukaryoticcells(Chapter9).
equalto the pH at which half the moleculesare dissociatedand
The many degradative enzymeswithin lysosomesfunction
half are neutral (undissociated). This is becausewhen [A-] :
optimally in an acidic environment,whereastheir action is
[HA], then log ([A-]/[HA]) : O, and thus pK" : pH. The
inhibited in the near neurral environmentof the cytoplasm.
Henderson-Hasselbalch equarionallows us to calculatethe de-
This illustratesthat mainrenanceof a specificpH is essential
gree of dissociationof an acid if both the pH of the solution
for proper functioning of some cellular strucrures.On the
and the pK, of the acid are known. Experimentally,by meas,
other hand, dramatic shifts in cellular pH may play an im-
uring the [A ] and [HA] as a function of the solution'spH, one
portant role in controlling cellularactivity.For example,the
can calculatethe pK. of the acid and thus the equilibrium con-
pH of the cytoplasmof an unfertilizedeggof the seaurchin,
stant Ka for the dissociationreacion (Figure2-26).
an aquatic animal, is 6.6. Within 1 minute of fertilization,
however,the pH risesto 7.2; that is, the [H+l decreases to
about one-fourth its original value, a change necessaryfor
B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l a r
subsequentgrowth and division of the egg. a n d E x t r a c e l l u l aFr l u i d s
A growing cell must maintain a consrant pH in the cyto-
Hydrogenlons Are Releasedby plasm of about 7.2-7.4 despitethe metabolic production of
many acids,suchas lactic acid and carbon dioxide;the latter
A c i d sa n d T a k e nU p b y B a s e s
reactswith water to form carbonicacid (H2CO3).Cellshave
In general, an acid is any molecule, ion, or chemical group a reservoir of weak basesand weak acids, called buffers,
that tendsto releasea hydrogenion (H+), suchas hydrochlo- which ensure that the cell's pH remains relatively constant

52 . c H A p r E R 2| c H E M t c A L F o u N D A T t o N S
 H2CO3:HCO3-+H*

H2C03

-';i9 1 1 0 0
= =)
.oO

6P
OG 50
tsc

-o 0L
0 7.48
OH

A FfGURE 2-26 lhe relationshipbetween pH, pKa,and the

dissociation of an acid.Asthe oHof a solutionof carbonicacid
risesfrom0 to 8 5, the percentageof the compound in the o 0.2 0.4 0.6 0'8 1.0
undissociated, form(HzCO:)
or un-ionized, decreases from100 CH3COOH
of dissociated
Fraction
percent andthatof the ionized formincreasesfrom0 percentWhen
AddedOH- -----)
the pH(6.4)isequalto the acidspKa,halfof the carbonic acidhas
ionizedWhenthe pH risesto above8, virtually allof the acidhas FfGURE 2-27 The titration curveof the buffer aceticacid
ionized to the bicarbonateform(HCOr) (CH3COOH). of aceticacidto hydrogen
ThepK,for the dissocration
andacetateionsis4 75 At thispH,halftheacidmolecules are
despitesmall fluctuationsin the amountsof H* or OH- be- Because
dissociated pHismeasured on a logarithmicscale,thesolution
ing generatedby metabolism or by the uptake or secretionof changesfrom91 percent CH3COOH at pH3.75to 9 percent CH3COOH
moleculesand ions by the cell. Buffers do this by "soaking at pH5 75.Theacid hasmaximum bufferingcapacityin thispH range
up" excessH* or OH when theseions are addedto the cell
or are producedby metabolism. in maintaining, or buffering, the pH of the cytoplasm. Phos-
If additional acid (or base)is added to a buffered solu-
phoric acid (H3POa) has three protons that are capable of
tion whose pH is equal to the pK, of the buffer (tHAl : dissociating,but they do not dissociatesimultaneously.Loss
[A ]), the pH of the solution changes,but it changesless of each proton can be describedby a discretedissociationre-
than it would if the buffer had not been present.This is be- action and pKo, as shown in Figure 2-28.The titration curve
causeprotons releasedby the addedacid are taken up by the
ionized form of the buffer (A- ); likewise, hydroxyl ions gen-
14
er:atedby the addition of baseare neutralized by protons re- P K a =1 2 ' 7
leasedby the undissociatedbuffer (HA). The capacity of a
12 HPO42-+ PO4
substanceto releasehydrogen ions or take them up depends
partly on the extent to which the substancehas already
taken up or releasedprotons, which in turn dependson the
pH of the solution relative to the pK" of the substance.The
ability of a buffer to minimize changesin pH, its buffering
PKa=7'2
capacity,dependson the concentration of the buffer and the I
o_
H2PO4-* HPO+2-+H*
relationship between its pK" value and the pH, which is ex-
pressedby the Henderson-Hasselbalch equation.
The titration curve for acetic acid shown inFigure 2-27
illustratesthe effect of pH on the fraction of moleculesin the
un-ionized(HA) and ionizedforms (A ). At one pH unit be- PKa=2'1
H3PO4+HrPOf+H+
low the pK" of an acid, 91 percentof the moleculesare in the
HA form; at one pH unit above the pK^, 9L percent are in
the A form. At pH values more than one unit above or
below the pK", the buffering capacity of weak acids and Added OH- ----->
basesdeclines rapidly. In other words, the addition of the A FIGURE2-28 The titration curve of phosphoric acid (H3PO4),
same number of moles of acid to a solution containing a a common buffer in biological systems.Thisbiologically ubiquitous
mixture of HA and A that is at a pH near the pK" will cause moleculehasthreehydrogenatomsthat dissociate at differentpH
lessof a pH changethan it would if the HA and A- were not values;thus phosphoricacidhasthreepK" values,as notedon the
present or if the pH were far from the pK. value. graph The shadedareasdenotethe pH ranges-withinone pH unit
All biological systemscontain one or more buffers. Phos- of the three pKuvalues-where the bufferingcapacityof phosphoric
phate ions, the ionized forms of phosphoric acid, are present acidis high In theseregions,the additionof acid(or base)will cause
in considerablequantitiesin cellsand are an important factor smallchangesin the PH
relatively

EQUILIBRIUM
CHEMICAL 53
 for phosphoric acid shows that the pK" for the dissociationof
the secondproton is 7.2. Thus at pH7.2, about 50 percentof
cellula^rphosphate is H2POa- and about 50 percent is
HPO4'- according to the Henderson-Hasselbalchequation.
For this reason,phosphateis an excellentbuffer at pH values
around 7.2, the approximate pH of the cytoplasm of cells,
and at pH 7.4, the pH of human blood.
C h e m i c a lE q u i l i b r i u m
r A chemical reaction is at equilibrium when the rate of
the forward reaction is equal to the rate of the reversere-
action (no net changein the concentration of the reactants
or products).
The equilibrium constant K.o of a reaction reflects the
tio of products to reacrantsai equilibrium and thus is a
measure of the extent of the reaction and the relative sta-
bilities of the reactantsand products.
r The K.o depends on the temperature, pressure, and
chemical properties of the reactants and products but is
independent of the reaction rate and of the initial concen-
trations of reactantsand products.
For any reaction, the equilibrium constant K.o equalsthe
tio of the forward rate constant to the reversi rate con-
stant (krl&r).The rates of conversion of reactantsto prod-
ucts and vice versa depend on the rate constants and the
concentrationsof the reactantsor products.
'Sfithin
r cells, the linked reactions in metabolic pathways
generallyare at steadystate,not equilibrium, at which rate
of formation of the intermediatesequals their rate of con-
sumption (seeFigure 2-23) and thus the concentrations of
the intermediatesare not changing.
r The dissociation constant K6 for the noncovalent bind-
ing of two molecules is a measure of the stability of the
complex formed betweenthe molecules(e.g.,ligand-receptor
or protein-DNA complexes).
r The pH is the negativelogarithm of the concentration of
hydrogen ions (-log [H+]). The pH of the cytoplasm is nor-
mally about 7.2-7.4, whereasthe interior of lysosomeshas
a pH of about 4.5.
r Acids releaseprotons (H+) and basesbind them. In bio-
logical molecules,the carboxyl and phosphate groups are
the most common acidic groups; the amino group is the
most common basicgroup.
r Buffers are mixrures of a weak acid (HA) and its corre-
sponding baseform (A ), which minimize the changein pH
of a solution when acid or alkali is added. Biological sys-
tems use various buffers to maintain their pH within a very
narrow range.
W Biochemical
Energetics
The production of energy,its storage,and its use are central
to the economy of the cell. Energy may be defined as the
ability to do work, a concept applicable to automobile
54 . c H A p r E R 2| c H E M t c A L F o u N D A T t o N s
engines and electric power plants in our day-to-day world
and to cellular enginesin the biological world. The energy
associatedwith chemical bonds can be harnessedto support
chemical work and the physical movements of cells.
SeveralFormsof EnergyAre lmportant
i n B i o l o g i c aS
l ystems
There are two principal forms of energy:kinetic and poten-
tial. Kinetic energyis the energyof movement-the motion of
molecules,for example.The secondform of energy,potential
energy, or stored energy, is particularly important in the
study of biological or chemical systems.
Thermal energy, or heat, is a form of kinetic energy-the
energy of the motion of molecules.For heat to do work, it
must flow from a region of higher temperature-where the
averagespeedof molecularmotion is greater-to one of lower
temperature. Although differencesin temperature can exist
betweenthe internal and external environmentsof cells,these
thermal gradientsdo not usually serveas the sourceof energy
for cellular activities.The thermal energy in warm-blooded
animals, which have evolveda mechanismfor thermoregula-
tion, is usedchiefly to maintain constantorganismictempera-
tures. This is an important function becausethe rates of many
cellular activities are temperature-dependent.For example,
cooling mammalian cellsfrom their normal body temperature
of 37 "C to 4 "C can virtually "freeze" or stop many cellular
processes(e.g.,intracellularmembranemovements).
Radiant energyis the kinetic energyof photons, or waves
of light, and is critical to biology. Radiant energycan be con-
verted to thermal energy,for instancewhen light is absorbed
by moleculesand the energy is converted to molecular mo-
tion. Radiant energy absorbed by moleculescan also change
the electronic structure of the molecules, moving electrons
into higher-energystates (orbitals), whence it can later be
recoveredto perform work. For example, during photo-
synthesis,light energy absorbed by specializedmolecules
(e.g.,chlorophyll) is subsequentlyconverted into the energy
of chemical bonds (Chapter 12).
Mechanical energy,a major form of kinetic energy in bi-
ology usually resultsfrom the conversionof stored chemical
energy.For example, changesin the lengths of cytoskeletal
filaments generate forces that push or pull on membranes
and organelles(Chapters 17 and 18).
Electric energy-the energy of moving electrons or other
charged particles-is yet another major form of kinetic energy.
Several forms of potential energy are biologically signifi-
cant. Central to biology is chemicalpotential energy,the energy
stored in the bonds connecting atoms in molecules.Indeed,
most of the biochemicalreactionsdescribedin this book in-
volve the making or breaking of at least one covalent chemical
'We
bond. recognize this energy when chemicals undergo
energy-releasingreactions. For example, the high potential
energy in the covalent bonds of glucosecan be releasedby
controlled enzymaticcombustion in cells (Chapter 12). This
energyis harnessedby the cell to do many kinds of work.
A secondbiologically important form of potential energyis
the energyin a concentration gradient. \X/henthe concentration
of a substanceon one side of a barrier,such as a membrane,is (a)
different from that on the other side, a concentration gradient Endergonic
exists. All cells form concentration gradients between their
interior and the external fluids by selectivelyexchangingnutri-
ents, waste products, and ions with their surroundings.Also,
1
A
I
organelleswithin cells (e.g., mitochondria, lysosomes)fre- I
quently contain different concentrationsof ions and other mol- (\ ah

ecules;the concentration of protons within a lysosome, as we
saw in the last section,is about 500 timesthat of the cytoplasm.
A third form of potential energy in cells is an electric
potential-the energy of charge separation. For instance,
there is a gradient of electric charge of =200,000 volts per
cm acrossthe plasma membrane of virtually all cells.We dis-
cuss how concentration gradients and the potential differ-
enceacrosscell membranesare generatedand maintained in
Chapter 11 and how they are converted to chemical poten-
tial energyin Chapter 12.
CellsCanTransformOne Type
of Energyinto Another
According to the first law of thermodynamics,energy is nei-
ther creatednor destroyedbut can be converted from one form
to another.(In nuclear reactions,massis convertedto energy,
but this is irrelevantto biological systems.)In photosynthesis,
for example,the radiant energyof light is transformedinto the
chemicalpotential energy of the covalent bonds betweenthe
atoms in a sucroseor starch molecule.In musclesand nerves,
chemical potential energy stored in covalent bonds is trans-
formed, respectively, into the kinetic energyof musclecontrac-
tion and the electricenergyof nerve transmission.In all cells,
potential energy,releasedby breaking certain chemicalbonds,
is used to generatepotential energyin the form of concentra-
tion and electricpotential gradients.Similarly,energystoredin
chemical concentration gradients or electric potential gradi-
ents is usedto synthesizechemicalbonds or to transport mol-
eculesfrom one side of a membraneto another to generatea
concentrationgradient. The latter processoccurs during the
transport of nutrients such as glucoseinto certain cells and
transport of many wasteproducts out of cells.
Becauseall forms of energyare interconvertible,they can
be expressedin the sameunits of measurement.Although the
standard unit of energy is the joule, biochemistshave tradi-
tionally used an alternative unit, the calorie (1 joule : 0.239
calorie). Throughout this book, we use the kilocalorie to
measureenergychanges(1 kcal : 1000 cal).
The Changein FreeEnergyDetermines
the Directionof a ChemicalReaction
Becausebiological systemsare generally held at constant
temperatureand pressure,it is possibleto predict the direc-
tion of a chemicalreaction from the changein the free energy
G, named after J. V. Gibbs, who showed that "all systems
changein such a way that free energy [G] is minimized." In
the caseof a chemical reaction, reactants * products, the
free-energychangeAG is given by
AG : Goroaucts - Greactants
j t
o 6
o) c)
(!) 0)
0)
u
E
LI
Reactants
Progressof reaction----->
Progressof reaction----->
FIGURE 2-29 Changesin the free energy(AG)of exergonic
and endergonicreactions.(a)In exergonic reactions,the freeenergy
islowerthanthatof the reactants Consequently,
of the products
thesereactions occurspontaneouslyandenergyisreleased asthe
reactionsproceed(b)In endergonic the freeenergyof the
reactions,
productsisgreaterthanthat of the reactantsandthesereactions do
notoccurspontaneously An externalsourceof energymustbe
areto be converted
suppliedif the reactants into products
The relation of AG to the direction of any chemical reaction
can be summarizedin three statements:
r If AG is negative,the forward reaction will tend to occur
spontaneouslyand energy usually will be releasedas the re-
action takes place (exergonicreaction) (Figure2-291.
r If AG is positive, the forward reaction will not occur
spontaneously:energywill have to be added to the systemin
order to force the reactantsto becomeproducts (endergonic
reaction).
r If AG is zero, both forward and reversereactions occur
at equal rates and there will be no spontaneousconversion
of reactantsto products (or vice versa);the systemis at
equilibrium.
By convention, the standard free-energychange of a re-
action AGo' is the value of the change in free energy under
the conditionsof 298 K (25 "C)' 1 atm pressure,pH 7.0 (as
in pure water), and initial concentrationsof 1'M for all reac-
t"nts proJucts exceptprotons' which are kept at 10-7 M
"nd
(pH 7.0). Most biological reactions differ from standard
conditions, particularly in the concentrations of reactants'
which are normally lessthan 1 M.
The free energy of a chemical system can be defined as
G : H - TS, where H is the bond energy'or enthalpy' of the
system; 7 is its temperature in degreesKelvin (K); and S is
the entropy, a measureof its randomnessor disorder' If tem-
perature remains constant' a reaction proceeds sponta-
neously only if the free-energychange AG in the following
equation is negative:
()-6\
AG: AH- TAS

B I O C H E M I C AELN E R G E T I C S . 55
 In an exothermic reaction, the products contain less bond
energy than the reactanrs, the liberated energy is usually
converted to heat (the energy of molecular motion), and
AH is negative.In an endothermic reaction, the products
contain more bond energy than the reactants,heat is ab-
sorbed during the reaction, and AH is positive. The com-
bined effects of the changes in the enthalpy and entropy
determine if the AG for a reaction is positive or negarive.
An exothermic reaction (AH < 0) in which enrropy in-
creases(AS > 0) occurs spontaneously(AG < 0). An en-
dothermic reaction (AH > 0) will occur spontaneouslyif
AS increasesenough so that the T AS term can overcome
the positive AH.
Many biological reactions lead to an increase in order
and thus a decreasein entropy (AS < 0). An obvious exam-
ple is the reaction that links amino acids to form a protein.
A solution of protein molecules has a lower entropy than
does a solution of the same amino acids unlinked because
the free movementof any amino acid in a protein is restricted
when it is bound into a long chain. Often cells compensate
for decreases in entropy by "coupling" suchsynthetic,entropy-
lowering reactions with independent reactions that have a
very highly negariveAG (seebelow). In this way cells can
convert sources of energy in their environment into the
building of highly organized structures and metabolic path-
ways that are essentialfor life.
The actual changein free energy AG during a reacrion is
influenced by temperature, pressure,and the initial concen-
trations of reactants and products and usually differs from
AG''. Most biologicalreactions-like othersthat take place
in aqueoussolutions-also are affected by the pH of the so-
'We
lurion. can estimate free-energy changes for different
temperaturesand initial concentrationsusing the equation
AG: AC.'-r RTln Q: fproducts]
AC"'+ RTln Q-7)
I reacrantsl
where R is the gas constanrof 1.987 call(degree.mol),
T is
the temperature (in degreesKelvin), and Q is the initial rctio
of products to reactants. For a reaction A + B == C. in
which two molecules combine to form a third, e in Equa-
tion2-7 equalstcl/lAltBl.In this case,an increasein the ini-
tial concentration of either [A] or [B] will result in a larger
negative value for AG and thus drive the reaction toward
more formation of C.
Regardlessof the AG"' for a particular biochemicalreac-
tion, it will proceed spontaneouslywithin cells only if AG is
negative, given the intracellular concentrations of reactants
and products. For example, the conversion of glyceralde-
hyde 3-phosphate (G3P) to dihydroxyacetonephosphate
(DHAP), two inrermediatesin the breakdownof glucose,
.^DHAP
G3P .
has a AGo' of -1840 caVmol.If the initial concentrationsof
G3P and DHAP are equal, then AG : AGo' becauseRT ln
1 : 0; in this situation, the reversiblereaction G3P : DHAP
will proceedspontaneouslyin the direction of DHAp forma-
tion until equilibrium is reached. However, if the initial
56 CHAPTER2 I CHEMICALFOUNDATIONS
[DHAP] is 0.1 M and the initial [G3P] is 0.001 M, with other
conditions standard,then Q in Equation 2-7 equals0.1/0.001
: 100, giving a AG of *887 caVmol.Under theseconditions,
the reaction will proceed in the direction of formation of G3P.
The AG for a reaction is independent of the reaction rare.
Indeed, under usual physiological conditions, few if any of the
biochemical reactions neededto sustain life would occur with-
out some mechanism for increasing reaction rates. As we de-
scribe below and in more detail in Chapter 3, the rates of reac-
tions in biological systemsare usually determinedby the activity
of enzymes,the protein catalyststhat acceleratethe formation
of products from reactantswithout altering the value of AG.
The Ad' of a ReactionCan Be
Galculatedfrom lts K.o
A chemical mixture at equilibrium is in a stable state of
minimal free energy.For a system at equilibrium (AG : 0,
Q : K"o), we can write
AGo': -2.3RTlogK.o: -1362logK.o Q-8)
under standard conditions (note the changeto base 10 loga-
rithms). Thus if we determinethe concentrationsof reactants
and products at equilibrium (i.e., the K.o), we can calculate
the value of AGo'. For example,the K.o for the inrerconver-
sion of glyceraldehyde 3-phosphate to dihydroxyacetone
phosphate (G3P : DHAP) is 22.2 under standard condi-
tions. Substitutingthis value into Equation 2-8, we can easily
calculatethe AGo' for this reaction as - 1840 callmol.
By rearranging Equation 2-8 and taking the antiloga-
rithm, we obtain
Keq : 10-(aG"'/2'3RT) (2-e)
From this expression,it is clear that if AGo' is negative,the
exponent will be positive and henceK.o will be greater than
1. Therefore at equilibrium rhere will be more products than
reactants; in other words, the formation of products from
reactants is favored. Conversely,if AG'' is positive, the
exponent will be negativeand K.o will be lessthan 1.
The Rateof a ReactionDependson the
Activation EnergyNecessary to Energize
the Reactantsinto a TransitionState
As a chemical reaction proceeds, reactants approach each
otherl some bonds begin to form while others begin to
break. One way to think of the state of the moleculesduring
this transition is that there are strains in the electronic con-
figurations of the atoms and their bonds. In order for the
collection of atoms to move from the relatively stable state
of the reactants to this intermediate state during the reac-
tion, an introduction of energy is necessary.This is illus-
trated in the reaction energy diagram in Figure 2-30. Thus
the collection of atoms is transiently in a higher-energysrare
at some point during the course of the reaction. The state
during a chemicalreaction at which the systemis at its high-
est energy level is called the transition state or transition-
 Transitionstate Catalystssuch as enzymes(Chapter 3) acceleratereaction
+ (uncatalyzed) rates by lowering the relative energy of the transition state
II and so the activation energy (seeFigure 2-30). The relative
energiesof reactantsand products will determineif a reaction
Transitionstate
is thermodynamically favorable (negativeAG), whereas the
(catalyzed) activation energy will determine how rapidly products form
6 (reaction kinetics). Thermodynamically favorable reactions
0)
0) will not occur if the activation energiesare too high.
0)
LI

Products
Progressof reaction---->
A FIGURE 2-30 Activationenergy of uncatalyzedand
catalyzedchemicalreactions.Thishypothetical reactionpathway
(blue)depicts proceeds A
thechanges in freeenergyG asa reaction
reactionwilltakeplacespontaneously if thefreeenergy(G)of the
productsislessthanthatof the reactants (AG< 0). However, all
chemicalreactions proceed throughone(shownhere)or morehigh-
energytransition andthe rateof a reaction
states, isinversely
proportionalto theactivation energy(AG*),whichisthe difference
freeenergybetween the reactants andthetransition stateIn a
catalyzedreaction (red),thefreeenergies of the reactantsand
productsareunchanged but thefreeenergyof thetransition stateis
lowered,thusincreasing thevelocityof the reaction
L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l e
C h e m i c aR l e a c t i o nw
s ith Energetically
FavorableReactions
Many processesin cellsareenergetically unfavorable(AG > 0)
and will not proceed spontaneously. Examples include the syn-
thesis of DNA from nucleotides and transport of a substance
acrossthe plasma membrane from a lower to a higher concen-
tration. Cells can carry out an energy-requiring, or endergonic,
reaction (AGr > 0) by coupling it to an energy-releasing,or ex-
ergonic, reaction (AGz < 0) if the sum of the two reactionshas
an overall net negativeAG.
in Suppose,for example,that the reactionA = B + X has a
AG of + 5 kcaUmoland that the reactionX = Y + Zhas a L,G
of -10 kcal/mol:
(1) A B+X AG:+Skcal/mol

state intermediate.The energy neededto excite the reactants
to this higher-energystate is called the activation energy of
the reaction. The activation energy is usually representedby
AGt, analogousto the representationof the changein Gibbs
free energy (AG) already discussed.From the transition
state, the collection of atoms can either releaseenergy as the
reaction products are formed or releaseenergy as the atoms
go "backward" and re-form the original reactants.The ve-
locity (V) at which products are generated from reactants
during the reaction under a given set of conditions (temper-
ature, pressure,reactant concentrations)will depend on the
concentration of material in the transition state, which in
turn will depend on the activation energy and the character-
istic rate constant (z) at which the transition state ls con-
verted to products. The higher the activation energy, the
lower the fraction of reactantsthat reach the transition state
and the slower the overall rate of the reaction. The relation-
ship between the concentration of reactants,z and V is
(AG+/2
3RT)
v : u lreactants]x 10
( 2\ x Y+Z AG:-10kcal/mol
^B+Y+Z
Sum: A. AG'' :-5kcal/mol
In the absenceof the secondreaction' there would be much
more A than B at equilibrium. However, becausethe conver-
sion of X to Y + Z is such a favorable reaction, it will pull
the first process toward the formation of B and the con-
sumption of A. Energetically unfavorable reactions in cells
often are coupled to the energy-releasinghydrolysis of ATP'
as we discussnext.
Hydrolysisof ATPReleasesSubstantialFree
E n e r g ya n d D r i v e sM a n y C e l l u l a rP r o c e s s e s
In almost all organisms, adenosinetriphosphate, or AIP, is
the most important molecule for capturing' transiently stor-
ing, and subsequentlytransferring energy to perform work
(e.g.,biosynthesis,mechanicalmotion). The useful energyin
an ATP molecule is contained in phosphoanhydride bonds,
which are covalent bonds formed from the condensationof
two moleculesof phosphateby the loss of water:

From this equation, we can seethat lowering the activation OO

ti
o- -i-on + Ho-P-o- . *
energy-that is, decreasingthe free energy of the transition
stateAG+-leads to an accelerationof the overall reactionrate ll
V. A reduction in AGt of 1.36 kcal/mol leads to a tenfold o- o-
increasein the rate of the reaction, whereas a 2.72 kcal/mol o
reduction increasesthe rate 100-fold. Thus relatively small llll
o--P-O-?-O- + H2O
changesin AGt can lead to large changesin the overall rate I I
of the reaction. o- o

B I O C H E M I C AELN E R G E T I C S 57
 NH" pH. During synthesis of ATP, a large input of energy is
required to force the negative charges in ADP and P1 to-
*zc'-a-N gether.ConverselSmuch energy is releasedwhen ATP is hy-
Phosphoanhydride bonds rtl \u drolyzed to ADP and P;. In comparison, formation of the
HC__*,rC_;1
o to to phosphoester bond between an unchargedhydroxyl in glyc-
- o - P -i ol |- lL|Pl - O _ | . P - O - erol and P1requires less energy, and less energy is released
cH2-o-
tll when this bond is hydrolyzed.
oo-o H Cells have evolved protein-mediated mechanismsfor
transferringthe free energyreleasedby hydrolysis of phos-
HO OH phoanhydride bonds to other molecules, thereby driving
Adenosine triphosphate reactions that would otherwise be energeticallyunfavor-
able. For example, if the AG for the reaction B + C -+ D is
A FIGURE 2-31 Adenosinetriphosphate(ATP).Thetwo positive but less than the AG for hydrolysis of ATP, the re-
phosphoanhydride bonds(red)in ATP, whichlinkthethreephosphate action can be driven to the right by coupling it to hydroly-
groups,eachhasa AG'of about-7 3 kcal/mol for hydrolysis sis of the terminal phosphoanhydride bond in ATP. In one
Hydrolysisof thesebonds,especially theterminal one,rsrnesource common mechanism of such energy coupling, some of the
of energythatdrivesmanyenergy-requiring reactionsin biological energy stored in this phosphoanhydride bond is transferred
systems. to one of the reactants by breaking the bond in ATP and
forming a covalent bond between the releasedphosphate
An ATP molecule has two key phosphoanhydride (also group and one of the reactants.The phosphorylatedinter-
calledphosphodiester)bonds (Figure 2-31). Hydrolysis of a mediate generatedin this way can then react with C to
phosphoanhydride bond (-) in each of the following reac- form D * P; in a reaction that has a negativeAG:
tions has a highly negativeAG'' of about -7.3 kcal/mol:
B+ATPTB-p+ADP
Ap-p-p + H2O ----+ Ap-p + Pi + H+
B-p+C-+D+P;
(ATP) (ADP)

Ap-p-p+H2O- Ap+PPi+H+ The overallreaction

(ATP) (AMP)
Ap-p + H2O ----+ Ap + P; * H+
(ADP) (AMP)
In thesereactions,Pi standsfor inorganic phosphate(pO+, )
and PP;for inorganic pyrophosphate,two phosphategroups
linked by a phosphoanhydride bond. As the top two reac-
tions show, the removal of a phosphate or a pyrophosphate
group from ATP leaves adenosinediphosphate (ADp) or
adenosinemonophosphate (AMP), respecively.
A phosphoanhydride bond or other high-energy bond
(commonly denoted by -) is not intrinsically different from
other covalent bonds. High-energy bonds simply releasees-
pecially large amounts of energywhen broken by addition of
water (hydrolyzed). For instance,the AG"' for hydrolysis of
a phosphoanhydride bond in ATP (-7.3 kcal/mol) is more
than three times the AGo' for hydrolysis of the phosphoester
bond (red) in glycerol3-phosphate(-Z.2kcallmol):
ooH
Ho-p-o-cHr-lH - cH2oH
I where [C1"] is the initial concentration of the substancein-
U side the cell and [Co",] is its concentration outside the cell.
Glycerol3-phosphate
A principal reason for this differenceis that ATp and its hy-
drolysis products ADP and P1are highly charged at neutral
B+C+ATP:-D+ADP+P.
is energeticallyfavorable (AG < 0).
An alternative mechanism of energy coupling is to use
the energy releasedby ATP hydrolysis to changethe confor-
mation of the molecule to an "energy-rich" stressedstate. In
turn, the energy stored as conformational stresscan be re-
leased as the molecule "relaxes" back into its unstressed
conformation. If this relaxation processcan be mechanisti-
cally coupled to another reaction, the releasedenergycan be
harnessedto drive important cellular processes.
As with many biosynthetic reactions,transport of mole-
cules into or out of the cell often has a positive AG and thus
requires an input of energy to proceed. Such simple trans-
port reactions do not directly involve the making or break-
ing of covalent bonds; thus the AGo' is 0. In the case of a
substancemoving into a cell, Equation 2-7 becomes
AG: Rrt"+."9* (2-10)
'We
can see from Equation 2-10 that AG is positive for
transport of a substanceinto a cell against its concentration
gradient (when [Cr"] > [C"",]); the energy to drive such
"uphill" transport often is supplied by the hydrolysis of

58 CHAPTER2 I CHEMICALFOUNDATIONS
ATP. Conversely,when a substancemoves down its concen-
tration gradient ([C",,] > [C,"] ), AG is negative. Such
"downhill" transport releasesenergythat can be coupledto
an energy-requiringreaction, say, the movement of another
substanceuphill acrossa membrane or the synthesisof ATP
itself (seeChapters 1I and 12).
ATPls GeneratedDuring Photosynthesis
and Respiration
Clearlg to continue functioning, cells must constantly replen-
ish their ATP supply. In nearly all cells,the initial energysource
whose energyis ultimately transformed into the phosphoanhy-
dride bondsof ATP and bonds in other compoundsis sunlight.
In photosynthesis,plants and certain microorganismscan trap
the energy in light and use it to synthesizeAIP from ADP and
P1.Much of the ATP produced in photosynthesisis hydrolyzed
to provide energyfor the conversionof carbon dioxide to six-
carbon sugars,a processcalledcarbon fixation:
ATP ADP + P.
6co2 + 6 Hro V c 6 H 1 2 o+6 o 0 2
In animals, the free energy in sugars and other molecules
derived from food is releasedin the processof respiration.
All synthesisof ATP in animal cells and in nonphotosyn-
thetic microorganismsresultsfrom the chemicaltransforma-
tion of energy-richcompounds in the diet (e.g., glucose,
starch). We discussthe mechanismsof photosynthesisand
cellular respiration in Chapter 12.
The completeoxidation of glucoseto yield carbon dioxide,
c6Flpo6 + 6 02 --->6 co2 + 6 H2o
has a AG'' of -686 kcal/mol and is the reverseof photosyn-
thetic carbon fixation. Cells employ an elaborate set of pro-
tein-mediated reactions to couple the oxidation of 1 mole-
cule of glucoseto the synthesisof as many as 30 moleculesof
ATP from 30 molecules of ADP. This oxygen-dependent
(aerobic) degradation (catabolism) of glucose is the major
pathway for generating ATP in all animal cells, nonphoto-
syntheticplant cells, and many bacterial cells. Catabolism of
fatty acids can also be an important source of ATP.
Light energy captured in photosynthesisis not the only
source of chemical energy for all cells. Certain microorgan-
isms that live in or around deepoceanvents,where adequate
sunlight is unavailable,derive the energyfor converting ADP
and Pi into ATP from the oxidation of reduced inorganic
compounds.Thesereducedcompounds originate deepin the
earth and are releasedat the vents.
N A D * a n d F A DC o u p l eM a n y B i o l o g i c a l
Oxidation and ReductionReactions
In many chemical reactions, electrons are transferred from
one atom or molecule to another; this transfer may or may
not accompanythe formation of new chemical bonds or the
releaseof energythat can be coupled to other reactions.The
loss of electronsfrom an atom or a molecule is called oxida-
tion, and the gain of electrons by an atom or a molecule is
called reduction. Becauseelectrons are neither created nor
destroyedin a chemical reaction, if one atom or molecule is
oxidized, another must be reduced. For example' oxygen
draws electronsfrom ps2+ (ferrous) ions to form Fe3* (fer-
ric) ions, a reaction that occurs as part of the process by
which carbohydrates are degraded in mitochondria. Each
oxygen atom receivestwo electrons, one from each of two
Fe"- ions:
2 Fe2* + Yz oz -->2 Fe3* + 02-
Thus Fe2*is oxidized, and 02 is reduced.Suchreactionsin
which one molecule is reduced and another oxidized often
are referred to as redox reactions. Oxygen is an electron
acceptor in many redox reactions in cells under aerobic
conditions.
Many biologically important oxidation and reduction re-
actions involve the removal or the addition of hydrogen
atoms (protons plus electrons) rather than the transfer of
isolated electronson their own. The oxidation of succinate
to fumarate, which also occurs in mitochondria, is an exam-
ple (Figure 2-32). Protons are soluble in aqueous solutions
(as H3O+), but electrons are not and must be transferred
directly from one atom or molecule to another without a
water-dissolvedintermediate.In this rype of oxidation reaction'
electrons often are transferred to small electron-carrying
molecules, sometimes referred to as coenzymes.The most
common of theseelectron carriers are NAD* (nicotinamide
adeninedinucleotide),which is reducedto NADH' and FAD
(flavin adenine dinucleotide), which is reduced to FADH2
(Figure 2-33). The reduced forms of these coenzymescan
transfer protons and electrons to other molecules, thereby
reducing them.
o o
c-o- c-o
I I
H-C-H c-H
| ---\----\---2
H-C-H v v c-H
I ze 2H* I
c- o- c-o
o o
Succinate Fumarate
A FIGURE 2-32 Conversion of succinateto fumarate.Inthis
reaction,
oxidation whichoccurs in mitochondriaaspartof the citric
losestwo electrons
acidcycle,succinate andtwo protonsTheseare
to FAD,reducing
transferred it to FADH2.
B I O C H E M I C AELN E R G E T I C S 59
 (b)
Reduced: FADH2
Reduced: NADH

o
c-NH2
+ le

Hll
I I I
Ribose Ribose Ribitol Ribitol
I I I I
2P 2P 2P 2P
..1 I I I
A O e n o sn
re Adenosine Adenosine Adenosine

NAD++H++2e-.- NADH FAD+2H- +2e- i- FADH2

FIGURE 2-33 Theelectron-carryingcoenzymesNAD+and FAD. adeninedinucleotide)

isreducedto FADH2 bythe additionof two
(a)NAD*(nicotinamide adeninedinucleotide)
isreducedto NADHby andtwo protons,
electrons asoccurswhensuccinateisconverted to
the addition
of two electrons
andoneprotonsimultaneously In fumarate(seeFigure2-32)ln thistwo-stepreaction,
addition of one
manybiological redoxreactions,
a pairof hydrogenatoms(two electron
togetherwith oneprotonfirstgenerates
a short-lived
protonsandtwo electrons)areremoved froma moleculeIn some semiquinoneintermediate(notshown),whichthenaccepts a second
cases,oneof the protons
andbothelectrons aretransferred
to andproton.
electron
NAD-;the otherprotonisreleased intosolution(b)FAD(flavin

To describeredox reactions, such as the reaction of fer-
rous ion (Fe2+)and oxygen (O2), it is easiestto divide them
into two half-reactions:
Oxidation of Fe2n: 2 Fez* --+ 2 Fe3* -t 2 e-
Reduction of 02: 2 e * 1/z02 -- 02-
In this case,the reducedoxygen (O2-) readily reactswith
two protons to form one water molecule(HzO). The readi-
nesswith which an atom or a molecule gains an electron is
its reduction potential E. The tendencyto lose electrons,the
oxidation potential, has rhe same magnitude but opposite
sign as the reduction potential for the reversereaction.
Reduction porentials are measuredin volts (V) from an
arbitrary zero point set ar the reduction potential of the
following half-reactionunder standard conditions (25 "C.
1 atm, and reactantsat 1 M):
H+ + e- IY'Z *t,
oxidation
The value of E for a molecule or an atom under standard
conditions is its standardreduction potential, E'6 A mole-
cule or an ion with a positive E'e has a higher affinity for
electronsthan the H* ion does under standardconditions.
Conversely,a molecule or ion with a negative E'6 has a
lower affinity for electronsthan the H* ion does under stan-
dard conditions.Like the valuesof AGo', standardreduction
potentials may differ somewhat from those found under the
conditions in a cell becausethe concentrations of reactants
in a cell are not 1 M.
In a redox reaction,electronsmove spontaneouslytoward
atoms or moleculeshaving more positiuereductionporentials.
In other words, a compound having a more negativereduc-
tion potential can transfer electrons spontaneouslyto (i.e.,
reduce)a compound with a more positivereduction potential.
In this type of reaction, the changein electricpotential AE is
the sum of the reduction and oxidation potentialsfor the two
half-reactions.The AE for a redox reaction is related to the
changein free energyAG by the following expression:
AG (callmol) : -n (23,064)AE (volts) (2-11)
wheren is the number of electronstransferred.Note that a re-
dox reactionwith a positiveAE value will have a negativeAG
and thus will tend to proceedspontaneouslyfrom left to right.
BiochemicalEnergetics
r The changein free energy AG is the most useful measure
for predicting the direction of chemicalreactionsin biolog-
ical systems.Chemical reactions tend to proceed sponta-
neouslyin the direction for which AG is negative.The mag-
nitude of AG is independentof the reaction rate.
r The chemicalfree-energychangeAGo' equals -2.3 RT
log K.o. Thus the value of AGo' can be calculatedfrom the
experimentally determinedconcentrationsof reactantsand
products at equilibrium.
r The rate of a reaction dependson the activation energy
neededto energizereactantsto a rransition state. Catalysts
such as enzymesspeedup reactions by lowering the activa-
tion energyof the transitionstate.
r A chemical reaction having a positive AG can proceed if
it is coupled with a reaction having a negariveAG of larger
magnitude.
r Many otherwise energetically unfavorable cellular
processesare driven by the hydrolysis of phosphoanhy-
dride bonds in ATP (seeFigure2-31).

60 CHAPTER2 I CHEMICALFOUNDATIONS
 r Directly or indirectlS light energycaptured by photosyn- containing fatty acylgroups, (c) in the cytosolic domain of the
thesisin plants and photosynthetic bacteria is the ultimate protein, and (d) in the extracellulardomain of the protein?
source of chemical energy for almost all cells. 3. V-M-Y-F-E-N: This is the single-letteramino acid abbre-
r An oxidation reaction (loss of electrons)is always cou- viation for a peptide. What is the net charge of this peptide
pled with a reduction reaction (gain of electrons). at pH 7.0? An enzyme called a protein tyrosine kinase can
'Sfhat
attach phosphatesto the hydroxyl groups of tyrosine.
r Biological oxidation and reduction reactions often are
is the net charge of the peptide at pH 7.0 after it has been
coupled by electron-carryingcoenzymessuch as NAD-
phosphorylated by a tyrosine kinase? \What is the likely
and FAD (seeFigure2-33).
source of phosphateutilized by the kinase for this reaction?
r Oxidation-reduction reactionswith a positive AE have a 4. Disulfide bonds help to stabilize the three-dimensional
negativeAG and thus tend to proceed spontaneously. structure of proteins. What amino acids are involved in the
formation of disulfide bonds?Does the formation of a disul-
fide bond increaseor decreaseentropy (AS)?
KeyTerms 5. In the 1960s, the drug thalidomide was prescribed to
acid 52 hydrophobic 3 1 pregnant women to treat morning sickness. However'
a carbon atom (C*) 41 hydrophobic effect 38 thalidomide caused severe limb defects in the children of
some women who took the drug, and its use for morning
amino acids41 ionic interactions 36
sicknesswas discontinued.It is now known that thalidomide
amphipathic 31 molecular
was administered as a mixture of two stereoisomericcom-
base52 complementarity 39 pounds, one of which relieved morning sicknessand the
'Sfhat
buffers 52 monosaccharides44 other of which was responsible for the birth defects'
chemicalpotential energy54 44
nucleosides Why might two such closely related com-
are stereoisomers?
covalentbond 32 nucleotides44 pounds have such different physiologic effects?

dehydration r eaction 4 0 oxidation 59 6. Name the compound shown below.

AG (free-energy change)55 pH 51
disulfide bond 43 p h o s p h o a n h y d r i dbeo n d s5 7
endergonic55 phospholipid btlayers41 HrrriS)c-)x
polar 34 ll :, ;; ll ll se C
i H
endothermic 55
energy coupling 58 polymer 40 Hrl',t-c\fi)c-i/
enthalpy (H) 55 redox reaction 59
reduction 59 o
entropy (S)55 5',
-o- P-o-o-P-o-o-?-o-9Hz
equilibrium constant49 saturated 47 -o
I I I
exergonic 55 steadystate50 o- o- o
exothermic56 stereoisomers
33 2',
fatty acids 47 unsaturated 47 OH OH
hydrogen bond 37 van der $0aals
interactions37 Is this nucleotide a component of DNA, RNA, or both?
hydrophilic 31
Name one other function of this compound.
7. The chemical basis of blood-group specificity residesin
the carbohydratesdisplayedon the surfaceof red blood cells.
Review the Concepts Carbohydrateshave the potential for great structural diver-
sity. Indeed,the structural complexity of the oligosaccharides
1,. The gecko is a reptile with an amazing ability to climb that can be formed from four sugarsis greater than that for
smooth surfaces,including glass.Recentdiscoveriesindicate oligopeptidesfrom four amino acids. Sfhat propertiesof car-
that geckosstick to smooth surfacesvia van der'lfaals inter- bohydratesmake this great structural diversity possible?
actions between septaeon their feet and the smooth surface. 8. Ammonia (NH:) is a weak basethat under acidic condi-
How is this method of stickinessadvantageousover covalent tions becomesprotonated to the ammonium ion in the fol-
interactions?Given that van der Sfaalsforces are among the lowing reaction:
weakest molecular interactions, how can the gecko's feet
stick so effectively? NH, + H- -+ NH+-
2. The K* channel is an example of a transmembranepro-
tein (a protein that spans the phospholipid bilayer of the NH3 freely permeates biological membranes, including
plasmamembrane).What typesof amino acidsare likely to be those of lysosomes.The lysosomeis a subcellular organelle
found (a) lining the channel through which K* passes,(b) in with a pH of about 4.5-5.0; the pH of cytoplasmis -7'0.
contact with the hydrophobic core of the phospholipid bilayer lWhat is the effect on the pH of the fluid content of lysosomes

R E V I E WT H E C O N C E P T S 61
 when cells are exposedto ammonia? No/e: Protonated ammo-
nia does not diffuse freely acrossmembranes.
9. Consider the binding reaction L + R -+ LR, where L is
'S7hen
a ligand and R is its receptor. 1 x 10 3 M L is added
to a solution containing5 x 10-2 M R, 90% of the L binds
to form LR. rVhat is the K.o of this reaction? How will the
K.o be affected by the addition of a protein that catalyzes
this binding reaction?V/hat is the K6?
10. Vhat is the ionization state of phosphoric acid in the
cytoplasm? Vhy is phosphoric acid such a physiologically
important compound?
1 1 . T h e A G o ' f o r t h e r e a c t i o nX + Y - + X Y i s - 1 0 0 0
callmol. What is the AG at 25 'C (298 Kelvin) starting with
0.01 M each X, I and XY? Suggesttwo ways one could
make this reaction energeticallyfavorable.
12. According to health experts,saturatedfatty acids,which
come from animal fats, are a major factor contributing to
coronary heart disease.What distinguishesa saturated fatty
acid from an unsaturated fatty acid, and to what does the
term saturated refer?RecentlS trans unsaturatedfatty acids,
or trans fats, which raise total cholesterollevelsin the bodv.
have also been implicated in heart disease.How does the cis
stereoisomerdiffer from the trans configuration, and what
effect does the cis configuration have on the structure of the
fatty acid chain?
13. Chemicalmodifications to amino acidscontribute to the di-
versity and function of proteins. For instance,7-carboxylation
of specific amino acids is required to make some proteins
biologically active. What particular amino acid undergoes
this modification, and what is the biological relevance?War-
farin, a derivative of coumarin, which is present in many
plants, inhibits 7-carboxylation of this amino acid and was
used in the past as a rat poison. At present, it is also used
clinically in humans. What patients might be prescribedwar-
farin and whv?
62 CHAPTER2 I CHEMICALFOUNDATIONS
References
Albertg R. A., and R. J. Silbey.2005. PhysicalChemistry,
4th ed. Wiley.
Atkins, P.,and J. de Paula. 2005. The Elementsof Physical
Chemistry,4th ed. \7. H. Freemanand Company.
Berg,J. M., J. L. Tymoczko, and L. Stryer.2007. Biochemistry,
5th ed. W. H. Freemanand Company.
Cantor, P. R., and C. R. Schimmel.1980. BiophysicalChem-
istry.V. H. Freemanand Company.
Davenport,H.W. 1974. ABC of Acid-BaseChemistry,6rh ed..
University of ChicagoPress.
Eisenberg,D., and D. Crothers.1,979.PhysicalChemistryuith
Applications to the Life Sciences.Benjamin-Cummings.
Guyton, A. C., and J. E. Hall. 2000. Textbook of Medical Physi-
ology, 10th ed. Saunders.
Hill, T. J. 1977. FreeEnergy Transductionin Biology. Academic
Press.
Klotz, I. M. 1978. Energy Changesin BiochemicalReactions.
AcademicPress.
Murray, R. K., et al. 1999.Harper'sBiocbemistry,25thed. Lange.
Nicholls, D. G., and S.J. Ferguson.1992. Bioenergetics2. Aca-
demic Press.
Oxtoby, D., H. Gillis, and N. Nachtrieb. 2003. Principlesof
Modern Chemistry,Sth ed. Saunders.
Sharon,N. 1980. Carbohydrates. Sci.Am.243(5):90-116.
Tanford, C. 1980. The Hydropl;obic Effect: Formation of Mi-
cellesand Biological Membranes,2d ed. \filey.
Tinoco, I., K. Sauer,and J. Wang. 200L. PhysicalChemistry-
Principlesand Applications in Biological Sciences,4thed. Prentice
Hall.
Van Holde, K., W. Johnson,and P.Ho. 1998. Principlesof
PhysicalBiochemistry.PrenticeHall.
Voet, D., and J. Voet. 2004. Biochemistry,3d,ed. \7iley.
Wood, !(. B., et al. 1981,.Biochemistry:A ProblemsApproach,
2d ed. Benjamin-Cummings.
 CHAPTER

STRUCTURE
PROTEIN
AND FUNCTION

Ribbondiagramof a betapropeller domainfrom the humansignaling

proteinKeaplTenwatermolecules (spheres)areboundto eachof
the sixbladesof the propeller.
lvlanyproteinsarebuiltfrom multiple,
independently stableproteindomains[FromL J Beamet X Li,C A
Bottoms, andM Hannink,2005,ActaCrystallogr D: Biol.Crystallogr
51(10):1335-1342I Credit.Courtesy
of RobertHube[Martinsried

roteins, which are polymers of amino acids, come in ordered arrays to perform specific functions more efficiently
many sizesand shapes.Their three-dimensionaldiversity than if those proteins were not assembledtogether. Enzymes
reflects underlying structural differences: principally are proteins that catalyze chemical reactions. Membrane
variations in their lengths and amino acid sequences,and in transport proteins permit the flow of ions and molecules
some cases,differencesalso in the number of disulfide bonds acrosscellular membranes. Regulatoryproteins act as siSnals'
or the attachment of small moleculesor ions to their amino sensors,and switches to control the activities of cells by alter-
acid side chains. In general,the linear, unbranched polymer ing the functions of other proteins and genes.These include
of amino acidscomposing any protein will fold into only one signaling proteins, such as hormones and cell-surfacerecep-
or a few closely related three-dimensional shapes-called tors that transmit extracellular signals to the cell interior'
conformations. The conformation of a protein together with
the distinctive chemical properties of its amino acid side OUTLIN E
chains determines its function. As a consequence,proteins
can perform a dazzling array of distinct functions inside and 3.1 HierarchicalStructureof Proteins 64
outside of cells that either are essentialfor life or provide se- 74
3.2 P r o t e i nF o l d i n g
lective evolutionary advantageto the cell or organism that
contains them. It is, therefore, not surprising that character- 3.3 ProteinFunction 78
izing the structuresand activities of proteins is a fundamen-
tal prerequisitefor understanding how cells work. Much of 3.4 RegulatingProteinFunction
this textbook is devoted to examining how proteins act to- ProteinDegradation
gether to enablecells to live and function properly.
3.5 R e g u l a t i n gP r o t e i nF u n c t i o nl l :
Many proteins can be grouped into just a few broad func-
Noncovalentand CovalentModifications
tional classes. Structural proteins, for example,determinethe
shapesof cellsand their extracellularenvironments,and serve 3.6 Purifying,Detecting,and
as guide wires or rails to direct the intracellularmovement of CharacterizingProteins 92
moleculesand organelles.They usually are formed by the as-
semblyof multiple protein subunitsinto very large,long struc- 3.7 Proteomics 105
nres. Scaffold proteins bring other proteins together into

63
 Motor proteins are responsiblefor moving other proteins, MOLECULAR STRUCTURE
organelles,cells-even whole organisms.Any one protein can Primary (sequence)
be a member of more than one protein class,as is the caseof
some cell-surfacesignaling receptorsthat are both enzymes
and regulator proteins becausethey transmit signals from S e c o n d a r y( l o c a lf o l d i n g )
outside to inside cells by catalyztngchemical reactions. To
accomplishefficientlytheir diversemissionssomeproreinsas- Tertiary Supramolecular
sembleinto largecomplexes,often calledmolecularmachines. ( l o n g - r a n gf e
olding) (large-scale
assembly)
How do proteinsmediateso many diversefunctions?They
do this by exploiting a few simple activiries.Most fundamen- Ouaternary
(multimericstructure)
tally, proteins bind-to one anorher,to other macromolecules,
suchas DNA, and to smallmoleculesand ions.In many cases
such binding can induce a conformational changein the pro-
(b)
tein and thus influenceits activity. Binding is basedon molec-
ular complementaritybetweena protein and its binding part- Regulation
ner, as describedin Chapter 2. A second key activity is @
enzymatic catalysis.Appropriate folding of a protein will
place some amino acid side chainsand carboxyl and amino
groups of the backboneinto positions that permit the cataly- FUNCTION
sis of covalent bond rearrangements.A third activity involves
Transport i
folding into a channel or pore within a membrane through
which moleculesand ions flow. Although theseare especially
crucial protein activities,they are not the only ones. For ex- C a t a l y s i s4
ample,fish that live in frigid waters-rhe Antarctic borchsand
Arctic cods-have antifreezeproteins in their circulatory sys-
tems to preventwater crystallizationat subzerotemperatures.
A completeunderstandingof how proteins permit cells
to live and thrive requiresthe identificationand characteri- FIGURE 3-1 Overviewof proteinstructureand function.
zation of all the proteins used by a cell. In a sense,molecu- (a)Proteins
areassembled according to a hierarchy of structures A
lar cell biologistswant to compile a completeprotein 'parts polypeptide l i 'nse asr e q u e n coef a m i n oa c i d sl i n k e db y p e p t i d e
list' and consrructan all-inclusive"users manual" that de- bonds(primary structure) foldsinto localhelices or sheets
scribeshow theseprclteinswork. Compiling a comprehen- (secondarystructure) thatpackrntolarge(longer-range) complex
sive protein parts list has become feasiblein recent years three-dimensional structures (tertiary structu re) Someindividual
with the sequencingof entire genomes-complete sets of polypeptides
associate intomultichain complexes (quaternary
g e n e s - o f m o r e a n d m o r e o r g a n i s m s .F r o m a c o m p u r e r structure),
whichin somecases canbeverylarge,consisting of tensto
hundredsof subunits (supramolecular assemblies) (b)Protein function
analysisof genome sequences,researcherscan deducethe
includes
organization of thegenome, otherproteins, lipidbilayer
number of amino acidsand their sequenceof most of the en-
membranes, andcytoplasm (structure); controlof proteinactivity
coded proteins (Chapter5). The rerrn proreomewas coined
(regulation),
monitoring of theenvironment andtransmitting resultant
to refer to the entire protein complementof an organism.The (signaling),
information flowof smallmolecules andionsacross
human genome conrains 20,000-25,000 genes(only four membranes (transport); (viaenzymes);
catalysis of chemical reactions
times that of the single-cellyeast Saccharomycescereuisiae). andgeneration of forcefor movement (viamotorproteins) These
However, it encodesabout 33.000 different protein because functions
andothersarisefromspecifrc bindinginteractions and
of variation in mRNA producrion (e.g..alteinativesplicing conformatronal changes in thestructure of a properly foldedprotein
(Chapter8)). Even more variation is generaredby 100 types
of protein modification that can produce hundreds of thou- how proteins fold into thesestructures.\(e then rurn to protein
sandsof distinct human proteins.By comparingprotein se- function, focusingon enzymes,the specialclassof proteinsthat
quencesand structures of proteins of unknown function to catalyzechemicalreactions.Variousmechanismsthat cellsuse
those of known function, scientistscan often deduce much to control the activitiesand life spansof proteinsare coveredin
about their functions. In the past, characterizationof protein the next two sections.Next comesa sectionon commonly used
function by genetic, biochemical, or physiological methods techniquesin the biologist'stool kit for isolating proteins and
often precededthe identification of particular proteins. In the characterizingtheir properties.The chapter concludeswith a
moderngenomicand proteomicera,a protein is usuallyiden- discussionof the burgeoningfield of proteomics.
tified prior to determining its function.
In this chapter,we begin our study of how the structureof
a protein givesriseto its function, a themethat recursthrough- ff,t Hierarchical
Structureof Proteins
out this book (Figure 3-1). The first secrion examineshow A protein chain folds into a distinct three-dimensionalshape
chains of amino acid building blocks are arrangedin a three- that is stabilizedby noncovalent interacrionsberween re-
dimensional structural hierarchy.The next section discusses gions in the linear sequenceof amino acids.A kcy concept rn

64 CHAPTER
3 | PROTEIN
S T R U C T U RAEN D F U N C T I O t T
(a) Primarystructure
-Ala -Glu -Val-Thr-Asp- Pro-Gly-
(c) Tertiarystructure
Domain
(d) Ouaternarystructure
(b) Secondarystructure bond formation between the amino group of one amino acid
s heli and the carboxyl group of another results in the net releaseof
a water molecule (dehydration) (Figure 3-3a). The repeated
amide N, cr carbon (C*), carbonyl C and oxygen atoms of each
amino acid residue form the backbone of a protein molecule
from which the various side-chaingroups project (Figure 3-3b,
c). As a consequenceof the peptide linkage, the backbone ex-
hibits directionality becauseall the amino groups are located
on the same side of the Co atoms. Thus one end of a protein
has a free (unlinked) amino group (the N-terminus), and the
other end has a free carboxyl group (the C-terminus). The
sequenceof a protein chain is conventionally written with its
(a)

HO HO
ttl tll
*HsN- Cd-C- O- + *H3N- Co-C- O-

R1
3l n'
ll
lN t,o
\7
HOHO
riltll

PePtide
FIGURE 3-2 Fourlevelsof proteinhierarchy. (a)Thelinear bond
sequence of amino acids
linked together by peptide bondsisthe
(b)Folding (b)
primarystructure. of the polypeptide chainintolocala
or B sheets
helices representssecondary structure.(c)Secondary
structural
elements togetherwith various loopsandturnsin a single
polypeptide chainpackintoa largerindependently stablestructure,
whichmayinclude distinct
domains; thisistertiarystructure(d)
Someindividual polypeptides with theirown tertiary can
structures
intoa quaternary
associate structuredefininga multichain complex. (N-terminus) (C-terminus)

understandinghow proteins work is that function is deriued (c)

from three-dimensional structure, and three-dimensional
structure, which is determined primarily by noncoualent
interactions betueen regions in the linear sequenceof amino
acids, is specifiedby amino acid seqwence.Indeed,principles
relating biologicalstructureand function initially were formu-
lated by the biologistsJohannvon Goethe(1'749-1,832),Ernst
Haeckel (1834-1.91.9),and D'Arcy Thompson \1'860-1'948).
They greatly influencedthe school of "organic" architecture
pioneeredin the early rwentieth century that is epitomizedby
the dicta "form follows function" (Louis Sullivan)and "form
is function" (Frank Lloyd'Wright). Here, we considerthe ar-
A FIGURE 3-3 Structureof a polypeptide.(a)Individual amino
chitectureof proteins at four levelsof organization: primar5 whichform via reactions
acidsarelinked together by peptidebonds,
secondary,tertiary, and quaternary (Figure 3-2). the
thatresultin a lossof water(dehydration).R1,R2,etc.,represent
sidechains ("Rgroups")of aminoacids.(b)Linear polymers of
The PrimaryStructureof a Proteinls lts Linear oeptidebond-linked aminoacidsarecalledpolypeptides, whichhave
a freeaminoend (N-terminus)anda free end
carboxyl (C-terminus)
Arrangemeno t f AminoAcids (c)A ball-and-stickmodelshowspeptidebonds(yellow) linkingthe
As discussedin Chapter 2, proteins are constructed by the aminonitrogen atom(blue)of oneaminoacid(aa)with thecarbonyl
polymerizationof 20 differenttypesof amino acids.Individual carbonatom(gray) onein thechain.TheR groups
of an adjacent
amino acids are linked together in linear, unbranched chains by (green)extendfromthe cr atoms
carbon (black)
of theaminoacids
covalent amide bonds, called peptide bonds, with occasional These sidechains largelydeterminethe properties
distinct of
disulfide bonds covalently linking side chains together.Peptide individual
oroteins

EF P R O T E I N S
H I E R A R C H I C ASLT R U C T U RO 65
 N-terminal amino acid on the left and its C-terminal amino A m i n ot e r m i n u s
acid on the right, and the amino acids are numbered sequen-
tially starting from the amino terminus (number 1).
The primary structure of a protein is simply the linear
arrangement, or sequence,of the amino acid residuesthat
compose it. Many terms are used to denote the chains
formed by the polymerization of amino acids. A short chain
of amino acids linked by peptide bonds and having a defined
sequenceis called an oligopeptide, or just peptide; longer
chains are referred to as polypeptides. peptides generally
contain fewer than 20-30 amino acid residues, whereas
polypeptides are often 200-500 residueslong. The longest
protein described to date is the muscle protein titin with
25,926 residues.We generally reservethe term protein for a
polypeptide (or complex of polypeptides) that has a well-
defined three-dimensionalstructure. It is implied that pro-
teins and peptides are the natural products of a cell.
The size of a protein or a polypeptide is reported as its
massin daltons (a dalton is 1 atomic massunit) or as its mo-
lecular weight (M\7), which is a dimensionlessnumber. For
example,a 10,000-MI7 protein has a mass of 10,000 dal-
tons (Da), or 10 kilodaltons (kDa). In the penultimatesec-
tion of this chapter, we will consider different methods for
measuringthe sizesand other physical characteristicsof pro-
teins. The known and predicted proteins encoded by the
yeast genome have an averagemolecular weight of 52,728
and contain, on average,466 amino acid residues.The aver-
age molecular weight of amino acids in proteins is 113, tak-
ing into account their averagerelative abundances.This
value can be used to estimate the number of residuesin a
Carboxylterminus
protein from its molecular weight or, conversely,its molecu-
lar weight from the number of residues. A FIGURE 3-4 The ct helix, a commonsecondarystructurein
proteins.Thepolypeptide backbone (seenasa ribbon)isfoldedinto
a spiralthat isheldin placeby hydrogen bondsbetweenbackbone
SecondaryStructuresAre the Core Elements oxygenandhydrogen atomsOnlyhydrogens involvedin bondingare
of ProteinArchitecture shown.Theoutersurface of the helixiscovered
bv theside-chainR
groups(green)
The secondlevel in the hierarchy of protein strucrure is sec-
ondary structure. Secondary structures are stable spatial
arrangementsof segmentsof a polypeptide chain held to- The a Helix In a polypeptide segmentfolded into an o he-
gether by hydrogen bonds between backbone amide and lix, the backbone forms a spiral structure in which the car-
carbonyl groups and often involving repeatingstructural pat- bonyl oxygen atom of eachpeptide bond is hydrogen-bonded
terns. A singlepolypeptide may contain multiple types of sec- to the amide hydrogen atom of the amino acid four residues
ondary structure in various portions of the chain, depending farther along the chain (in the direction of the C-terminus)
on its sequence.The principal secondarystructures are the (Figure 3-4). Within an a helix, all the backbone amino and
alpha (c) helix, the beta (p) sheet,and a short U-shapedbeta carboxyl groups are hydrogen-bondedto one another,exceptat
(F) turn. Portions of the polypeptide that don't form these the very beginning and end of the helix. This periodic
structures,but neverthelesshave a well-defined,stableshape, arrangementof bonds confersan amino-to-carboxy-terminal
are said to have anirregular structure.The term random ioil directionality on the helix becauseall the hydrogen bond ac-
appliesto highly flexible portions of a polypeptide chain that ceptors (e.g.,the carbonyl groups) have the same orientation
have no fixed three-dimensionalstructure.In an averagepro- (pointing in the downward direction in Figure 3-4) and re-
tein, 50 percent of the polypeptide chain exists as ct helices sults in a structure in which there is a complete turn of the
and B sheets;the remainder of the molecule is in coils and spiral every 3.6 residues.An ct helix 36 amino acids long has
turns. Thus, ct helicesand B sheetsare the major internal sup- 10 turns of the helix and is 5.4 nm long (0.54 nm/turn).
portive elementsin most proteins. In this section,we exploie The stable arrangement of hydrogen-bonded amino
the shapesof secondarystructuresand the forces that iavor acids in the a helix holds the bar:kbone in a straight, rod-
their formation. In later sections,we examine how linear ar- like cylinder from which the side chains point outward.
rays of secondary structure fold together into larger, more The relative hydrophobic or hydrophilic quality of a par-
complex arrangementscalled tertiary structure. ticular helix within a protein is determined entirely by the

66 . c H A p r E 3R | pRorEtN
s r R u c r u RA
EN DF U N c l o N
characteristicsof the side chains, becauseall the polar amino
and carboxyl groups of the peptide backboneare engagedin
hydrogen bonding with one another in the helix. In water-
soluble proteins, the hydrophilic helicestend to be found on
the outside surfaces,where they can interact with the aque-
ous environment, whereas hydrophobic helices tend to be
buried within the core of the folded protein. The amino acid
proline is usually not found in cr helices,becausethe covalent
bonding of its amino group with a carbon in the side chain
preventsits participation in stabilizingthe backbonethrough
normal hydrogen bonding. While the classic ct helix is the
most intrinsically stable, and most common helical form in
proteins, there are variations, such as more tightly or loosely
twisted helices.For example, in a specializedhelix called a
coiled coil (describedseveralsectionsfarther on), the helix is A FIGURE 3-6 Structureof a p turn' Composed of four residues,
more tightly wound (3.5 residuesand 0.51 nm per turn). B turnsreverse thedirection chain(=180"U-turn)The
of a polypeptide
C. carbons of thefirstandfourthresidues <0.7 nmapart,
areusually
The p Sheet Another type of secondarystructure, the B andthoseresidues areoftenlinkedby a hydrogenbond. B turns
sheet,consistsof laterally packed B strands.Each B strand is a the
facilitate foldingof longpolypeptides intocompact structures
short (5- to 8-residue),nearly fully extended polypeptide seg-
ment. Unlike in the ct helix (where hydrogen bonding between
the amino and carboxyl groups in the backbone occursbetlveen Nveenthe B strand segments'or on different polypeptide chains.
nearly adjacentresidues),hydrogenbonding in the B sheetoc- Figure 3-5b shows how two or more B strands align into adia-
curs between backbone atoms in separate,but adjacent, B cent rows, forming a nearly two-dimensional B pleatedsheet(or
strands (Figure 3-5a). Thesedistinct B strandsmay be either simply pleated sheet), in which hydrogen bonds within the
within a single polypeptide chain, with short or long loops be- plane of the sheethold the B strandstogether as the side chains
itick out aboveand below the plane. Like a helices,B strands
have a directionality defined by the orientation of the peptide
(a) Topview bond. Therefore, in a pleated sheet,adjacent p strands can be
oriented in the same (parallel) or opposite (antiparallel) direc-
tions with respectto eachother' In someproteins, B sheetsform
the floor of a binding pocket or a hydrophobic core; in other
'lo oroteins embedded in membranes the B sheetscurve around
and form a hydrophilic central pore through which ions and
small moleculesmay flow (Chapter11).

e "{r p Turns Composedof four residues,B turns are locatedon

...)
the surface of a protein, forming sharp bends that reversethe
direction of the polypeptide backbone, often toward the pro-
tein's interior. These short, U-shaped secondarystructures are
often stabilizedby a hydrogenbond betweentheir end residues
(Figure 3-6). Glycine and proline are commonly present in
Amino Carboxyl turns. The lack of alargeside chain in glycine and the presence
termrnus termrnus of a built-in bend in proline allow the polypeptidebackboneto
fold into a tight U shape.B turns help large proteins to fold into
(b) Side highly compact structures.There are six types of well-defined
turns, their detailed structures depending on the arrangement
of H-bonding interactions.A polypeptide backbonealso may
contain longer bends, or loops' In contrast with tight B turns,
which exhibit just a few well-defined conformations' longer
loops can have many different conformations.
A FIGURE 3-5 The p sheet,anothercommonsecondary
structurein proteins.(a)Topviewof a simplethree-stranded B O v e r a l lF o l d i n go f a P o l y p e p t i d eC h a i nY i e l d sl t s
p strandsThestabilizing
sheetwith antiparallel rrydrogenbonds TertiaryStructure
between the B strandsareindicatedby greendashed lines(b)Side
viewof a B sheetThe projection
of the R groups (green)
aboveand Tertiary structure refers to the overall conformation of a
belowthe planeof thesheetisobvious in thisview Thefixedbond polypeptide chain-that is, the three-dimensionalarrange-
anglesin the polypeptide
backbone produce a pleatedcontour -..ri oi all its amino acid residues'In contrastwith secondary

EF P R O T E I N S
H I E R A R C H I C ASLT R U C T U RO 67
 Animation:Oil Drop Model of ProteinStructurefntt
Unfoldedprotein Nativeprotein
FIGURE 3-7 Oil drop modelof proteinfolding.The
hydrophobic residuesof a polypeptidechaintendto cluster
together,somewhat likean oil drop,on the inside,
or core,of a
foldedprotein,drivenawayfromthe aqueous surroundings bythe
hydrophobic effect(Chapter 2) Charged anduncharged polarside
chainsappearon the protein's surfacewheretheycanform
stabilizing
interactions
with surroundinq waterandions
structures, which are stabilized only by hydrogen bonds,
tertrary structure is primarily stabilized by hydrophobic
interactions between nonpolar side chains, together with
hydrogen bonds between polar side chains and peptide
bonds. These stabilizing forces compactly hold together ele-
ments of secondary structure-o helices, B strands, turns,
and coils. Becausethe stabilizing interactions are weak,
however, the tertiary structure of a protein is not rigidly
fixed but undergoescontinual, minute fluctuations, and
some segmentswithin the tertiary structure of a protein can
be so very mobile they are consideredto be disordered (that
is, lacking well-defined, stable,three-dimensionalstructure).
This variation in structure has important consequencesfor
the function and regulation of proteins.
_ Chemical properties of amino acid side chains help de-
fine tertiary structure. Disulfide bonds between the side
chains of cysteineresiduesin some proteins covalently link
regions of proteins, thus restricting the mobility of proteins
and increasingthe stability of their tertiary structures.Amino
acids with charged hydrophilic polar side chains tend to be
on the outer surfacesof proteins; by interacting with water,
they help to make proteins soluble in aqueoussolutions and
can form noncovalent interactions with other water-soluble
molecules,including other proteins. In contrast, amino acids
with hydrophobic nonpolar side chains are usually se-
questeredaway from the water-facing surfacesof a protein,
in many casesforming a water-insolublecentral core (called
the oil drop model of globular proteins, becauseof the rela-
tively hydrophobic, or 'oily', core, Figure 3-7). Uncharged
hydrophilic polar side chains are found on both the surface
and inner core of proteins.
58 o c H A p r E 3R | pRorEtN
s r R u c r u RA
EN DF U N c l o N
do not readily dissolvein water, usually play a structural role or
participate in cellular movements.Globwlar proteins are gener-
ally water-soluble, compactly folded structures, often but not
exclusively spheroidal, that comprise a mixture of secondary
structures (seethe structure of myoglobin, below). Integral
membrane proteins are embedded within the phospholipid
bilayer of the membranes that serve as the walls of cells and
organelles.The three broad categoriesof proteins noted here
are not mutually exclusive-some proteins are made up of
combinations of two or even all three of thesecategories.
Different Ways of Depictingthe Conformation
of ProteinsConveyDifferent Typesof
Information
The simplest way to representthree-dimensionalprotein
structure is to trace the courseof the backbone atoms, some-
times only the C. atoms, with a solid line (called a Co trace,
Figure 3-8a); the most complex model shows every atom
(Figure 3-8b). The former shows the overall fold of the
polypeptide chain without consideration of the amino acid
side chains; the latter, a ball-and-stick model, details the in-
teractions between side-chain atoms, including those that
stabilize the protein's conformation and interact with other
molecules, as well as the atoms of the backbone. Even
though both views are useful, the elements of secondary
structure are not always easily discernedin them. Another
type of representationusescommon shorthand symbols for
depicting secondarystructure-for example, coiled ribbons
or solid cylinders for o' helices,flat ribbons or arrows for B
strands,and flexible thin strandsfor B turns, coils, and loops
(Figure3-8c). In variations of ribbon diagrams,ball-and-stick
or space-fillingmodels of side chains can be attached to the
backbone ribbon, while ribbon and cylinder models make
the secondarystructuresof a protein easyto see.
However, none of these three ways of representingpro-
tein structure conveys much information about the protein
surface,which is of interest becauseit is where other mole-
cules usually bind to a protein. Computer analysiscan iden-
tify the surface atoms that are in contact with the watery
environment. On this water-accessible surface,regionshaving
a common chemical character (hydrophobicity or hy-
drophilicity) and electricalcharacter (basic or acidic) can be
indicated by coloring (Figure 3-8d). Such models reveal the
topography of the protein surface and the distribution of
charge, both important features of binding sites, as well as
clefts in the surface where small molecules bind. This view
representsa protein as it is "seen" by another molecule.
S t r u c t u r aM
l o t i f s A r e R e g u l a rC o m b i n a t i o n o
sf
Secondaryand TertiaryStructures
Particular combinations of secondary and tertiary struc-
tures, called structural motifs or folds, appear often as seg-
ments within many different proteins. Structural motifs
(a) Co backbonetrace (b) Ball and stick
( c )R i b b o n s (d) Solvent-accessible
surface
contribute to the global structure of the entire protein' and
any particular structural motif often performs a common
function in different proteins (e.g., binding to a particular
small molecule or ion). The primary sequencesresponsible
for any given structural motif may be very similar to one an-
other. In other words, a common sequencemotif can result
in a common three-dimensionalstructural motif. However,
it is possible for seemingly unrelated primary sequencesto
result in folding into a common structural motif. Conversely,
it is possiblethat a commonly occurring sequencemotif does
not fold into a well-defined structural motif. Sometimes
short sequencemotifs that have an unusual abundanceof a
particular amino acid, e.g., proline or aspartateor gluta-
mate, are called "domains"l however,these and other short
contiguous segmentsare more appropriately called motifs
than domains (which are defined below).
Many proteins, including fibrous proteins and DNA-
regulating proteins called transcription factors (Chapter 7),
assembleinto dimers or trimers by using an ct helix-based
coiled coil, or heptad-repeat,structural motif. In this struc-
tural motif, ct helicesfrom two, three, or even four separate
polypeptide chains coil about one another-resulting in a
coil of coils, hence the name (Figure 3-9a). The individual
helicesbind tightly to one another becauseeach helix has a
strip of aliphatic (hydrophobic, but not aromatic) side
chains (leucinevaline, etc.) running along one side of the he-
lix that interacts with a similar strip in the adjacent helix,
thus sequesteringthe hydrophobic groups away from water
< FIGURE 3-8 Fourways to visualizeprotein
structure. a monomertc
Ras, guanine
nucleotide-binding protein, isshownin allfour
panels,with guanosine diphosphate (GDP) always
depicted (a)
in blue TheC* backbone trace
demonstrates howthe polypeptide istightly
packedintoa smallvolume. (b)A ball-and-stick
representation revealsthe locationof allatoms.
(c)A ribbonrepresentation emphasizes how B
strands(lightblue)ando helices (red)are
organized in the proternNotetheturnsand
loopsconnecting andstrands.
pairsof helices (d)
A modelof the water-accessible surfacereveals
the numerous lumps,bumps,andcrevices on the
proteinsurfaceRegions of positivecharge are
shaded purple;regions of neqativechargeare
shaded red
and stabilizing the assemblyof multiple independenthelices'
These hydrophobic strips are generatedalong only one side
of the helix becausethe primary sequencesof the helicesex-
hibit a motif of repeating segmentsof seven amino acids
(heptads) in which the sides chains of the first and fourth
residuesare aliphatic and the other side chains are often hy-
drophilic (Figure 3-9a). Becausehydrophilic side chains ex-
t..,J fro- one side of the helix and hydrophobic side chains
extend from the opposite side,the overall helical structure is
amphipathic. Becauseleucine frequently appearsin the fourth
poritionr and the hydrophobic side chains merge together
iike the teeth of a zipper, these structural motifs are also
called leucine zippers.
Many other structural motifs employ a helices' A com-
mon calcium-binding motif called the EF hand uses two
short helicesconnectedby a loop (Figure 3-9b). This struc-
tural motif found in more than 100 proteins is used for sens-
ing the calcium levels in cells. The bindittg of a Ca2* ion to
oxygen atoms in conservedresiduesin the loop dependson
the concentration of Caz* and often induces a conforma-
tional change in the protein, altering its activity. Thus, cal-
cium concentrationscan directly control proteins' structures
and functions. Somewhat different helix-turn-helix and ba-
sic helixloop-helix (bHLH) structural motifs are used for
protein binding to DNA and consequentlythe regulation of
geneactivity. Yet another motif commonly found in proteins
ihat bind RNA or DNA is the zinc finger, which contains
three secondary structures-an ct helix and two B strands
EF P R O T E I N S .
H I E R A R C H I C ASLT R U C T U RO 69
 (a) Coiled-coilmotif (b) EF-hand/helix-loop-helix (c) Zinc-fingermotif
motif
Ca2*

Leu (4)

Asn(1)

FIGURE 3-9 Motifs of protein secondarystructure.(a)The proteinssuchascalmodulin, oxygenatomsfromfiveresidues in the

parallel
two-stranded motif(left)ischaracterized
coiled-coil by two o acidicAlutamate- andaspartate-rich loopandonewatermolecule
heliceswoundaroundeachother.Helixpacking isstabilized
by formionrcbondswith a Ca2*ion.(c)Thezinc-finger motifispresent
interactionsbetweenhydrophobic sidechains (redandblue)present in manyDNA-binding proteins
that helpregulatetranscription.
A
at regularintervals
alongeachstrand,andfoundalongtheseamof 7n'- ionis heldbetween a pairof B strands
(blue)anda singleo
the intertwinedhelices.Eacho helixexhibitsa characteristic
heotad helix(red)by a pairof cysteine
residues anda pairof histidine
repeatsequence with a hydrophobic residueoften,but not always,at residuesThetwo invariant cysteineresidues
areusuallyat positions3
positions1 and4, asindicated Thecoiled-coil natureof this and6, andthetwo invariant histidine areat positions
residues 20
structuralmotifismoreapparent in longcoiledcoils(Rrghfdrawnat and24 in this25-residue motif.[SeeA. Lewit-Bentley
andS Rety,2000,
differentscale)(b)An EFhanda typeof helix-loop-helix motif, EF-handcalcium-bindingproteins,
Curr.OpinStrucBiol.10:637-643;SA
consistsof two helicesconnected by a shortloopin a specific W o l f e , L N e k l u d o v aa, n d C O P a b o ,2 0 0 0 , D N A r e c o g n i t i o nb y C y s 2 H r sz2i n c
conformation commonto manyproteins, including manycalcium- finger proteins,Ann Rev.Biophys Biomol. Struc.29:1g3-2121
bindingandDNA-binding regulatoryproteinsIn calcium-bindinq

with an antiparallel orienrarion-that form a fingerlike bun-
dle held together by a zinc ion (Figure 3-9c).
rWewill encounter numerous
additional modfs in later
discussionsof other proteins in this and other chapters.The
presenceof the same structural motif in different proteins
with similar functions clearly indicates that these useful
combinations of secondary structures have been conserved
in evolution.
S t r u c t u r aal n d F u n c t i o n aD
l o m a i n sA r e M o d u l e s
of TertiaryStructure
Distinct regions of protein tertiary structure are often referred
group to another molecule) or binding ability (e.g.,a DNA-
binding domain or a membrane-bindingdomain). Functional
domains are often identified experimentally by whittling
down a protein to its smallestactive fragment with the aid of
proteases,enzymesthat cleaveone or more peptide bonds in
a target polypeptide.Alternatively, the DNA encoding a pro-
tein can be modified so that when the modified DNA is used
to generatea protein, only a particular region, or domain, of
the full-length protein is made. Thus it is possible to deter-
mine if specificportions of a protein are responsiblefor par-
ticular activities exhibited by the protein. Indeed, functional
domains are often also associatedwith correspondingstruc-
tural domains.
A structural dornain is a region =40 or more amino acids in
length, arrangedin a stable,distinct secondaryor tertiary struc-
ture, that often can fold into its characteristic structure inde-
pendently of the rest of the protein. As a consequence.distinct
structural domains can be linked together-sometimes by
short or long spacers-to form a large, multidomain protein.
Each of the subunits in hemagglutinin, for example, contains

70 . c H A p r E R3 p R o r E t Ns r R U c r u R EA N D F U N c l o N
|
 < FIGURE 3-10 Tertiaryand quaternarylevels
of structure.Theprotetnpicturedhere,
hemagglutinin (HA),isfoundon the surface of
influenza virus.Thislong,multimeric molecule has
threeidentical subunits,eachcomposed of two
Globular
domain polypeptide chains, HA1andHA2(a)Tertiary
structure of each HA subunitcomprises thefolding
of itshelices andstrands intoa compact structure
thatis 13.5nm longanddivided intotwo domains'
Themembrane-distal domain(silver) isfoldedintoa
globular conformation The membrane-proximal
domain(gold)hasa fibrous, stemlike conformation
owingto thealignment of two longcthelices
(cylinders) of HA2with B strands in HAl Shortturns
and longerloops,oftenat the surfaceof the
molecule, connect the helices andstrands in each
chain.(b)Quaternary structure of HAisstabilized by
PROXIMAL Fibrous lateralrnteractionsbetweenthe longhelices
domain (cylinders)in thefibrousdomains of thethree
subunits (gold,blue,andgreen), forminga triple-
stranded stalk.Eachof the distalglobular
coiled-coil
domains in HA bindssialicacid(red)on thesurface
of targetcellsLikemanymembrane proteins, HA
contains several covalently linkedcarbohydrate
chains (notshown).
N
t
External
,
Viral I
I
membrane t
I
lnternal I
I

a globular domain and a fibrous domain (Figure 3-10a)' functions in developmentally important signaling (Chapter
Like structural motifs (composed of secondary structures)' 16). Besidesthe EGF domain, theseproteinshave other do-
structural domains (composed of secondary and tertiary mains in common with other proteins. For example, TPA
structures) are incorporated as modules into different pro-
teins. The modular approach to protein architecture is par-
ticularly easyto recognizein large proteins, which tend to be
mosaics of different domains that confer distinct activities
and thus can perform different functions simultaneously.
Structural domains frequently are also functional domains in
that they can have an activity independentof the rest of the
protein. In Chapter 6 we consider the mechanism by which
EGF
the gene segmentsthat correspond to domains becameshuf- precursor
fled in the course of evolution, resulting in their appearance
in many proteins.
The epidermal growth factor (EGF) domain is a struc-
tural domain presentin severalproteins (Figure3-11). EGF
is a small, soluble peptide hormone that binds to cells in the EGF
embryo and in skin and connectivetissue in adults, causing
them to divide. It is generatedby proteolytic (breaking of
peptide bond) cleavagebetween repeated EGF domains in
the EGF precursor protein, which is anchored in the cell
membraneby a membrane-spanning domain. EGF domains
A FIGURE3-11 Modular nature of protein domains' Epidermal
with sequencessimilar to, but not identical with, those in
the EGF peptide hormone are present in other proteins and
can be liberated by proteolysis.Theseproteins include tis-
sue plasminogenactivator (TPA), a proteasethat is used to
dissolve blood clots in heart attack victims; Neu protein,
which takes part in embryonic differentiation; and Notch andP Bork,1993'Curr'
shapeand color.lndaptedfromI D Campbell
protein, a receptor protein in the plasma membrane that Opin StrucBiol 3.3851

EF P R O T E I N S O
H I E R A R C H I C ASLT R U C T U RO 71
 the domains in all proteins. Structural domains can be rec-
ognized in proteins whose structures have been determined
by x-ray crystallography or nuclear magnetic resonance
(NMR) analysis or in images captured by electron mi-
croscopy.
Regions of proteins that are defined by their distinctive
spatial relationshipsro the rest of the protein aretopological
domains. For examplc, some prorerns associatedwith iell-
surfacemembranescan have a portion extending inward
into the cytoplasm (cytoplasmic domain), a portion embed-
ded within the phospholipid bilayer membrane (membrane-
spanningdomain), and a portion extending outward into the
extracellular space(extracellulardomain). Each of thesecan
comprlse one or more structural motifs and structural and
functional domains.
ProteinsAssociateinto MultimericStructures
a n d M a c r o m o l e c u l aArs s e m b l i e s
Multimeric proteins consisr of two or more polypeptide
chains or subunits. A fourth level of structur;l orga-niza-
tion, quaternary structure, describes the number (stoi_
chiometry) and relative positions of the subunits in multi-
menc proteins. Hemagglutinin, for example,is a trimer of
three identical subunits (homotrimer) held together by
noncovalent bonds (Figure 3-10b). Other multimeric pro_
teins can be composed of various numbers of ideniical
(homomeric) or different (hereromeric)subunits (see the
discussionof hemoglobin, below). Often, the individual
monomeric subunits of a multimeric protein cannot func_
tion normally unlessthey are assembledinto the multimeric
protein. In some cases,assemblyinto a multimeric protein
(oligomerization)permits proreins that actsequentiallyin a
pathway to increasetheir efficiencyof operaiion owing to
their juxtaposition in space.
The highest level in the hierarchy of protein strucrure is
the associationof proteinsinto macromolecularassemblies.
Typicallg such structures are very large, in some casesex-
ceeding1 MDa in mass,approaching30-300 nm in size,and
containing tens to hundreds of polypeptide chains, and
sometimesother biopolymers such as nucleic acids.The cap_
sid that encasesthe nucleic acidsof the viral genomeis an ex_
ample of a macromolecular assemblywith a'structural func-
tion. The bundles of cytoskeletalfilaments that support and
give shape to the plasma membrane are another examole.
Other macromolecular assembliesact as molecular ma-
chines, carrying out the most complex cellular processesbv
integrating individual functions into one coordinared
tional components including general transcription factors,
promoter-binding proteins, helicase,and other protein com-
p l e x e s( F i g u r e3 - 1 2 ) . R i b o s o m e sa. l s o d i s c u s s e d
in Chapter
4, are complex multiprotein and multi-nucleic acid machines
that synthesizeproteins.
72 . c H A p r E 3R I pRorEtN
s r R U c r u RAEN DF U N c l o N
Generaltranscriptionfactors
R N Ap o l y m e r a s
DNA
Promoter
I
Y
I
Transcription
preinitiation
comprex
A FfGURE 3-12 A macromolecular machine:the transcription-
initiationcomplex.ThecoreRNApolymerase, general transcription
factors,a mediator
complex containing about20 subunits, andother
proteincomplexes not depicted hereassemble at a promoter in DNA
Thepolymerase carriesout transcriptionof DNA;theassociated
proteinsarerequiredfor initialbindingof polymerase to a specific
promoterThemultiple components functiontoqetherasa machine
M e m b e r so f P r o t e i nF a m i l i e sH a v ea C o m m o n
E v o l u t i o n a r yA n c e s t o r
Studiesof myoglobin and hemoglobin, the oxygen-carrying
proteins in muscle and red blood cells,respectively,provided
early evidencethat a protein's function derivesfrom its three-
dimensional structure, which in turn is specified by amino
acid sequence.X-ray crystallographic analysis showed that
the three-dimensionalstructuresof myoglobin (a monomer)
and the cr and B subunitsof hemoglobin (a o2g2retramer)
are remarkably similar. Sequencingof myoglobin and the
hemoglobin subunits revealedthat many identical or chem-
ically similar residues are found in identical positions
throughout the primary structures of both proteins. A mu-
tation in the gene encoding the B chain that resultsin the
substitution of a valine for a glutamic acid disturbs the fold,
ing and function of hemoglobin and causes sickle-cell
anemla.
Similar comparisonsbetweenother proteins conclusively
confirmed the relation between the amino acid sequence,
three-dimensionalstructure, and function of proteins. Use of
sequencecomparisons to deduce protein function has ex_
panded substantiallyin recent years as the genomesof more
and more organismshave been sequenced.
The molecular revolution in biology during the last
decadesof the twentieth century also created a new scheme
of biological classification based on similarities and differ-
encesin the amino acid sequencesof proteins. proteins that
have a common ancestor are referred to as homologs. The
main evidencefor homology among proteins, and hence for
their common ancestry,is similarity in their sequencesor
 Dicot Monocot
hemoglobin hemoglobin

Annelid

Hemoglobin

Protozoan

Algal

F un g aI
Bacterial

Ancestral
ygen-binding
Ps u b u n i t Myoglobin
of hemoglobin
duplicationgaveriseto theo andB subunits of hemoglobinRrght:
FIGURE 3-13 Evolutionof the globin proteinfamily.leff: A
primitive
monomeric oxygen-bindingglobinisthoughtto bethe Hemoglobin isa tetramer of two a andtwo B subunitsThe
hemoglobins, musclemyoglobins, and similarity
structural of thesesubunits with leghemoglobin and
ancestorof modern-day blood
haverevealedthat myoglobin, both of which are monomers, is evidentA heme
plantleghemoglobins Sequence comparisons
parallels
of the globinproteins of animals
the evolution and molecule (red)noncovalently with eachglobinpolypeptide
associated
evolution
plantsMajorjunctions with thedivergence
occurred of plantglobins responsible
isdirectly for oxygen-binding in theseproteins[(relt)
fromhemoglobin Latergene AdaptedfromR C Hardison, 1996,Proc AcadSciIJSA
Nat'l 93:5675l
fromanimalglobins andof myoglobin

'We example, the amino acid sequencesof globins, the proteins

structures. can therefore describehomologous proteins
as belonging to a "family" and can trace their lineage from
comparisonsof their sequences. The folded three-dimensional
structuresof homologous proteins are similar evenif parts of
their primary structure show little evidenceof homology. Ini-
tially, proteins with relatively high sequencesimilarities
(>50 percent exact matches,or "identities") and related
functions or structureswere defined as an evolutionarily re-
lated family, while a swperfamilyencompassedtwo or more
families in which the interfamily sequencesmatched lesswell
(=30-40 percent identities)than within one family. It is gen-
erally thought that proteins with 30 percent sequenceiden-
tity are likely to have similar three-dimensionalstructures;
however, proteins with far less sequencematching can have
very similar structures.Recently,revised definitions of fam-
ily and superfamily have been proposed, in which a family
comprisesproteins with a clear evolutionary relationship
(>30 percentidentity or additional structural and functional
information showing common descent but <30 percent
identity), while a superfamily comprisesproteins with only a
probable common evolutionary origin (e.g., lower percent
sequenceidentities).Often investigatorsconsider proteins to
constitute a common superfamily (have a common evolu-
tionary origin) when they contain one or more common
motifs or domains.
The kinship among homologous proteins is most easily
visualizedby a tree diagrambasedon sequenceanalyses.For
of hemoglobin and myoglobin and their relatives from bac-
teria, plJnts, and animals, suggestthat they evolved from an
a.r.esiral monomeric, oxygen-binding protein (Figure 3-13)'
With the passageof time, the gene for this ancestralprotein
slowly changed, initially diverging into lineagesleading to
animal and plant globins. Subsequentchangesgave rise to
myoglobin. the monomeric oxygen-storingprotein in mus-
c1., ind to the a and B subunits of the tetrameric hemoglo-
bin molecule (cr2B2)of the circulatory system'
HierarchicalStructure of Proteins
r A protein is a linear polymer of amino.acids linked to-
gether by peptide bonds. Various, mostly noncovalent,
interactions between amino acids in the linear sequence
stabilizea protein's specificfolded three-dimensionalstruc-
ture, or conformation'
r The cr helix, B strand and sheet' and B turn are the most
prevalent elementsof protein secondarystructure' Sec-
tndary structuresare stabilized by hydrogen bonds be-
tween atoms of the peptide backbone'
r Protein tertiary structure resultsfrom hydrophobic inter-
actions betweennonpolar side groups and hydrogen bonds
between polar side groups and the polypeptide backbone'

EF P R O T E I N S .
H I E R A R C H I C ASLT R U C T U RO 73
 These interactions stabilize folding of the secondarystruc-
ture into a compact overall arrangement.
r Certain combinations of secondarystructuresgive rise to
different motifs, which are found in a variety of pioteins and
are often associatedwith specificfunctions (seeFigure 3-9).
Proteins often contain distinct domains, independently
Ided regions of secondaryor tertiary structure *ith .h.r-
acteristicstructural, functional, and topological properties
( s e eF i g u r e3 - 1 0 ) .
r The incorporation of domains as modules in different
proteins in the course of evolution has generateddiversity
in protein structure and function.
r The number and organizationof individual polypeptide
subunits in multimeric proteins define their quaternary
structure.
r Cellscontain largemacromolecularassemblies in which all
the_necessary participantsin complex cellular processes(e.g.,
DNA, RNA, and protein synthesis;photosynthesis;signal
transduction)are integratedto form molecular machines.
Homologous proteins, which have similar sequences,
ructures, and functions, evolved from a common ances_
tor. They can be classifiedinto families and superfamilies.
f[ ProteinFolding
As noted above,when it comesto biologicalstructuressuch as
proteins, "form follows function" and .,form is function."
Thus it is essentialthat when a polypeptideis synthesizedwith
its particular primary structure (sequence),it folds into the
tricaciesof translation are consideredin Chapter 4. Here, we
describethe key determinantsof the proper folding of a'nas_
cent (newly formed or forming) polypeptide chain.
P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o
Which ProteinsCan Fold
A critical structural feature of polypeptides that limits how
the chain can fold is the planar p.piid. bond. Figure 3_3 il_
lustratesthe amide group in peptide bonds in a
folypeptide
chain. Becausethe peptide bond itself behave,puni"lty tit.
a double bond,
OO-
llt
a-a.
Pi"\1-P2 <-> r--u-':fi-P,
I terms, the native state is usually the conformation with the
HH
the carbonyl carbon and amide nitrogen and those atoms
directly bonded to them must all lie rn a fixed plane (Fig_
74 o c H A p r E 3R | p R o r E t N
s r R U c r u RAEN DF U N c l o N
A FIGURE 3-14 Rotationbetweenplanarpeptidegroupsin
proteins.Rotationaboutthe Co-amino nitrogenbond(the@angle)
andthe C"-carbonyl carbonbond(thery'angle)permitspolypeptide
backbones,in principle,
to adopta verylargenumberof potential
conformations However stericrestraints
dueto thestructure
of the
polypeptide
backbone andthe propertiesof theaminoacidside
chainsdramatically
restrict
the potential
conformationsthatcanbe
adoptedby anygivenprotein
ure 3-14); there is no rotation possibleabout the peptide
bond itself. As a consequence,the only flexibility in a
polypeptide chain backbone, allowing it to adopt varying
conformations (twists and turns to fold into different three-
dimensional shapes),is rotation of the fixed planes of pep-
tide bonds with respectto one another about two bonJs-
the C.-amino nitrogen bond (rotational angle called d) and
the C"-carbonyl carbon bond (rotational angle caled r/).
Yet a further constraint on the potential conformations
that a polypeptide backbone chain can adopt is that onlv a
limited number of $ and ry'angles porribl., b..",rr" io.
most d and t! angles the backbone "..
or side chain atoms
would come too closeto one another and thus the associated
conformation would be highly unstable or even physically
impossibleto achieve.
Information Directinga protein,sFoldingls
E n c o d e di n l t s A m i n o A c i d S e q u e n c e
While the consrraintsof backbonebond anglesseemvery re-
strictive, any polypeptide chain containing only a few
residuescould, in principle,still fold inro many conforma-
tions. For example,if the @and ry'angleswere limited to only
eight combinations, an a-residue-longpeptide would poten-
tially have 8" conformations-a very large number for evena
small polypeptide of only 10 residueslong (about g.6 million
possibleconformations)! In general,however,any particular
protein adopts only one or just a few very closely related
characteristic functional conformations called the natiue
state; for the vast majority of proteins, the native state is the
most stably folded form of the molecule. In thermodynamic
lowest free energy. lfhat features of proteins limit their
folding from very many conformationi to iust one? The
propertiesof the sidechains (e.g.,size,hydrophobicitg ability
to form hydrogen and ionic bonds)' together with their par-
ticular sequencealong the polypeptide backbone,impose key
restrictions. For example, a large side chain such as that of
tryptophan might prevent (stericallyblock) one region of the
chain from packing closelyagainstanother region, whereasa
side chain with a positive charge such as arginine might at-
tract a segmentof the polypeptide that has a complementary
negatively charged side chain (e.g., aspartic acid). Another
example we have already discussed is the effect of the
aliphatic side chains in heptad repeatson the formation of
coiled coils. Thus, a polypeptide's primary structure deter-
mines its secondary,tertiary, and quaternary structure.
The initial evidencethat the information necessaryfor a
protein to fold properly is encodedin its sequencecame from
in vitro studieson the refolding of purified proteins. Various
(c)
perturbations (such as thermal energy from heat' extremes
of pH that alter the chargeson amino acid side chains, and
chemicals, called denaturants, such as urea or guanidine
hydrochloride at concentrations of 6-8 M) can disrupt the
weak noncovalent interactions that stabilize the native con-
formation of a protein, leading to its denaturation. Treat-
ment with the reducing agents)such as B-mercaptoethanol,
that break disulfide bonds can further destabilizedisulfide-
containing proteins. Under such unfolding or denaturing
conditions, entropy increaseswhen a population of uni-
formly folded moleculesis destabilizedand converted into a
collection of many unfolded, or denatured, molecules that
have many different non-native and biologically inactive
conformations. As we have seen,there are very many possi-
ble non-nativeconformations(e.g.,8"-1).
The spontaneousunfolding of proteins under denaturing
conditions is not surprising,given the substantialincreasein
entropy. What is striking, however,is that when a pure sam-
ple of a singletype of unfolded protein is shifted back to nor-
mal conditions (body temperature' normal pH levels,reduc-
tion in the concentration of denaturantsby dilution or their
removal), some denatured polypeptides can spontaneously A FIGURE 3-15 Hypotheticalprotein-foldingpathway'Folding
proteinfollowsthestructural
of a monomeric of primary
hierarchy
renature (refold) into their native' biologically active states. of small
(b-d)+ tertiary(e)structureFormation
(a)+ secondary
This kind of refolding experiment, as well as studies that of morestable
motifs(c)appears
structural to precedeformation
show synthetic proteins made chemically can fold properly, (d)andthefinaltertiary (e)
structure
domains
showed that sufficient information must be contained in the
protein's primary sequenceto direct correct refolding. Newly
synthesizedproteins appear to fold into their proper confor-
mations just as denaturedproteins do. The observedsimilar-
ity in the folded, three-dimensional structures of proteins
with similar amino acid sequences, noted in Section3.1, pro- tide. However the conditions inside a cell are not the same
vided additional evidencethat the primary sequencealso de- as those in test tubes used for in vitro refolding experi-
terminesprotein folding in vivo. It appearsthat formation of
secondarystructuresand structural motifs occursearly in the
folding process,followed by assemblyof more compact and
complex domains, which then associateinto more complex
tertiary and quaternary structures(Figure 3-15).

Foldingof Proteinsin Vivo ls Promoted

by Chaperones
The refolding of a denatured protein is presumed to mimic
many aspectsof the folding of a newly synthesizedpolypep- vides. lfithout such help, they would waste much energy in

p R o T E t NF 6 L D I N G o 75
 Foldingfllt ;
FocusAnimation:lhaperone-Mediated

< FIGURE 3-16 Chaperone-mediated protein

ibosome
folding.Manyproteins foldintotheirproperthree-
dimensional structures
with theassistance of Hsp70-like
U n f o l d e dp r o t e i n proteinsThesemolecular chaperones transiently bind
N u c l e o t i d e - b i n d i ndgo m a i n to a nascentpolypeptide asit emerges froma ribosome
or to proteins
thathaveotherwise unfoldedInthe
UT
Hsp70cycle,a substrate unfolded proteinbindsin rapid
equilibriumto theopenconformation of the substrate-
bindingdomain(SBD) of Hsp7O, to whichan ATpis
boundin the nucleotide-bindingdomain(NBD) (s1sp a;
Accessory proteins
(DnaJ/Hsp4O) stimulate the hydrolysis
of ATPandconformational changein Hsp70,resulting
in theclosedform,in whichthesubstrate islockedinro
the SBD;hereproperfoldingisfacilitated (stepZ)
Exchange of ATPfor the boundADp,stimulated by
otheraccessory proteins(GrpE/BAG1),
E converts
Hsp70backto the openform(stepE), releasing
the
the
GrpE/BAG1
properlyfoldedsubstrate (step4)
\aATP ADP

the synthesisof improperly folded, nonfuncrional proteins. to the exposureof hydrophobic side chains that have not yet
which would have to be destroyedto prevenrtheir disrupt_ had a chanceto be buried in the inner core ofthe folded pio-
ing cell function. Cells clearly have such mechanisms,since tein. These exposed hydrophobic side chains on diffeient
more than 95 percent of the proteins present within cells moleculeswill stick to one another owing to the hydropho-
have been shown to be in their native ionformations. The bic effect (Chapter 2) and thus promote aggregation.'Vfh.r,
explanation for the cell's remarkableefficiencyrn Dromot_ a newly synthesizedmolecule begins to fold, it is at risk of
ing the proper protein folding encodedin primary *orr.r..
is that cells make a ser of proteins, called chaperon.r, th"t
facilitate protein folding. The importance of chaperonesis
highlightedby the observationsthat they are evolutionarily
conserved,they are found in all organismsfrom bacteriato
humans, and some are highly homologous and use almost
identical mechanisms to assist p.ot.in folding. Chaper_ Molecular Chaperones The heat-shockprotein Hsp70
ones, which in eukaryotes are located in every cellular and its homologs (Hsp70 in the cytosol and mitochondrial
compartment and organelle, bind to rhe target proteins matrix, BiP in the endoplasmicreticulum, and DnaK in bac_
whose folding they will assist. Two general-families of
chaperonea s re recognized:

r Molecular chaperones,which bind and stabilize unfolded

or partly folded proteins, thereby preventing theseproteins
from aggregatingand being degraded
r Chaperonins,which form a small folding chamber into
which an unfolded protein can be sequesteied,giving it
time and an appropriate environment to fold p-p..ly

One reasonthat chaperonesare neededfor intracellular pro_

tein folding is that they help prevent aggregationof unfoided
proteins. Unfolded and partly folded proteins tend to aggre_
causesa conformational change in the chaperone rhat re_
gate into large, often water insoluble masses,from which it
leasesthe target protein.
is extremely difficult for a protein to dissociateand then fold
Additional proteins, such as the co-chaperoneHsp40 in
into its proper conformation. In part this aggregationis due eukaryotes (DnaJ in bacteria), help increise efficiency of

76 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
Hsp70-mediatedfolding of many proteins by stimulating
the hydrolysis of ATP by Hsp70/DnaK (seeFigure 3-15).
An additional protein called GrpE in bacteria (similar
activity of BAG1 in mammals) also interacts with the
Hsp70/DnaK, promoting the exchange of ATP for ADP.
Multiple molecular chaperonesare thought to bind all nas-
cent polypeptide chains as they are being synthesizedon
ribosomes.In bacteria, 85 percent of the proteins are re-
leasedfrom their chaperonesand proceedto fold normally;
an even higher percentageof proteins in eukaryotesfollow
this pathway.
Chaperonins The proper folding of a large variety of
newly synthesizedproteins also requires the assistanceof
another class of proteins, the chaperonins. These huge
cylindrical macromolecular assembliesare formed from
two rings of oligomers, which can exist in a "tight" peptide-
binding state and a "relaxed" peptide-releasingstate. The
eukaryotic chaperonin TriC consistsof eight subunits per
ring. In the bacterial, mitochondrial, and chloroplast chap-
eronin, known as GroEL, each ring contains sevenidentical
subunits (Figure 3-1'7a).The GroEL folding mechanism,
which is better understood than TriC-mediated folding,
servesas a general model (Figure 3-1'7b).A partly folded or
misfolded polypeptide is inserted into the cavity of the
barrel-like GroEL. where it binds to the inner wall and
folds into its native conformation. In an MP-dependent
step, GroEL undergoesa conformational change and re-
leasesthe folded protein, a processassistedby a co-chaper-
onin, GroES, which caps the ends of GroEL. The binding
of ATP and the co-chaperoninGroES to one of the rings in
the tight state of GroEL causesa twofold expansion of its
cavity, shifting the equilibrium toward the relaxedpeptide-
folding state. There is a striking similarity between the
capped-barreldesign of GroELiGroES, in which proteins
are sequesteredfor folding, and the structure of the 265
proteasomethat participatesin protein degradation (dis-
cussedin Section3.4).
AlternativelyFoldedProteinsAre lmplicated
in Diseases
As noted earlier, each protein normally folds into a sin-
Eil gl., energeticallyfavorableconformationthat is speci-
fied by its amino acid sequence.Recentevidencesuggests'
however, that a protein may fold into an alternative three-
dimensional structure as the result of mutations, inappropriate
covalent modifications made after the protein is synthesized,or
other as-yet-unidentifiedreasons.Such "misfolding" not only
leads to a loss of the normal function of the protein but often
marks it for proteolytic degradation. However, when degrada-
tion isn't complete or doesn't keep pace with misfolding, the
subsequentaccumulation of the misfolded protein or its prote-
olytic fragments contributes to certain degenerative diseases
characterizedby the presenceof insoluble protein plaques in
various organs, including the liver and brain.
Some neurodegenerativediseases,including Alzheimer's
diseaseand Parkinson'sdiseasein humans and transmissible
ments or the soluble alternativelyfolded proteins are toxic to
the cell is unclear. I

ffi viu"o:GroEl Pasecycle

(b) Properly
- Protein folded
Ribosome Partiallyfolded or protein
misfoldedprotein

proteinfolding.Proper to a moreopen,"relaxed"state,which
Bindingof ATPshiftsGroEL
A FIGURE 3-17 Chaperonin-mediated is
thefoldedprotein.
releases oneendof GroEL
Duringthisprocess,
foldingof someproteins depends on chaperonins suchasthe
blocked
transiently bytheco-chaperoninGroES,an of
assembly
prokaryoticGroEL(a)GroEL isa hollow,barrel-shapedcomplex of 14
(b) 1O,OO0-MW (a)fromA Roseman
subunits[Part Cell87:241'
etal, 1996'
60,000-MW
identical arranged
subunits in two stacked rings In
the absence of ATPor presenceof ADBGroEL in a "tight"
exists of
courtesy H Saibil
l
conformational statethatbindspartlyfoldedor misfolded proteins.

P R O T E I NF O L D I N G 77
 (a)
-*si
t2oPtt
FIGURE3-18 Alzheimer,sdiseaseis characterizedby the
formation of insoluble plaques composed of amyloid protein.
( a )A t l o w r e s o l u t i o na ,n a m y l o i dp l a q u ei n t h e b r a i no f a n
patientappearsas a tangleof filaments.(b)The reqularstructureof
frlaments f rom p.aquesrsrevealedrn rhe atomicforcemicroscope.
Proteolysis of the naturallyoccurringamyloidprecursorproteinyields
a shortfragment,cailedp-amyloidprotern,that for unKnownreasons
changesfrom an a-helicalto a B-sheetconformationThisalternative
degenerative
diseases[Courtesv
of K Kosk I
P r o t e i nF o l d i n g
r The sequence of a proteindeterrnines its three-dimensional
structure, which detern.rinesits function. In short, function
derivesfrom structure;structurederivesfrom sequence.
Becauseprotein function derivesfrom protern srructure,
wly synthesized proteinsmust fold into the correctshaoe
f u n c r i o np r o p e r l y .
r The planar structureof the peptidebond limits the num_
ber of conformationsa polypeptidecan have.
The amino acid sequenceof a protein dictatesits foldine
to a specificthree-dimensional
conformarion.rhe native
state. Proteins will unfold, or denature,if treated under
conditionsthat disrupt rhe noncovalenrinteractionsstabi_
lizing their three-dimensional
structures.
olding in vivo occurswith assistance
from chao-
ich bind ro nascentpolypeptidesemergingfrom
and prevenrtheir misfoldine.
78 . cHAprER
3 p R o r E l Ns r R U c r u R A
I
j r Some neurodegenerativediseasesare causedby aggre-
j S"t."r of proteins that are s r a b l y f o l d e d i n a n a l t e r n a r i v e
conlormatlon.
I
ff,l ProteinFunction
Although proteins have many different shapesand sizesand
mediate an extraordinarily diverse array of activitiesboth in-
side and outside of cells, most of thesediversefunctions are
basedon rheabilityof proteinsro engagein a commonactiviry,
the binding to themselves,other macromolecules,small mole,
cules,and ions. Here we will describesomeof the key features
underlyingprotein binding, and then turn to look at one group
of proteins, enzymes,in greater detail. The activities of the
other functional classesof proteins (structural,scaffold,trans-
port, regulatory,motor) will be describedin other chapters.
S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e
F u n c t i o n so f M o s t P r o t e i n s
-:* The molecule to which a protein binds is often calleclits
100nm ligand. In some casesligand binding causesa changein the
, , shape of a protein. Ligand-binding-drivenconformational
changesare integral to the mechanismof action of many
proteins and are important in regulating protein activity.
Two propertiesof a protein characterizehow it binds lig-
Alzheimers
ands.Specificity refersto the ability of a protein to bind one
ure of affinity (Chapter 2). The stronger the interaction be-
virus) or other foreign substances(e.g.,proteinsor polysaccha_
ridesin pollens).Different antibodiesare generatedin response
to differentanrigens,and theseantibodieshavethe remarkable
characteristicof binding specificallyto (..recognizing,')a por_
tion of the antigen,called an epitope,which initially induced
the production of the antibody, and not to other molecules.
Antibodies act as specificsensorsfor antigens,forming anti-
body-antigen complexesthat initiate a cascadeof proiective
r e e c t i o nisn c e l l so f t h e i m m u n es y s r e m .
E N DF U N c l o N
 Light chain CDR
lnterchain
disulfide "t"
bonds

periments discussedin subsequentchapters'

Heavychain \7e will see many examples of protein-ligand binding
throughout this book, including hormones binding to receptors
(Chapter 15), regulatory moleculesbinding to DNA (Chap-
t , i\. cell-adhesion molecules binding to extracellular
matrix (Chapter 1'9),to name just a few. Next we will con-
sider how the binding of one class of proteins, enzymes,to
Carbohydrate
their ligands results in the catalysisof the chemical reactions
essentialfor the survival and function of cells.

EnzymesAre HighlYEfficientand
SpecificCatalysts
Proteins that catalyze chemical reactions, the making and

catalyzed by a specific efizyme. In many ways' enzymes are

the cell's chemists,performing many of a cell's chemical re-
actions. (An additional form of catalytic macromolecule in
cells is made from RNA. TheseRNAs are called ribozymes')
Thousands of different types of enzymes'each of which
catalyzesa single chemical reaction or set of closely related
reactions,have beenidentified. Certain enzymesare found in
the majority of cells becausethey catalyze the synthesisof
A FIGURE 3-19 Protein-ligandbindingof antibodies.(a)Ribbon
common cellular products (e.g.,proteins, nucleic acids, and
modelof an antibody. Every antibodymolecule of the immunoglobulin
phospholipids) or take part in the production of energy (e'g''
lgGclassconsistsof two identicalheavychains(lightanddarkred)and
bondsThe ty th. .ottu.rsion of glucoseand oxygen into carbon dioxide
lightchains(blue)covalently
two rdentical linkedby disulfide
diagram of the overall
structure containlng the tvuoheavy and water). Other enzymesare present only in a particular
insetshowsa
andtwo lightchains(b)Thehand-in-glove fit betweenan antibody and
thesiteto whichit binds(epitope) on itstargetantrgen-inthiscase,
chickenegg-white lysozyme Regionswherethetwo molecules make
contactareshownassudaces. Theantibody contacts theantigenwith
residuesfromallitscomplementarity-determining regions(CDRs).Inthis
view,the molecular complementarity of theantigenandantibodyis tract. or even outside the organism (e.g., toxic enzymesin
apparent
especially where"fingers"extending fromtheantigensurface the venom of poisonous snakes).
areopposed to "clefts"in theantibody surface. Like all catalysts,enzymesincreasethe rate of a reaction
but do not affect the extent of a reaction, which is deter-
AII antibodies are Y-shapedmoleculesformed from two
identical heavy chains and two identical light chains (Fig-
ure 3-19a). Each arm of an antibody moleculecontains a
singlelight chain linked to a heavy chain by a disulfide bond.
Near the end of each arm are six highly variable loops' called
complementarity-determiningregions (CDRs/, which form
the antigen-binding sites.The sequencesof the six loops are
highly variable among antibodies, generating unique com-
plementary ligand-binding sites that make them specific for
different epitopes (Figure 3-19b). The intimate contact be-
tween thesetwo surfaces,stabilizedby numerousnoncovalent
interactions, is responsiblefor the extremely precisebinding
specificity exhibited by an antibody.

P R O T E I NF U N C T I O N O 79
 -__Transitionstate (a) Catalyticsite (b) Catalyticsite Binding pocket
(uncatalyzed)

Transitionstate
0) t (catalyzed)
q) . A/l.+
."cat
o
o
'-I
B i n d i n gp o c k e t

Products
A FIGURE 3-21 Active site of the enzymetrypsin. (a)An
Progressof reaction-----> enzyme's activesiteiscomposed of a bindingpocket, whichbinds
A FIGURE 3-20 Effectof an enzymeon the activationenergy specifically
to a substrate,anda catalytic site,whichcarnes out
of a chemicalreaction.Thishypothetical catalysis(b)A surfacerepresentation of the serineprotease trypsin
reactionpathwaydepicts
the changes in freeenergyG asa reaction proceeds Activesitecleftscontaining thecatalytic site(sidechainsof the
A reactionwill
takeplacespontaneously onlyif the totalG of the products catalytic
triadSer-195, Asp-102, andHis-57 shownasstickfigures)
isless
thanthatof the reactants(negative andthe substrate sidechainspecificitybindingpocketareclearly
AG) However, allchemical
reactionsproceed throughoneor morehigh-energy visible[Part(b)courtesy
of PTeesdale-spittte
]
transitionstates,
andthe rateof a reactionisinversely proportional to the activation
energy(AG+), whichisthedifference in freeenergybetween the
reactantsandthetransition state(highest pointalongthe pathway). substrate-bindingsite and the substrate,which is mediated
Enzymes andothercatalysts accelerate the rateof a reactionby by multiple weak noncovalent interactions and is very sensi-
reducingthefreeenergyof thetransition stateandthusAG+. tive to the shapesof substrates.Usually only one or a few
substratescan fit preciselyinto a binding site.
rates of reactions 106-1012times that of the corresponding The idea that enzymesmight function by binding to rheir
uncatalyzedreactions under otherwise similar conditions. substratesin the manner of a key fitting into a lock was sug-
gestedfirst by Emil Fischerin1894.Ln1913 Leonor Michaelis
An Enzyme'sActive Site BindsSubstrates and Maud Leonora Menten provided crucial evidence sup-
porting this hypothesis.They showed that the rate of an enzy-
a n d C a r r i e sO u t C a t a l y s i s
matic reaction was proportional to the substrateconcentration
at low substrateconcentrations, but that as the substratecon-
centrations increased,the rate reached a maximal velocity
V-o and became substrate concentration-independent, with
the value of V-"" being directly proportional to the amount of
enzymepresent in the reaction mixture (Ftgure 3-22).
They deducedthat this saturation at hieh substratecon-
sitesmake up only a small fraction of the total protein, with
the rest involved in folding of the polypeptide, iegulation of
the active site, and interactions with other molecules.

termediate step in the ultimately irreversible conversion of

substrateto product (P) (Figure3-23):

substratehas bound. In some enzymes,the catalytic and sub- E + S i------^ES-+E + p

strate-binding sites overlap; in others, the two regions are
structurally as well as functionally distinct. and that the rate Ve of formation of product at a particular
substrateconcentration [S] is given by what is now called the
Mich aelis-Mentenequahon:

vo:v-"*;J!-
+ Km
(3-1)
L)l

where the Michaelis constant K-, a measureof the affinity

zyme. As noted above, this specificity of enzymesis a conse_ of an enzyme for its substrate (seeFigure 3-22), is the sub-
quenceof the precisemolecular complementarity betweenits strate concentration that yields a half,maximal reaction rate

80 o c H A p r E 3R | p R o r E t N
s r R u c r u RA
EN DF U N c l o N
(a)

c
.=6

2.0
oJ

b.> t.5
^=
. : : J /

6-
E(L 1.0
Enzyme

0.5
o:9
c().
t

Concentrationof substrateIS] etnoino

no.t"t
f I
(b)

1.0
High-affinity
o nR substrate

I 0.6 L o w - a f fi n i t y

g 0.4
o
s u b s t r a t e( S ' )
I
o.2
0
(tSlor tS'l)
of substrate
Concentration
A FfGURE 3-22 K^ and V."* for an enzyme-catalyzed reaction'
K, andVru"aredetermined fromanalysis of the dependence of the Enzyme
initialreaction velocity on substrate concentration Theshapeof
thesehypothetical kineticcurves ischaracteristic of a simpleenzyme-
catalyzed reaction in whichonesubstrate (S)isconverted into
product(P)Theinitralvelocity is measured immediately after
A FIGURE 3-23 Schematicmodel of an enzyme'sreaction
additionof enzyme to substrate beforethe substrate concentration (E)bind
mechanism. Enzyme kineticssuggestthatenzymes
changes appreciably (a)Plotsof the initialvelocrty at two different
molecules
substrate (5)througha fixedandlimitednumberof sites
concentrations of enzyme [E]asa functionof substrate concentration tsknownasan
on theenzymes Theboundspecies
(theactivesites).
[S] The lsl that yields a half-maximal reaction rateisthe Michaelis in with
equilibrium
enzyme-substrate(ES)complex. The EScomplex is
constant K., a measure of theaffinityof Efor turningS intoP andisan intermediate stepin
the unboundenzyme andsubstrate
Quadrupling theenzyme concentration causes a proportronal increase
of substrate
the conversion to products(P)
in the reaction rate,andso the maximal velocity V.u" isquadrupled;
theK., however, isunaltered. (b)Plotsof the initialvelocity versus
substrate concentration witha substrate Sfor whichtheenzyme hasa
highaffinityandwitha substrate S' for whichtheenzyme hasa lower
affinity.Notethatthe V.u"isthe samewith bothsubstrates, because
IE]isthesame,but that Km is higher for S',the low-affinitysubstrate

substrate complexes (ES' ES', ES", etc.) generatedprior to

(i.e.,112V-.*), and thus is analogousto the dissociationcon- the final releaseof the Products:
stant K6 (Chapter 2). The smaller the value of K-, the more --^ ES' - '"'E+P
. ^ ^ S' -
E S F . . . . .E
effectivethe enzyme is at making product from dilute solu- E+S
tions of substrateand the smaller the substrateconcentration
neededto reach half-maximal velocity.The concentrationsof The energyprofiles for such multistep reactionsinvolve mul-
the various small moleculesin a cell vary widely, as do the K- tiple hills and valleys (Figure 3-24), and methods have been
valuesfor the different enzymesthat act on them. A good rule developedto trap the intermediatesin such reactionsto learn
of thumb is that the intracellular concentrationof a substrate more about the details of how enzymescatalyzereactions'
is approximately the same as or somewhat greater than the
K^ value of the enzymeto which it binds' SerineProteasesDemonstrateHow an Enzyme's
The rates of reaction at substratesaturation vary enor-
Active Site Works
mously among enzymes.The maximum number of substrate
moleculesconvertedto product at a singleenzymeactivesiteper Serineproteases,a large family of proteolytic enzymes' are
secondis called the turnouer nwmber,which can be lessthan 1 used throughout the biological world-to digest meals (the

FUNCTION
PROTEIN 81
 (a) (b)

Enzyme-transition
() (5 statecomplex
j
EX+
6 o) E n z y m e+ Enzyme-
o q) substrate substrate
o o
0) o) comprex
IL LI

E+S
E+P
Progressof reaction-----> Progressof reaction----->
S<-X++P E+S*ES+EX+------+E+p
A FIGURE 3-24 Free-energyreactionprofilesof uncatalyzed formation of an EScomplex followedby conversion
viaa single
and multistepenzyme-catalyzed reactions.(a)Thefree-energy state(EX+)
transition to thefreeenzyme (E)andp Theacrrvatron
reactionprofileof a hypothetical
simpleuncatalyzed reaction energyfor eachof thesestepsissignificantly
lessthanthe activation
convertingsubstrate (S)to product(p)viaa singlehigh_energy energyfor the uncatalyzedreaction;
thustheenzyme dramatically
state.(b)Manyenzymes
transition catalyze suchreactions by dividing enhances the reactionrate.
the processintomultiple discrete
steps,in thiscasethe initial
pancreatic enzymestrypsin, chymotrypsin, and elastase),to Figure 3-25a shows how a substratepolypeptide binds to
the substrate-bindingsite in the active site of trypsin. There
are two key binding interactions.First, the substrateand en-
zyme form hydrogen bonds that resemblea B sheet.Second,
a key side chain of the substratethat determineswhich pep-
tide in the substrate is to be cleaved extends into the en-
consider how trypsin and its two evolutionarily closely re- zyme's side-chain-specificitybinding pocket, at the bottom
lated pancreatic proteases,chymotrypsin and elastase,cat- of which resides the negatively charged side chain of the
alyze cleavageof a peptide bond:
o (a)

x ,9 -:' p,,-C...- Catalyticsite

p(""'"tr1-P2 + HrO + NH3+-P2
His-57
l*o
H Ho' Peptidebond
to be cleaved
where P1is the portion of the protein on the N-terminal sideof \r'r
the peptide bond, and P2is the portion on rhe C-terminalside. C: O-
Oxyanion
S7efirst consider how serineproreasesbind specificallyto their hole
substratesand then show in detail how catalysistakesplace.
A r g i n i n es i d e
c h a i n( R 3 )i n
substrate
> FIGURE 3-25 Substratebindingin the activesite of typsin_
N
like serineproteases. (a)Theactivesiteof trypsin(bluemolecule) H
wrtha boundsubstrate (blackmolecule) Thesubstrate formsa two_
stranded B i n d i n gs i t e Side-chain-
B sheetwith the bindingsite,andthe sidechainof an
arginine (Rr)in thesubstrate specificity
is boundin the side-chain-specificitv binding pocket
binding p o c k e tl .t sp o s i t i v ecl h
y a r g egdu a n i d i n i ugmr o u pi s
stabilized by the negative chargeon the sidechainof the enzyme,s
A s p - 1 8 9T h i sb i n d i n g a l i g n tsh ep e p t i d be o n do f t h ea r g i n i n e Asp-189
appropriately for hydrolysis catalyzed by theenzyme,s active_site (b)
c a t a l y tti rci a d( s i d ec h a i nos f S e r - 1 9 5H,i s - 5 a7 n dA s p - 1 0 2()b. )T h e
a m i n oa c i d sl i n i n gt h es i d e - c h a i n - s p e c ibf i nc idt iyn g
pocket
d e t e r m i ni e t ss h a p ea n dc h a r g ea,n dt h u si t sb i n d i n g properties
Trypsin accommodates the positively charged sidechainsof arginine
and lysine; chymotrypsin, large,hydrophobic sidechainssuchas
p h e n y l a l a n ianned; e l a s t a ssem , a lsl r d ec h a i nssu c ha sg l y c i naen d
alanine.[Part(a)modified
from.J.J. perona
andC. S. Craik,1991.
J. Biol.
Chen 272(48).29987-29990 I Trypsin Chymotrypsin Elastase

82 . c H A p r E R3 | p R o r E t Ns r R U c r u R EA N D F U N c l o N
enzyme'sAsp-189. Trypsin has a marked preferencefor hy-
drolyzing proteins (black in Figure 3-25a) at the carboxyl
side of a residue with a long positively charged side chain
(arginine or lysine), becausethe side chain is stabilizedin the
specificity binding pocket by the negativeAsp-189.
Slight differencesin the structures of otherwise similar
specificity pockets help explain the differing substratespecifici-
ties of the two related serine proteases:chymotrypsin prefers
large aromatic groups (asin Phe,Tyr, Trp), and elastaseprefers
the small side chains of Gly and Ala (Figure 3-25b1.The un-
chargedSer-189in chymotrypsinallows large, uncharged,hy-
drophobic side chains to bind stably in the pocket. The
branchedaliphatic side chainsof valine and threoninein elas-
tase replace glycines in the sides of the pocket in trypsin and
thus prevent large side chains in substratesfrom binding, but
allow stablebinding of the short alanineor glycinesidechain.
In the catalytic site, all three enzymesuse the hydroxyl
group on the side chain of a serine in position 195 to cat-
alyzethe hydrolysis ofpeptide bonds in protein substrates.A
catalytic triad formed by the three side chains of Ser-195,
His-57, Asp-102 participates in what is essentiallya two-
step reaction. Figure 3-26 shows how the catalytic triad co-
operates in breaking the peptide bond, with Asp-102 and
His-57 supporting the attack of the hydroxyl oxygen of Ser-195
on the carbonyl carbon in the substrate.This attack initially
forms an unstable transition state with four groups attached
to this carbon (tetrahedral intermediate). Breaking of the
C-N peptide bond then releasesone part of the protein
(NH3-P2), while the other part remains covalently attached
to the enzymevia an ester bond to the serine'soxygen, form-
ing a relatively stable intermediate (the acyl enzyme). The
subsequentreplacementof this oxygen by one from water, in
a reaction involving another unstable tetrahedral intermedi-
ate, leadsto releaseof the final product (P1-COOH). The
tetrahedral intermediates are partially stabilized by hydro-
gen bonding from the enzyme'sbackbone amino groups in
what is called the oxyanion hole.The large family of serine
proteasesand related enzymeswith an active-siteserine il-
lustrates how an efficient reaction mechanism is used over
and over by distinct enzymesto catalyzesimilar reactions.
The serineproteasemechanismpoints out severalgeneral
key featuresof enzymaticcatalysis:(1) enzymecatalytic sites
are designedto stabilizethe binding of a transition state' thus
lowering the activation energy and acceleratingthe overall

( a ) E Sc o m p l e x (b) Tetrahedralintermediate (c) Acyl enzyme

(transitionstate) (ES'complex)

D o H
'r'r.,..-N
, rH; N
/
"c
Oxyanion
P.,/ P,,,
hole
H

T,o
(f) EP complex (e) Tetrahedralintermediate (d) Acyl enzyme
(transitionstate) (ES'complex)

<-

Oxyanion
hole

His-57side chain
,,\ _ . N ,,,.\
_ HN. ,_-NH
HN. \- \l
\J
Active Inactive (lowpH)

A FIGURE 3-26 Mechanismof serineprotease-mediated of oneof the reaction products (NH2-P2), andformationof the acyl
hydrolysisof peptidebonds.Thecatalytic triadof Ser-195, His-57, enzyme(ES'complex)(d)An oxygenfroma solvent watermolecule
proteases employs a multistep thenattacks the carbonyl carbon of the acyl enzyme (e)Thisattack
andAsp-102 in theactivesitesof serine
mechanism to hydrolyzepeptidebondsin targetproteins(a)Aftera resultsin theformation of a second tetrahedral intermediate
polypeptidesubstrate bindsto the activesite(seeFigure3-23)forming (fl Additionalelectronmovements resultin the breaking of the Ser-
the hydroxyloxygenof Ser-1 95 attacks the carbonyl 195-substrate bond(formation of the EPcomplex) andrelease of the
an EScomplex,
targetedpeptidebond(yellow)Movements finalreaction product (P1-COOH). The side chain of His-57,which is
carbonof the substrate's
areindicated by arrows.(b)Thisattackresults in the heldin the proper orientationby hydrogen bonding to the sidechain
of electrons
intermediate, in of Asp-102, catalysis
facilitates bywithdrawing anddonating protons
statecalledlhe tetrahedral
formationof a transition
whichthenegative charge on thesubstrates oxygen isstabilizedby throughout the reaction(inset)lf the pH istoo low andthe sidechain
s oxyanion hole.(c)Additional of His-57isprotonated, it cannotparticipate andthe
in catalysis
hydrogen bondsformedwiththe enzyme
electronmovements resultin the breakingof the peptidebond,release enzyme isinactive

FUNCTION
PROTEIN 83
 reaction, (2) multiple side chains, togerherwith the polypep- hydrolytic enzymesthat function in acidic conditions must
tide backbone,carefully organizedin three dimensions,work employ a different catalytic mechanism.This is the casefor
togetherto chemicallytransform substrateinto product, often enzymeswithin the stomach (pH = 1) such as the protease
by multistepreactions,and (3) acid-basecatalysismediatedby pepsin or those within lysosomes(pH = 4.5), which play a
one or more amino acid side chainsis often used by enzymes, key role in degrading macromoleculeswithin cells (seeFig-
as when the imidazolegroup of His-57 in serineproteasesacts ure 3-27, left).Indeed, lysosomal hydrolasesthat degrade a
as a base to remove the hydrogen from Ser-195,shydroxyl wide variety of biomolecules(proteins, lipids, etc.) are rela-
group. As a consequence,often only a particular ionization tively inactive at the pH in the cytosol (=7), and that helps
state (protonated or nonprotonated) of one or more amrno protect a cell from self-digestionshould theseenzymesescape
acid side chains in the catalytic site is compatible with cataly- the confinesof the membrane-boundlysosome.
sis,and thus the enzyme'sactivity is pH-dependent. One key feature of enzymatic catalysisnot seenin serine
For example,the imidazoleof His-57 in serineproreases, proteases,but found in many other enzymes,is a cofactor,or
whose pK" is =5.8, can help the Ser-195hydroxyl attack the prosthetic (helper) group. This is a nonpolypeptide small
substrateonly if it is not protonated. Thus, the activity of the molecule or ion (e.g., iron, zinc, copper, manganese)that is
proteaseis low at pH < 6.8, and the shapeof the pH activ- bound in the active site and plays an essentialrole in the
ity profile in the pH range 4-8 matches the titration of the reaction mechanism. Small organic prosthetic groups in
His-57 side chain, which is governed by the Henderson- enzymesare also caIIedcoenzymes.Someof theseare chem-
Hasselbalchequation,with an inflection near pH 6.8 (seeFig- ically modified during the reaction and thus need to be re-
ure 3-27, right, and Chapter 2). The activity drops at higher placed or regeneratedafter each reaction; others are not.
pH values,generatinga bell-shapedcurve, becausethe proper Examplesof the former include NAD* (nicotinamideadenine
folding of the protein is disrupted when the amrno group ar dinucleotide) and FAD (flavin adenine dinucleotide) (see
the protein's amino rerminus is deprotonated (pK" = 9); the Figure 2-33), whereas heme groups that bind oxygen in
conformation near the active site changesas a consequence. hemoglobin or transfer electrons in some cytochromes are
The pH sensitivity of an enzyme'sactivity can be due to examples of the latter (Figure 12-14). Thus, the chemistry
changesin the ionization of catalytic groups, groups that par- catalyzedby enzymesis not resrrictedby the limited number
ticipate directly in substratebinding, or groups that influence of amino acids in polypeptide chains. Many vitamins-e.g.,
the conformation of the protein. Pancreaticproteasesevolved the B vitamins, thiamine (B1), riboflavin (B2), niacin (B3),
to function in the neutral or slightly basic conditions in the and pyridoxine (86), and vitamin C-which cannot be syn-
intestines;hence,their pH oprima are =8. proteasesand other thesized in higher animal cells, function as or are used to
generatecoenzymes.That is why supplementsof vitamins
must be added to the liquid medium in which animals cells
Lysosomalenzvme are grown in the laboratory (Chapter9).

Small moleculesthat can bind to active sitesand disruot

the reactionsare called enzymeinbibitors. Suchinhibitois
o are useful tools for studying the roles of enzymesin cells and
0)
E whole organisms by allowing analysis of the consequencesof
N
c the loss of the enzyme'sactivity. Thus, inhibitors complement
o)
0) the use of mutations in genesfor probing an enzyme'sfunction
G
in cells (seeChapter 5). However, interpreting results of in-
o hibitor studies can be complicated if, as is often the case,rhe
inhibitors block the activity of more than one protein. Small-
2345678910
molecule inhibition of protein activity is the basis for mosr
oH
drugs (e.g., aspirin inhibits enzymescalled cyclooxygenases)
A FfGURE 3-27 pH dependence of enzymeactivity.lonizable and also for chemical warfare agents. Sarin and other nerve
(pH-titratable)
groupsin theactivesitesor elsewhere in enzymes gasesreact with the active serinehydroxyl groups of both serine
oftenmustbe eitherprotonated or deprotonated to permitproper proteasesand a related enzym%acetylcholineesterase,which is
substratebindingor catalysis or to permitthe enzyme to adoptthe a key enzymein regulating nerve conduction (seeChapter 23). I
correctconformation Measurement of enzyme activity asa function
of pHcanbe usedto identify the pKa'sof thesegroupsThe
pancreaticserineproteases, suchaschymotrypsin (rightcurve), Enzymesin a CommonPathwayAre Often
exhibitmaximum activityaroundpHg because of titrationof the PhysicallyAssociatedwith One Another
activesiteHis-57 (requiredfor catalysis,pKu= 6 8) andof theamino
terminus of the proteln(required for properconformation, pK"= 9) Enzymes taking part in a common metabolic process (e.g.,
Manylysosomal hydrolases haveevolved to exhibita lowerpH the degradation of glucoseto pyruvate) are generallylocated
optimum(=45, leftcurve) to matchthe low internal pH in lysosomes in the same cellular compartment (e.g., in the cytosol, at a
in whichtheyfunction. [Adapted fromp Lozano, T.DeDiego, andJ L membrane,or within a parricularorganelle).'Withina com-
l b o r r a ,1 9 9 7 ,E u r J B i o c h e m2 4 8 ( 1) : 8 0 - 8 5 ,a n d W A J u d i c ee t a l , 2 0 0 4 . E u r . partment, products from one reaction can move by diffusion
J B i o c h e m2 7 1 ( 5 ) : 1 0 4 6 - 1 0 5l3 to the next enzyme in the pathway. However, diffusion

84 C H A P T E R3 |
p R O T E t NS T R U C T U RAEN D F U N C T T O N
(a) Reactants
/ stops its forward movement within a fraction of a nanome-
Reactants Products
Products
(c)
A FIGURE 3-28 Assemblyof enzymesinto efficient
multienzymecomplexes. Inthe hypothetical reaction pathways
herethe initialreactants
illustrated areconverted intofinalproducts
bythe sequential actionof threeenzymes: A, B,andC. (a)Whenthe
enzymes arefree in solutionor even constrained withinthesame
compartment,
cellular the intermediates in the reaction sequence
mustdiffusefromoneenzyme to the next,an inherently slow
process.(b)Diffusion isgreatlyreduced or eliminated whenthe
individualenzymes associateintomultisubunit complexes, eitherby
themselves or with the aidof a scaffoldprotein(c)Theclosest
integrationof differentcatalytic occurs
activities whentheenzymes
arefusedat the genetic level,becoming domains in a single
polypeptide chain
entailsrandom movement and can be a slow, relatively ineffi-
cient processfor moving moleculesbetweenwidely dispersed
enzymes(Figure 3-28a). To overcomethis impediment, cells
have evolved mechanismsfor bringing enzymesin a com-
mon pathway into closeproximity.
In the simplest such mechanism, polypeptides with dif-
ferent catalytic activities cluster closely together as subunits
of a multimeric enzyme or assembleon a common "scaf-
fold" that holds them together(Figure3-28b). This arrange-
ment allows the products of one reaction to be channeled
directly to the next enzyme in the pathway. In some cases,
independentproteins have been fused together at the genetic
level to createa singlemultidomain, multifunctional enzyme
(Figure3-28c).
E n z y m e sC a l l e dM o l e c u l a rM o t o r s C o n v e r t
Energyinto Motion
At the nanoscaleof cells and molecules,movement is influ-
enced by forces that differ from those in the macroscopic
world. For example, the high protein concentration (200-
300 mglml) of the cytoplasm preventsorganellesand vesicles
from diffusing faster than 100 pm/3 hours. Even a micrometer-
sized bacterium experiencesa drag force from water that
ter when it stops actively swimming. To generatethe forces
necessaryfor many cellular movements,cells dependon spe-
cialized enzymes commonly called molecular motors, or
motor proteins. These mechanochemicalenzymes convert
energyreleasedby the hydrolysis of ATP or contained within
ion gradients into a mechanical force, usually generating
either linear or rotary motion.
From the observed activities of motor proteins' we can
infer three generalproperties that they possess:
r The ability to transducea source of energy'either ATP or
an ion gradient, into linear or rotary movement
r The ability to bind and translocatealong a substrate
r Net movement in a given direction
\(/e will see many examples of such motors in subsequent
chapters.
Protein Function
r The functions of nearly all proteins depend on their abil-
ity to bind other molecules(ligands).
r The specificity of a protein for a particular ligand refers
to the preferential binding of one or a few closely related
ligands.
r The affinity of a protein for a particular ligand refers to
the strength of binding, usually expressedas the dissocia-
tion constant K6.
r Ligand-binding sites on proteins and the corresponding
ligands themselvesare chemically and spatially comple-
mentary.
r Enzymes are catalyticproteins that acceleratethe rate of
cellular reactions by lowering the activation energy and
stabilizing transition-stateintermediates(seeFigure 3-20).
r An enzyme active site, which is usually only a small part
of the protein, comprisestwo functional parts: a substrate-
binding site and a catalytic site. The amino acids compos-
ing the active site are not necessarilyadjacent in the amino
acid sequencebut are brought into proximity in the native
conformation.
r The substrate-bindingsite is responsiblefor the exquisite
specificity of enzymesowing to its molecular complemen-
tarity with the substrateand the transition state.
r The initial binding of substrates(S)to enzymes(E) results
in the formation of an enzyme-substratecomplex (ES)'
which then undergoesone or more reactions catalyzed by
the catalytic groups in the active site until the products (P)
are formed and diffuse away from the enzyme.
r From plots of reaction rate versus substrate concentra-
tion, two characteristicparametersof an enzymecan be de-
termined: the Michaelis constant K-, a rough measureof
P R O T E I NF U N C T I O N 85
 the enzyme'saffinity for converting substrateinto product,
and the maximal velocity V*^*, d measureof its catalytic
power (seeFigure 3-22).
r The rates of enzyme-catalyzedreactions vary enormously,
with the turnover numbers (number of substratemolecules
converted to products at a singleactive site at substratesat-
uration) ranging between<1 to 6 x 10s molecules/s.
r Many enzymes catalyzethe conversion of substratesto
products by dividing the process into multiple discrete
chemical reactions that involve multiple distinct enzyme
substratecomplexes(ES',ES",etc.).
r Serineproteaseshydrolyze peptide bonds in protein sub-
stratesusing as catalyticgroups the sidechainsof Ser-195,
H i s - 5 7 ,a n d A s p - 1 0 2 .
r Amino acids lining the specificity binding pocket in the
binding site of serine proteasesdetermine the residue in a
protein substrate that will be hydrolyzed and account for
differencesin the specificity of trypsin, chymotrypsin, and
elastase.
r Enzymesoften use acid-basecatalysismediated by one or
more amino acid side chains,such as the imidazolegroup
of His-57 in serineproteases,to catalyzereactions.
r The pH dependenceof protonation of catalytic groups
(pK") is often reflected in the pH-rate profile of the en-
zyme's activity. The pH sensitivity of an enzyme'sactivity
can be due to changesin the ionization of catalytic groups,
of groups that participate directly in substrate binding, or
of groups that influence the conformation of the protein.
r In some enzymes,nonpolypeptide small moleculesor
ions, called cofactors or prosthetic groups, can bind to
the active site and play an essentialrole in enzymaric
catalysis.Small organic prostheticgroups in enzymesare
also called coenzymes;vitamins, which cannot be synthe-
sized in higher animal cells, function as or are used to gen-
erate coenzymes.
r Enzymesin a common pathway are located within spe-
cific cell compartments and may be further associatedas
domains of a monomeric protein, subunits of a multimeric
protein, or componentsof a protein complex assembledon
a common scaffold(seeFigure 3-28).
r Motor proteins are mechanochemicalenzymesthat con-
vert energy releasedby ATP hydrolysis into either linear or
rotary movement.
fll Regulating
ProteinFunction
l:
ProteinDegradation
Most processesin cells do not take place independently of
one another or at a constant rare. The activities of all oro-
teins and other biomoleculesare regulated to integrate their
functions for optimal performance for survival. For exam-
ple, the catalytic activity of enzymesis regulated so that the
amount of reaction product is lust sufficient to meet the
86 . c H A p r E 3R | p R o r E t N
s r R U c r u RAEN DF U N c l o N
needsof the cell. As a result, the steady-stateconcentrations
of substratesand products will vary, depending on cellular
conditions. Regulation of nonenzymaticproteins-the open-
ing or closing of membrane channels or the assembly of a
macromolecular complex, for example-is also essential.
In general,there are three ways to regulateprotein activ-
ity. First, cells can increaseor decreasethe steady-statelevel
of the protein by altering its rate of synthesis, its rate of
degradation, or both. Second,cells can change the intrinsic
activity, as distinct from the amounr, of the protein (e.g.,the
affinity of substratebinding, the fraction of time the protein
is in an active versus inactive conformation). Third. there
can be a change in location or concentration within the cell
of the protein itself, the target of the protein's activity (e.g.,
an enzyme'ssubstrate),or some other molecule required for
the protein's activity (e.g., an enzyme'scofactor). All three
types of regulation play essentialroles in the lives and func-
tions of cells.
RegulatedSynthesisand Degradationof
Proteinsls a FundamentalPropertyof Cells
Controlof ProteinSynthesis The rateof synthesis
of
proteins is determined by the rate at which the DNA encod-
ing the protein is converted to mRNA (transcription), the
steady-stateamount of the active mRNA in the cell, and the
rate at which the mRNA is converted into newly synthesized
protein (translation). These important pathways are de-
scribed in detail in Chapter 4.
Control of Protein Degradation The life span of intra-
cellular proteins varies from as short as a few minutes for
mitotic cyclins, which help regulate passagethrough the
mitotic stage of cell division, to as long as the age of an
organism for proteins in the lens of the eye. Protein life span
is controlled primarily by regulatedprotein degradation.
There are two especiallyimportant rolesfor protein degra-
dation. First, degradation removes proteins that are poten-
tially toxic, improperly folded or assembled,or damaged-
including the products of mutated genes and proteins
damaged by chemically active cell metabolites. Despite the
existenceof chaperone-mediatedprotein folding, it is esti-
mated that as many as 30 percent of newly made proteins
are rapidly degraded becausethey are misfolded, their as-
sembly into complexes is defective, or they are otherwise
unsuitable. Most other proteins are degraded more slowlS
about'l,J percent degradationper hour in mammalian cells.
Second,the controlled destruction of otherwise normal oro-
teins provides a powerful mechanism for maintaining the
appropriate levelsofthe proteins and their activities,and for
permitting rapid changes in these levels to help the cells
respond to changing conditions.
Eukaryotic cells have several pathways for degrading
proteins. One major pathway is degradation by enzymes
within lysosomes, membrane-limited organelles whose
acidic interior (pH =4.5) is filled with a host of hydrolytic
enzymes. Lysosomal degradation is directed primarily to-
ward aged or defective organelles of the cell-a process
llll+ Animation:The Proteasome

< FIGURE 3-29 Ubiquitin-and proteasome-mediated

proteolysis. (a)Computer-generated imagereveals thata
Cytosolic proteasome hasa cylindrical structure with a 195 cap(blue)at
target protern eachendof a 20Score Proteolysis of ubiquitin-tagged proteins
occurswithinthe innerchamber of the core(b)Proteins are
targetedfor proteasomal degradation by polyubiquitination
E1isactivated by attachment of a ubiquitin(Ub)
E Enzyme
molecule (step E) and then transfersthis Ub molecule to a
E 1 = u b i q u i t i n - a c t i v a t i negn z y m e
o residue
cysteine in E2(stepZ) Ubiquitin ligase (E3)transfers
the boundUb molecule on E2to theside-chain -NH2 of a
E 2 = u b i q u i t i n - c o n j u g a t i negn z y m e -NH -C- Ub
residue
lysine in a targetprotein (step B) Additional Ub
E 3 = u b i q u i t i nl i g a s e
molecules areadded to the targetprotein by repeatlng
Ub = u[iqui1i6
steps[-El, forminga polyubiquitin chain(step4) The
I polyubiquitinylated targetis recognized bythe proteasome cap,
s ti,iii;J;i,' whichusesATPhydrolysis
unfolding,andtransfer
to driveremoval
of the unfolded
of the Ubgroups,
proteinintothe
t proteolysis
chamber in the core,from which the shortpeptide
- U b - U b - U b n + 1 digestionfragments arelaterreleased (stepE) lPart (a)fromw
Baumeisteretal, 1998,Cell92.357; courtesy of W Baumeister l

ATPrl

ADP</l
Release
Recognition
uo
,,n
-".j0
ub
orrJ
t-/
called autophagy (seeFigure 9-2)-and toward extracellular
proteins taken up by the cell. Lysosomeswill be discussedat
length in later chapters. Here we will focus on cytoplasmic
protein degradationby proteasomes.
TheProteasomel s a C o m p l e xM o l e c u l a r
MachineUsedto DegradeProteins
Proteasomesare very large macromolecularmachinesconsist-
ing of =50 protein subunitsand having a massof 2-2.4 ', 1'06
Da. They have a cylindrical, barrel-like catalytic core called
the 20S protedsome(where S is a Svedbergunit basedon the
sedimentationproperties of the particle and is proportional
to its size).Bound to the ends of this core are one or two cap
complexesthat regulate proteasomal activity. There are ap-
proximately 30,000 proteasomesin a typical mammalian
cell. There are multiple forms of proteasomes.The best stud-
ied of theseis the 265 proteasome(Figure3-29a1,which has
a catalyticcore approximately14.8 nm tall and 11.3 nm in
diameter and a 19S cap regulatory particle at each end.
)s There are severaldistinct regulatory cap complexeswith dif-
ferent activities.The 195 cap has 16-18 protein subunits'6
of which can hydrolyze ATP (i.e., they are ATPases)to pro-
U b the energy needed to unfold protein substratesand
uu vide
uo selectivelytransferthem into the inner chamber of the protea-
some. Genetic studiesin yeast have shown that cells cannot
\ unfotding survive without functional proteasomes,thus demonstrating
their importance. Furthermore, proper proteasomal activity
\ is so important that cells will expend as much as 30 percent
cleauage
of the energy neededto synthesizea protein to degradeit in
a Droteasome.
\ The proteasomal catalytic core comprises two inner
Dir"hurg"
rings, with six proteolytic active sitesfacing toward the inner
chamber of the =1..7-nm-diameterbarrel' and two outer
rings that control substrateaccess.Proteasomescan degrade
most proteins thoroughly becausethey have active sites(two
each) that cleaveafter hydrophobic residues,acidic residues,
and basic residues. Polypeptide substrates must enter the
chamber via a regulated aperture at the center of the outer
rings. In the 265 proteasome,the opening of the aperture,
which is narrow and often allows the entry of only unfolded
proteins, is controlled by ATPasesin the 195 cap. The short
peptide products of proteasomal digestion (2-24 residues
long) exit the chamber and are further degradedrapidly by
cytosolic peptidases,eventually being converted to individ-
ual amino acids. Some have quipped that a proteasomeis a
"cellular chamber of doom" in which proteins suffer a
"death of a thousandcuts'"
Inhibitors of proteasomefunction can be used thera-
peutically.Becauseof the global importance of pro-
teasomes for cells, continuous, complete inhibition of
proteasomeskills cells. However, partial' discontinuous
p.ot.u.o-. inhibition has been introduced as an approach
io .".r.., chemotherapy.To survive and groq cells normally

DEGRADATION
l: PROTEIN
FUNCTION
R E G U L A T I NPGR O T E I N 87
require the robust activity of a regulatory protein called
NF^B, as well as other similar "pro-survival" proteins. In
turn, NF*B can function fully and promote survival only
when its inhibitor, I^B, is disengagedand degradedby pro-
teasomes(Chapter 16). Partial inhibition of proteasomal
activity by a small-moleculeinhibitor drug results in in-
creasedlevels of I*B and, consequently,reducedNF^B ac-
tivity (loss of pro-survival activity). Cells subsequentlydie
by a mechanismcalled apoptosis (programmedcell death,
Chapter 21). Becauseat leasrsome types of tumor cells are
more sensitive to being killed by proteasome inhibitors
than normal cells are, controlled administration of protea-
some inhibitors (at levelsthat kill the cancer cells but not
normal cells) has proved ro be an effectivetherapy for at
least one type of lethal cancer,multiple myeloma. I
U b i q u i t i nM a r k sC y t o s o l i cP r o t e i n sf o r
Degradationin Proteasomes
If proteasomesare to rapidly degradeonly thoseproteins that
are either defectiveor scheduledto be removed,they must be
able to distinguish between those proteins that need to be
degradedfrom most of the proteins that don't. To solve this
problem, cells identify proteins that should be degraded by
covalently attaching multiple copiesof a76-residuepolypep-
tide called ubiquitin that is highly conservedfrom yeast to
humans. A complex sensingsystemhas evolved to determine
which proteins are to be degraded,and then a three-step
processis usedto polyubiquitinylatethe targetproteins.The
195 regulatory cap of the 265 proteasomethen recognizes
the ubiquitin-labeledproreins, and unfolds and transports
them into the proteasomefor degradation. The ubiquitina-
tion process(Figure 3-29b) involves:
1. Activation of wbiquitin-actiuatingenzyme(E1)by the
addition of a ubiquitin molecule,a reactionthar requires
ATP
2. Transfer of this ubiquitin molecule to a cysteineresidue
in ubiquitin- conjugating enzyme (E2)
3. Formation of an isopeptidebond betweenthe carboxyl
terminus of the ubiquitin bound to E2 and the amino group
of the side chain of a lysine residuein rhe targer prorein, a
reaction catalyzedby ubiquitin-protein ligase (E3). Subse-
quent ligasereactionscovalently attach additional ubiqui-
tins to the side chain of lysine 48 of the previously added
ubiquitin to generatea linear polymer of ubiquitins, or a
polyubiquitin-modifiedtargetprotein.
Specificity of Degradation Thrgetingof specificproteinsis
primarily achievedthrough the substrate specificity of the E3
ligase.Thereare hundredsof E3 ligasesin mammaliancells,en-
suring that the wide variety of proteins to be polyubiquitiny-
lated can be modified when necessary.
An example of the control of the activity of a key cellular
protein by the ubiquitin-proteasomesystem is the regulated
88 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
degradationof proteins called cyclins,which control the cell
cycle (Chapter 20). Cyclins contain the internal sequence
Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn (X can be any amino
acid), which is recognizedby specificubiquitinylating enzyme
complexes.At a specifictime in the cell cycle, each cyclin is
phosphorylated by a cyclin kinase. This phosphorylation is
thought to causea conformational change that exposesthe
recognition sequenceto the ubiquitinylating enzymes,leading
to polyubiquitination and proteasomaldegradation.
Multifunctional Ubiquitin Tagging Someubiquitination
performs cell functions other than the degradation of a t^r-
getedprotein. Examplesof alternativeubiquitination schemes
include (1) the covalentaddition of a singleubiquitin molecule
(monoubiquitination) to a lysine on a rarget protein, (2) the
addition of multiple single ubiquitins (multiubiquitination),
(3) Iinking the ubiquitin to rhe N-terminus of the target pro-
tein, and (4) polyubiquitination in which the ubiquitins are
linked to one another via their Lys-63residueinsteadof at the
Lys-48 position. These modifications can influence the traf-
ficking (sorting) of proteins within a cell (e.g.,internalization
from the cell surface),control DNA repair and regulation of
transcription,and undoubtedlyperform numerousother func-
tions yet to be discovered.Cells also have a variery of deubiq-
uitinylating enzymes that can remove ubiquitins from the
targetproteinsand thus introducethe possibilityin somecases
of reversingthe regulationcausedby the initial ubiquitination.
Regulating Protein Function l: Protein Degradation
r Proteins may be regulated at the level of protein synthe-
sis,protein degradation,or the intrinsic activity ofproteins
through noncovalent or covalent interactions.
r The life span of intracellular proteins is largely deter-
mined by their susceptibilityto proteolytic degradation.
r Many proteins are marked for destruction with a poly-
ubiquitin tag and then degradedwithin proteasomes,large
cylindrical complexeswith multiple proreasesin their inte-
riors (seeFigure3-29).
r Variations in the nature of the covalent attachment of
ubiquitin to proteins are involved in cellular functions
other than proteasome-mediateddegradation, such as
changesin the location or activity of proteins.
RegulatingProteinFunctionll:
f[
Noncovalent
and CovalentModifications
The intrinsic activities of proteins are modulated by both
noncovalent and covalent changesin the protein. Noncova-
lent modifications usually involve the binding or dissociation
of a molecule and a consequentchangein the conformation
of the protein. Often, in such cases,protein activation in-
volves the releaseor rearrangementof an inhibitory subunit
or domain.
 Covalentmodifications include hydrolysisof the polypep-
tide chain or addition of a molecule to the side chain of one
or more residuesor to the N- or C-terminus of the protein.
Such modifications can cause a conformational change in
c
the protein that can alter its activity (form is function). Co-
valent modifications can also modify the shape of a protein =5U
without changing the conformation of the polypeptide and
U)
its side chains, for example, by adding a charge or bulky a
group that can alter the ability of the protein to bind to other
molecules.Lastly, covalent modifications can direct the pro-
tein to particular locations in a cell (e.g.,the cytoplasmic sur-
face of the plasmamembrane). 100
0 20 40 60 80
Many noncovalent and covalent modifications are re-
4 Poz(torr) t
versible,thus allowing the activity of an individual protein It
to be enhancedor suppressedmultiple times during the life- p O 2i n c a P i l l a r i e s P O 2i n a l v e o l i
of activemuscles of lungs
time of the protein. Others,suchas proteolysis,are irreversible
and can be supersededonly by degradationof the modified a EXPERIMENTAL FIGURE 3-30 Hemoglobinbindsoxygen
protein and synthesis of a replacement. In the case of cooperatively. Eachtetrameric hemoglobin proteinhasfour
enzymes,these regulatory modifications alter K-, V-"*, or oxygen-binding sites;at saturationallthesitesareloadedwith
oxygen. Theoxygen concentration iscommonly measured asthe
both. Nature has devisedmany different strategiesfor non-
partialpressure(pOz) Pso isthe pO2 at which halfthe oxygen-
covalent and covalent regulation of activity. Here we dis-
bindingsitesat a givenhemoglobin concentration areoccupied; it is
cuss some common mechanisms for regulating protein
somewhat analogous to the K' for an enzymatic reaction. Thelarge
function; additional examples will be describedin other
changein theamountof oxygenboundovera smallrangeof pO2
chapters. of oxygenin peripheral tissues
valuespermitsefficient unloading
suchas muscle The sigmoidal shape of a plotof percent saturation
t i n d i n gP e r m i t sA l l o s t e r i c ,
N o n c o v a l e nB versusligandconcentration isindicativeof cooperative bindingIn
the absenceof cooperative binding, a bindingcurveisa hyperbola,
or Cooperative,Regulationof Proteins fromL Stryer,1995,
to thecurves
similar in Figure 3-22 [Adapted
One of the most important mechanismsfor regulatingpro- ed,
Biochemistru,4th W H Freeman and Company.l
tein function is through allosteric interactions. Broadly
speaking,allostery (from the Greek "other shape") refers to
any changein a protein's tertiary or quaternary structure, or
in both, induced by the noncovalent binding of a ligand.
lfhen a ligand binds to one site (A) in a protein and induces
a conformational changeand associatedchangein activity spreadsto the other subunits, lowering the K- (increasing
of a different site (B), the ligand is called an allosteric effec- the affinity) for the binding of additional oxygen molecules
tor of the protein, while site A is called an allosteric binding to the remaining hemes and yielding a sigmoidal oxygen-
site, and the protein is called an allosteric protein. By defi- binding curve (Figure3-30)' Becauseof the sigmoidalshape
nition, allostericproteins have multiple binding sitesfor ei- of the oxygen-saturationcurve, it takes only a fourfold in-
ther a single type of ligand or for multiple different ligands. creasein oxygen concentration for the percent saturation of
The allosteric change in activity can be positive or negative' the oxygen binding sitesin hemoglobin to go from 10 to 90
i.e., can induce an increaseor a decreasein protein activity. percent- Conversely,if there were no cooperativity and the
Allosteric regulation is particularly prevalent in multimeric shapeof the curve was typical of that for Michaelis-Menten-
enzymesand other proteins where conformational changes type binding, it would take an 81-fold increase in oxygen
in one subunit are transmitted to an adiacent subunit. concentration to accomplish the same increasein loading.
Cooperatiuity is a term often used synonymously with This cooperativity permits hemoglobin to take up oxygen
allostery,and usuallyrefersto the influence(positiveor neg- very efficiently in the lungs where the oxygen concentration
ative) that the binding of a ligand at one site has on the is high, and unload it in tissueswhere the concentration is
binding of another moleculeof the sametype of ligand at a low. Thus, cooperativity amplifies the sensitivity of a system
different site. to concentration changesin its ligands, providing in many
Hemoglobin presentsa classicexample of positive coop- casesselectiveevolutionary advantage.
erative binding in that the binding of a singleligand, oxygen, Negative cooperativity often involves the end product of
increasesthe affinity of the binding of the next oxygen mol- a multistep biochemical pathway, which binds to and re-
ecule.Each of the four subunits in hemoglobin contains one ducesthe activity of an enzymethat catalyzesan early, rate-
heme molecule. The heme groups are the oxygen-binding controlling step for that pathway. In this way excessive
componentsof hemoglobin(seeFigure3-13).The binding of buildup of the product is prevented.This kind of regulation
oxygen to the heme molecule in one of the four hemoglobin of a metabolic pathway is also called end-product inhibition
subunits inducesa local conformational changewhose effect or feedback inhibition.

T N D C O V A L E N TM O D I F I C A T I O N S
P R O T E I NF U N C T I O NI I : N O N C O V A L E N A
REGULATING
89
 N o n c o v a l e nB
t i n d i n go f C a l c i u ma n d G T pA r e ( a ) C a l m o d u l i nw i t h o u t c a l c i u m
Widely UsedAs AllostericSwitchesto Control
ProteinActivity
Unlike oxygen, which causesgraded allostericchangesin the
activity of hemoglobin, other allosteric effectors act as
switches,turning the activity of many different proteins on
or off. Two important allosteric switches that we will en-
counter many times throughout this book are Ca2* and
GTP.

Ca2+/Calmodulin-Mediated Switching The concentra-

tion of Ca2* fteein the cytosol (not bounJto moleculesother
than water) is kept very low (=10 7 M) by specializedmem-
brane transport proteins that continually pump Ca2+ out of
the^cytosol.However,as we learn in Chapter 11, the cytosolic
Ca'- concentrationcan increasefrom 10- to 100-fold when
Ca2*-permeablechannelsin the cell surfacemembranesopen
and allow extracellularCa2* ro flow into the cell. This risi in
cytosolicCa2* is sensedby specializedCa2*-binding proteins,
which alter cellular behavior by turning other prorelns on or
off. The importanceof extracellular Ca2* for cell activity was
first documentedby S. Ringer in 1883, when he discovered
that isolated rat hearts suspendedin a NaCl solution made
with 'hard' (Ca2*-rich) London rap warer contracted beauti-
fully whereasthey beatpoorly and stoppedquickly if distilled
water was used.
Many of the Ca2*-bindingproteinsbind Ca2* using the
EF hand/helix-loop-helix structural motif discussedearlier
(seeFigure3-9b). The prototype EF hand prorein, calmodulin,
is found in all eukaryotic cells and may exist as an individual
monomenc protein or as a subunit of a multimeric protein.
A dumbbell-shapedmolecule, calmodulin contains four
Ca'--binding EF hands with K6's of =10-6 M. The binding
of Ca2* to calmodulin causesa conformational changethal
permits Ca2*/calmodulin to bind to conservedsecuencesin
various target proteins, thereby swirching their activities
on or off (Figure 3-31). Calmodulin and similar EF hand
proteins thus function as sulitch proteins, acting in concert
with changes in Ca2* levels to modulate the activity of
other proteins.
Switching Mediated by Guanine Nucleotide-Binding
Proteins Another group of intracellular switch proteins con-
stitutes the GTPase superfamily. As the name suggests,these
proteins are enzymes, GTPases, that can hydrolyze GTp
(guanosinetriphosphate) to GDp (guanosinediphosphate).
They include the monomeric Ras protein (seeFigure 3-8) and
the G. subunit of the trimeric G proteins, both discussedat
length in Chapter 15. Both Ras and Go can bind to the plasma
membrane,function in cell signaling,and play a key role in cell
proliferation and differentiation. Other membersof the GTpase
superfamily function in protein synthesis,the transport of pro-
teins between the nucleus and the cytoplasm, the formation of
coated vesiclesand their fusion with target membranes,and re-
arrangemenrsof the actin cytoskeleton.The Hsp70 chaperone
proteln we encounteredearlier is an example of an ATp/ADp
switch, similar in many respectsto a GTp/GDp switch.
(bl Caz*/calmodulinbound to target peptide
A FIGURE 3-31 Conformational changesinducedby Ca2*
bindingto calmodulin.Calmodulin isa widelydistributedcytosolic
proteinthatcontainsfourCa2*-brnding sites,onein eachof itsEF
handsEachEFhandhasa helix-loop-helix motif At cytosolicCa2+
concentrations
aboveabout5 x 10-7M, bindingof Ca2*to
calmodulinchanges the protein'sconformation fromthe dumbbell-
shaped, unboundform(a)to onein whichhydrophobic sidecharns
becomemoreexposed to solvent.TheresultingCa2*/calmodulin can
wraparoundexposed helices targetproteins
of various (b),thereby
altering
theiractivity.
AII the GTPase switch proteins exist in two forms, or
conformations(Figure 3-32):(1) an active("on")form with
bound GTP that modulates the activity of specific target
proteins to which they bind and (2) an inactive ( ,.off ', ) form
with bound GDP, which is generatedby the relatively slow
hydrolysis of the GTP bound to the active form. The
amount of time any given GTPaseswitch remains active de-
pends on the rate of its GTPase activity. Thus the GTpase
activity acts as a timer to control this switch. Cells contain
a variety of proteins that can modulate the baseline (or in-
trinsic) rate of GTPase activity for any given GTpase
switch. For example, GTPase activity can be enhanced by
specific GTPase-activating proteins, called GAPs, or de-
pressedby other proteins acting as allosteric regulators.
After the switch has beenturned off (GTP hydrolysis), it can
be turned back on by GTP exchange factor (GEF), which
replacesthe bound GDP with a different GTp molecule
from the surrounding fluid. Thus, cells can control when
the switch is turned on and how long the switch remains on.
'We
examine the role of various GTPase switch proteins in
regulating intracellular signaling and other processesin sev-
eral later chaoters.

90 C H A P T E R3 | P R O T E | NS T R U C T U RAEN D F U N C T T O N
 Active ("on") Nearly 3 percent of all yeast proteins are protein kinases
or phosphatases,indicating the importance of phosphoryla-
tion and dephosphorylation reactions even in simple cells.
All classes of proteins-including structural proteins,
* GAPs scaffolds, enzymes, membrane channels' and signaling
GEFs4+ <* nGSs molecules-have members regulated by kinase/phosphatase
* GDls switches.Different protein kinases and phosphatasesare
Inactive("off") specific for different target proteins and can thus regulate a
variety of cellular pathways, as discussedin later chapters.
Some of these enzymesact on one or a few target proteins,
whereas others have many targets. The latter are useful in
integrating the activities of proteins that are coordinately
A FIGURE 3-32 The GTPaseswitch. Conversion of the active, controlled by a single kinase/phosphatase switch.
GTP-bound GTPase intothe inactive
formby hydrolysisof GTPis Frequently,the target of the kinase (and phosphatase)is yet
by GAPs(GTPase-activating
accelerated proteins)
andRGSs another kinase or phosphatase' creating a cascade effect.
(regulators
of G proteinsignaling)
andinhibitedby GDls(guanine There are many examples of such kinase cascades,which
nucleotide inhibitors)
dissociation Reactivation
by replacingGDPwith permit amplification of a signal and many levels of fine-
GTPis promoted by GEFs(guanine nucleotide
exchange factors). tuning control (seeChapter 15).

ProteolyticCleavagelrreversiblyActivatesor
lnactivatesSomeProteins
P h o s p h o r y l a t i oann d D e p h o s p h o r y l a t i o n
Unlike phosphorylation, which is reversible, the activation
CovalentlyRegulateProteinActivity
or inactivation of protein function by proteolytic cleavageis
One of the most common mechanismsfor regulating protein an irreversible mechanism for regulating protein activity.
activity is phosphorylation, the addition of phosphate For example,many polypeptidehormones,such as insulin,
groups to hydroxyl groups on serine,threonine, or tyrosine are synthesizedas long precursors, and prior to secretion
residues. Protein kinases catalyze phosphorylation, and from cells some of their peptide bonds must be hydrolyzed
phosphatases catalyze deph osph orylation. The counteract- for them to fold properly. In some cases'a single long pre-
ing activities of kinasesand phosphatasesprovide cells with cursor prohormone polypeptidecan be cleavedinto several
a "switch" that can turn on or turn off the function of vari- distinct active hormones. To prevent the pancreatic serine
ous proteins (Figure3-33). Phosphorylationchangesa pro- proteasesfrom inappropriately digesting proteins before
tein's chargeand generallyleadsto a conformational change; they reach the small intestines, they are synthesizedas ey-
these effects can significantly alter ligand binding or other mogens, inactive precursor proteins. Cleavageof a peptide
featuresof the protein, leading to an increaseor decreasein bond near the N-terminus of trypsinogen (the zymogen of
lts actlvlty. trypsin) by a highly specific protease in the small intestine
generatesa new N-terminal residue (Ile-16), whose amino
group can form an ionic bond with the carboxylic acid side
chain of an internal aspartic acid. This causesa conforma-
Active tion changethat opens the substrate-bindingsite' activating
o the enzyme. The active trypsin can then activate trypsino-
tl
R-O-P-O- gen, chymotrypsinogen, and other zymogens. Similar' but
Hzo more elaborate,proteasecascades(one proteaseactivating
inactive precursorsof others) that can amplify an initial sig-
nal play important roles in several systems' such as the
Protein
phosphatase blood-clotting cascade.The importance of carefully regulat-
ing such systemsis clear-inappropriate clotting could fa-
tally clog the circulatory system, while insufficient clotting
P.
could lead to uncontrolledbleeding.
R-OH An unusual and rare type of proteolytic processing'
Inactive termed protein self-splicing,takes place in bacteria and some
eukaryotes.This processis analogousto editing film: an in-
A FIGURE 3-33 Regulationof protein activity by the
ternal segmentof a polypeptide is removed and the ends of
kinase/phosphatase switch.Thecyclicphosphorylation and
dephosphorylationof a proteinisa commoncellular mechanism for the polypeptide are reioined (ligated). Unlike other forms of
regulatingproteinactivityIn thisexample,the targetproteinR is proteolytic processing, protein self-splicing is an autocat-
active(top)whenphosphorylated andinactive(bottom)when alytic process,which proceedsby itself without the partici-
dephosphorylated; someproteins havetheopposite responsesto pation of enzymes.The excisedpeptide appearsto eliminate
phosphorylation itself from the protein by a mechanismsimilar to that usedin

T N D C O V A L E N TM O D I F I C A T I O N S
P R O T E I NF U N C T I O NI I : N O N C O V A L E N A
REGULATING 91
the processingof some RNA molecules (Chapter 8). In ver-
tebrate cells, the processingof some proteins includes self-
cleavage,but the subsequentligation step is absent. One
such protein is Hedgehog, a membrane-bound signaling
molecule that is critical to a number of develoomental
processes (Chapter 15).
H i g h e r - O r d eRr e g u l a t i o nI n c l u d e sC o n t r o l
of ProteinLocationand Concentration
All the regulatory mechanismsheretofore describedaffect
a protein locally at its site of action, turning its activity on
or off. Normal functioning of a cell, however, also re-
quires the segregationof proteins to particular compart-
ments such as the mitochondria, nucleus, and lysosomes.
In regard to enzymes,compartmentationnot only provides
an opportunity for controlling the delivery of substrateor
the exit of product, but also permits compering reactions
to take place simultaneously in different parts of a cell.
We describethe mechanismsthat cells use to direct vari-
ous proteins to different compartmenrs in Chapters 12
and 13.
Protein Regulation ll: Noncovalent
and Covalent Modifications
r In allostery,the noncovalentbinding of one ligand mol,
ecule, the allosteric effector, induces a conformational
change that alters a protein's activity or affinity for other
ligands. The allosteric effector can be identical in structure
to or different from the other ligands, whose binding it af-
fects.The allostericeffector can be a substrate.acuvaror.or
inhibitor.
r In multimeric proteins, such as hemoglobin, that bind
multiple identicalligand molecules(e.g.,oxygen),the bind-
ing of one ligand molecule may increase or decreasethe
binding affinity for subsequentligand molecules.This type
of allosteryis known as cooperativiry.
r Several allosteric mechanisms act as switches, turning
protein activity on and off in a reversiblefashron.
r Two classesof intracellular switch proteins regulatea va-
riety of cellular processes:
(1) Ca2+-bindingproteins (e.g.,
calmodulin) and (2) members of the GTpase superfamily
(e.g.,Ras), which cycle between active GTP-bound and in-
activeGDP-boundforms (seeFigure 3-32).
r The phosphorylation and dephosphorylationofhydroxyl
groups on serine,threonine, or tyrosine residueside chains
by protein kinases and phosphatasesprovide reversible
on/off regulation of numerous proteins.
I Many types of covalent and noncovalent regulation are
reversible, but some forms of regulation, like proteolytic
cleavage,are irreversibre.
r Higher-order regulation includes compartmentation of
proteins and control of protein concentration.
92 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
fp Purifying,
Detecting,
and
Characterizing Proteins
A protein often must be purified before its structure and the
mechanism of its action can be studied in detail. However,
becauseproteins vary in size, charge, and water-solubility,
no single method can be used to isolate all proteins. To iso-
late one particular protein from the estimated 10,000 differ-
ent proteins in a particular type of cell is a daunting task that
requires methods both for separating proteins and for
detectingthe presenceof specificproteins.
Any molecule,whether protein, carbohydrate,or nucleic
acid, can be separated,or resolued,from other moleculeson
the basisof their differencesin one or more physical or chem-
ical characteristics.The larger and more numerous the differ-
ences between two proteins, the easier and more efficient
their separation.The two most widely usedcharacteristicsfor
separatingproteins are size,defined as either length or mass,
and binding affinity for specific ligands. In this section, we
briefly outline several important techniques for separating
proteins; these separation techniquesare also useful for the
separationof nucleic acids and other biomolecules.(Special-
ized methods for removing membrane proteins from mem-
branesare describedin Chapter 10 after the unique properties
of theseproteins are discussed.)\Wethen consider the use of
radioactive compounds for tracking biological activity. Fi-
nallS we considerseveraltechniquesfor characterizinga pro-
tein's mass,sequence,and three-dimensionalstructure.
CentrifugationCan SeparateParticlesand
MoleculesThat Differ in Massor Density
The first step in a typical protein purification schemeis cen-
trifugation. The principle behind centrifugation is that two
particles in suspension(cells, cell fragments, organelles,or
molecules)with different massesor densitieswill settleto the
bottom of a tube at different rates. Remember,mass is the
weight of a sample (measuredin grams), whereas density is
the ratio of its weight to volume (grams/liter).Proreinsvary
greatly in mass but not in density.Unlessa protein has an at-
tached lipid or carbohydrate, its density will not vary by
more than 15 percentfrom I .37 glcm3,rhe averageprotein
density.Heavier or more densemoleculessettle,or sediment.
more quickly than lighter or lessdensemolecules.
A centrifuge speedssedimentationby subjectingparticles
in suspensionto centrifugal forces as great as 1,000,000
times the force of gravity g, which can sedimentparticles as
small as 10 kDa. Modern ultracentrifugesachieve these
forces by reaching speedsof 150,000 revolutions per minute
(rpm) or greater.However, small particles with massesof
5 kDa or lesswill not sedimentuniformly even at such high
speeds.
Centrifugation is used for two basic purposes: (1) as a
preparativetechniqueto separateone type of material from oth-
ers and (2) as an analytical technique to measurephysical prop-
erties (e.9., molecular weight, densiry shape, and equilibrium
binding constants) of macromolecules.The sedimentation
 (a) Differentialcentrifugation (b) Rate-zonalcentrifugation

I Sample is poured into tube I Sample is layered on top of density gradient

Largerparticle
Largerparticle S m a l l e rp a r t i c l e

S m a l l e rp a r t i c l e

Low density
(low sucrose
concentration)

Centrifuge Sucrose
Particlessettle gradient High density

.l
accordingto ( h i g hs u c r o s e
mass concentration)

Centrifuge
Particlessettle
Centrifugal+ according to
mass

Stop centrifuge
Collect fractions
E and do assay

HI
Decreasing massof Particles
EXPERIMENTAL FIGURE 3-34 Centrifugation techniques (b)In rate-zonal a mixtureisspun(stepE) justlong
centrifugation,
separateparticlesthat differ in massor density.(a)In differential enoughto separate molecules thatdifferin massbut maybe similar
a cellhomogenate
centrifugation, or othermixtureisspunlong in shapeanddensity (e.g, globular
proterns, RNAmolecules)into
enoughto sediment (eg , cellorganelles,
the largerparticles cells), drscretezoneswithina densitygradientcommonly formedby a
whichcollectasa pelletat the bottomof thetube(stepE) The concentratedsucrose solution. areremoved
Fractions fromthe
smaller (e g , soluble
particles proteins, nucleicacids)remainin the bottomof the tubeandsubjected to testing(assayed).
whichcanbetransferred
liquidsupernatant, to another tube(stepB)

constant s of a protein is a measureof its sedimentationrate.
The sedimentationconstant is commonly expressedin sved-
bergs (S), where a typical, large protein complex is about
3-5S, while a eukaryoticribosomeis 80S.
Differential Centrifugation The most common initial step
in protein purification for cells or tissuesis the separation of
water-solubleproteins from insolublecellular material by differ-
ential centrifugation. A starting mixture, commonly a cell
homogenate(mechanicallybroken cells),is poured into a tube
and spun at a rotor speedand for a period of time that forces
cell organellessuch as nuclei and large unbroken cells or large
cell fragments to collect as a pellet at the bottom; the soluble
proteinsremain in the supernatant(Figure3-34a1.The super-
natant fraction then is poured off' and either it or the pellet can
be subjectedto other purification methods to separatethe many
differentproteinsthat they contain.
Rate-Zonal Centrifugation On the basisof differencesin
their masses, proteins can be separated by centrifugation
through a solution of increasingdensity called a density gradi-
ent. A concentratedsucrosesolution is commonly usedto form
density gradients.'Whena protein mixture is layeredon top of a
sucrosegradient in a tube and subjectedto centrifugation, each
protein in the mixture migrates down the tube at a rate con-
irolled by the factors that affectthe sedimentationconstant.All
the proteins staft from a thin zone at the top of the tube and
,.pui"t. into bands, or zones (actually, disks)' of proteins of

P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S 93

 Technique {lttt
Animation:SDSGel Electrophoresis

- I Denature sample with < EXPERIMENTAL FIGURE 3-35 SDS-polyacrylamide gel

[ | sodium dodecylsulfate electrophoresis (SDS-PAGE) separatesproteinsprimarilyon the
+ basisof their masses. (a)Initialtreatment with SDS,a negatively
? charged detergent, dissociatesmultimeric proteinsanddenatures all
aPa.D, ? Y the polypeptide chains (steptr). Duringelectrophoresis,the SDS-
& ^.. sDS-coated proteincomplexes migrate throughthe polyacrylamide gel(stepE).
N
-J l & P &Proteins
2\ Smallcomplexes areableto movethroughthe poresfasterthan
w:,q- Pj largeronesThusthe proteins separate intobandsaccording to their
sizes astheymigrate. Theseparated proteinbandsarevisualized by
staining with a dyeGtepB). (b)Example of SDS-PAGE separation of
allthe proteins in a whole-celllysate (detergent solublized
cells):
(/eft)the manyseparate stained proteins, appearing almostasa
continuum; (right)a proteinpurified fromthe lysateby a singlestep
Cross-linked of antibody-affinity chromatography. Theproteins werevisualizedby
polyacrylamide staining with a silver-based dye lPart(b)modified fromB LiuandM
gel Krieger,2002,J BiolChem277(31):34125-34135 l

(b)
E o
ElectrophoresisSeparatesMoleculeson the
Basisof Their Charge-to-Mass
Ratio
Electrophoresisis a technique for separatingmoleculesin a
mixture under the influence of an applied elecric field and is

Decreasing
J::;'*1tffi,"."17 !'{
9X
3 t-Y
one of the most frequently usedtechniquesto study proteins
and nucleic acids. Dissolved molecules in an electric field
stze
kDa o" q-5 move, or migrate, at a speeddetermined by their charge-to-
200 mass (charge:mass)ratio. For example, if two molecules
116 have the same mass and shape,the one with the greater net
97
charge will move faster toward an electrodeof the opposite
66 polarity.

45 SDS-Polyacrylamide Gel Electrophoresis Becausemany

31
different masses.In this separationtechnique,called rate-zonal
centrifugation, samplesare centrifuged just long enough to
separatethe moleculesof interest into discretezones (Fig-
ure 3-34b). If a sample is centrifuged for too short a time,
the different protein moleculeswill not separatesufficiently.
If a sample is centrifuged much longer than necessary,all
the proteins will end up in a pellet at the bottom of the tube.
Although the sedimentation rate is strongly influenced
by particle mass, rate-zonal centrifugation is seldom effec-
tive in determining precise molecular weights becausevari-
ations in shape also affect sedimentation rate. The exact ef-
fects of shape are hard to assess,especially for proteins or
other molecules,such as single-strandednucleic acid mole-
cules,that can assumemany complex shapes.Nevertheless,
rate-zonal centrifugation has proved to be the most practi-
cal method for separatingmany different types of poiy-"r,
and particles.A second density-gradienttechnique,called
equilibrium density-gradient centrifugation, is used mainly
to separateDNA, lipoproteins that carry lipids through the
crrculatory system,or organelles(seeFigure 9-26).
proteins or nucleic acidsthat differ in sizeand shapehave nearly
identical charge:massratios, electrophoresisof thesemacromol-
eculesin solution results in little or no separation of molecules
of different lengths.However, successfulsepararionof proteins
and nucleic acidscan be accomplishedby electrophoresisin var-
ious gels (semisolid suspensionsin water similar to the con-
gealedgelatin found in desserts)rather than in a liquid solution.
Electrophoretic separation of proteins is most commonly per-
formed in polyacrylamide gels. .il(i'hena mixture of proteins is
placedin a gel and an electriccurrent is applied, smallerproteins
migrate faster through the gel than do larger proteins because
the gel acts as a sieve,with smaller speciesable to maneuver
more rapidly through the pores in the gel than larger species.
The shapeof a moleculecan also influence its rate of migration
(long asymmetricmoleculesmigrate more slowly than spherical
onesof the samemass).
Gels are cast betweena pair of glassplates by polymeriz-
ing a solution of acrylamide monomers into polyacrylamide
chains and simultaneously cross-linking the chains into a
semisolidmatrix. The pore size of a gel can be varied by ad-
justing the concentrations of polyacrylamide and the cross-
Iinking reagent.The rate at which a protein moves through
a gel is influenced by the gel's pore size and the strength of
the electric field. By suitable adjustment of theseparamerers,

94 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
proteins of widely varying sizescan be resolved(separated
from one another) by polyacrylamide gel electrophoresis
(PAGE).
In the most powerful technique for resolving protein
mixtures, proteins are exposedto the ionic detergentSDS
(sodiumdodecylsulfate)beforeand during gel electrophore-
sis (Figure3-35). SDSdenaturesproteins,in part becauseit
binds to hydrophobic side chains, destabilizing the hy-
drophobic interactions in the core of a protein that con-
tribute to its stableconformation. (SDStreatmentis usually
combined with heating in the presenceof reducing agents
that break disulfide bonds.) As a consequence,multimeric
proteins dissociateinto their subunits,and all polypeptide
chains are forced into extended conformations with simi-
lar charge:massratios. SDS treatment thus eliminates the
effect of differencesin shapein native structures;therefore,
chain length, which correspondsto mass, is the principal
determinant of the migration rate of proteins in SDS-
polyacrylamide electrophoresis(SDS-PAGE).Even chains
that differ in molecular weight by lessthan 10 percentcan
be resolved by this technique. Moreover, the molecular
weight of a protein can be estimatedby comparing the dis-
tance that it migratesthrough a gel with the distancesthat
proteins of known molecular weight migrate (there is
roughly a linear relationship between migration distance
and the log of the molecular weight). Proteins within the
gelscan be extractedfor further analysis(e.g.'identification
by methodsdescribedbelow).
Two-Dimensional Gel Electrophoresis Electrophoresis of
all cellularproteinsby SDS-PAGEcan separateproteinshaving
relatively large differencesin mass but cannot readily resolve
proteinshaving similar masses(e.g.,a 41-kDa protein versusa
42-kDa protein). To separateproteins of similar masses'an-
other physical characteristicmust be exploited. Most com-
monlg this characteristicis electric charge' which is determined
by the pH and the relative number of the protein's positively
and negatively charged groups, which is in turn dependenton
the pKu'sof the ionizablegroups(seeChapter2). Two unrelated
proteins having similar massesare unlikely to have identical net
chargesbecausetheir sequences,and thus the number of acidic
and basicresidues,are different.
In two-dimensionalelectrophoresis,proteins are sepa-
rated sequentially,first by their charges and then by their
masses(Figure3-36al.In the first step'a cell or tissueextract
is fully denatured by high concentrations (8 M) of urea and
then layered on a gel strip that contains a continuous pH
gradient. SDS cannot be used, becauseits binding changes
the charge of the protein. The gradient is formed by
ampholytes, a mixture of polyanionic and polycationic
molecules, that are cast into the gel' with the most acidic
ampholyte at one end and the most basic ampholyte at the

(b) lsoelectricfocusing (E l ->

(a) Protein pH 4.0
mixture

Separate lsoelectric
in first E f o c u s i n g( l E F ) ET
dimension .66 I

by charge .2
o
X o,
o
pH10.0
34r 0
Apply first gel
to top of second z 6
f
OJU
I
o
o
o

q)

pH 4.0 pH 10.0
16 v

Separate

s
lil,i??"?l"
by size

A EXPERIMENTAL FIGURE3-36 Two-dimensionalgel two-dimensionalgelof a proteinextractfromculturedcells,each

separates proteins on the basis of charge and a singlepolypeptide
spotrepresents canbe detected
Polypeptides by
electrophoresis
dyes,ashere,or by othertechniques suchasautoradiography Each
mass.(a) In this technique,proteinsarefirst separatedinto bandson
the basisof their chargesby isoelectric focusing(step[) The polypeptide
ischaracterized point(pl)andmolecular
by itsisoelectric
(b)courtesy
weiqht IPart of J Celis
]
resultinggel strip is appliedto an SD5-polyacrylamide9el (stepZ),
and the proteinsare separatedinto spotsby mass (step B) (b) In this

AN
P U R I F Y I N GD,E T E C T I N G PG
, D CHARACTERIZIN ROTEINS 95
 opposite end. A charged protein will migrate through the
gradient until ir reachesits isoelectricpoint (pI), the pH at
which the net charge of the protein is zero. This technique,
called isoelectric focusing (IEF), can resolve proteins that
differ by only one charge unit. Proteins that have been sepa-
rated on an IEF gel can then be separatedin a second di-
mension on the basis of their molecular weights. To accom-
plish this separation,the IEF gel strip is placed lengthwiseon
one outside edge of a sheetlike (two-dimensional, or slab)
polyacrylamide gel, this time saturated with SDS. When an
electric field is imposed, the proteins will migrate from the
IEF gel into the SDS slab gel and then separateaccording to
their masses.
The sequential resolution of proteins by charge and
mass can achieve excellent separation of cellular proteins
(Figure 3-35b). For example, two-dimensional gels have
been very useful in comparing the proteomes in undifferen-
tiated and differentiated cells or in normal and cancer cells
becauseas many as 1000 proteins can be resolvedas indi-
vidual spots simultaneously.Sophisticatedmethods have
been developed to permit the comparison of complex pat-
terns of proteins in two-dimensional gels from related, but
distinct,samples(e.g.,tissuefrom a normal versusa mutant
individual) to permir identification of differencesin rhe
types or amounts of proteins in the samples (seesection on
proteomics,below).
Liquid ChromatographyResolvesproteins
b y M a s s ,C h a r g e o
, r B i n d i n gA f f i n i t y
A third common technique for separating mixtures of pro-
teins or fragments of proteins, as well as other molecules,is
basedon the principle that moleculesdissolvedin a solution
can differentially interact (bind and dissociate)with a partic-
ular solid surface, depending on the physical and chemical
properties of the molecule and the surface.If the solution is
allowed to flow across the surface, then molecules that in-
teract frequently with the surface will spend more time
bound to the surface and thus flow past the ,,rrfac. -or.
slowly than molecules that interact infrequently with it. In
this technique, called liquid chromatography (LC), the sam-
ple is placed on rop of a tightly packed column of spherical
beadsheld within a glassor plastic cylinder.The samplethen
flows down the column, usually driven by gravitational or
hydrostatic forces alone or with the assistanceof a pump,
and small aliquots of fluid flowing out of the column, called
fractions, are collected sequentially for subsequentanalysis
for the presenceof the proteins of interest.The nature of the
beads in the column determineswhether the separation of
proteins dependson differencesin mass, charge, or binding
affinity.
Gel Filtration Chromatography proteins that differ in
masscan be separatedon a column composedof porous beads
made from polyacrylamide, dextran (a bacterial polysaccha-
ride), or agarose(a seaweedderivative)-a technique called gel
filtration chromatography. Although proteins flow around the
spherical beads in gel filtration chromatography they spend
96 o c H A p r E 3R | pRorEtN
s r R u c r u RA
EN DF U N c l o N
some time within the large depressionsthat cover a bead'ssur-
face. Becausesmaller proteins can penetrate into thesedepres-
sions more readily than larger proteins can, they travel through
a gel filtration column more slowly than larger proteins (Figure
3-37a). (In contrast, proteins migrate throwgh the pores in an
electrophoreticgel; thus smaller proteins move faster than
larger ones.) The total volume of liquid required to elute (or
separateand remove) a protein from a gel filtration column de-
pends on its mass: the smaller the mass, the more time it is
trapped on the beads,the greater the elution volume. By use of
proteins of known mass as standards to calibrate the column,
the elution volume can be usedto estimatethe massof a protein
in a mixture. A protein's shapeas well as its masscan influence
the elution volume.
lon-Exchange Chromatography In ion-exchangechro-
matographg a secondtype of liquid chromatography proteins
are separatedon the basis of differencesin their charges.This
techniquemakesuseof speciallymodified beadswhose surfaces
are coveredby amino groups or carboxyl groups and thus carry
either a positive charge (NHr*) or a negativecharge(COO ) at
neutral pH.
The proteins in a mixture carry various net charges at
any given pH. When a solution of a protein mixture flows
through a column of positively charged beads,only proteins
with a net negative charge (acidic proteins) adhere to the
beads; neutral and positively charged (basic) proteins flow
unimpeded through the column (Figure 3-37b). The acidic
proteins are then eluted selectivelyfrom the column by pass-
ing a solution of increasingconcentrationsof salt (a salt
gradient) through the column. At low sah concentratrons,
protein moleculesand beads are attracred by their opposite
charges. At higher salt concentrations, negative salt ions
bind to the positively charged beads, displacing the nega-
tively charged proteins. In a gradient of increasing salt
concentration, weakly bound proteins, those with relatively
low charge, are eluted first and highly charged proteins are
eluted last. Similarly, a negatively charged column can be
used to retain and fractionate basic (positively charged)
protelns.
Affinity Chromatography The ability of proteins to bind
specifically to other molecules is the basis of affinity chro-
matography. In this technique, ligand or orher molecules
that bind to the protein of interest are covalently attached to
the beads used to form the column. Ligands can be enzyme
substrates,inhibitors or their analogues,or other small mol-
eculesthat bind to specificproteins. In a widely usedform of
this techniqu e-anti b o dy -aff in ity, or immu n oaffinity, ch r o -
matography-the attached molecule is an antibody specific
for the desiredprotein (Figure 3-37c). (\7e discussantibod-
ies as tools to study proteins next).
An affinity column in principle will retain only those
proteins that bind the molecule attached to the beads; the
remaining proteins, regardlessof their charges or masses,
will pass through the column because they do not bind.
However, if a retained protein is in turn bound to other
molecules, forming a complex, then the entire complex is
(a) Gel filtrationchromatography (c)Antibody-affi
nity chromatography

Largeprotein Load in
pH 7 buffer
Small protein
O Protein
Layer Add buffer recognized Elute
to wash by antibody with
sample +
on proteins pH3
o Proteinnot
column through buffer
recognized
column by antibody

EIuted
Polymergel bead 2 1 fractions Antibody
1 2
Eluted
(b) lon-exchange
chromatography f ractions

Negativelycharged
proteino
Positivelycharged Anions
proternO retained Elute negatively
by beads charged protein
Layer with salt solution
sample (Nacl)fl fl
on
column

Eluted
fractions

Positively
charged
gel bead 3 2 1 fractions

EXPERIMENTAL FTGURE 3-37 Threecommonlyusedliquid the opposite chargebindto the beadsmoreor lesstightly,
chromatographic techniquesseparateproteinson the basisof depending on theirstructures.Boundproteins-inthiscase,
mass,charge,or affinity for a specificbinding partner.(a)Gel negativelycharged-are subsequentlyelutedby passing a salt
chromatography
filtration separatesproteinsthatdifferin sizeA gradient(usuallyof NaClor KCI)through the column As the ions
bindto the beads, they the
displace proteins (moretightlybound
mixture of proteins layered
iscarefully on thetop of a cylinder
packed with porousbeadsSmaller proteins
travelthroughthe proteinsrequire highersaltconcentration in orderto be released).
columnmoreslowlythanlargerproteinsThusdifferent proteins (c)In antibody-affinity
chromatography, a mixture of proteinsis
emerging in theeluateflowingout of the bottomof thecolumnat passed througha columnpacked with beadsto whicha specific
differenttimes(different elutionvolumes) canbe collected in antibody iscovalentlyattachedOnlyproteinwith highaffinityfor the
tubes,calledfractions(b)lon-exchange chromatography antibody isretained by thecolumn;allthe nonbinding proteinsflow
separate
separates proteins thatdifferin netchargein columns packed with through.Afterthe columniswashed, the boundproteiniseluted
beads thatcarry eithera positive
charge(shown here) or a with an acidicsolutionor someothersolutionthat disrupts the
special
negative charge. Proteins havingthe samenetchargeasthe beads antigen-antibody complexes; the released proteinthen flows out of
arerepelled andflow throughthe column, whereas proteins having thecolumn and iscollected

retained on the column. The proteins bound to the affinity disrupted. The ability of this technique to separatepartrcu-
column are then eluted by adding an excessof a soluble lar proteins dependson the selectionof appropriate binding
form of the ligand or by changing the salt concentration or partners that bind more tightly to the protein of interest
pH such that the binding to the moleculeon the column is than to other proteins.

. 97
P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S
 H i g h l y S p e c i f i cE n z y m ea n d A n t i b o d yA s s a y s
Can DetectIndividualProteins
The purification of a protein, or any other molecule, re-
quires a specific assay that can detect the presenceof the
molecule of interest as it is separatedfrom other molecules
(e.g.,in column or density-gradientfractions or gel bands or
spots).An assaycapitalizeson somehighly distinctivechar-
acteristicof a protein: the ability to bind a particular ligand,
to catalyze a particular reaction, or to be recognized by a
specific antibody. An assaymust also be simple and fast to
minimize errors and the possibilitythat the protein of inter-
est becomesdenaturedor degradedwhile the assayis per-
formed. The goal of any purification scheme is to isolate
sufficient amounts of a given protein for study; thus a use-
ful assaymust also be sensitiveenough that only a small
proportion of the available material is consumed by it.
Many common protein assaysrequire just 10 e to 1.0 12g
of material.
Chromogenic and Light-Emitting Enzyme Reactions
Many assaysare tailored to detect some functional aspectof a
protein.For example,enzymaticactivity assaysare basedon the
ability to detect the loss of substrateor the formation of prod-
uct. Some enzymatic assaysutilize chromogenic substrates,
which changecolor in the courseof the reaction. (Somesub-
strates are naturally chromogenic; if they are not, they can be
linked to a chromogenic molecule.)Becauseof the specificiryof
an enzyme for its substrate,only samples that contain the
enzyme will change color in the presenceof a chromogenic
substrate; the rate of the reaction provides a measure of the
quantity of enzymepresent.Enzymesthat catalyzechromogenic
reactionscan also be fused or chemically linked to an antibody
and used to "report" the presenceor location of an antigen to
which the antibody binds (seebelow).
Antibody Assays As noted earlier, antibodies have the
distinctive characteristicof binding tightly and specificallyto
antigens.As a consequence,preparations of antibodies that
recognizea protein antigen of interest can be generatedand
used to detect the presenceof the protein, either in a com-
plex mixture of other proteins (finding a needle in a
haystack, as it were) or in a partially purified preparation of
a particular protein. The tight binding of the antibody to its
antigen, and thus the presenceof the antigen, can be visual-
ized by labeling the antibody with an enzyme)a fluorescent
molecule, or radioactive isotopes. For example, luciferase,
an enzyme present in fireflies and some bacteria, can be
linked to an anribody. In the presenceof ATp and the sub-
strate luciferin, luciferase catalyzesa light-emitting reaction.
In either case,after the antibody binds to the protein of in-
98 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
naturally fluorescent protein found in jellyfish (seeFigure
9-12). Alternatively after the first antibody binds to its target
protein, a second, labeled antibody is used to bind to the
complex of the first antibody and its target. This combina-
tion of two antibodiespermits very high sensitivity in the de-
tection of a targetprotein.
To generatethe antibodies, the intact protein or a frag-
ment of the protein is injected into an animal (usually a rab-
bit, mouse, or goat). Sometimesa short synthetic peptide of
10-15 residuesbasedon the sequenceof the protein is used
as the antigen to induce antibody formation. A synthetic
peptide, when coupled to a large protein carrier, can induce
an animal to produce antibodies that bind to that portion
(the epitope) of the full-sized, natural protein. Biosyntheti-
cally or chemically attaching the epitope to an unrelatedpro-
tein is called epitope tagging. As we'll see throughout this
book, antibodies generatedusing either peptide epitopes or
intact proteins are extremely versatilereagentsfor isolating,
detecting,and characterizingprotelns.
Detecting Proteins in Gels Proteins embedded within a
one- or two-dimensionalgel usually are not visible. The two
general approachesfor detecting proteins in gels are either to
Iabel or stain the proteins while they are still within the gel or
to electrophoreticallytransfer the proteins to a membrane
made of nitrocellulose or polyvinylidene difluoride and then
detect them. Proteinswithin gels are usually stainedwith an
organic dye or a silver-basedstain, both detectedwith normal
visible light, or with a fluorescent dye that requires specialized
detection equipment. Coomassieblue is the most commonly
usedorganic dye, typically usedto detect=1000 ng of protein,
with a lower limit of detectionof =4-1,0 ng. Silver stainingor
fluorescencestainingare more sensitive(lower limit of =1 ng).
Coomassie and other stains can also be used to visualize
proteins after transfer to membranes;however.the most com-
mon method to visualize proteins in these membranes is
immunoblotting, commonly called'Westernblotting.
Western blotting combines the resolving power of gel
electrophoresisand the specificityof antibodies.This mul-
tistep procedure is commonly used to separateproteins
and then identify a specificprotein of interest. As shown
in Figure 3-38, two different antibodies are used in this
method, one that is specific for the desiredprotein and a
secondthat binds to the first and is linked to an enzymeor
other molecule that permits detectionof the first antibody
(and thus the protein of interesr to which it binds). En-
zymesto which the secondantibody is attachedcan either
generatea visible colored product or, by a processcalled
chemiluminescence,produce light that can readily be
recorded by film or a sensitive detector. The two different
antibodies, sometimescalled a "sandwich," are used to
amplify the signalsand improve sensitivity.If an antibody
is not available,but the geneencodingthe protein is avail-
able and can be used to expressthe protein, recombinant
DNA methods (Chapter 5) can irrcorporatea small peptide
epitope (epitope tagging) into the normal sequenceof the
protein that can be detected by a commercially available
antibody to that epitope.
Illl+ Technique
Animation:lmmunoblotting
Electrophoresisand transfer Antibody detection ! Chromogenic detection
I

S D S - p o l y a c r y l a m i d e g e lM e m b r a n e lncubate with Reactwith substrate

A b ,( J ) ; Incubatewith enzYme- for Ab2-linkedenzyme
washexcess linkedAb2 (Y);
wash excess

A EXPERIMENTAL FIGURE 3-38 Westernblotting u n b o u nAd b ' . S t e pE : T h e m e m b r a ni sei n c u b a t ewdi t ha s e c o n d

(immunoblotting) combinesseveraltechniquesto resolveand antibody (Ab2)thatspecifically recognizes andbindsto thefirstAb1
Thissecond antibody iscovalently linked eitheran enzyme
to (e.9.,
detect a specificprotein. StepE: Aftera proteinmixturehasbeen
throughan SDSgel,theseparated bands(orspots, alkaline phosphatase, which can catalyze a chromogenic reaction),
electrophoresed
for a two-dimensionalgel)aretransferred(blotted)
fromthe gelonto radioactive isotope, or someothersubstance whosepresence can
removedStepZ: be detected with greatsensitivity Step4: Finally, the location and
a porousmembraneJrom whichit isnot readily
isfloodedwith a solution of antibody(Abr)specific amountof boundAb2aredetected (e.g, by a deep-purple
Themembrane
protern
for the desired andallowedto incubate for a while.Onlythe precipitate fromchromogenic reaction), permitting the
membrane-bound bandcontaining thisproteinbindsthe antibody, electrophoretic mobility (and therefore the mass) of the destred
(whoseposition cannotbe oroteinto be determined, aswellasitsquantity (based on band
forminga layerof antibody molecules
seenat thispoint) Thenthe membrane iswashedto remove intensrtv).

RadioisotopesAre lndispensableTools Radioisotopes Useful in Biological Research Hundreds

f o r D e t e c t i n gB i o l o g i c aM
l olecules
A sensitivemethod for tracking a protein or other biological
molecule is by detecting the radioactivity emitted from ra-
dioisotopesintroduced into the molecule. At least one atom
in a radiolabeled molecule is present in a radioactive form,
called a radioisotope.
of biological compounds (e.g.,amino acids,nucleosides,and
numerous metabolic intermediates)labeled with various ra-
dioisotopes are commercially available. These preparations
vary considerably in their specific actiuity, which is the
amount of radioactivity per unit of material, measured in

ISOTOPE }|ALF-tIIE

Phosphorus-32 1'4.3days

60.4 days dom/mmol) are available.Likewise' commercialpreparations

Iodine-125 tH-l"b.l.d nucleic acid precursorshave much ligher spe-
oi
1aC-labeled
Sulfur-35 87.5days cific activities than those of the corresponding
preparations.In most experiments'the former are preferable
Tritium (hydrogen-3) 12.4 years t".uot. they allow RNA or DNA to be adequatelylabeled
alter a shorter time of incorporation or require a smaller cell
Carbon-14 5730.4years sample.Various phosphate-containingcompounds in which
every phosphorus atom is the radioisotope phosphorus-32

P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S 99

 a^rereadily available. Becauseof their high specific activity,
32P-labeled
nucleotides are routinely ur.d to label nucleic
acids in cell-freesystems.
Labeled compounds in which a radioisotope replaces
atoms normally present in the molecule have virtually the
same chemical properties as the corresponding nonlabeled
compounds. Enzymes,for instance,generally cannot distin-
guish betweensubstrateslabeledin this way and their nonla-
beled substrates.The presenceof such radioactive atoms is
indicatedwith the isotopein brackets(no hyphen) as a prefix
(e.g., [3H]leucine).In .ontrurt, labeling almost all biomole-
cules (e.g., protein or nucleic acid) with the radioisotope
iodine-125 (12sI)requires the covalent addition of 12sIto a
molecule that normally does nor have iodine as part of its
structure.Becausethis labelingproceduremodifies the chemi-
cal structure, the biological activity of the labeled molecule
may differ somewhat from that of the nonlabeledform. The
presenceof such radioactive atoms is indicated with the iso-
tope as a prefix with a hyphen (no bracket) (e.g.,125l-trypsin).
Standardmethods for labeling proteins with 12sIresult in co-
valent attachmentof the 125Iprimarily to the aromatic rings of
tyrosine side chains (mono- and diiodotyrosine).
Labeling Experiments and Detection of Radiolabeled
Molecules Whether labeledcompoundsare detectedby au-
toradiography, a semiquantitativevisual assay or their radioac-
tivity is measuredin an appropriate ',counter," a highly quanti- protein of interestcan be usedto precipitatethe protein away
tative assaythat can determine the amount of a radiolabeled
compound in a sample, depends on the nature of the experi-
ment. In some experiments,both types of detection are used.
In one use of autoradiographS a tissue,cell, or cell con-
stituent is labeledwith a radioactivecompound, unassociated periment, a fluorescent compound
radioactivematerial is washed away, and the structure of the
sample is stabilized either by chemically cross-linking the
macromolecules('fixation') or by freezingit. The sample is
then overlaid with a photographic emulsion sensitiveto radi-
ation. Developmentof the emulsion yields small silver grains
whose distribution correspondsto that of the radioactivema-
terial and is usuallydetectedby microscopy.Autoradiographic
studiesof whole cellswere crucial in determiningthe intracel-
lular siteswhere various macromoleculesare synthesizedand
the subsequentmovements of these macromoleculeswithin
Quantitative measurementsof the amount of radioactiv-
ity in a labeledmarerial are performed with severaldifferent
100 . c H A p r E3R | p R o r E t N
s r R U c r u RAEN DF U N c l o N
disintegrations per minute per small pixel of surface area.
These instruments, which can be thought of as a kind of
reusable electronic film, are commonly used to quantitate
radioactive molecules separated by gel electrophoresisand
are replacingphotographic emulsionsfor this purpose.
A combination of labeling and biochemical techniques
and of visual and quantitative detectionmethods is often em-
ployed in labeling experiments.For instance,to identify the
major proteins synthesizedby a particular cell type, a sample
of the cells is incubated with a radioactive amino acid (e.g.,
[35S]methionine)for a few minutes, during which time the L-
beled amino acid mixes with the cellular pool of unlabeled
amino acids and some of the labeled amino acid is biosyn-
thetically incorporated into newly synthesizedprotein. Subse-
quently unincorporatedradioactive amino acid is washed
away from the cells. The cells are harvested,the mixture of
cellular proteins is extractedfrom the cells (for example, by a
detergentsolution), and then separatedby gel electrophore-
sis; and the gel is subjectedto autoradiography or phospho-
rimager analysis.The radioactive bands correspondto newly
synthesizedproteins, which have incorporatei the radiola-
beled amino acid. Alternatively, the proteins can be resolved
by liquid chromatographS and the radioactivity in the eluted
fractions can be determinedquantitatively with a counter.To
detect only one specificprotein, rather than all the proteins
biosyntheticallylabeled this way, a specific antibody to the
from the other proteins in the sample (immunoprecipitation).
The precipitate is then solubilized in a detergenr,separating
the antibody from the protein, and the sampleis subjectedto
SDS-PAGEfollowed by autoradiography.In this type of ex-
that is activated by the
radiation is often infusedinto the gel so that the light emitted
can be used to detect the presenceof the labeled protein,
either using film or a two-dimensional electronicdetector.
Pulse-chase experimentsare particularly usefulfor tracing
changesin the intracellular location of proreins or the modi-
fication of a protein or metabolit. ou.i time. In this experi-
mental protocol, a cell sample is exposed to a radiolabeled
compound that can be incorporated or otherwiseattachedto
a cellular molecule of interest-the "pulse"-for a brief pe-
riod of time, then washedwith buffer to remove the unincor-
porated label, and finally incubated with an unlabeled form
of the compound-the "chase" (Figure 3-39). Samplestaken
periodically during the chaseperiod are assayedto determine
the location or chemicalform of the radiolabel as a function
of time. Often, pulse-chaseexperiments,in which the protein
is detected by autoradiography after immunoprecipitation
and SDS-PAGE,are usedto follow the rate of synthesis,mod-
ification, and degradation of proteins by adding radioactive
amino acid precursorsduring the pulse and then detectingthe
amounts and characteristicsof the radioactiveprotein during
the chase.One can thus observepostsyntheticmodifications
of the protein that changeits electrophoreticmobility and the
rate of degradation of a specificprotein. A classicuse of the
pulse-chasetechniquewas in studiesto elucidatethe pathwav
traversedby secretedproteins from their site of synihesisin
the endoplasmicreticulum to the cell surface(Chapter 14).
 key component, which provides a measure of the relative
abundancesof each of the ions in the sample.The fourth es-
sential component is a computerized data systemthat is used
to calibrate the instrument; acquire,store, and processthe re-
Normal sulting data; and often direct the instrument automatically
protein to collect additional specific types of data from the sample'
p-e.
based on the initial observations. This type of automated
feedback is used for the tandem MS (MS/MS) peptide-
(b) sequencingmethods describedbelow.
m The two most frequently usedmethods of generatingions
Mutant
'.,& "' of proteins and protein fragmentsare (1) matrix-assistedlaser
'Drotein o- desorption/ionization(MALDI) and (2) electrospray(ES)' In
MALbI (Figure 3-40) the peptide or protein sampleis mixed
l"j,\'J,"
;:::,'J."'Ll3,'
il?HT":',i"H "".'
with a low-molecular-weight,W-absorbing organic acid (the
matrix) and then dried on a metal tatget. Energy from a laser
ionizes and vaporizes the sample producing singly charged
A EXPERTMENTAL FIGURE 3-39 Pulse-chase experimentscan
molecular ions from the constituentmolecules'In ES (Figure
track the pathway of protein modificationor movement
3-41.a),the sample of peptidesor proteins in solution is con-
within cells.(a)Tofollowthefateof a specific newlysynthesized
proteinin a cell,cellswereincubated with [3sS]methionine for 0 5 hr verted into a fine mist of tiny droplets by spraying through a
(thepulse) to labelallnewlysynthesized proteins,andthe radioactive
aminoacidnot incorporated intothe cellswasthenwashedaway
Thecellswerefurtherrncubated (thechase) for varying timesup to
24 hours,andsamples from each timeof chase weresubjected to
immunoprecipitation to isolate onespecific protein(here,the low-
density lipoprotein receptor) SDS-PAGE of the immunoprecipitates
followedby autoradiography permitted visualizationof the one
specificprotein, whichis initiallysynthesized asa smallprecursor (p) arated by the mass analyzeron the basis of their mlz.
andthen rapidlymodified to a larger matureform (m) by addition of The two most frequently used mass analyzersare time-
carbohydrates Abouthalfof the labeled proteinwasconverted from of-flight (TOF) instruments and ion traps. TOF instruments
p to m duringthe pulse,the restwasconverted after0.5 hourof e*ploit the fact that the time it takes an ion to passthrough
chaseTheproteinremains stablefor 6-8 hoursbeforeit begins to
the length of the analyzer before reaching the detector is
be degraded (indicated by reduced band (b)
intensity). The same
experiment wasperformed in cellsin whicha mutantformof the
proteinismadeThemutantp formcannotbe properly converted to
the m form,andit is morequicklydegraded thanthe normalprotein
lAdapted fromK F Kozarsky, H A Brush, andM Krieger, 1986,J CellEloi
102(5)1567-1s7s I Roshanl(eab 021-66950639 Metal
I lonization
target
+++
MassSpectrometryCan Determinethe Mass O@o

and Sequenceof Proteins

Mass spectrometry (MS) is a powerful technique for charac- Sample
tertzingproteins.MS is particularly useful in determining the
'With
mass of a protein or fragments of a protein. such in-
formation in hand, it is also possibleto determinepart of or
@

Lightestions
t
q)

all the protein's sequence.This method permits the very c arrive at

highly accuratedirect determination of the ratio of the mass detector first
(m) of a charged molecule (ion) to its charge (z), ot mlz. Time
Techniquesare then used to deducethe absolutemass of the
a EXPERIMENTAL FIGURE 3-40 Molecularmasscan be
ion. There are four key features of all mass spectrometers. laserdesorption/ionization
determinedby matrix-assisted
The first is an ion source,from which charge, usually in the massspectrometry'ln a MALDI-TOF
time-of-flight (MALDI-TOF)
form of protons, is transferred to the peptide or protein massspectrometer, pulses of lightfroma laserionizea proteinor
molecules.The formation of these ions occurs in the pres- peptidemixture that isabsorbed on a metaltarget(stepE) An
enceof a high electric field that then directs the chargedmo- fieldaccelerates
electric the ionsin the sample towardthe detector
lecular ions into the second key component' the mass ana- (steosEl andEl).Thetimeto the detector to the
isproportional
lyzer. The mass analyzer,which is always in a high vacuum squarerootof the mass-to-charge (mlz) For
ratio. ionshaving the
chamber, physically separatesthe ions on the basis of their samecharge, the smaller ionsmovefaster(shorter timeto the
differing mass-to-charge(mlz) ratios. The mass-separated detector).Themolecular weightiscalculated usingthetimeof flight
ions are subsequentlydirected to strike a detector,the third of a standard

AN
P U R I F Y I N GD,E T E C T I N G PG
, D CHARACTERIZIN ROTEINS O 101
 (a) Electrospray
needle Atmosphere
Vacuum

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Mass Detector
analyzer
Droplets rons
containing Mass spectrometer
solvatedions

Electrosprayionization

(b) 568.65
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20 473.15 549.46 1124.44 1398.48
q) 261.30
CC 10
0 1536.14
300 400 500 600 700 800 900 1000 1100 1200 1300 14oO 15oO 1600
m/z
EXPERIMENTAL FIGURE 3-41 Molecularmassof proteins specificpeptideioncanbe selected for fragmentatron intosmaller
and peptidescan be determinedby electrosprayionization ionsthatarethenanalyzed anddetected. TheMS/MS (also
spectrum
ion-trap massspectrometry.(a)Electrospray (ES)ionization calledthe product-ion spectrum) provides detailedstructural
convertsproteins andpeptides in a solution intohighlycharged information aboutthe parention,including sequence informationfor
gaseous ionsby passing the solutionthrougha needle(forming the peptidesHere,the ionwith a mlzof 836.47wasselected,
droplets)thathasa highvoltageacross it (chargingthe droplets). fragmented andthe m/z massspectrumof the productionswas
Evaporation of thesolvent produces gaseous ionstharenrera mass measured Notethereis'nolongeran ionwith an m/zoI g36.47
spectrometer.Theionsareanalyzed by an ion-trapmassanalyzer that present because it wasfragmented. Fromthevarying sizesof the
thendirectsionsto the detector.(b) Toppanet:Massspectrumof a productions,the understanding that peptidebondsareoftenbroken
mixtureof threemajorandseveral minorpeptides ispresented asthe in suchexperiments, the knownmlzvalues for individualaminoacid
relative
abundance of the ionsstrikingthe detector (y axis)asa fragments, anddatabase information, the sequence of the peptide,
functionof the mass-to-charge (m/z)ralio(x axis).Bottornpanel:ln FIIVGYVDDTQFVR, canbe deduced. [part(a)based ona fiourefrom
an MS/MSinstrument (suchasthe iontrapshownin part(a)),a 5 Carr;part(b),unpublished
datafromS Carr I

102 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
proportional to the square root of mlz (smaller ions move from the polypeptide and identified by high-pressureliquid
fasterthan larger oneswith the samecharge,seeFigure 3-40). chromatography.The polypeptide is left one residueshorter,
In ion-trap analyzers,tunable electric fields are used to cap- with a new amino acid at the N-terminus. The cycle is re-
'trap,' ions with a specific mlz and to sequentially peated on the ever shortening polypeptide until all the
ture, or
pass the trapped ions out of the analyzer onto the detector residueshave been identified.
(seeFigure 3-4ta). By varying the electric fields, ions with a Before about 1985' biologistscommonly used the Edman
wide range of mlz values can be examined one by one, pro-
ducing a massspectrum,which is a graph of mlz (x axis) ver-
sus relative abundance(y axis) (Figure 3-41'b,top panel).
In tandem, or MS/MS, instruments, any given parent ion in
the original mass spectrum (Figure 3-41b, top panel) can be
mass-selected, broken into smallerions by collisionwith an in- merous model organismsis expanding rapidly. As discussed
ert gas, and then the mlz and relative abundancesof the result- in Chapter 5, the sequencesof proteins can be deducedfrom
ing fragment ions measured (Figure 3-41b, bottom panel), all DNA sequencesthat are predictedto encodeproteins'
within the samemachinein about 0.1 s per selectedparent ion. A powerful approach for determining the primary struc-
This secondround of fragmentationand analysispermits the t,r.. o? an isolated protein combines MS and the use of se-
sequencesof short peptides (<25 amino acids) to be deter- quencedatabases.First' the peptide "mass fingerprint" of the
peptide mass fingerprint is the
mined, becausecollisional fragmentation occurs primarily at irot.i.r is obtained by MS. A
peptide bonds, so the differencesin massesbetweenions corre- iist of the molecular weights of peptides that are generated
spond to the in-chain massesof the individual amino acids,
permitting deductionof the sequencein conjunctionwith data-
basesequenceinformation (Figure3-41'b,bottom panel).
Mass spectrometryis highly sensitive,able to detectas lit-
tle as 1 x 10-15 mol (100 attomoles)of a peptideor 10 x
10-15 mol (10 femtomoles)of a protein of 200,000 M$(. Er-
rors in mass measurementaccuracyare dependentupon the
specific mass analyzer used, but are typically =0.01 percent ProteinConformationls Determined
for peptidesand 0.05-0.1 percent for proteins. As described
by SophisticatedPhysicalMethods
in the proteomicssectionthat follows, it is possibleto useMS
to analyze complex mixtures of proteins as well as purified In this chapter,we have emphasizedthat protein function is
proteins. Most commonly, protein samples are digested by dependent on protein structure. Thus, to figure out how a
proteases,and the peptide digestionproducts are subjectedto prot.i.t works, its three-dimensional structure must be
analysis. An especiallypowerfully application of MS is to Lno*n. Determining a protein's conformation requiresso-
take a complex mixture of proteins from a biological speci- phisticated physical methods and complex analysesof the
men, digestit with trypsin or other proteases'partially sepa- .*p.ri-..tt"l data. \(e briefly describethree methods used
rate the componentsusing liquid chromatography (LC), and to generatethree-dimensionalmodels of proteins'
then transferthe solution flowing out of the chromatographic
column directly into an ES tandem mass spectrometer.This X-ray €rystallography The use of x-ray crystallographyto
technique, called LC-MSIMS, permits the nearly continuous determine the three-dimensionalstructuresof proteins was pio-
analysisof a very complex mixture of proteins'
The abundancesof ions determined by mass spectrome-
try in any given sample are relative, not absolute' values.
Therefore, if one wants to use MS to compare the amounts
of a particular protein in two different samples(e.g.,from a
normal versusa mutant organism), it is necessaryto have an
internal standard in the sampleswhose amounts do not dif- atoms of the crystal scatterthe x-rays' which produce a drfftac-
fer between the two samples. One then determines the tion pattern of discretespots when they are interceptedby pho-
amounts of the protein of interestrelative to that of the stan- tographic film or an electronicdetector (Figure 3-42)' Suchpat-
dard in each sample.This permits quantitatively accuratein- t...r, extremely complex---composedof as many as 25,000
"..
tersamplecomparisonsof protein levels.

ProteinPrimaryStructureCan Be Determinedby
ChemicalMethods and from Gene Sequences
The classicmethod for determining the amino acid sequence
of a protein is Edman degradation. In this procedure, the
free amino group of the N-terminal amino acid of a polypep- three-dimensionalelectron density map in hand' one then "fits"
tide is labeled, and the labeled amino acid is then cleaved a molecular model of the protein to match the electron density,

I 103
P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N s
 (a) proteins. To date, the detailed three-dimensionalstructures of
more than 10,000 proteins have beenestablished.

Cryoelectron Microscopy Although someproteinsreadily

crystallize, obtaining crystals of others-particularly large
multisubunit proteins and membrane-associatedproteins-
requires a time-consuming trial-and-error effort to find just the
right conditions,if they can be found at all. (Growing crystals
suitablefor structural studiesis as much an art as a science.)
There are several ways to determine the structures of such
difficult-to-crystallize proteins. One is cryoelectron mi-
croscopy. In this technique, a protein sample is rapidly frozen
in liquid helium to preserveits structure and then examined in
the frozen, hydrated state in a cryoelectron microscope. pic-
tures of the protein are taken at various anglesand recorded on
film using a low dose of electronsto prevent radiation-induced
damageto the structure. Sophisticatedcomputer programs an-
alyze the imagesand reconstruct the protein's structure in three
dimensions. Recent advancesin cryoelectron microscopy per-
mit researchersto generatemolecular models that can help
provide insight into how the protein functions. The use of cry-
oelectron microscopy and other types of electron microscopy
for visualizing cell structures is discussedin Chapter 9.

NMR Spectroscopy The three-dimensionalstrucures of

small proteins containing as many as 200 amino acidscan be
studied with nuclear magneric resonance (NMR) spec-
troscopy. In this technique, a concentratedprotein solution
is placed in a magnetic field, and the effects of different ra-
dio frequencieson the nuclear spin statesof different atoms
are measured.The spin state of any atom is influenced by
neighboring atoms in adjacent residues,with closely spaced
residues having a greater influence than distant residues.
From the magnitude of the effect, the distances between
residues can be calculated by a triangulation-like process;
these distances are then used to generate a model of the
three-dimensionalstructure of the protein.
Although NMR does not requiie the crystallizarion of a
protein, a definite advantage, this technique is limited to
a proteincan be determined.(a)Basic components proteins smaller than about 20 kDa. However. NMR analv-
of an x_ray
crystallographicdetermination. Whena narrowbeamof x_rays strikes sis can also be applied to isolated protein domains, which
a crystal,partof it passesstraightthroughandthe restrsscattered can often be obtained as stable structures and tend to be
(diffracted)
in various directionsTheintensity of thediffracted small enough for this technique.
waves, whichformperiodic arrangements of diffraction
spots,is
recorded on an x-rayfilm or with a solid-stateelectronic
detector.
(b)X-raydiffractionpatternfor a proteincrystalcollected on a solid_
statedetector. Fromcomplex analyses of patternsof spotslikethis
one,the location Purifying, Detecting, and Characterizingproteins
of the atomsin a proteincanbe determined. [part
(a)adaptedfrom L Stryer,1995,Biochemistry,
4Ihed, W H Freeman
and r Proteins can be separated from other cell components
Company,p 64; part(b)courtesy of J Berger.j and from one another on the basis of differencesin their
physicaland chemicalproperties.
r Various assaysare used to detect and quantify Droteins.
Some assaysuse a light-producing reactironto generatea
readily detectedsignal. Other assaysproduce an amplified
colored signal with enzymesand chromogenic substrates.
r Centrifugation separatesproteins on the basis of their
rates of sedimentation, which are influenced by their
massesand shapes.

104 . c H A p r E R3 p R o r E t Ns r R U c r u R EA N D F U N c l o N
|
r Electrophoresis separatesproteinson the basisoftheir rates
of movement in an applied electric field. SDS-polyacrylamide
gel electrophoresis(PAGE) can resolve polypeptide chains
differing in molecular weight by 10 percentor less(seeFig-
ure 3-35). Two-dimensional gel electrophoresisprovides
additional resolution by separating proteins first by charge
(first dimension)and then by mass(seconddimension).
r Liquid chromatography separatesproteins on the basis
of their rates of movement through a column packed with
spherical beads.Proteins differing in mass are resolved on
gel filtration columns; those differing in charge, on ion-
exchange columns; and those differing in ligand-binding
properties, on affinity columns, including antibody-based
affinity chromatography(seeFigure 3-37).
r Antibodies are powerful reagentsused to detect, quan-
tify, and isolate proteins.
'Western
r Immunoblotting, also called blotting, is a fre-
quently usedmethod to study specificproteins that exploits
the high specificity and sensitivity of protein detection by
antibodies and the high-resolution separation of proteins
by SDS-PAGE.
r Radioisotopesplay a key role in the study of proteins and
other biomolecules.They can be incorporated into mole-
cules without changing the chemical composition of the
molecule,or as add-on tags.They can be usedto help detect
the synthesis,location, processing,and stability of proteins.
r Autoradiography is a semiquantitativetechnique for de-
tecting radioactively labeled molecules in cells, tissues,or
electrophoreticgels.
r Pulse-chaselabeling can determine the intracellular fate
of proteinsand other metabolites(seeFigure 3-39).
r Mass spectrometry is a very sensitiveand highly precise
method of detecting, identifying, and characterizing pro-
teins and peptides.
r Three-dimensionalstructuresof proteins are obtained by
x-ray crystallography,cryoelectron microscopS and NMR
spectroscopy.X-ray crystallography provides the most
detailedstructuresbut requiresprotein crystallization. Cry-
oelectron microscopy is most useful for large protein com-
plexes, which are difficult to crystallize. Only relatively
small proteins are amenableto NMR analysis.
Proteomics
El
For most of the twentieth century,the study of proteins was
restrictedprimarily to the analysisof individual proteins. For
example,one would study an enzymeby determiningits enzy-
matic activity (substrates,products.rate of reaction,require-
ment for cofactors,pH, etc.),its structure' and its mechanism
of action. In some cases,the relationshipsberweena few en-
zymesthat participate in a metabolic pathway might also be
studied.On a broader scale,the localizationand activity of an
enzymewould be examined in the context of a cell or tissue.
The effectsof mutations, diseases,or drugs on the expression
and activity of the enzyme might also be the subject of
investigation.This multipronged approach provided deep in-
sight into the function and mechanismsof action of individual
interacting proteins'
-Ho*.u.r,or relatively small numbers of
proteins
this one-by-oneapproach to studying proteins does
not efficiently provide insights into a global picture of what is
happening in the proteome of a cell, tissue, or entire organism'
Proteomicsls the Study of All or a LargeSubset
of Proteinsin a BiologicalSYstem
The advent of genomics (sequencingof genomic DNA and its
associatedtechnologies' such as simultaneous analysis of the
levels of all mRNAs in cells and tissues)clearly showed that a
studies:
In a given sample (whole organism, tissue,cell' subcellu-
r compartment), what fraction of the whole proteome is
expressed(i.e.,which proteinsare present)?
r Of those proteins presentin the sample,what are their
relative abundances?
r What are the relative amounts of the different splice
forms and chemically modified forms (e.g.,phosphorylated,
methylated, fatty acylated) of the proteins?
r Which proteins are presentin large multiprotein com-
'lfhat
plexes,anl which proteins are in each complex? are
interact?
the functions of thesecomplexesand how do they
r 'When the state (e.g.'growth rate' stageof cell cycle, dif-
ferentiation, stresslevel) of a cell changes'do the proteins in
the cell or secretedfrom the cell changein a characteristic
r Can such fingerprint-like changesbe used for diagnostic
purposes?For example, do certain cancersor heart disease
iurrr. .hur".teristic changesin blood proteins? Can the
proteomic fingerprint help determine if a given cancer is re-
,irr".t, or sensitiveto a particular chemotherapeuticdrug?
Proteomic fingerprints can also be the starting point for
studiesof the meihanisms underlying the changeof state'
Proteins (and other biomolecules)that show changesthat
are diagnostic of a particular state are calledbiomarkers'
Can changesin the proteome help define targetsfor drugs
suggestmechanismsby which that drug might induce
*ic side effects?If so, it might be possibleto engineer
modified versionsof the drug with fewer side effects'
o
PROTEOMTCS 105
 Theseare just a few of the questionsthat can be addressedus-
ing proteomics.The methods used to answer thesequestions
are as diverseas the questionsthemselves,and their numbers
are growing rapidly.
AdvancedTechniquesin MassSpectrometry
Are Criticalto ProteomicAnalysis
Advances in proteomics technologies(e.g., mass specrrome-
try) are having a profound effect on the types of questionsthat
can be practically studied. For many years,rwo-dimensional
gel electrophoresishas allowed researchersto separate,dis-
plaS and characterizea mixture of proteins (Figure 3-36).
The spots on a two-dimensional gel can be excised,the pro-
tein fragmentedby proteolysis(e.g.by trypsin digestion),and
the fragments identified by MS. An alternative to this two-
dimensional gel method is high throughput LC-MS/MS.
Figure 3-43 outlines the general LC-MS/MS approach in
which a complex mixture of proteins is digested with a
protease, the myriad resulting peptides are fractionated by
LC into multiple, less complex fractions, which are slowly
but continuously injected by electrosprayionization into a
tandem mass spectrometer.The fractions are then sequen-
tially subjectedto multiple cyclesof MS/MS until sequences
of many of the peptidesare determinedand used to identify
from databasesthe proteins in the original biological sample.
An example of the use of LC-MS/MS to identify many of
the proteinsin eachorganelleis seenin Figure 3-44. Cellsfrom
murine (mouse)liver tissuewere mechanicallybroken to re-
lease the organelles, and the organelles were partially sepa-
rated by density-gradient centrifugarion. The locations of
the organelles in the gradient were determined using im-
munoblotting with antibodies that recognize previously iden-
tified, organelle-specificproteins. Fractions from the gradient

Podcast:Use of Mass Spectrometryin Cell Biology

Electrospray
ionizationmassspectrometer
1 s tM S ^ooH Collision 2 n dM S Detector
uun fragmentation
Complex D{L
mixtureof NHz
peptides

'. .;
4321
I
tn
I
separation
into fractionsII
J prot"ur" of less o-c\a
| ( e . g . t, r y p s i n ) complex NI TJ

mixtures g
cooH

coNH2
Selectmassof a Sequence
Protein i,-eptidrion from seiecte{lpeFl;ij*
mixture in a fraction
biological
sample Repeatfor other peptidesin the fraction

Repeatfor multiplefractionsfrom the LC outflowto sequencemost peptides

in the startingcomplexpeptidemixture

Compareresultswith databases
t
to identifyproteinsin the original
b i o l o g i c asla m p l e

electrospray
ionization
into a tandemmassspectrometer The
fractions
arethensequentially subjected to multiple
cyclesof MS/MS
untilmassesandsequences of manyof the peptides aredetermined
andusedto identifythe proteinsin the original
biological
sample
throughcomparison with proteindatabases. ona fiqure
[Based provided
by5 Carr.l

106 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
(a) < EXPERIMENTALFIGURE 3-rt4 Density-gradientcentrifugation
can be usedto identify manyof the proteinsin
and LC-MS/MS

Mitochondria

c previously
thatrecognize identified, proteinsFractions
organelle-specific
o
were
fromthe gradient subjectedto proteolysis
andLC-MS/MS to
.9 i>

vc
o
t--rnontottins
f
F1 ATP synthase
E a r l ye n d o s o m e
E5 a n t i g e n1 125(1):187-199]
organelle. L J Fosteretal
[From ,2006,Cell
1,2-a-mannosidase
Proteolysisand LC-MS/MS
eukaryotic cell, the yeastSaccharomycescereuisiae'Approxt-
Localizationof proteins
mately 500 complexeshave been identified, with an average
of 4.9 distinct proteins per complex' and thesein turn are in-
roo
o volved in at least400 complex-to-complexinteractions'Such
systematicproteomic studiesare providing new insights into
c
0)
c
tire organiiation of proteins within cells and how proteins
work together to permit cellsto live and function'

(b)
32 gradientfractions

l. Proteomics
22,260peptides

L
2 , 19 7 p r o t e i n s

I
1,500 p r o t e i n sq u a n t i f i e d subcellularlevels.
.t. r Proteomicsprovides insightsinto the fundamental organ-
1,404proteinslocalized* Nucleus196 izationof proteins within cells,and how this organization is
.1. influencedby the state of the cells (e.g.,differentiation into
Cytosol 488 + 1,258cytoplasmicProteins distinct cell types; responseto stress'disease,and drugs)'
.t. r A wide variety of methods are used for proteomic analy-
ses,including two-dimensional gel electrophoresis,density-
Proteosome
Mitochondrion
< cytoplasmic
50 gradient centrifugation, and mass spectroscopy(MALDI-
zgt organelles
TOF and LC-MS/MS)'
omics has helped begin to identify the proteomesof
les ("organelleproteomeprofiling") and the organ-
of individual proteins into multiprotein complexes
eract In a complex network to support life and cel-
lular function

ER/Golgi Early
vesicles enoosomes
220 76

Proteomics combined with molecular geneticsmethods

are currently being usedto identify all protein complexesin a

P E R S P E C T I VF
EOS RT H E F U T U R E O 107
 motif 68 quaternary str]uctvre 72
motor proteins 85 r ate-zonal centrifugation 93
peptide bond 55 secondarystructure 65
polypeptides 65 tertiary structure 67
primary structure 66 ubiquitin 88
proteasomes54 v^^*go
protern 66 'Western
blotting 98
proteome 64 x-ray crystallogr aphy 10 3
proteomics105

Review the Concepts

7, The three-dimensional structure of a protein is deter_

built. Becausesubunits of the complex may already be solved
by crystallographSa composite,tru.tur. consistingof the
x-ray-derived subunit structuresfit to the EM-deriveJ model
will be generated.An example of this approach for an elec_
tron-transport "supercomplex" is describedin Chapter 12.
Methods for rapid structure determination combined
with identificarion of novel substratesand inhibitors will
mined by its primary, secondary,and tertiary structures.De-
fine the primary, secondary, and tertiary structures. What
are some of the common secondary structures? ril/hat are
the forces that hold together the secondary and tertiary
structures?
2. Proper folding of proteins is essentialfor biological ac_
tivity. Describethe roles of molecular chaperonesand chap_
eronins in the folding of proteins.
3. Enzymes can catalyzechemical reactions. How do en_
zymes increasethe rate of a reaction? What constitutes the
active site of an enzyme?For an enzyme-catalyzedreaction,
what are K- and V-""? For enzymeX, the K- for substrrte
A is 0.4 mM and for substrate B is 0.01 mM. \Which sub_
strate has a higher affinity for enzyme X?
4. Motor proteins convert energy into a mechanicalforce.
Describethe three generalproperties characteristicof motor
proterns,

6, The function of proteins can be regulatedin a number of

ways. What is cooperativitg and how does it influence pro_
range. Routine analysisof specimenswith such diversecon_ tein function? Describe how protein phosphorylation and
centrations should dramatically improve the mechanistic proteolytic cleavagecan modulate protein function.
and diagnostic value of blood plasma proteomics. 7. A number oftechniques can separateproteins on the ba_
sis of their differencesin mass. Describe the use of two of
KeyTerms thesetechniques,cenrrifugation and gel electrophoresis.The
blood proteins rransferrin (M\X/ 76 kDa) and lysozyme(MW
15 kDa) can be separated by rate-zonal centrifugation or
cr helix 65 conformations 63 SDS-polyacrylamidegel electrophoresis.Which oi the two
activation energy 79 cooperativity 89 proteins will sediment faster during centrifugation? Which
active site 80 (tomarn /u will migrate faster during electrophoresis?
allostery 89 electrophoresis94 8. Chromatography is an analytical method used to sepa_
amyloid frlament 77 lr
nomotogy /Z rate proteins. Describethe principles for separatingproteins
autoradiography100 by gel filtration, ion-exchange,and affinity ihromatography.
K- 90
9, Various methods have beendevelopedfor detectingpro_
B sheet 66 ligand 78
teins. Describehow radioisotopesand autoradiography can
B turn 66 liquid chromatography 9G
be used for labeling and detectingproteins. Ho* doe, Ifr.rt_
chaperones75 molecular machine 64 ern blotting detectproteins?

108 o C H A P T E R 3I P R O T E I NS T R U C T U RAEN D F U N C T I O N
10. Physical methods are often used to determine protein mic fractions by differential centrifugation. Two-dimensional
conformation. Describe how x-ray crystallography,cryo- gels were run, and the stained gels are shown.below' \fhat do
electron microscopy,and NMR spectroscopycan be used to you conclude about the cellular localization of proteins 1-7?
d e t e r m i n et h e s h a p eo f p r o t e i n s . Control
1L. Mass spectrometryis a powerful tool in proteomics.What Nu c l e a r Cytoplasmic
are the four key features of a mass spectrometer?Describe 4pH 10 4 pH 10
briefly how MALDI and two-dimensionalpolyacrylamidegel
electrophoresis(2D-PAGE)could be usedto identify a protein o a

expressedin cancercellsbut not in normal healthycells.

a
a
Analyze the Data a o
a a
Proteomicsinvolvesthe global analysisof protein expression.
+ Drug
In one approach, all the proteins in control cells and treated
cells are extracted and subsequentlyseparatedusing two-di- Nuclear Cytoplasmic
pH 10 4 pH 10
mensional gel electrophoresis.TypicallS hundreds or thou-
sandsof protein spots are resolvedand the steady-statelevels
of each protein are compared between control and treated
o o
cells. In the following example, only a few protein spots are a
shown for simplicity. Proteins are separated in the first o
o o
dimension on the basis of charge by isoelectricfocusing
o a
(pH a-10) and then separatedby sizeby SDS-polyacrylamide
gel electrophoresis.Proteinsare detectedwith a stain such as
Coomassieblue and assignednumbers for identification. d. Summarize the overall properties of proteins 1-7,
combiningthe data from parts (a), (b), and (c)' Describehow
a. Cellsare treatedwith a drug ("+ Drug") or left un-
you could determinethe identity of any one of the proteins'
treated ("Control"), and then proteins are extractedand sep-
arated by two-dimensional gel electrophoresis.The stained References
gels are shown below. What do you concludeabout the effect
of the drug on the steady-statelevelsof proteins 1-7?
General References
Berg,J. M., J. L. Tymoczko, and L. Stryer.2007' Biochemistry,
Control + Drug
6th ed. W. H. Freemanand ComPanY.
4pH10 4pH10
Nelson, D. L., and M. M. Cox. 2005. LehningerPrinciplesof
12 Biochemistry,4thed. !(/. H. Freemanand Company'
O. o
Web Sites
o Entrv site into proterns,structures'genomes'and taxonomy:
4"-a

^o t
av
-o o o http://www.ncbi.nlm.nih.govlEntrezl
aa a a The protein 3D structuredatabase:http://www'rcsb'org/
Structuralclassificationsof proteins: http://scop'berkeley'edu/
Sitescontaininggeneralinformation about protetns:
b. You suspectthat the drug may be inducinga protein httpy'/www.exp"tyiti; http://www.proweb'org/; http://scop'berkeley'
kinaseand so repeatthe experimentin part (a) in the pres- edu/intro.html
32P-labeled inorganicphosphate.In this experiment
enceof PROSITEDatabaseof protein families and domarns:
the two-dimensional gels are exposedto x-ray film to detect te/
http://www.expasy.org/Prosr
32P-labeledproteins. The x-ray films are Domain organizationof proteins and large collection of multi-
the presenceof arelPfanl
ple sequencealignments:http://www.sanger'ac'uk/Softw
shown below. \fhat do you conclude from this experiment
about the effect of the drug on proteins 1-7? Hierarchical Structure of Proteins and Protein Folding
Branden,C., and J. Tooze. 1999. Introduction to Protein Strwc-
Control + Drug ture. Garland.
4 pH 10 4 pH 10 BrodsknJ. L., and G. Chiosis.2006'Hsp7Omolecularchaper-
on.r' .-.tji"g ,oies in human diseaseand identificationof small
a a molecule-lod"ulrrotr. Curr. Top. Med- Chem' 6(11'\:1215-1225'
Bukau, B, J. Veissman, and A. Horwich' 2006 Molecular chap-
eronesand ptot.i.t quality control. Cell 125(3):443451"
Cohen,F.E. 1999.Proteinmisfoldingand prion diseases' /'
Mol. B iol. 293:313-320.
a
Coulson,A. F., and J. Moult. 2002' A unifold, mesofold' and
superfoldmodel of protein fold use.Proteins46:61'-7"1 '
there a unifying mecha-
c. To determinethe cellular localizationof proteins 1-7, Daggett,V, and A' R. Fersht.2003' Is
nism foi-protein folding? TrendsBiocbem' Sci' 28(\l:18-25 '
the cellsfrom part (a) were separatedinto nuclearand cytoplas-

R E F E R E N C E S. 10 9
 Dobson, C. M. 1999. Protein misfolding, evolution, and disease.
TrendsBiochem. Sci.24:329-332.
Gimona, M.2006. Protein linguistics-a grammar for modular
protein assembly?Nat. Reu.Mol. Ceil Biol.7(Il:69_73.
Gough,J. 2006. Genomic scalesub-family assignmentof pro_
tein domains.Nucl. Acids Res.34(13):3625-3633.
Koonin, E. V., Y. I. Wolf, and G. p. Karev.2002. The srructure
of the protein universeand genomeevolution. Nature 420:21,g_223.
Lesk, A. M. 2001. Introduction to protein Architecture.Oxford.
and H. S. Rye. 2005. GroEl-mediated protein folding:
,\i", V,
making the impossible,possible.Crit. Reu.Biochem.Mol. Biol.
4l(4):21.1-239.
Orengo, C. A., D. T. Jones,and J. M. Thornton. 1994. protein
superfamiliesand domain superfoldi. Nature 372: 631_634.
Patthn L. 1999. Protein Euolution. Blackwell Science.
. Rochet,J.-C., and P.T. T andsbury.2000. Amyloid fibrillogene_
sis:themesand variations. Curr. Opin. Struc. Bioi. l}:60_6g.
. . Voge!,C, and C. Chothia. 2006. prorein family expansionsand
biological complexity. PLoS Comput. Biol. 2(5.;e4g.
Yaffe,M. 8.2006. "Bits" and pieces.SclSTKE.
Jun 20 (3401:pe2g.
Y,oung,J. C., et aL.2004.pathways of chaperone-mediated pro_
tein folding in the cytosol. Nat. Reu.Mol. Cet[ glol. 5:7g1._797.
Protein Function
^ Dressler,D. H., and H. Potter. 1997. DiscoueringEnzymes.
Scientific American Libra ry.
Fersht,A. 1999. Enzyme Structureand Mechanism,3d ed.
'!7.
H. Freemanand Company.
Jeffery,C. J.2004. Molecular mechanismsfor multitasking:
recentcrystal structuresof moonlighting proteins. Cwrr.Opin."Struc.
Biol. l4(61:663-668.
Marnett, A. B., and C. S. Craik. 2005. papa'sgot a brand new
tag: advancesin identificarionof proteasesani the]. substrates.
TrendsB iotechnol. 23 2\: 59-64.
L. 2005. The catalytic triad of serinepeptidases.Cell
_Polgar,
Mol. Life Sci.62(1.9-20\:2t 6I-217 2.
Radisky,E. S., et aL.2006.Insightsinto the serineprotease
mechanismfrom atomic resolutionltructures of trypsin reacrion
inrermediates. Proc. Nat'l Acad. Sci.USA 103(1g);6g35_6g40.
Schenone,M, B. C. Furie, and B. Furie. 2004. The blood
coagulationcascade.Curr. Opin. H ematol. ll(4\ :272_277.
Schramm,V. L. 2005. Enzymatictransition staresand transition
stateanalogues. Cun Opin. Struc.Biol. 15(6):604_613.
Regulating Protein Function l: protein Degradation
Glickman, M. H., and A. Ciechanover.2002Theubicuitin_
proteasomeproteolytic pathway: destructionfor the sakeof
construction.Physiol. Reu. 82(2\:373-42g.
Goldberg_, A.L.2003. Protein degradationand protection
againstmisfolded or damagedproteinl. Nature 426:.g95_g99.
Goldberg,.{. L, S.J. Elledge,and J. If. Harper. 2001. The cellu_
,
r a rc h a m b e o
r t d o o m . S c i .A m . 2 8 4 1 1 1 : O g _ 7 3 .
Groll, M., and R. Huber. 2005. purification, crystallization,and
]^rff^f1a!fsi9 of the yeast20S proteasome.Met'h. Enzymol.
398:329-336.
Kisselev,A. F., A. Callard, and A. L. Goldberg.2006. Impor_
tanceof rhe.differentproteolytic sitesof the prot;some and the effi-
cacy ot rnhrbrrorsvarieswith the protein subsfrate. Biol. Cbem.
J.
281(13):8582-8590.
Rechsteiner, M., and C. p. Hilt. 2005. Mobilizing the proteolytic
machine:cell biologicalrolesof proreasome
acrivatorsand in_
hibitors.TrendsCell Biol. l5(l);27-J3.
Zhou. P.2006. REGgamma:a shorrcutto destruction.Cel/
r24(2\:256-257.
Zolk, O., C. Schenke, and A. Sarikas .2006.The ubiquitin_
proteasomesysrem:focuson the heart.Cardiouasc.Res.7Oi3\:410_12t.
110 C H A P T E R3 | p R o T E t NS T R U C T U RAEN D F U N C T T O N
Regulating Protein Function ll: Noncovalent and Covalent
Modifications
Bellelli,A., et al. 2005. The allostericpropertiesof hemoglobin:
insightsfrom narural and site directed-uiants. Cwrr.prot. pZo. Sci.
7(t):1745.
Burack, \7. R., and A. S. Shaw-2000. Signaltransducrion:hang_
ing on a scaffold.Curr. Opin. Cell Biol. 12:271-216.
_ Halling, D. B, P. Aracena-parks,and S. L. Hamilton. 2006. Reg_
uljrtion of voltage-gatedCaz+ channelsby calmodulin. Sci STKE
lXn
1.7(3181:er1..
Horovitz, A., et al. 2001. Review: allosteryin chaperonins.
/.
Struc. Biol. 135:104-1,14.
Kern, D., and E. R. Zuiderweg. 2003. The role of dynamicsin
allostericregulation.Curr. Opin. Struc. Biol. 13(6):748-757.
I..ane,K. T., and L. S. Beese.2006. Thematicreview series:lipid post_
translationalmodifications.Structuralbiology of protein farnesyitrans_
feraseand geranylgeranyltransferase rypeL j. Lip;d Res.4Z(41:tg1,-691.
Lim, W. A.2002. The modular logic of signalingproteins:build_
ing allostericswitchesfrom simple binding domains. Curr. Opin.
Struc.Biol. 12:61,-68.
Martin, C., Y. Zhang.2005. The diversefunctions of histone
lysinemethylation.Na/. Reu.Mol. Cell Biol.6(11):838-849.
Sawyer,T. K., et al. 2005. Proreinphosphorylationand signal
transductionmodulation: chemistryperspeitivesfor small-mo'iecule
drug discovery.Med. Chem. l(3)2%-3I9.
Xia,Z., and D. R. Storm.2005. The role of calmodulinas a sisnal
integrator for synapticplasticity. Nat. Reu.Neurosci. 6(41:267-27 d.
Yap, K. L., et al. 1999. Diversiry of conformational statesand
changeswithin the EF-hand protein superfarnlly.proteins 37:499_507.
Purifying, Detecting, and Characterizing proteins
Domon, B., and R. Aebersold.2006. Mass spectrometryand
protein analysis.Science3t2(5771):212-217.
. Fncarnacion,S., et al. 2005. Comparativeproteomicsusing 2_D
gel electrophoresisand massspectrometryas tools to dissectstimu_
lons and regulonsin bacteriawith sequencedor partially sequenced
genomes.Biol. Proc. Online 7:ll 7-135.
Hames, B. D. A Practical Approach. Oxiord Universiry press.A
methods seriesthat describesprotein purification methods and assays.
Patton, W. F.2002. Detection,technologies in proteome analysis.
J. Chromatogr.B. Analyt. Technol.Biomed. Life SZl.ZZtlt.Zy:l_Zt.
White, I. R., et al. 2004. A staristicalcomparisonof silver and
!IP\O Ruby staining for proreomic analysis.Electrophoresis
25(17):3048-3054.
Proteomics
Foster,L. J., et al. 200 . A mammalian organellemap by pro_
tein correlation profiling. Cell 125(1\:187-195.
Fu, Q., and J. E. Van Eyk.2006. proteomicsand heart disease:
identifying biomarkers of clinical vtj\ty. Expert Reu. proteomics
3(2):237-249.
_ Gavin. A. C., et a|.2006. Proreomesurveyrevealsmodularity of
the yeastcell machinery.Nature 440(7084):631-636.
Kislinger,T., et al. 2006. Global surveyof organ and organelle
protern expre_ssion in mouse:combinedproteomicand trans&iptomic
profiling. Cell 125(1):173-186.
Kolker, E, R. Higdon, and J. M. Hogan. 2006. protein identifi_
cation.andexpressionanalysisusing mass spectrometry.Trends
Mi crobi ol. I4\5 l:229-235
. Krogan, N. J., et al. 2006. Global landscapeof protein complexes
in the yeastSaccb aromycescereuisiae. Nature-440(i0g4):637_623.
Ong, S. E., and M. Mann. 2005. Mass spectrometry_based pro_
teomrcsturns quanritarive.Nar. Chem. Biol. l(5):252_262.
Rifai, N., M. A. Gillette, and S. A. Carr.2006. protein bio_
T."lk.f dis.coveryand validation: the long and uncertainparh ro
clinical utility. Nat. Biotecb. 24(8\971-t$.
 C H A P T ER

BASICMOLECULAR
GENETIC
MECHANISMS
Colored transmissionelectron micrograph of one ribosomal
RNA transcription unit from a Xenopus oocyte. Transcription
proceeds from left to right,with nascentribosomalribonucleoprotein
complexes (rRNPs) growingin lengthas eachsuccessive RNA
polymerase I moleculemovesalongthe DNAtemplateat the center
ln thisoreoaration eachrRNPis orientedeitheraboveor belowthe
centralstrandof DNAbeingtranscribed, so that the overallshapeis
similarto a feather.In the nucleolusof a livingcell,the nascent rRNPs
extendin alldirections,likea bottlebrushlProfessorOscar L MillerAcience
PhotoLibrary
l

he extraordinary versatility of proteins as molecular

machines and switches, cellular catalysts' and compo-
nents of cellular structureswas describedin Chapter 3.
In this chapter we consider how proteins are made' as well
as other cellular processesthat are critical for the survival of
an organism and its descendents.Our focus will be on the
vital molecules known as nucleic acids, and how they ulti-
mately are responsible for governing all cellular function. stored in the sequenceof the four possible nucleotides that
As introduced in Chapter 2, nucleic acids are linear poly-
mers of four types of nucleotides(seeFigures 2-13' 2-1'6
and 2-1,7).These macromolecules( 1 ) contain in the precise
sequenceof their nucleotidesthe information for determin-
OUTLINE
ing the amino acid sequenceand hence the structure and 113
4.',| Structureof NucleicAcids
function of all the proteins of a cell, \2) are critical func-
tional components of the cellular macromolecular factories 4.2 Transcriptionof Protein-CodingGenes
that select and align amino acids in the correct order as a and Formationof FunctionalmRNA 120
polypeptide chain is being synthesized,and (3) catalyze a
number of fundamental chemical reactions in cells, includ- 4.3 The Decodingof mRNA bY tRNAs 127
ing formation of peptide bonds between amino acids during
protein synthesis. 4.4 StepwiseSynthesisof Proteinson Ribosomes 132
Deoxyribonucleicacid (DNA)is an informational mol- 4.5 DNA Replication 139
ecule that contains in the sequenceof its nucleotidesthe
information required to build all the proteins of an organ- 4.6 DNA Repairand Recombination 145
ism, and hencethe cells and tissuesof that organism. It is
4.7 of the Cellular
Viruses:Parasites
ideally suited to perform this function on a molecular
level. Chemically, it is extraordinarily stable under most GeneticSYstem
terrestrial conditions, as exemplified by the ability to

111
 make up the -3 x 10e base pairs in the human genome.
Becauseof the principles of basepairing discussedin this
chapter, the information is readily copied with an error
rate of <1 in 10e nucleotides per geniration. The exact
replication of this information in any speciesassuresits
genetic continuity from generation to generation and is
critical to the normal developmenrof an individual. DNA
fulfills these functions so well that it is the vessel for
genetic information in all forms of life known. (One
exception is RNA viruses; however, these are limited to
extremely short genomesbecauseof the relative instability
of RNA compared with DNA, as we will see.)The discov-
ery that virtually all forms of life use DNA to encodetheir
genetic information, and also use nearly the same nucleic
acid sequencecode to specifyamino acid sequence,implies
that all forms of life descendedfrom a common ancesror
on the basis of storageof information in nucleic acid
sequence.This information is accessedand replicated by
specific base pairing between bases. The information
stored in DNA is arrangedin hereditary units, now known
as genes,which control identifiable traits of an organism.
In the processof transcription, the information stored in
DNA is copied into ribonucleic acid (RNA), which has
three distinct roles in protein synthesis.
Portions of the DNA nucleotide sequenceare copied into
lation becausethe nucleotide sequencelanguage of DNA
and RNA is translated into the amino acid sequencelan_
guageof proteins.
Discovery of the structure of DNA in 1953 and subse_
quent elucidation of how DNA directs synthesisof RNA,
which then directs assemblyof proteins-the so-called,cen_
decoded into a variety of proteins in the correct cells at the
correct times in development. This regulation of gene
expressionallows hemoglobin to be expressedonly in cells
of the bone marrow (reticulocytes)destinedto develop into
1',t2 CHAPTER
4 I B A S t CM O L E C U L A G
R E N E T TM
CE C H A N t s M s
circulating red blood cells (erythrocytes),and directs devel-
oping neurons to make the proper synapses(connections)
with 1011other developingneurons in the human brain. The
fundamental molecular generic processesof DNA replica-
tion, transcription, and translation must be carried out with
extraordinary fidelity, speed,and accurate regulation in or,
der for organismsas complex as prokaryotes and eukaryotes
to develop normally. This is achievedby chemical processes
that operate with extraordinary accuracycoupled with mul-
tiple layers of checkpoint, or surveillance,mechanismsthat
test whether critical steps in these processeshave occurred
correctly before the next step is initiated. The highly regu-
lated expressionof genesnecessaryfor the developmento1 a
multicellular organism requires integrating information
from signalssent by distant cellsin the developingorganism,
as well as from neighboring cells, and an intrinsic develop-
mental program determined by earlier stepsin embryogene-
sis taken by that cell's progenitors. All this regulation is
dependent on control sequencesin the DNA that function
with proteins called transcription factors to coordinate the
expressionof every gene. RNA sequenceswe discussin
Chapter 8 that regulateRNA processingand translation also
are encoded in DNA originally. Thus nucleic acids function
as the "brains and central nervous system" of the cell, while
proteins carry out the functions they specify.
In this chapter,we first review the structuresand proper-
ties of DNA and RNA, and explore how the different char-
acteristicsof each type of nucleic acid make them suited for
their respectiveroles in the cell. In the next severalsections
we discussthe basic processessummarizedin Figure 4-1:
transcription of DNA into RNA precursors, processing of
these precursorsto make functional RNA molecules.trans-
lation of mRNAs into proteins, and the replication of DNA.
After outlining the individual roles of -RNA, IRNA, and
rRNA in protein synthesis,we presenta detailed description
of the components and biochemical stepsin translation. .We
also consider the molecular problems involved in DNA
replication and the complex cellular machinery for ensuring
accuratecopying of the geneticmaterial. Along the way, we
compare theseprocessesin prokaryotes and eukaryotes.The
next section describeshow damageto DNA is repaired, and
how regions of different DNA molecules are exchangedin
the processof recombination to generatenew combinations
of traits in the individual organisms of a species.The final
section of the chapter presents basic information about
viruses, which, in addition to being significant pathogens,
are important model organisms for studying macromolecu-
lar synthesisand other cellular processes.Viruses have rela-
tively simple structures compared with cells, and small
genomesthat made them tractable for historic early studies
of the basic processesof DNA replication, transcription,
translation, recombination, and gene expression.Viruses
continue to teach important lessonsin molecular cell biology
today and have been adapted as experimental tools for
introducing any desired genes into cells, tools that are
currently being tested for their effectivenessin human gene
rnerapy.
 RNA
vtrus

I rranscript,""\
ry^'

a9 *4si
Cytosol

o Protein
R i b o s o m- a lI ".
.rtr"lt. n'mlno
acios
* Translation $rFh
I factors
tRNA

p m R N At r a n s l a t i o n

A FIGURE 4-1 Overviewof four basicmoleculargenetic translation(B), thefour-base codeof the mRNAisdecoded intothe
processes. In thischapter we coverthethreeprocesses that leadto 2O-amino acidlanguage of proteinsRibosomes, the macromolecular
productionof proteins (n-B) andthe process for replicatingDNA machines thattranslatethemRNAcode,arecomposed of two subunits
(4) Because machinery, theyhavebeen assembled fromribosomal
in the nucleolus RNAs (rRNAs) andmultiple
viruses utilizehost-cell
important models for studyingtheseprocesses Duringtranscriptton proteins (/eft).
Aftertransportto the cytoplasm,ribosomal subunits
geneby RNApolymerase (n), thefour-base DNA associate with an mRNA and carryout proteinsynthesis with the help
of a protein-coding
codespecifying the aminoacidsequence of a proteiniscopied, or of transferRNAs (tRNAs) andvarious translationfactorsDuringDNA
transcribed,into a precursor messenger RNA(pre-mRNA) by the replication(4), whichoccurs onlyin cellspreparingto divide,
polymerization triphosphate monomers (rNTPs) deoxyribonucleoside triphosphatemonomers (dNTPs) arepolymerized
of ribonucleoside
Removal of noncoding sequences andothermodifications to the to yieldtwo identicalcopiesof eachchromosomal DNA molecule'
pre-mRNA (Z), collectively knownasRIVA processing,producea Eachdaughter oneof the identical
cellreceives copies
functionalmRNA, whichistransported to the cytoplasm During

ff Structureof NucleicAcids
DNA and RNA are chemically very similar. The primary
structuresof both are linear polymerscomposedof monomers
called nucleotides.Both function primarily as informational
molecules,carrying information in the exact sequenceof
their nucleotides.Cellular RNAs range in length from fewer
than one hundred to many thousandsof nucleotides.Cellu-
lar DNA moleculescan be as long as severalhundredmillion
nucleotides.These large DNA units in associationwith pro-
teins can be stained with dyes and visualized in the light
microscope as chromosomes,so named becauseof their
stainability. Though chemically similar, DNA and RNA
exhibit some very important differences.For example, RNA
can also function as a catalytic molecule.As we'll see,it is
the different and unique properties of DNA and RNA that
make them each suited for their specificroles in the cell'
A N u c l e i cA c i d S t r a n dl s a L i n e a rP o l y m e r
w i t h E n d - t o - E n dD i r e c t i o n a l i t Y
In all organisms, DNA and RNA each comprise only four
different nucleotides. Recall from Chaptet 2 that all nu-
cleotidesconsist of an organic base linked to a five-carbon
sugar that has a phosphate group attached to carbon 5' In
RNA, the sugar is ribose; in DNA, deoxyribose(seeFig-
,r,re2-16). The nucleotides used in synthesisof DNA and
RNA contain one of five different bases.The basesadenine
(A) and guanine(G) arepurines,whichcontainapaftof fused
rings; the basescytosine (C), tbymine (T), and uracil (U) are

ACIDS
S T R U C T U ROEF N U C L E I C 113
 (a) (b) erty of the molecule. The chemical linkage berweenadjacent
o
I nucleotides,commonly called a phosphodiesterbond, actu-
5'end O-P:O ally consistsof two phosphoesterbonds, one on the 5, side
o of the phosphate and another on the 3' side.
I The linear sequenceof nucleotideslinked by phosphodi-
t,?'2o,.-? esterbonds constitutesthe primary structureof nucleic acids.
OH
HH Like polypeptides, polynucleotides can twist and fold into
H three-dimensionalconformations stabilized by noncovalent
3',
o bonds. Although the primary structures of DNA and RNA
are generally similar, their three-dimensionalconformations
5', C-A-G 3', are quite different. Thesestructural differencesare critical to
the different functions of the two types of nucleic acids.

3'end OH H
A FIGURE 4-2 Chemicaldirectionalityof a nucleicacidstrand.
Shownherearealternative representations of a singlestrandof DNA
containing onlythreebases: (C), (A),
cytosine adenine andguanine (G)
(a)Thechemical structureshowsa hydroxyl groupat the 3, endand
a phosphate groupat the 5, end Notealsothattwo phosphoester
bondslinkadjacent nucleotides; thistwo-bondlinkage commonly rs
referredto asa phosphodiester bond.(b)In the ,,stick,'diagram
(top),thesugars areindicated asvertical linesandthe phosphodiester tween the two strands: A is paired with T through two hy-
bondsasslanting lines;the bases aredenotedbytheirsingle_letter
abbreviations Inthe simplest representation (bottom), onlythe bases bonds (Figure 4-3b). This base-paircomplementarityis a
areindicatedByconvention, a polynucleotide sequence tsatways
writtenin the 5'-+3'direction (leftto righ0unless otherwise indicated the bases.The presenceof thousands
pyrimidines, which contain a single ring (see Figue 2-17).
Three of thesebases-A, G, and C-are found in both DNA
and RNA; however, T is found only in DNA, and U only in
RNA. (Note that the single-letterabbreviationsfor these
basesare also commonly usedto denotethe entire nucleotides
in nucleic acid polymers.)
A single nucleic acid strand has a backboae composedof
repeating pentose-phosphateunits from which the purine
and pyrimidine basesextend as side groups. Like a poiypep-
tide, a nucleic acid strand has an end-to-endchemical oiien-
polynucleotide sequencesare written and read in the 5'--+3,
direction (from left to right); for example,the sequenceAUG
is assumedto be (5')AUG(3,). As we will see, the 5,_+3,
directionality of a nucleic acid strand is an important prop-
N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y
A n t i p a r a l l eS
l trands
The modern era of molecular biology began in 1953 when
'Watson
James D. and Francis H. C. Crick proposed that
DNA has a double-helical structure. Their proposal, based
on analysis of x-ray diffraction patterns generated by
Rosalind Franklin and Maurice Wilkins, and coupled with
careful model building, proved correct and paved the way
for our modern understandingof how DNA functions as the
geneticmaterial.
DNA consists of two associatedpolynucleotide strands
that wind together to form a double helix. The two sugar-
phosphate backbonesare on the outside of the double helix,
and the bases project into the interior. The adjoining basesin
each strand stack on top of one another in parallel planes
(Figure 4-3a). The orientation of the two strands is antipar-
allel; that is, their 5'-+3' directions are opposite.The strands
are held in precise register by formation of base pairs be-
drogen bonds; G is paired with C through three hydrogen
consequenceof the size,shape,and chemicalcomposition of
of such hydrogen
bonds in a DNA molecule contributes greaiy to the stability
of the double helix. Hydrophobic and van der N7aalsinter-
actions between the stacked adjacent basepairs further sta-
bilize the double-helicalstrucure.
In natural DNA, A always hydrogen bonds with T and
G with C, forming A.T and G.C basepairs, as shown in Fig-
ure 4-3b. These associations,always between a larger
purine and smaller pyrimidine are often called '\X/atson-
Crick base pairs. Two polynucleotide strands, or regions
thereof, in which all the nucleotides form such base pairs
are said to be complementary. However, in theory and in
synthetic DNAs, other base pairs can form. For example,
guanine(a purine) could theoreticallyform hydrogenbonds
with thymine (a pyrimidine), causing only a minor distor-
tion in the helix. The spaceavailable in the helix also would
allow pairing between the two pyrimidines cytosine and
thymine. Although the nonstandard G.T and C.T basepairs
are normally not found in DNA, G.U basepairs are quite
common in double-helical regions that form within other-
wise single-strandedRNA. Nonstandard base pairs do not
occur naturally in duplex DNA becausethe DNA copying

114 . c H A p r E4R | B A S tM
c o L E c u L AGRE N E TMt cE c H A N t s M s
 < FIGURE 4-3 The DNAdoublehelix.(a)Space-
fillingmodelof B DNA,the mostcommonformof
C.Hs o..
DNAin cells.Thebases (lightshades) projectinward
fromthesugar-phosphate backbones (darkredand
blue)of eachstrand,buttheiredgesareaccessible
throughmajorandminorgrooves. Arrowsindicate
of eachstrand.Hydrogen
the 5'-+3'direction bonds
betweenthe basesarein the centerof the structure
Themajorandminorgrooves arelinedby potential
hydrogen bonddonorsandacceptors (highlighted
in (b)
yellow). Chemical structureof DNA double
helixThisextended schematic showsthe two sugar-
CH, \^_o phosphate backbones andhydrogen bondingbetween
u .r
/t-o
theWatson-Crick basepairs,A T andG C. lPart(a)from
oo=p1 'o
^ ln R Wingetal, 1980,Nature287i755: part(b)
f romR E
9Hz
\ffi,\-' i983,5ciAm 249:941
Dickerson,

cHz
ft
o ),'oo
o
o-!
9Hz

5'CH2
5', ,1oo
3',

enzyme, which is described later in this chapter, does not By far, the most important modifications in the structure
permit them. of standard B form DNA come about as a result of protein
Most DNA in cells is a right-handed helix. The x-ray binding to specificDNA sequences.Although the multitude
diffraction pattern of DNA indicatesthat the stacked bases of hydrogen and hydrophobic bonds between the basespro-
are regularly spaced0.34 nm apart along the helix axis. The vide stability to DNA, the double helix is flexible about
helix makes a complete turn every 3.4 nm; thus there are its long axis. Unlike the ct helix in proteins (seeFigure 3-4)'
about 10.1 pairs per turn. This is referredto as the B form of
DNA, the normal form present in most DNA stretchesin ( c ) ZD N A
( a )B D N A ( b ) AD N A
cells. On the outside of B form DNA, the spacesbetweenthe
intertwined strands form two helical grooves of different
widths describedas the maior groove and the minor groove
(seeFigure 4-3a). As a consequence,the atoms on the edges
of each basewithin thesegroovesare accessiblefrom outside
the helix, forming two types of binding surfaces.DNA-binding
proteins can read the sequenceof basesin duplex DNA by
contacting atoms in either the major or the minor grooves.
Other structuresof DNA have beendescribedin addition
to the major B form. Two of these are compared with B
DNA in Figure 4-4. Under laboratory conditions in which
most of the water is removed from DNA, the crystallo-
graphic structure of B DNA changesto the A form, which is
wider and shorter than B DNA, with basepairs tilted rather
than perpendicular to the helix axis. RNA-DNA and RNA-
RNA helices exist in this form in cells and in vitro. Short 4-4 Modelsof variousknown DNA structures.The
A FIGURE
DNA moleculescomposed of alternating purine-pyrimidine sugar-phosphate backbones whichareon the
of thetwo strands,
nucleotides(especiallyG's and C's) adopt an alternative left- outsrdein allstructures,areshownin redandblue;the bases (lighter
handed helix configuration instead of the normal right- shades)areoriented inward(a)TheB formof DNAhas :10.1 base
handed helix. This structure is called Z DNA becausethe pairsperhelical turn.Adjacent stackedbasepairs
are 0.34nm apart'
basesseemto zigzag when viewed from the side. Some evi- (b)Themorecompact A formof DNAhas11 basepairsperturnand
dence suggeststhat Z DNA may occur in cells, although its exhibits to the helixaxis
a largetilt of the basepairswith respect
function is unknown. (c)Z DNAisa left-handed doublehelix

E F N U C L E I CA C I D S
S T R U C T U RO 115
 \fhy did DNA evolve to be the carrier of genetic informa-
tion in cellsas opposedto RNA? The hydrogen at the 2' posi-
tion in the deoxyriboseof DNA makes it a far more stable
molecule than RNA, which instead has a hydroxyl group
at the 2' position of ribose (SeeFigure2-16).The 2'-hydroxyl
groups in RNA participatein the sloq OH--catalyzedhydrol-
ysis of phosphodiesterbonds at neutral pH (Figure4-6).The
absenceof 2'-hydroxyl groups in DNA preventsthis process.
Therefore, the presenceof deoxyribosein DNA makes it a
more stablemolecule-a characteristiccritical to its function
in the long-term storageof geneticinformation.

D N A C a nU n d e r g oR e v e r s i b l e
S t r a n dS e p a r a t i o n
During replication and transcription of DNA, the strands
of the double helix must separateto allow the internal
FIGURE 4-5 Proteininteractioncan bend DNA.Theconserved edgesof the basesto pair with the basesof the nucleotides
C-terminal domainof theTATAbox-binding protein(TBp) bindsto being polymerizedinto new polynucleotidechains.In later
theminorgrooveof specific DNAsequences richin A andl, untwisting sections,we describethe cellular mechanismsthat separate
andsharply bendingthedoublehelixTranscription
of mosteukaryotic and subsequentlyreassociateDNA strandsduring replica-
genesrequires participation
of TBPlndapted
fromD B Nikolov andS K tion and transcription. Here we discuss fundamental
Burley,1997, ProcNat'lAcad
SciU5A94:151 factors influencing the separation and reassociation of
DNA strands.Thesepropertiesof DNA were elucidatedby
in vitro expenments.
thereare no hydrogenbondsparallelto the axis of the DNA The unwinding and separation of DNA strands,referred
helix. This properry allows DNA to bend when complexed to as denaturation, or "melting," can be induced experi-
with a DNA-binding protein (Figure4-5). Bendingof DNA mentally by increasing the temperature of a solution of
is critical to the dense packing of DNA in chromatin, the DNA. As the thermal energy increases, the resulting
protein-DNA complex in which nuclearDNA occursrn eu- increasein molecular motion eventually breaks the hydro-
karyotic cells(Chapter6). gen bonds and other forces that stabilizethe double helix;

npn

I I
n_D-at o
o CH2-O- Basen
Hzo ."\
I KUU)
9H,_O- Basen \" "./|
." \] H\+H
I\tlu) tl
r\f-Jl'i oa ooH oHo
.ro
I
o o-H OH ,,P\ o P:o O-P:O
oo I I
I OH
-_---------> OH
3' monophosphate 2'monophosphate
o.
I
CH, o Base-.
t

1..' \
"/"

\H H)
n\_/n
l1
ooH ooH
I - O - PI: O
o-P:O
o o
FIGURE 4-6 Base-catalyzedhydrolysisof RNA.The2,-hydroxyl phosphodiester bondhydrolysis cannotoccurin DNA,whichlacks2,
groupin RNAcanactasa nucleophile,
attackinqthe phosohodiester hydroxyl groupslAdapted fromNelson principles
etal, Lehninger of
bond The2',3' cyclic
monophosphatederivative
isfurtherhydrolyzed Biochemistry,4thed , W H Freemanand Company.l
to a mixture
of 2' and3' monophosphates
Thismechantsm of

116 CHAPTER
4 | B A S I CM O L E C U L A G
R E N E T TM
CE C H A N T S M S
 (a)
S i n gl e - s t r n
aded
E
N
0.75
c E+o
o
0.5
75 80 90
('C)
Temperature
EXPERIMENTAL FIGURE 4-7 G.Ccontentof DNAaffects
meltingtemperature.Thetemperature at whichDNAdenatures
increases of G.Cpairs(a)Meltingof doubled-
with the proportion
stranded DNAcanbe monitored of ultraviolet
by theabsorption
lightat 260 nm.As regions DNAunpair,
of double-stranded the
absorptionof lightby thoseregions almosttwofoldThe
increases
the strandsthen separate,driven apart by the electrostatic
repulsion of the negatively charged deoxyribose-phosphate
backbone of each strand. Near the denaturation tempera-
ture, a small increasein temperaturecausesa rapid, near
simultaneousloss of the multiple weak interactions holding
the strands together along the entire length of the DNA
molecules.Becausethe stackedbasepairs in duplex DNA
absorb lessultraviolet (UV) light than the unstackedbases
in single-stranded DNA, this leadsto an abrupt increasein
the absorption of UV light, a phenomenon known as hyper-
chromicity (Figure 4 -7a).
The mebing temperature (T-) at which DNA strands
will separatedependson severalfactors.Moleculesthat con-
tain a greater proportion of G'C pairs require higher tem-
peratures to denature becausethe three hydrogen bonds in
G'C pairs make thesebasepairs more stablethan A'T pairs,
which have only two hydrogen bonds. Indeed, the percent-
age of G'C base pairs in a DNA sample can be estimated
from its T- (Figure4-7b\.The ion concentrationalso influ-
ences the T- becausethe negatively charged phosphate
groups in the two strands are shieldedby positively charged
ions. When the ion concentration is low, this shielding is de-
creased,thus increasingthe repulsive forces between the
strandsand reducingthe T-. Agentsthat destabilizehydrogen
bonds, such as formamide or urea, also lower the 7-.
Finalln extremesof pH denature DNA at low temperature.
At low (acid) pH, the basesbecomeprotonated and thus
positively charged, repelling one another. At high (alkaline)
pH, the baseslose protons and become negatively charged,
again repelling one another becauseof the similar charge.In
cells,pH and temperatureare,for the most part, maintained.
These features of DNA separation are most useful for
manipulating DNA in a laboratory setting.
The single-strandedDNA molecules that result from
denaturation form random coils without an organized
(b)
100
.g 80
G
660
c)
E
0)
s20
"
lo 80 90 1oo 110
r- (.c)
temperatureat whichhalfthe bases in a double-stranded DNA
sample havedenatured isdenotedI' (for"temperature of melting")
Lightabsorption DNAchanges
by single-stranded muchlessasthe
temperature isincreased (b)TheI. is a of
function the G'Ccontent
of the DNA;the higherthe G+C percentage, the greaterthe I.
structure. Lowering the temperature, increasing the ion
concentration, or neutralizing the pH causes the two
complementarystrandsto reassociateinto a perfect double
helix. The extent of such renaturation is dependenton time,
the DNA concentration, and the ionic concentration. Two
DNA strands not related in sequencewill remain as random
coils and will not renature; most importantly, they will not
inhibit complementary DNA partner strands from finding
each other and renaturing. Denaturation and renaturation
of DNA are the basis of nucleic acid hybridization, a pow-
erful technique used to study the relatednessof two DNA
samplesand to detect and isolate specificDNA moleculesin
a mixture containing numerous different DNA sequences
( s e eF i g u r e5 - 1 6 ) .
TorsionalStressin DNA ls Relievedby Enzymes
Many prokaryotic genomic DNAs and many viral DNAs are
circular molecules. Circular DNA molecules also occur in
mitochondria, which are present in almost all eukaryotic
cells, and in chloroplasts, which are present in plants and
some unicellular eukaryotes'
Each of the two strands in a circular DNA molecule
forms a closed structure without free ends' Localized
unwinding of a circular DNA molecule, which occurs dur-
ing DNA replication, induces torsional stress into the re-
maining portion of the molecule becausethe ends of the
strands are not free to rotate. As a result, the DNA molecule
twists back on itself, like a twisted rubber band, forming
supercoils(Figure 4-8a). In other words, when part of the
DNA helix is underwound, the remainder of the molecule
becomes overwound. Bacterial and eukaryotic cells, how-
ever, contain topoisomerase .I, which can relieve any tor-
sional stressthat developsin cellular DNA moleculesduring
replication or other processes. This enzymebinds to DNA
ACIDS
S T R U C T U ROEF N U C L E I C . 117
 (a) Supercoiled (b) Relaxedcircle
at random sites and breaks a phosphodiesterbond in one
strand. Such a one-strand break in DNA is called a nick.
The broken end then unwinds around the uncut strand,
leadingto loss of supercoils(Figure4-8b). FinallS the same
enzyme joins (ligates)the two ends of the broken strand.
Another type of enzyme, topoisomerase11,makes breaks in
both strands of a double-stranded DNA and rhen religates
them. As a result, topoisomerase II can both relieve tor-
sional stressand link together two circular DNA molecules
a s i n t h e I i n k so f a c h a i n .
Although eukaryotic nuclear DNA is linear,long loops of
DNA are fixed in place within chromosomes (Chapter 6).
Thus torsional stressand the consequentformation of su-
percoils also could occur during replication of nuclear DNA.
As in bacterial cells, abundant topoisomeraseI in eukaryotic
nuclei relieves any torsional stressin nuclear DNA that
would develop in the absenceof this enzyme.
DifferentTypesof RNA Exhibit Various
ConformationsRelatedto Their Functions
The primary structure of RNA is generally similar to that of
DNA with two exceptions: the sugar component of RNA,
ribose, has a hydroxyl group at the 2, position (seeFig-
ure 2-16b), and thymine in DNA is replaced by uracil in
RNA. The presenceof thymine rarher rhan uracil in DNA is
important to the long-term stability of DNA becauseof its
function in DNA repair (seeSection 4.6). As noted earlier,
the hydroxyl group on C2 of ribose makes RNA more chem-
ically labile than DNA. As a result of this labilitS RNA is
cleaved into mononucleotides by alkaline solution (see
Figure 4-6), whereas DNA is not. The C2 hydroxyl of RNA
also provides a chemically reactive group that takes part in
118 . cHAprER
4 I B A s t cM o L E c u L A G
R E N E T tMc E c H A N t s M S
< EXPERIMENTAT FIGURE 4-8 Topoisomerase I
relievestorsionalstresson DNA.(a)Electron
micrograph of SV40viralDNA.Whenthecircular
DNAof the SV40virusisisolated andseoarated
fromitsassociated protein, the DNAduplexis
underwound andassumes thesupercoiled config-
uration. (b)lf a supercoiled DNAisnicked(i e.,
onestrandcleaved), the strands canrewind,
leadingto lossof a supercoil. Topoisomerase I
catalyzes thisreaction andalsoreseals the broken
endsAll thesupercoils in isolated SV40DNAcan
be removed bythe sequential actionof thisenzyme,
producing the relaxed-circle conformation For
clarity,
theshapes of the molecules at the bottom
havebeensimplified
RNA-mediated catalysis.Like DNA, RNA is a long polynu-
cleotide that can be double-strandedor single-stranded,
linear or circular. It can also participate in a hybrid helix
composed of one RNA strand and one DNA strand. As
discussedabove, RNA-RNA and RNA-DNA double helices
have a compact conformation like the A form of DNA (see
Figure4-4b).
Unlike DNA, which exists primarily as a very long dou-
ble helix, most cellular RNAs are single-strandedand
exhibit a vaiety of conformations (Figure 4-9). Differences
in the sizesand conformations of the various types of RNA
permit them to carry out specific functions in a cell. The
simplest secondary structures in single-strandedRNAs are
formed by pairing of complementary baseswithin a linear
sequence."Hairpins" are formed by pairing of baseswithin
:5-10 nucleotidesof eachother,and "stem-loops" by pair-
ing of basesthat are separatedby >10 to severalhundred
nucleotides.These simple folds can cooperate to form more
complicated tertiary structures, one of which is termed a
"pseudoknot."
As discussedin detail later, IRNA molecules adopt a
well-defined three-dimensional architecture in solution
that is crucial in protein synthesis.Larger rRNA molecules
also have locally well-defined three-dimensional struc-
tures, with more flexible links in between.Secondaryand
tertiary structures also have been recognized in mRNA,
particularly near the ends of molecules. Clearly, then,
RNA moleculesare like proteins in that they have struc-
tured domains connecred by less structured, flexible
stretches.
The folded domains of RNA molecules not only are
structurally analogous to the a helicesand B strands found
in proteins, but in some casesalso have catalytic capacities.
(a) Secondarystructure In this chapter, we focus on the functions of mRNA,
tRNA, and rRNA in gene expression.In later chapters we
will encounter other RNAs, often associatedwith proteins'
that participate in other cell functions.

Structure of NucleicAcids
Stem-loop r Deoxyribonucleic acid (DNA), the geneticmaterial, car-
(blTertiarystructure ries information to specify the amino acid sequencesof
proteins. It is transcribed into severaltypes of ribonucleic
Ca,
U- acid (RNA), including messengerRNA (mRNA), transfer
G un RNA (IRNA), and ribosomal RNA (rRNA), which func-
U GC tion in protein synthesis(seeFigure4-1).
UA r Both DNA and RNA are long' unbranched polymers of
c
U UA nucleotides, which consist of a phosphorylated pentose
U UA
linked to an organic base,either a purine or pyrimidine'
U^ Aa
The purines adenine (A) and guanine (G) and the pyrim-
; ine cytosine (C) are presentin both DNA and RNA. The
nA
;c pyrimidine thymine (T) present in DNA is replaced by the
5', GC -A pyrimidine uracil (U) in RNA.
s'GC"AA
Pseudoknot r Adiacent nucleotides in a polynucleotide are linked by
A FIGURE 4-9 RNAsecondaryand tertiary structures.(a)Stem- phosphodiester bonds. The entire strand has a chemical
loops,hairpins, andothersecondary structures canformby base directionality with 5' and 3' ends (seeFigwe 4-2).
pairingbetweendistantcomplementary segments of an RNAmolecule. r Natural DNA (B DNA) contains two complementary
In stem-loops, thesingle-stranded loopbetween the base-paired antiparallel polynucleotide strands wound together into a
helicalstemmaybe hundreds or eventhousands of nucleotrdes regular right-handed double helix with the baseson the
long,whereas in hairpins,theshortturn maycontainasfew asfour inside and the two sugar-phosphatebackbones on the
nucleotides. (b)Pseudoknots, onetypeof RNAtertiarystructure, are
outside (seeFigure 4-3). Basepairing betweenthe strands
formedby interaction of secondary loopsthroughbasepairing
and hydrophobic interactions between adjacent base
betweencomplementary basesThestructure shownformsthe core
pairs stacked perpendicular to the helix axis stabilize this
domainof the humantelomerase RNAlefti Secondary-structure
with base-paired nucleotides in greenandblueandsingle- natlve structure.
diagram
strandedregionsin red.Middle:Sequence of the telomerase RNA r The bases in nucleic acids can interact via hydrogen
coredomain,colored to correspond to thesecondary-structure diagram bonds. The standard Watson-Crick base pairs are G'C,
at the left.R/ghtiDiagramof the telomerase coredomainstructure A.T (in DNA), and G'C, A'U (in RNA). Basepairing stabi-
determined by 2D-NMR, showingbase-paired bases onlyanda tube lizes the native three-dimensionalstructures of DNA and
for the sugar-phosphate backbone, colored to correspond to the RNA.
diagrams (b),middle
to the left [Part andright,adapted fromC A Theimer
et al , 2005, Mol Cell 17:671]l r Binding of protein to DNA can deform its helical struc-
ture, causing local bending or unwinding of the DNA
molecule.
r Heat causesthe DNA strandsto separate(denature).The
Such catalytic RNAs are called ribozymes. Although ri-
melting temperature7- of DNA increaseswith the percent-
bozymes usually are associatedwith proteins that stabilize
age of G'C basepairs. Under suitableconditions, separated
the ribozyme structure, it is the RNA that acts as a catalyst.
Some ribozymes can catalyzesplicing, a remarkable process complementarynucleic acid strandswill renature.
in which an internal RNA sequenceis cut and removed, and r Circular DNA moleculescan be twisted on themselves'
the two resulting chains then ligated. This process occurs forming supercoils (see Figure 4-8). Enzymes called
during formation of the majority of functional mRNA topoisomerasescan relieve torsional stressand remove su-
moleculesin multicellular eukaryotes,and also occurs in sin- percoils from circular DNA molecules' Long linear DNA
gle-celled eukaryotes such as yeast, bacteria, and archaea. ian also experiencetorsional stressbecauselong loops are
Remarkably, some RNAs carry out self-splicing, with the fixed in place within chromosomes'
catalytic activity residing in the sequencethat is removed. r Cellular RNAs are single-strandedpolynucleotides,
The mechanismsof splicing and self-splicingare discussedin some of which form well-defined secondary and tertiary
detail in Chapter 8. As noted later in this chapter, rRNA structures (seeFigure 4-9). Some RNAs, called ribozymes'
plays a catalytic role in the formation of peptide bonds dur- have catalytic activitY.
ing protein synthesis.

E F N U C L E I CA C I D S
S T R U C T U RO 119
@ Transcription
of Protein-Coding
Genesand Formationof Functional
mRNA
The simplest definition of a gene is a "unir of DNA that
contains the information to specify synthesisof a single
polypeptide chain or functional RNA (such as a IRNA)."
The DNA moleculesof small viruses contain only a few
genes,whereas the single DNA molecule in each of the
chromosomesof higher animals and plants may contain
severalthousandgenes.The vast maiority of genescarry rn-
formation to build protein molecules,and it is the RNA
copies of such protein-coding genes that constitute the
mRNA moleculesof cells.
During synthesisof RNA, the four-base language of
DNA containing A, G, C, and T is simply copied, or tran-
scribed,into the four-baselanguageof RNA, which is iden-
tical except that U replacesT. In contrast, during protein
synthesis, the four-base language of DNA and RNA is
translatedinto the 20-amino acid languageof proteins. In
this section,we focus on formation of functional mRNAs
from protein-coding genes(seeFigure 4-1, A). A similar
process yields the precursors of rRNAs and tRNAs
encoded by rRNA and IRNA genes;these precursorsare
then further modified to yield functional rRNAs and
tRNAs. In addition, thousands of recently discovered
micro RNAs (miRNAs) that function ro resulare transla-
t i o n o f s p e c i f i ct a r g e tm R N A s a n d r r a n s c r i p t - i oonf s p e c i f i c
target genesare transcribedinto precursorsby RNA poly-
merasesand processedinto functional miRNAs. Transcrip-
tion and processingof these other types of RNA is dis-
cussed in Chapter 8. Regulation of transcription allows
distinct setsof genesto be expressedin the many different
types of cellsthat make up a multicellular organism.It also
allows different amounrs of mRNA to be transcribedfrom
different genes, resulting in differences in the amounts of
the encodedproteins in a cell. Regulation of transcription
is addressedin Chapter 7.
A TemplateDNA Strand ls Transcribedinto a
y N AC h a i nb y R N Ap o l y m e r a s e
C o m p l e m e n t a rR
During transcription of DNA, one DNA strand acs as a
template, determining the order in which ribonucleoside
triphosphate (rNTP) monomers are polymerized to form a
complementary RNA chain. Basesin the template DNA
strand base-pair with complementary incoming rNTps,
which then are joined in a polymerization reaction catalyzed
by RNA polymerase.Polymerization involves a nucleophilic
attack by the 3' oxygen in the growing RNA chain on the ct
phosphate of the next nucleotide precursor to be added,
resulting in formation of a phosphodiesterbond and release
of pyrophosphate(PP;).As a consequenceof this mecha-
nism, RNA moleculesare always synthesizedin the 5,-+3,
direction (Figure 4-10a).
The energeticsof the polymerization reaction strongly
favor addition of ribonucleotides to the growing RNA
120 . c H A p r E4R | B A s t cM o L E c u L AGRE N E TM
t cE c H A N t s M s
chain becausethe high-energybond betweenthe cr and B
phosphate of rNTP monomers is replaced by the lower-energy
phosphodiesterbond betweennucleotides.The equilibrium
for the reaction is driven farther toward chain elongation by
pyrophosphatase,an enzyme that catalyzescleavageof the
releasedPP; into two molecules of inorganic phosphate.
Like the two strands in DNA, the template DNA strand and
the growing RNA strand that is base-pairedto it have
opposite5'-+3' directionality.
By convention, the site on the DNA at which RNA
polymerasebegins transcription is numbered *1. Down-
stream denotes the direction in which a template DNA
strand is transcribed;upstreamdenotesthe opposite direc-
tion. Nucleotide positions in the DNA sequencedown-
stream from a start site are indicated by a positive (+)
sign; those upstream,by a negative(- ) sign. BecauseRNA
is synthesized5'-+3', RNA polymerase moves down the
template DNA strand in a 3'-+5' direction. The newly
synthesizedRNA is complementary to the template DNA
strand; therefore, it is identical with the nontemplate
DNA strand, except with uracil in place of thymine (see
F i g u r e4 - 1 0 b ) .
Stages in Transcription To carry out transcription, RNA
polymerase performs several distinct functions, as depicted
in Figure 4-11. During transcription initiation, RNA poly-
meraserecognizesand binds to a specific site, called a pro-
moter, in double-strandedDNA (step [). RNA polymerases
require various protein factors, called general transcription
factors, to help them locate promoters and initiate transcrip-
tion. After binding to a promoter, RNA polymerase sepa-
rates the DNA strandsin order to make the basesin the tem-
plate strand available for basepairing with the basesof the
ribonucleosidetriphosphatesthat it will polymerize rogether.
RNA polymerasesmelt 12-t4base pairs of DNA around the
transcription start site, which is located on the template
strand within the promoter region (step [). This allows the
template strand to enter the active site of the enzyme that
catalyzesphosphodiester bond formation between ribonu-
cleoside triphosphates that are complementary to the pro-
moter template strand at the start site of transcription. The
12-14-base-pairregion of melted DNA in the polymeraseis
known as the "transcription bubble." Transcription initia-
tion is considered complete when the first two ribonu-
cleotides of an RNA chain are linked by a phosphodiester
bond (stepS).
After several ribonucleotides have been polymerized,
RNA polymerase dissociatesfrom the promoter DNA and
generaltranscription factors.During the stageof strand elon-
gation, RNA polymerasemoves along the template DNA one
baseat a time, opening the double-strandedDNA in front of
its direction of movement and guiding the strandstogetherso
that they hybridize at the upstream end of the transcriprion
bubble (Figure4-11, step@). One ribonucleotideat a time is
added to the 3' end of the growing (nascent)RNA chain
during strand elongation by the polymerase. The enzyme
maintains a melted region of approximately 1.4 base pairs,
the transcription bubble. Approximately eight nucleotidesat
(a) the 3' end of the growing RNA strand remain base-pairedto
the template DNA strand in the transcription bubble. The
5'+ 3' RNA
strand growth elongation complex, comprising RNA polymerase,template
DNA, and the growing (nascent)RNA strand, is extraordi-
narily stable. For example, RNA polymerasetranscribesthe
longestknown mammalian gene,containing about 2 million
basepairs, without dissociatingfrom the DNA template or
releasingthe nascentRNA. SinceRNA synthesisoccurs at a
oHo rate of about 1000 nucleotidesper minute at 37 "C, the elon-
I gation complex must remain intact for more than 24 hours
O-P:O
to assurecontinuous RNA synthesisof the pre-mRNA from
this very long gene.
During transcription termination, the final stagein RNA
D
N synthesis,the completed RNA molecule, or primary transcript,
A is releasedfrom the RNA polymerase, and the polymerase
t
oHo dissociatesfrom the template DNA (Figure4-11, step El).
e - O - P : IO
Specific sequencesin the template DNA signal the bound
m I RNA polymeraseto terminate transcription. Once released,
?1tr
a
o
t 5'-+3'.(a)Polymerization of
e < FIGURE 4-10 RNAis synthesized
ribonucleotides by RNApolymerase duringtranscription Theribonu-
s cleotideto be addedat the 3' endof a growingRNAstrandis
t
f specifiedby base-pairing between the nextbasein thetemplate DNA
a strandandthecomplementary incoming ribonucleoside triphosphate
n (rNTP)A phosphodiester bondisformedwhenRNApolymerase
d
catalyzesa reaction between the 3' O of the growingstrandandthe
trl crphosphate base-paired
of a correctly rNTPRNAstrands always are
ooo andareopposite in polarityto
synthesized in the 5'--+3'direction
OH OH theirtemplate DNAstrands(b)Conventions for describing RNA
transcription lop: TheDNAnucleotide whereRNApolymerase
IncomingrNTP + 1.Thedirection the polymerase
begins transcription isdesignated
travelson the DNAis "downstream," andbases aremarked with
positivenumbers. Theopposite direction is "upstream," and bases
arenotedwith negative numbersSomeimportant genefeatures lie
upstream of thetranscription startsite,including the promoter
sequence thatlocalizes RNApolymerase to thegene (Bottom) The
DNAstrandthat isbeingtranscribed isthe template strand;its
complement, the nontemplate strand. TheRNAbeingsynthesized is
- lt=-l
rl complementary to thetemplate strand,andtherefore identical with
the nontemplate strandsequence, exceptwith uracilin placeof
5', thymine[Part (b)adapted eral, Modern
fromGriffiths Genetic Analysis,
2 d e d . W H F r e e m a na n d C o m P a n Y l

(b) Transcription
Promoter C o d i n gs e q u e n c e
ffilis.l.rf,'

J
5',
-20 +20

Upstream Downstream

strand5' CTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGATCTGCGCTGC
Nontemplate ",')
6fl
I DNA
strand3, cAcccTAACAGTCTGTACATATGGGGCATGCAGAAGGGCTCGCTTTTGCTAGACGCGACG
Temptate
I
Y
5' 3 ' P r i m a r YR N A
transcriPt

GG
F PROTEIN-CODIN
TRANSCRIPTION O F F U N C T I O N AM
E N E SA N D F O R M A T I O N LRNA 121
 FocusAnimation:BasicTranscriptional
Mech"n,rr {lll
< FIGURE 4-11 Threestagesin transcription.
RNA polymerase Start site Stopsite
on template on template Duringinitiation of transcription, RNApolymerase
INITIATION formsa transcription bubbleandbegins polymerization
strand strand
of ribonucleotides (rNTPs) at thestartsite,whichis
I Polymerasebindsto located withinthe promoter region.Oncea DNA
promotersequence 5', regionhasbeentranscribed, theseparated strands
in duplexDNA. reassociate intoa doublehelix.Thenascent
"Closedcomplex"
RNAis
displaced fromitstemplate strandexceptat its3'
Promoter end.The5' endof the RNAstrandexitsthe RNA
polymerase througha channel in the enzyme.Termi-
I Polymerasemelts
nationoccurs whenthe polymerase encounters a
duplexDNA near 5f
transcriptionstart site, specifictermination sequence (stopsite)Seethetext
3,
forminga transcription for detailsForsimplicity,
thediagram depicts
transcrip-
bubble. "Ooen tionof fourturnsof the DNAhelixencoding =40
comolex" nucleotides of RNA.MostRNAsareconsiderably longer,
requiringtranscription of a longerregionof DNA
p Polymerase catalyzes
phosphodiester 5', 5',
linkage
e'
of twoinitialrNTPs.

ELONGATION

I Polymeraseadvances
3'+ 5'down template
strand,meltingduplex
DNA and addingrNTps
to growingRNA.

TERMINATION 5',
At transcriptionstop site, 3',
!
polymerasereleases
completedRNA and
dissociatesfrom DNA.
Completed
RNA strand

an RNA polymeraseis free to transcribethe samegeneagain Organizationof GenesDiffers in Prokaryotic

or another gene.
Structure of RNA Polymerases The RNA polymerases
of bacteria, archaea,and eukaryotic cells are fundamentally
similar in structure and function. Bacterial RNA
polymerasesare composedof two relatedlarge subunits(B,
and B), two copiesof a smallersubunit (cr),and one copy of
a fifth subunit (o) that is not essentialfor transcription or
cell viability but stabilizesrhe enzyme and assisrsin the as-
sembly of its subunits. Archaeal and eukaryotic RNA poly-
merases have several additional small subunits associated
with this core complex, which we describe in Chapter 7.
Schematic diagrams of the transcription process generally
show RNA polymerasebound to an unbent DNA molecule,
as in Figure 4-11. However, X-ray crystallography and
other studies of an elongating bacterial RNA poiymerase
indicate that the DNA bends at rhe transcription bubble
(Figwe 4-12).
a n d E u k a r y o t i cD N A
Having outlined the process of transcription, we now
briefly consider the large-scalearrangement of informa-
tion in DNA and how this arrangement dictates the re-
quirementsfor RNA synthesisso that information transfer
goes smoothly. In recent years, sequencingof the entire
genomes from several organisms has revealed not only
large variations in the number of protein-codinggenesbut
also differencesin their organization in prokaryotes and
eukaryotes.
The most common arrangementof protein-coding genes
in all prokaryotes has a powerful and appealing logic: genes
encoding proteins that function together, for example, the
enzymesrequired to synthesizethe amino acid tryptophan,
are most often found in a contiguous array in the DNA. Such
an arrangement of genesin a functional group is called an
operon becauseit operatesas a unit from a singlepromoter.
Transcription of an operon produces a continuous strand of

122 CHAPTER
4 | BASTC
MOLECULAG
R E N E T TM
CE C H A N T S M S
 mRNA that carriesthe messagefor a relatedseriesof proteins
(a)
(Figure 4-13a). Each section of the mRNA representsthe
unit (or gene)that encodesone of the proteins in the series.
This arrangementresults in the coordinate expressionof aIl
the genes in the operon. Every time an RNA polymerase
molecule initiates transcription at the promoter of the
operon, all the genesof the operon are transcribedand trans-
lated. In prokaryotic DNA the genesare closelypacked with
very few noncoding gaps, and the DNA is transcribed di-
rectly into mRNA. BecauseDNA is not sequesteredin a nu-
cleus in prokaryotes, ribosomes have immediate accessto
the translation start sitesin the mRNA as they emergefrom
the surface of the RNA polymerase. Consequently,transla-
t i o n o f t h e m R N A b e g i n se v e n w h i l e t h e 3 ' e n d o f t h e
mRNA is still being synthesizedat the active site of the
RNA polymerase.
This economic clustering of genes devoted to a single
metabolic function does not occur in eukaryotes, even sim-
ple ones like yeasts,which can be metabolically similar to
(b) B subunit..r bacteria. Rather, eukaryotic genes encoding proteins that
function together are most often physically separatedin the
DNA; indeed such genes usually are located on different
chromosomes.Each gene is transcribed from its own pro-
moter, producing one mRNA, which generally is translated
to yield a singlepolypeptide (Figure 4-13b).
When researchersfirst compared the nucleotide se-
quences of eukaryotic mRNAs from multicellular organ-
isms with the DNA sequencesencoding them, they were
-d surprised to find that the uninterrupted protein-coding
':' sequenceof a given mRNA was discontinuous in its corre-
B'subunit/ sponding section of DNA. They concluded that the eukary-
r-1 otic gene existed in pieces of coding sequence,the exons,
IRNAZ separated by non-protein-coding segments' the introns.
This astonishingfinding implied that the long initial pri-
n subunit \o subunit mary transcript-the RNA copy of the entire transcribed
DNA sequence-had to be clipped apatt to remove the in-
A FIGURE 4-12 BacterialRNApolymerase. Thisstructure corre- trons and then carefully stitched back together to produce
sponds to the polymerase molecule in theelongation phase(step4) eukaryotic mRNAs.
of Figure4-11 ln thesediagrams, transcriptton is proceeding in the Although introns are common in multicellular eukary-
leftwarddirectionArrowsindicate wheredownstream DNAenters otes, they are extremely rare in bacteria and archaeaand un-
the polymerase andupstream DNAexitsat an anglefromthe down- common ln many unicellular eukaryotes such as baker's
streamDNA;thecodingstrandisred,the noncoding strandblue, yeast. However, introns are present in the DNA of viruses
nascent RNAgreenTheRNApolymerase B' subunit isgold,B islight that infect eukaryotic cells. Indeed, the presenceof introns
gray,andthe o subunitvisible fromthisangleisbrown In (a)a was first discovered in such viruses, whose DNA is tran-
modelof theelongation
space-filling complex isvrewed froman scribedby host-cellenzymes.
anglethatemphasizes the bendin the DNAasit passes throughthe
polymerase Theelongation complex is rotatedin (b)asshown,and
proteinsaremadelargely transparent to reveal the structure of the EukaryoticPrecursormRNAsAre Processed
transcriptionbubbleinside the polymerase that isnotvisible in the t o F o r mF u n c t i o n am
l RNAs
modelNucleotides
space-filling complementary to thetemplate DNA
In prokaryotic cells, which have no nuclei, translation of an
areaddedto the 3'-endof the nascent RNAstrand(atthe left) The
mRNA into protein can begin from the 5' end of the mRNA
newlysynthesized nascent RNAexitsthe polymerase at the bottom
through a c h a n n ef ol r m e db e t w e etnh eB a n dB ' s u b u n i tTs h et r evenwhile the 3' end is still being synthesizedby RNA poly-
subunitandtheothercrsubunitarevisible fromthisangle[Courtesy merase,In other words, transcription and translation occur
o f S e t hD a r s t ;s e eN K o r z h e v ae t a l , 2 0 0 0 , S c i e n c e 2 8 9 , 6 1 9 - 6 2 5a,n d N concurrently in prokaryotes. In eukaryotic cells, however,
Opalkaet al , 2003, Cell'114:335-345I not only is the site of RNA synthesis-the nucleus-
separatedfrom the site of translation-the cytoplasm-but
also the primary transcripts of protein-coding genes are
precursor mRNAs (pre-mRNAs) that must undergo several

ON
TRANSCRIPTIO GG
F PROTEIN.CODIN O F F U N C T I O N AM
E N E SA N D F O R M A T I O N LRNA 123
 (a)Prokaryotes (b) Eukaryotes

Yeastchromosomes
Kb TRP| TRP4

E. coli genome

580 V

trp operon
-
: E I p I c I B l A -8kb 910 Vll

I
Staft site
for trp mRNA 680 Xl
synthesis

rranscrintion
f
frp mRNA 5'
fff++ trp
mRNAs
Start sitesfor
protein synthesis

I Translation Translation
v I
:-
-j- 1 2345
Proteins
-a B Proteins r IIII

FIGURE 4-13 Geneorganizationin prokaryotesand theencoded proteins

in thetryptophan pathway. (b)Thefivegenes
eukaryotes.(a)Thetryptophan(trp)operonisa continuous encoding theenzymes required for tryptophansynthesis in yeast
segment of the E.colichromosome, containing fivegenes(blue) (Saccharomyces cerevisiae)
arecarriedon four differentchromosomes
that encodethe enzymes necessaryfor the stepwise synthesis
of Eachgeneistranscribed fromitsown promoter to yielda primary
tryptophanTheentireoperonistranscribed fromonepromoter into thatisprocessed
transcript intoa functionalmRNAencoding a
onelongcontinuous frp mRNA(red)Translation of thismRNAbegins singleprotein.
Thelengths of thevarious chromosomes areqivenin
at fivedifferent
startsites,yielding
frveproteins (green)Theorderof (103bases)
kilobases
the genesin the bacterialgenomeparallels the sequential
functionof

modifications, collectively termed RNA processing,to yield
a functional mRNA (seeFigure 4-I,2). This mRNA then
must be exported to the cytoplasm before it can be trans-
lated into protein. Thus transcription and translation cannot
occur concurrently in eukaryotic cells.
All eukaryotic pre-mRNAs initially are modified at the
two ends, and these modifications are retained in mRNAs.
As the 5' end of a nascent RNA chain emergesfrom the
surface of RNA polymerase, it is immediately acted on
by several enzymes that together synthesize the 5, cap, a
7-methylguanylate that is connected to the terminal nu-
Processingat the 3' end of a pre-mRNA involvescleav-
age by an endonuclease to yield a free 3,-hydroxyl group to
which a string of adenylic acid residuesis added orr. ut
time by an enzyme called poly(A) polymerase. The result- "
ing poly(A) tail contains 100-250 bases,being shorter in
yeastsand invertebratesthan in vertebrates.Poly(A) poly-
meraseis part of a complex of proteins that can locate and
cleavea transcript at a specific site and then add the correct
number of A residues,in a processthat does not require a
template.
The final step in the processing of many different eu-
karyotic mRNA molecules is RNA splicing: the internal
cleavageof a transcripr to excisethe introns, followed by lig-
ation of the coding exons. Figure 4-15 summarizesthe basic
steps in eukaryotic mRNA processing, using the B-globin
gene as an example. rJTeexamine the cellular machinery for
carrying out processing of mRNA, as well as IRNA and
rRNA, in Chapter 8.
The functional eukaryotic mRNAs produced by RNA
processingretain noncoding regions,referred to as 5' and 3,
wntranslated regions (UTRs), at each end. In mammalian
mRNAs, the 5' UTR may be a hundred or more nucleotides
long, and the 3'UTR may be severalkilobasesin length.
Prokaryotic mRNAs also usually have 5' and 3' UTRs, but
these are much shorter than those in eukaryotic mRNAs.
generally containing fewer than 10 nucleotides.

124 . c H A p r E R4 B A s t cM o l E c u L A RG E N E I c M E C H A N t s M s
|
 < FIGURE 4-14 Structureof the 5' methylatedcap' Thedistin-
guishingchemical featuresof the 5' methylated capon eukaryotic
7-Methylguanylate mRNAare(1)the 5'-+5' linkage of 7-methylguanylate to the initial
nucleotideof the mRNAmolecule and(2)the methylgroupon the 2'
hydroxylof the riboseof thefirstnucleotide (base1) Boththesefea-
turesoccur in allanimalcellsand in of
cells higher yeasts
plants; lack
themethylgroupon nucleotide 1 Theribose of thesecond nucleotide
(base2) alsoismethylated in vertebrates [SeeAJ Shatkin,1976,Cell
9:645I
HH
I
o-P:o l2'
I OH OH A l t e r n a t i v eR N AS p l i c i n gI n c r e a s e s
o
- o - PI: o the Numberof ProteinsExpressed
5' -> 5'linkage
f r o m a S i n g l eE u k a r y o t i cG e n e
o
I In contrast to bacterial and archaealgenes,the vast majority
o-P:o of genesin higher, multicellular eukaryotescontain multiple
I
o introns. As noted in Chapter 3' many proteins from higher
eukaryoteshave a multidomain tertiary structure (seeFigure
Base 1
3-11). Individual repeatedprotein domains often are en-
1',
coded by one exon or a small number of exons that code for
H
2' identical or nearly identical amino acid sequences'Such re-
o-cH3 peated exons are thought to have evolved by the accidental
-o-P:o multiple duplication of a length of DNA lying between two
o sites in adiacent introns, resulting in insertion of a string of
I repeated exons) separatedby introns, between the original
two introns. The presenceof multiple introns in many eu-
karyotic genespermits expressionof multiple' related pro-
teins from a single gene by means of alternative splicing. In
o-cH3 higher eukaryotes,alternativesplicing is an important mech-
,ro
O-P:O anism for production of different forms of a protein' called
I isoforms, by different types of cells'

illll} overview Animation: Life le of an mRNA

106 147
> FIGURE 4-15 Overviewof RNAprocessing. RNAprocessing p-Globin
produces functionalmRNAin eukaryotes. TheB-globin genecontains genomrc
threeprotein-codingexons(constituting thecodingregion,red)and DNA Poly(A)
Start site for
two interveningnoncoding introns (blue)Theintronsinterrupt the RNA synthesis site
protein-codingsequence between thecodons for aminoacids31 and
32 and105 and106.Transcription of eukaryotic protein-codinggenes Primary 5'
startsbeforethesequence thatencodes thefirstaminoacidand RNA
extends beyond thesequence encoding thelastaminoacid,resulting transcnpt
in noncoding regions(gray)
at theendsof theprimary transcript.These
untranslatedregions(UTRs)areretained duringprocessing. The5' cap
(mtGppp) isaddedduringformation of theprimary RNAtranscript, Aln
whichextendsbeyondthe poly(A)site Aftercleavage at the poly(A)
siteandaddition A residues
of multiple to the3' end,splicing removes
the intronsandjoinstheexons. Thesmallnumbers referto positions
in the 147-amino acidsequence of B-globin.

p-Globin A),
mRNA

MRNA
T R A N S C R I P T I OONF P R O T E I N - C O D I NGGE N E SA N D F O R M A T I O NO F F U N C T I O N A L 125
 F i b r o n e c t ig
ne n e
Fibroblast
f i b r o n e c t i nm R N A
Hepatocyte
fibronectinmRNA
A FIGURE 4-16 Alternativesplicing.The=75-kbfibronectin gene
(fop)containsmultiple
exons;
splicing of frbronectin
varies
by cell
type TheElllBandElllAexons(green)encodebindingdomains for
proteins
specific on thesurfaceof fibroblasts.
Thefibronectin
mRNA
Fibronectin, a multidomain protein found in mammals,
providesa good exampleof alternativesplicing(Figure4-16).
Fibronectin is a long, adhesiveprotein secretedinto the ex-
tracellular spacethar can bind other proteins together.\7hat
and where it binds dependson which domains are splicedto-
gether. The fibronectin gene contains numerous exons,
grouped into several regions corresponding to specific
domains of the protein. Fibroblasts produce fibronectin
mRNAs that contain exons EIIIA r.rd EtIIn; these exons
encode amino acid sequencesthat bind tightly to proteins
in the fibroblast plasma membrane. Consequently,this fi-
bronectin isoform adheresfibroblasrsto the extiacellular
matrix. Alternative splicing of the fibronectin primary tran-
script in hepatocytes,the major type of cell in the liver, yields
mRNAs that lack the EIIIA and EIIIB exons. As a result, the
fibronectin secretedby hepatocyresinto the blood does not
adhere tightly to fibroblasts or most other cell types, allow-
ing it to circulate. During formation of blood clots, however,
the fibrin-binding domains of hepatocyte fibronectin binds
to fibrin, one of the principal constituentsof clots. The
bound fibronectin then interacts with integrins on the mem-
branesof passingplatelets,thereby expanding the clot by ad-
dition of platelets.
More than 20 different isoforms of fibronectin have been
identified, each encoded by a different, alternatively spliced
mRNA composed of a unique combination of fibronectin
gene exons. Recent sequencingof large numbers of mRNAs
isolated from various tissues and comparison of their se-
quenceswith genomic DNA has revealeJthat nearly 60 per-
cent of all human genesare expressedas alternativelyspliced
mRNAs. Clearly, alternative RNA splicing greatly expands
the number of proteins encoded by the genomesof higher,
multicellular organisms.
Transcriptionof Protein-CodingGenes and Formation
of Functional mRNA
Transcription of DNA is carried out by RNA polymerase,
hich adds one ribonucleotide ar a time to the 3, end of a
growing RNA chain (seeFigure4-11). The sequenceof the
template DNA strand determines the order in which
ribonucleotidesare polymerized to form an RNA chain.
126 CHAPTER
4 | BASTC
M O L E C U L AG
R E N E T TM
CE C H A N T S M S
produced in fibroblasts
includes the ElllAandElllBexons,
whereas
theseexonsarespliced out of fibronectinmRNAin hepatocytes.
In
thisdiagram, introns(blacklines)arenot drawnto scale;
mostof
themaremuchlongerthananyof theexons.
r During transcription initiation, RNA polymerase
binds to a specific site in DNA (the promoter), Iocally
melts the double-strandedDNA to reveal the unpaired
template strand, and polymerizes the first two nu-
cleotides complementary to the template strand. The
melted region of t2-14 basepairs is known as the "tran-
scription bubble."
r During strand elongation, RNA polymerase moves
down the DNA, melting the DNA aheadof the polymerase,
so that the template strand can enter the active site of the
enzyme,and allowing the complementary DNA strands of
the region just transcribed to reanneal behind it. The tran-
scription bubble moves with the polymeraseas the enzyme
adds ribonucleotidescomplementaryto the template strand
to the 3' end of the growing RNA chain.
'S7hen
r RNA polymerasereachesa termination sequence
in the DNA, the enzymestops rranscription, leading to re-
leaseof the completed RNA and dissociationof the enzyme
from the template DNA.
r In prokaryotic DNA, severalprotein-coding genescom-
monly are clustered into a functional region, an operon,
which is transcribed from a single promoter into one
mRNA encoding multiple proteins with related functions
(seeFigure 4-13a). Translation of a bacterial mRNA can
begin before synthesisof the mRNA is complete.
r In eukaryotic DNA, each protein-coding gene is tran-
scribedfrom its own promoter.The initial primary transcript
very often containsnoncoding regions(introns) interspersed
among coding regions (exons).
r Eukaryotic primary transcripts must undergo RNA pro-
cessingto yield functional RNAs. During processing,the
ends of nearly all primary transcripts from protein-coding
genesare modified by addition of a 5' cap and 3' poly(A)
tail. Transcripts from genescontaining introns undergo
splicing,the removal of the introns and joining of the exons
(seeFigure4-15).
r The individual domains of multidomain proteins found
in higher eukaryotesare often encodedby individual exons
or a small number of exons. Distinct isoforms of such pro-
teins often are expressedin specific cell types as the result
of alternative splicing of exons.
 the mRNA calls for it. The correct IRNA with its attached
!f, The Decodingof mRNAby tRNAs amino acid is selectedat each step becauseeach specific
Although DNA storesthe information for protein synthesis IRNA molecule contains a three-nucleotidesequence,an
and mRNA conveysthe instructions encodedin DNA, most anticodon, that can base-pairwith its complementary
biological activitiesare carried out by proteins.As we saw codon in the mRNA.
in Chapter 3, the linear order of amino acidsin eachprotein
determinesits three-dimensionalstructure and activity. For 3. Ribosomal RNA (rRNA) associateswith a set of pro-
this reason,assemblyof amino acids in their correct order, teins to form ribosomes.Thesecomplex structures,which
as encoded in DNA, is critical to production of functional physically move along an mRNA molecule' catalyzethe as-
proteins and hence the proper functioning of cells and sembly of amino acids into polypeptide chains. They also
organrsms. bind tRNAs and various accessoryproteins necessaryfor
Translation is the whole processby which the nucleotide protein synthesis.Ribosomesare composedof a large and a
sequenceof an mRNA is used as a template to join the small subunit, each of which contains its own rRNA mole-
amino acids in a polypeptide chain in the correct order (see cule or molecules.
Figure 4-1,,g).In eukaryoticcells,protein synthesisoccurs
in the cytoplasm, where three types of RNA moleculescome Thesethree types of RNA participate in the synthesisof pro-
together to perform different but cooperative functions teins in all organisms. Indeed, development of three func-
(Figue 4-1.7)z tionally distinct RNAs was probably the molecular key to
the origin of life. In this section,we focus on the decoding of
1,. MessengerRNA (mRNA) carries the geneticinforma- mRNA by IRNA adaptors, and how the structure of each of
tion transcribed from DNA in a linear form. The mRNA is these RNAs relates to its specific task. How they work to-
read in setsof three-nucleotidesequences,called codons, gether with rRNA, ribosomes, and other protein factors to
each of which specifiesa particular amino acid. synthesizeproteins is detailed in the following section.Since
translation is essentialfor protein synthesis,the two processes
2. Transfer RNA (IRNA) is the key to decipheringthe commonly are referred to interchangeably.However, the
codons in mRNA. Each type of amino acid has its own sub- polypeptide chains resulting from translation undergo post-
set of tRNAs, which bind the amino acid and carry it to the translational folding and often other changes(e.g.,chemical
growing end of a polypeptide chain when the next codon in modifications, association with other chains) that are
required for production of mature' functional proteins
(Chapter3).
aa7-tRNA7
arflvrng
H
Growing
Hrru-J-R, MessengerRNACarriesInformation from DNA
polypeptide
chain in a Three-LetterGeneticCode
H-9 I o
?=o As noted above, the genetic code used by cells is a triplet
code, with every three-nucleotidesequence'or codon, being
!--o o
"read" from a specifiedstarting point in the mRNA. Of the
o 54 possiblecodons in the geneticcode' 51 specify individual
tRNA4 amino acids and three are stop codons. Table 4-1 shows that
leaving most amino acids are encoded by more than one codon.
Only two-methionine and tryptophan-have a single
codon; at the other extreme, leucine,serine,and arginine are
mRNA
each specifiedby six different codons. The different codons
\____YJ q/J gYJ
for a given amino acid are said to be synonymous.The code
Codon Codon Codon itself is termed degenerate,meaning that a particular amino
d0t ddZ ElOg acid can be specifiedby multiple codons.
Synthesisof all polypeptide chains in prokaryotic and
M o v e m e n to f r i b o s o m e
eukaryotic cells begins with the amino acid methionine. In
a FIGURE 4-17 The three rolesof RNAin protein synthesis. bacteria, a specialized form of methionine is used with a
Messenger RNA(mRNA) istranslatedintoproteinbythejointaction formyl group linked to its amino group. In most mRNAs,
RNA(IRNA)
of transfer andthe ribosome, whichiscomposed of
the start (initiator) codon specifying this amino-terminal
numerous proteins
andtwo majorribosomal RNA(rRNA) molecules
methionine is AUG. In a few bacterial mRNAs, GUG is
(notshown)Notethe basepairingbetween IRNAanticodons and
of a peptidebond used as the initiator codon, and CUG occasionallyis used as
complementary codonsin the mRNA.Formation
between the amino-group N on the incomingaa-tRNA andthe an initiator codon for methionine in eukaryotes. The three
carboxy-terminalC on the growingproteinchain(green) iscatalyzed codons UAA, UGA, and UAG do not specify amino acids
by oneof the rRNAs.aa : aminoacid;R : sidegroup[Adapted from but, rather, constitute stop (termination) codons that mark
A J F G r i f f i t h s e t a l , 1 9 9 9 ,M o d e r n G e n e t i cA n a l y s i s , WH F r e e m a na n d the carboxyl terminus of polypeptide chains in almost all
C o m p a n yl cells. The sequenceof codons that runs from a specific start

T H E D E C O D I N GO F m R N A B Y t R N A s 127
 sEcoN0
P0stTt0N

U C A G
Phe Ser Tyr cys U
Phe Ser Tyr cyt C
U
Leu Ser Stop Stop A
Leu Ser Stop Ttp G

E
Leu Pro His Atg U
z.
g
-E
I
Leu Pro His Atg C E
- o
q

F Leu Pro Gln Arg A =

a
e
4

@
Leu (Met)'* Pro Gln Arg G
E
g
=
m

Ile Thr Asn Ser U

Ile Thr Asn Ser C
A
Ile Thr Lys Atg A
Met (Start) Thr Lys Arg G

Val Ala Asp Glv U

Val Ala Asp Glv C
G
Val Ala Glu Glv A
Val (Met)" Ala GIu Glv G
*AUG is the most
common initiator codon; GUG usually codes for valine and CUG for leucine, but.
rarely, these codons can also code for methionine to initiate a protein chain.

codon to a stop codon is called a reading frame. This precise
linear array of ribonucleotides in groups of three in mRNA
specifiesthe precise linear sequenceof amino acids in a
polypeptide chain and also signals where synthesis of the
chain starts and stops.
Becausethe genetic code is a non-overlapping triplet
code without divisions between codons, a particular
mRNA theoretically could be translated in three different
reading frames. Indeed some mRNAs have been shown to
contain overlapping information that can be translatedin
different reading frames, yielding different polypeptides
(Figure 4-18). The vast majority of mRNAs, however,can
be read in only one frame becausestop codonsencountered
in the other two possiblereading frames terminatetransla-
tion before a functional protein is produced. Very rarely,
another unusual coding arrangement occurs becauseof
frame-shiftins. In this case the protein-synthesizingma-
chinery may read four nucleotidesas one amino acid and
then continue reading triplets, or it may back up one base
and read all succeedingtriplets in the new frame until ter-
mination of the chain occurs. Only a few dozen such in-
stancesare known.
The meaning of each codon is the same in most known
organisms-a strong argument that life on earth evolved
only once. In fact, the geneticcode shown in Table 4-1 is
known as the uniuersal code. However, the geneticcode has
beenfound to differ for a few codons in many mitochondria,
in ciliated protozoans, and in Acetabwlaria, a single-celled
plant. As shown in Table 4-2, most of thesechangesinvolve
reading of normal stop codons as amino acids, not an

128 CHAPTER
4 | BASTC
M O L E C U L AG
R E N E T TM
CE C H A N T S M S
 F r a m e1 (Figure 4-19). The anticodon in the IRNA then base-pairs
with a codon in mRNA so that the activatedamino acid can
be added to the growing polypeptide chain (seeFigures4-17
Polypeptidel and4-18).
Frame2 Some 30-40 different tRNAs have been identified in
bacterialcells and as many as 50-100 in animal and plant
cells.Thus the number of tRNAs in most cellsis more than
Polypeptide 2 the number of amino acids used in protein synthesis(20)
and also differs from the number of amino acid codons in
FIGURE 4-18 Multiplereadingframesin an mRNA
the genetic code (61). Consequently,many amino acids
sequence. lf translation of the mRNAsequence shownbeginsat two
have more than one IRNA to which they can attach (ex-
different upstream startsites(notshown), thentwo overlapping
framesarepossible In thisexample, thecodonsareshifted plaining how there can be more tRNAs than amino acids);
reading
onebaseto the rightin the lowerframeAs a result, the same in addition, many tRNAs can pair with more than one
nucleotide sequence specifies different aminoacidsduringtranslation, codon (explaining how there can be more codons than
Althoughregions of sequence thataretranslated in two of thethree tRNAs).
possible reading framesarerare,thereareexamples in bothprokary- The function of IRNA molecules,which are 70-80 nu-
otesandeukaryotes, andespecially in theirviruses, wherethe same cleotideslong, dependson their precisethree-dimensional
sequence isusedin two alternative mRNAs expressed fromthe same structures.In solution, all IRNA moleculesfold into a sim-
region o f D N Aa , n dt h es e q u e n ci ser e a di n o n er e a d i nfgr a m ei n ilar stem-loop arrangement that resemblesa cloverleaf
o n em R N Aa n di n a n a l t e r n a t i vr eea d i nfgr a m ei n t h eo t h e rm R N A . when drawn in two dimensions (Figure 4-20a)' The four
Thereareevena few instances wherethe sameshortsequence is stems are short double helicesstabilizedby Watson-Crick
readin allthreepossible reading frames basepairing; three of the four stemshave loops containing
sevenor eight basesat their ends,while the remaining,un-
exchangeof one amino acid for another.Theseexceptionsto loooed stem contains the free 3' and 5' ends of the chain.
the universalcode probably were later evolutionary develop- The three nucleotidescomposingthe anticodon are located
ments; that is, at no single time was the code immutably at the center of the middle loop' in an accessibleposition
fixed, although massivechangeswere not tolerated once a that facilitates codon-anticodon base pairing. In all tRNAs,
generalcode began to function early in evolution. the 3' end of the unlooped amino acrdacceptor stem has the
sequenceCCA, which in most casesis added after synthesis
and processingof the IRNA are complete.Severalbasesin
The FoldedStructureof tRNA Promoteslts
most tRNAs also are modified after transcription, creating
D e c o d i n gF u n c t i o n s nonstandard nucleotidessuch as inosine, dihydrouridine,
Translation, or decoding, of the four-nucleotidelanguageof and pseudouridine. As we will see shortly, some of these
DNA and mRNA into the 2O-amino acid languageof pro- modified basesare known to play an important role in pro-
teins requires tRNAs and enzymes called aminoacyl-tRNA tein synthesis. Viewed in three dimensions, the folded
synthetases.To participate in protein synthesis,a IRNA mol- IRNA molecule has an L shape with the anticodon loop
ecule must become chemically linked to a particular amino and acceptor stem forming the ends of the two arms (Fig-
acid via a high-energy bond, forming an aminoacyl-tRNA ure4-20b\.

C()DON Uf'IIVERSAL
C0DE C(]DT-
UNUSUAL OCCURRENCE

UGA Stop Trp My cop lasma, Spir op lasma, mitochondria

of many species

CUG Leu Thr Mitochondria in yeasts

UAA, UAG Stop Gln A cetab ul ar ia, Tetr ah y m ena,

Paramecium, etc.

UGA Stop cys Euplotes

"Found in nuclear genesof the listed organisms and in mitochondrial genesas indicated.
souRCE:S. Osawa et al., 1.992,Microbiol. Reu. 56:229.

OF mRNABY tRNAs
THEDECODING 129
 A m i n o a c i d( P h e ) High-energy
HO ester bond
til
H 2 N- C - C -OH
r'',li,,

z
d E
Linkaqe of
P;t" 1i 131114Ptre
P h e - t R N A P h eb i n d s
to the UUU codon
Net result:
Phe is selected
by its codon

ATP AMP
+ PPt
Aminoacyl- AAA AAA AAA
tRNA synthetase tRNA specificfor Aminoacyl-tRNA
specificfor Phe Phe (tRNAPhe) mRNA
FIGURE 4-19 Decodingnucleicacidsequenceinto amino hydroxyl of theterminaladenosine in the corresponding
IRNA.
acidsequence. Theprocessfor translating nucleic
acidsequences StepE: A three-base sequence in theIRNA(theanticodon) then
in mRNAintoaminoacidsequences in proteins involvestwo steps. base-pairs with a codonin the mRNAspecifying theattached
amino
Step[: An aminoacyl-tRNA synthetase f irstcouplesa specificamino acid.lf an erroroccurs
in eitherstep,thewrongaminoacidmaybe
acid,viaa high-energy
esterbond(yellow), to eitherthe 2, or 3, incorporated intoa polypeptide chainPhe: phenylalanine

NonstandardBasePairingOften Occurs
BetweenCodonsand Anticodons
@ = dihYdrouridine
= inosine If perfect Watson-Crick base pairing were demanded be-
e
= ribothymidine tween codons and anticodons,cellswould have to contain at
e
= pseudouridine least 61 different types of tRNAs, one for each codon that
@
specifiesan amino acid. As noted above,however,many cells
m = methylgroup
contain fewer than 61 tRNAs. The explanation for the
smaller number lies in the capability of a single IRNA anti-
codon to recognizemore than one, but not necessarilyeverg
codon corresponding to a given amino acid. This broader
recognition can occur becauseof nonstandard pairing be-
tween bases in the so-called uobble position: that is, the
third (3') base in an mRNA codon and the corresponding
first (5') basein its IRNA anticodon.
The first and second basesof a codon almost always
form standard Watson-Crickbasepairs with the third and
secondbases,respectively,of the correspondinganticodon,
but four nonstandardinteractionscan occur betweenbases
in the wobble position. Particularly important is the G.U
base pair, which structurally fits almost as well as the
standard G.C pair. Thus, a given anticodon in IRNA with
G in the first (wobble) position can base-pairwith the two

T{/CG loop Acceptor stem

< FIGURE 4-20 Structureof tRNAs.(a)Althoughtheexact
nucleotidesequence variesamongtRNAs, theyallfold intofour
base-paired stemsandthreeloopsTheCCAsequence at the 3, end
alsoisfoundin alltRNAsAttachment of an aminoacidto the 3, A
yieldsan amrnoacyl-tRNA. Someof theA, C, G, andU residues are
modified post-transcriptionally
in mosttRNAs (seekey).Dihydrouridine
(D)is nearlyalwayspresent in the D loop;likewise,ribothymidine (T)
andpseudouridlne (V) arealmostalwayspresent in theTTITCG loop.
Yeast alanineIRNA,represented here,alsocontains othermodified
basesThetripletat thetip of theanticodon loopbase-pairs with the
corresponding codonin mRNA(b)Three-dimensional modelof the
generalizedbackbone of alltRNAsNotethe L shapeof the molecule.
[Part(a) see R W Hollyet al , 1965, Science147i1462;part (b) from J G
Arnez and D Moras, 1997, TrendsBiochem Sci 22:211 I

130 CHAPTER
4 | B A S | CM O L E C U L A G
R E N E T TM
CE C H A N T S M S
 tRNA nized by the sameIRNA with the anticodon 3'-GAI-S'; the
inosine in the wobble position forms nonstandard base
pairs with the third basein the four codons. In the caseof
in. UUR codon, a nonstandard G'U pair also forms be-
tween position 3 of the anticodon and position 1 of the
lf these basesare in
first, or wobble, positionof codon.
a nticodon
321 Amino Acids BecomeActivatedWhen
12 t h e nt h e t R N A m a y CovalentlyLinkedto tRNAs
5 ' m R N A3 ' recognizecodons in
m R N A h a v i n gt h e s e Recognition of the codon or codons specifying a given
b a s e si n t h i r d p o s i t i o n amino acid by a particular IRNA is actually the secondstep
in decoding the genetic message.The first step' attachment
lf these basesare in
t h i r d ,o r w o b b l e ,p o s i t i o n
5' mRNA of codon of an mRNA
12
321 then the codon may
be recognizedby a the 3' terminus of IRNA moleculesby an AlP-requiring re-
tRNA havingthese action. In this reaction, the amino acid is linked to the tRNA
basesin first position
of anticodon by a high-energybond and is thus said to be actiuated. The
energy of this bond subsequentlydrives formation of the
c
peptide bonds linking adjacent amino acids in a growing
poiypeptide chain. The equilibrium of the aminoacylation
3', i.u.tiotr is driven further toward activation of the amino
tRNA acid by hydrolysis of the high-energyphosphoanhydride
A FIGURE 4-21 Nonstandard basepairingat the wobble bond in the releasedpyrophosphate (seeFigute 4-19).
position.Thebasein thethird(orwobble)position of an mRNA Aminoacyl-IRNA synthetasesrecognize their cognate
codonoftenformsa nonstandard basepairwith the basein thefirst tRNAs by interacting primarily with the anticodon loop
(orwobble)position of a tRNAanticodonWobblepairingallowsa and acceptor stem, although interactions with other re-
IRNAto recognize morethanone mRNAcodon(top);conversely, it
gions of a IRNA also contribute to recognition in some
allowsa codonto be recognized by morethanonekindof IRNA
cases.Also, specific bases in incorrect tRNAs that are
(bottom), although eachIRNAwill bearthesameaminoacid Note
structurally similar to a cognate IRNA will inhibit charg-
thata IRNAwith l(inosine) in thewobbleposition can"read"(become
ing of the incorrect IRNA. Thus, recognition of the correct
pairedwith)threedifferent codons, anda IRNAwith G or U in the
canreadtwo codonsAlthoughA istheoretically tRNA dependson both positive interactions and the ab-
wobbleposition
possible in thewobbleposition of the anticodon, it isalmostnever senceof negative interactions. Still, becausesome amino
foundin nature acids are so similar structurally, aminoacyl-tRNA syn-
thetasessometimesmake mistakes. These are corrected'
however, by the enzymesthemselves,which have a proof-
correspondingcodonsthat have either pyrimidine (C or U) reading activity that checks the fit in their amino
in the third position (Figure4-21).For example,the pheny- acid-binding pocket. If the wrong amino acid becomesat-
lalanine codons UUU and UUC (5'-+3') are both recog- tached to a tRNA, the bound synthetase catalyzesremoval
nized by the IRNA that has GAA (5'-+3') as the anticodon. of the amino acid from the IRNA' This crucial function
In fact, any two codons of the type NNPyr (N : any base; helps guarantee that a IRNA delivers the correct amino
Pyr : pyrimidine) encode a single amino acid and are acid t" the protein-synthesizingmachinery. The overall
decoded by a single tRNA with G in the first (wobble) error rate for translation in E' coli is very loq approxi-
position of the anticodon. mately 1 per 50,000 codons, evidenceof both the fidelity
Although adenine rarely is found in the anticodon of IRNA iecognition and the importance of proofreading
wobble position, many tRNAs in plants and animals con- by aminoacyl-IRNA synthetases.
tain inosine (I), a deaminatedproduct of adenine, at this
position. Inosine can form nonstandardbasepairs with A,
C, and U. A IRNA with inosine in the wobble position
thus can recognizethe correspondingmRNA codons with The Decoding of mRNA bY tRNAs
A , C , o r U i n t h e t h i r d ( w o b b l e )p o s i t i o n ( s e eF i g u r e4 - 2 1 ) '
r Genetic information is transcribed from DNA into mRNA
For this reason,inosine-containingtRNAs are heavily em-
in the form of an overlapping,degeneratetriplet code'
ployed in translation of the synonymouscodons that specify
a single amino acid. For example, four of the six codons r Each amino acid is encoded by one or more three-
for leucine (CUA, CUC, CUU, and UUA) are all recog- nucleotide sequences(codons) in mRNA. Each codon

T H E D E C O D I N GO F m R N A B Y t R N A s 131
 specifiesone amino acid, but most amino acidsare encoded
by multiple codons(seeTable 4-1).
r The AUG codon for methionine is the mosr common
start codon, specifyingthe amino acid at the NH2-terminus
of a protein chain. Three codons (UAA, UAG, UGA) func-
tion as stop codons and specify no amino acids.
r A reading frame, the uninterrupted sequenceof codons
in mRNA from a specific starr codon to a stop codon, is
translated into the linear sequenceof amino acids in a
polypeptide chain.
r Decoding of the nucleotide sequencein mRNA into the
amino acid sequenceof proteins depends on tRNAs and
aminoacyl-IRNA synthetases.
r All tRNAs have a similar three-dimensionalstructure
that includes an acceptor arm for attachment of a specific
amino acid and a stem-loopwith a three-baseanticodon se-
quence at its ends (seeFigure 4-20). The anticodon can
base-pairwith its correspondingcodon in mRNA.
r Becauseof nonstandard interactions, a tRNA may base-
pair with more than one mRNA codon; converselSa par-
ticular codon may base-pairwith multiple tRNAs. In each
case,however,only the proper amino acid is insertedinto a
g r o w i n gp o l y p e p t i d ec h a i n .
Each of the 20 aminoacyl-tRNA synthetasesrecognrzesa
ngle amino acid and covalently links it to a cognare
rRNA
.9
23S 5S
:.E (2900rNTs) ( 1 2 0r N T s )
o
o-
165
(1500 rNTs)
q,
o
t
o
(J
o
J
lrj
( 1 9 0 0r N T s )
FfGURE4-22 Prokaryotic and eukaryotic ribosome
components.In all cells,eachribosomeconsists of a largeand a small
subunit.The two subunitscontainrRNAs(red)of differentlengths,as
well as a differentset of proteins.All ribosomescontaintwo maior
132 . cHAprER
4 | B A S t cM o L E c u L A G
R E N E T tMc E C H A N t s M s
IRNA, forming an aminoacyl-tRNA (seeFigure 4-19). This
reaction activates the amino acid, so it can participate in
peptide bond formation.
@ of proteins
StepwiseSynthesis
on Ribosomes
The previous sectionshave introduced two of the major par-
ticipants in protein synthesis-mRNA and aminoacylated
IRNA. Here we first describethe third key player in protein
synthesis-the rRNA-containing ribosome-before taking a
detailed look at how all three components are brought to-
gether to carry out the biochemical eventsleading to forma-
tion of polypeptide chains on ribosomes. Similar to tran-
scription, the complex processof translation can be divided
into three stages-initiation, elongation, and termination-
which we consider in order. \7e focus our description on
translation in eukaryotic cells, but the mechanismof transla-
tion is fundamentally the same in all cells.
R i b o s o m eA
s re Protein-Synthesizin
Mga c h i n e s
If the many components that participate in translating
mRNA had to interact in free solution. the likelihood of
simultaneouscollisions occurring would be so low that the
rate of amino acid polymerization would be very slow. The
Assembled
Proteins Subunits ribosomes
Total: 31
50s
Total: 21
70s
80s
40s
r R N Am o l e c u l e(s2 3 Sa n d 1 6 5r R N Ai n b a c t e r i a2;g Sa n d 1 g Sr R N Ai n
vertebrates) and a 55 rRNA The largesubunitof verteorate
ribosomesalsocontainsa 5 85 rRNAbase-paired to the 2gS rRNA
The numberof ribonucleotides (rNTs)in eachrRNAtvpe is indicated
efficiencyof translation is greatly increasedby the binding of
the mRNA and the individual aminoacyl-tRNAs to a ribo-
some. The ribosome, the most abundant RNA-protein com-
plex in the cell, directselongationof a polypeptideat a rate of
three to five amino acids added per second.Small proteins of
100-200 amino acids are thereforemade in a minute or less.
On the other hand, it takes 2-3 hours to make the largest
known protein, titin, which is found in muscle and contains
about 30,000 amino acid residues.The cellular machine that
accomplishesthis task must be preciseand persistent.
With the aid of the electron microscope,ribosomeswere
first discoveredas small, discrete,RNA-rich particles in cells
that secretelarge amounts of protein. However, their role in
protein synthesiswas not recognizeduntil reasonably pure
ribosome preparations were obtained. In vitro radiolabeling
experimentswith such preparationsshowed that radioactive
amino acids were first incorporated into growing polypep-
tide chains that were associated with ribosomes before
appearing in finished chains.
Though there are differencesbetween the ribosomes of
prokaryotes and eukaryotes,the great structural and func-
tional similarities between ribosomes from all speciesreflects
the common evolutionary origin of the most basic con-
stituents of living cells. A ribosome is composedof three (in
bacteria) or four (in eukaryotes)different rRNA molecules
and as many as 83 proteins,organizedinto a largesubunit and
a small subunit (Figure4-22).The ribosomal subunitsand the
rRNA moleculesare commonly designatedin svedbergunits
(S), a measureof the sedimentationrate of macromolecules
centrifuged under standard conditions----essentially, a measure
of size.The small ribosomal subunit contains a singlerRNA
molecule, referred to as small rRNA. The large subunit con-
tains a molecule of large rRNA and one molecule of 55 rRNA,
plus an additional moleculeof 5.8SrRNA in vertebrates.The
lengths of the rRNA molecules, the quantity of proteins in
eachsubunit, and consequentlythe sizesof the subunitsdiffer
between bacterial and eukaryotic cells. The assembledribo-
some is 70S in bacteriaand 80S in vertebrates.
The sequencesof the small and large rRNAs from several
thousand organismsare now known. Although the primary
nucleotide sequencesof these rRNAs vary considerablS the
sameparts of eachtype of rRNA theoreticallycan form base-
paired stem-loops, which would generate a similar three-
dimensional structure for each rRNA in all organisms.The
actual three-dimensionalstructuresof bacterial rRNAs from
E. coli recently have been determined by x-ray crystallogra-
phy of the 70S ribosome(Figure4-23).The multiple' much
smaller ribosomal proteins for the most part are associated
with the surfaceof the rRNAs. Although the number of pro-
tein molecules in ribosomes gready exceedsthe number of
RNA molecules,RNA constitutes about 60 percent of the
massof a ribosome.At the interfaceof the small and large ri-
bosomal subunits,three local domains are formed' known as
the A site, the P site, and the E site. As we'll seeshortly, these
are the main sitesof interaction for the aminoacyl-tRNA and
mRNA within the ribosome as protein synthesistakes place'
During translation, a ribosome moves along an mRNA
chain, interacting with various protein factors and tRNAs
and undergoing large conformational changes'Despite the
complexity of the ribosome, great progresshas beenmade in
deteimining the overall structure of bacterial ribosomesand
in identifying various reactive sites' X-ray crystallographic
studies on the T. thermophilus 705 ribosome, for instance,
have not only revealed the dimensions and overall shape
of the ribosomal subunits but also localized the positions of
tRNAs bound to the ribosome during elongation of a grow-
ing protein chain. In addition, powerful chemical techniques
r.r.h footprinting, which is describedin Chapter 7,have
"r
been used to identify specific nucleotide sequencesin rRNAs
that bind to protein or another RNA. Some 40 yearc after the
initial discovery of ribosomes' their overall structure and func-
tioning during protein synthesisare finally becoming clear.
the AUG
Recognizes
Methionyl-tRNA;M"t
Start Codon
As noted earlier,the AUG codon for methionine functions as
the start codon in the vast majority of mRNAs. A critical as-
pect of translation initiation is to begin protein synthesisat
ihe start codon, thereby establishing the correct reading
frame for the entire mRNA. Both prokaryotes and eukary-
otes contain two different methionine tRNAs: tRNAlM"'can
initiate protein synthesis, and IRNAM" can incorporate
methionine only into a growing protein chain. The same
aminoacyl-tRNA synthetase(MetRS) charges both tRNAs
with met'hionine;however, only Met-tRNA,M" (i.e., activated
methionine attached to tRNAiM't) can bind at the appropri-
ate site on the small ribosomal subunit, the P site, to begin
synthesisof a polypeptide chain' The regular Met-tRNAM"'
and all other chargedtRNAs bind only to another ribosomal
site, the A site, as described later. As mentioned earlier, in
bacteria, the initiating methionine has a formyl group linked
to its amino group, forming N-formylmethionine.
TranslationInitiation UsuallyOccursat the First
A U G f r o m t h e 5 ' E n do f a n m R N A
During the first stage of translation' the small and large ri-
bosomal subunits assemblearound an mRNA that has an
aminoacylated initiator IRNA correctly positioned at the
start codon. This processis mediated by a specialset of pro-
teins known as translation initiation factors (IFs). As each
individual component ioins the complex, it is accompanied
by one or more specific initiation factors. Interactions be-
tween theseinitiation factors help stabilizethe complex' Fur-
thermore, some initiation factors are coupled to GTP, and
the hydrolysis of GTP to GDP functions as a proofreading
switch that allows subsequentsteps to proceed only if the
the two ribosomal subunits once the small subunit with a
charged initiator IRNA (Met-tRNAiM"t) has bound to an
ON RIBOSOMES O
S T E P W I S sEY N T H E 5 I 5O F P R O T E I N S 133

initiation codon in an mRNA. The first step of translation
initiation is formation of a preinitiation complex. The
preinitiation complex is formed when the 40S subunit com-
plexed with the multisubunit eIF3 complex associateswith
eIFIA and a ternary complex consisting of the Met-
tRNAiM't and eIF2 bound to Ctp (Figure4-i4, steptr). The
initiation factor eIF2 alrernates between association with
GTP and GDP; it can bind Met-tRNA1M.. only when it is
associatedwith GTP. Cells can regulate protein synthesis
by phosphorylating a serine residue on the eIF2 bound to
GDP; the phosphorylated complex is unable to exchange
the bound GDP for GTP and therefore cannot bind Met-
tRNAiM"t, thus inhibiting protein synthesis.
The 5' cap of an mRNA to be translated is bound by the
eIF4 cap-binding complex. The eIF4 cap-binding complex
consists of several subunits with different functions; the
eIF4E subunit of the eIF4 complex binds the 5/ cap structure
on mRNAs (Figure 4-14).ThemRNA-eIF4 complix then as-
subunit so that it can remove short regions of RNA second-
ary structure in bound RNA using energy from ATp hydrol_
ysis. This multicomponent initiation complex then probably
slides along, or scctns,the bound mRNA as the helicase
activity of eIF4A unwinds RNA secondary structures that
might otherwise interfere with scanningalong the mRNA in
the 3' direction. Scanning srops when the tRNAiM.t anti-
codon recognizesthe start codon, which is the first AUG
134 . cHAprER
4 | B A s t cM o L E c u L A G
R E N E T tMc E C H A N t s M s
Podcast:Structure of the Ribosome C)
ZN
Rotating 3-D Model of a BacterialRibosome
< FfGURE4-23 Structureof E.coli 70Sribosomeas determined
by x-ray crystallography.Modelof the ribosome viewedalongthe
interface
between the large(50S)andsmall(30S)subunitsThe165
rRNAandproteins in thesmallsubunitarecolored lightgreenand
darkgreen,respectively;the23SrRNAandproteins in the largesubunit
arecoloredlightpurpleanddarkpurple,respectively; andthe 55rRNA
iscoloreddarkblue Thepositions of the ribosomalA, f andEsitesare
indicatedNotethatthe ribosomal proteins arelocatedprimarilyon the
surface
of the ribosome, andthe rRNAs on the inside,liningtheA, f
andEsites[From B S Schuwirth
etal.2005.Nature}l}:827 1
downstream from the 5' end in most eukaryotic mRNAs
(step E). Recognition of the start codon leads to hydrolysis
of the GTP associatedwith eIF2. an irreversible steD that
prevents further scanning.Selectionof the initiating AUG is
facilitated by specific surrounding nucleotides called the
Kozak sequence,for Marilyn Kozak, who defined it: (5,)
ACCAUGG (3'). The A precedingthe AUG (underlined)and
the G immediately following it are the most important nu-
cleotidesaffecting translation initiation efficiency.Once the
small ribosomal subunit with its bound Met-tRNAiM". is
correctly positioned at the start codon and the GTp bound
by elF2 is hydrolyzed to GDP, elF1.,2,3, and 4 dissociate
and the small subunit unites with the large (605) ribosomal
subunit in a processcatalyzedby eIF5 and 6, completing for-
mation of an 80S ribosome. Vith the entire complex assem-
bled, the Met-tRNA1M"'bound to the AUG codon is situated
in the P site. Recruitment of the large ribosomal subunit is
accompaniedby hydrolysis of a GTP bound by eIF5, another
proofreading step (step @). Coupling the ribosome-subunit-
joining reaction to GTP hydrolysis allows the initiation
process to continue only when the subunit interaction has
occurred correctly. It also makes this an irreversiblestep, so
that the ribosomal subunits do not dissociateuntil the entire
mRNA is translated and protein synthesisis terminated.
The eukaryotic protein-synthesizing machinery begins
translation of most cellular mRNAs within about 100 nu-
cleotides of the 5' capped end as just described.However,
somecellular mRNAs contain an internal ribosome entry site
(IRES) located far downsrream of the 5' end. In addition,
translation of some viral mRNAs, which lack a 5, cap, is ini-
tiated at IRES sequencesby the host-cell machinery of
infectedeukaryotic cells.Someof the sametranslation initia-
tion factors that assistin ribosome scanningfrom a 5, cap are
required for locating an internal AUG start codon, but ex-
actly how an IRES is recognizedis lessclear.Recentresultsin-
dicate that some IRES sequencesfold into an RNA structure
that binds to the E site on the ribosome (seebelow), thereby
positioning a nearby internal AUG start codon in the p site.
In bacteria, binding of the small ribosomal subunit to an
initiation site occurs by a different mechanism that allows ini-
tiation at internal sitesin the polycistronic mRNAs transcribed
from operons.In bacterial mRNAs, an :6-base sequencecom-
plementary to the 3' end of the small rRNA precedesthe AUG
 < FIGURE 4-24 lnitiationof translationin eukaryotes'/nset"
elF6 Whena ribosome dissociates at thetermination of translation, the
\
4OSand605subunits associate factorselF3andelF6,
with initiation
\,
formingcomplexes
respectively, thatcaninitiateanotherroundof
translationSteps Il and Z: Sequential addition of the indicated
components to the40Ssubunit-elF3 complex formsthe initiation
complexStepB: Scanning of the mRNAbythe associated initiation
complex leadsto positioning of thesmall subunit and bound Met-
tRNAiM"tat thestartcodon. Step 4: Association of the large subuntt
(605)formsan 805ribosome readyto translate the mRNATwoiniti-
ationfactors, elF2(step[) andelF5(step4) areGTP-binding proteins,
whoseboundGTPishydrolyzed duringtranslationinitiation.The precise
timeat whichparticular initiationfactorsare released is not yet well
characterized See the textfor a more detaileddiscussron lAdapted
f r o m R M e n d e za n d J D R i c h t e t2 0 0 1 , N a t u r eR e vM o l C e l lB i o l 2 : 5 2 1 l

start codon by 4-7 nucleotides.Basepairing betweenthis se-

quencein the mRNA, called the Shine-Dalgarnosequenceafter
iis discoverers,and the small rRNA placesthe small ribosomal
subunit in the proper position for initiation. Next' f-Met-
tRNAiM" and initiation factors comparableto eIFIA' eIF2'
and eIF3 associatewith the small subunit,followed by associa-
tion of the large subunit to form the complete bacterial ribo-
someby a mechanismsimilar to that in eukaryotes'

D u r i n gC h a i nE l o n g a t i o nE a c hI n c o m i n g
Preinitiation complex Aminoacyl-tRNAMovesThroughThree
AUG- (AAA)" R i b o s o m aSl i t e s
The correctly positioned ribosome-Met-tRNAiM't complex
e l F 4( c a p - b i n d i ncgo m p l e x )+ m R N A is now ready to begin the task of stepwiseaddition of amino
wret$cre acidsby the in-frame translation of the mRNA' As is the case
with initiation, a set of special proteins, termed translation
mTGppp
(AAA}"

Initiationcomplex
RNA ATP
u n w i n d in q , codon at a time along the mRNA.
s c a n n r n g ,a n d E ADP + P; At the completion of translation initiation, as noted
start srte
r e c o gn r tr or l e l F 1 A ,e l F 3 ,e l F 4c o m p l e x , akeady,Met-tRNA1M" is bound to the P site on the assem-
elF2.GDP + P; bled SOSribosome (Figure 4-25, top). This region of the
ribosome is called the P site becausethe IRNA chemically
linked to the growing polypeptide chain is located here' The
second aminoacyl-tRNA is brought into the ribosome as a
(AAAln 3',
ternary complex in associationwith EFla'GTP and becomes
6 0 5 s u b u n i t - e l F 6e,l F S ' G T P bound to the A site, so named becauseit is where aminoacy-
g( lated tRNAs bind (step E). EFlct'GTP bound to various
+ P;
e l F 6 ,e | F S ' G D P
l\

80S ribosome

SN R I B O S O M E S '
S T E P W I SSEY N T H E 5 IO5 F P R O T E I NO 135
 FocusAnimation:proteinSynthesis
flltt
< FIGURE 4-25 Peptidylchainelongationin eukaryotes. Once
the80Sribosome with Met-tRNA,M",in the ribosome p siteis
assembled (top),a ternary complex bearing thesecond aminoacid
(aar)codedbythe mRNAbindsto theA site(stepO) Following a
conformational changein the ribosome induced by hydrolysis of GTp
in EFIcTGTP (stepZ), thelargerRNAcatalyzes peptide bondformation
betweenMetiandaa2(step!) Hydrolysis of GTpin EF2.GTp causes
anotherconformational changein the ribosome that results in its
translocation onecodonalongthe mRNAandshiftsthe unacylated
tRNAiM"t to the EsiteandtheIRNAwiththe boundpeptrde to the p
Entry of next E site(step4) Thecyclecanbeginagainwith bjndingof a ternary
a a - t R N Aa t
A site complex bearing aa3to the now openA site In thesecono and
subsequent elongationcycles,theIRNAat the Esiteisejected during
stepI asa resultof theconformational change induced by hydrolysis
of GTPin EFIa.GTP[Adapted fromK H Nierhaus etal. 2OOO. inR A
Garrettet al, eds, TheRibosome:Structure,
Function,
Antibiotics,andCellular
lnteractions,
ASMPress, p 319l

Thus, GTP hydrolysis by EFlcr is another proofreading step

G T Ph y d r o l y s i s , L that allows protein synthesisto proceed only when the cor-
E | \ 6.oor-r,
iii,'"?il1,'""", rect aminoacylated IRNA is bound to the A site. This ohe-
change + nomenon contributes to the fidelity of protein synthesis.
\fith the initiating Met-tRNAiM" at the p site and the sec-
ond aminoacyl-tRNA tightly bound at the A site,the q amino
group of the secondamino acid reactswith the ,,activated"
(ester-linked)methionine on rhe initiator IRNA, forming a
peptidebond (Figure4-25, stepB; seealsoFigure4-17).This
peptidyltransferdse reaction is catalyzed by the large rRNA,
which preciselyorients the interacting atoms, permitting the
Peptidebond reaction to proceed.The catalytic ability of the large rRNA in
formation bacteriahas beendemonstratedby carefullyremoving the vast
majority of the protein from large ribosomal subunits. The
nearly pure bacterial23S rRNA can catalyzea pepridyltrans-
ferasereaction betweenanalogsof aminoacylated-tRNAand
peptidyl-tRNA. Further support for the catalytic role of large
rRNA in protein synthesiscomesfrom crystallographicstud-
ies showing that no proteins lie near the site of peptide bond
synthesisin the crystal structureof the bacteriallarge subunit.
Following peptide bond synthesis,the ribosomeis translo-
l'z +z'one
cated along the mRNA a distanceequal to one codon. This
ff*l:Ii"" E translocation step is monitored by hydrolysis of the GTp in
EF2.GDp*pi
Jl'.-- eukaryotic EF2.GTP. Once translocation has occurred cor-
rectly, the bound GTP is hydrolyzed, another irreversible
processthat prevents the ribosome from moving along the
RNA in the wrong direction or from translocatingan incor-
rect number of nucleotides.As a result of conformational
changesin the ribosomethat accompanyproper translocation
and the resulting GTP hydrolysis by EF2, tRNA;M.t, now
without its activatedmethionine,is moved to the E (exit) site
on the ribosome; concurrently,the secondtRNA, now cova-
lently bound to a dipeptide(a peptidyl-rRNA), is moved to the
P site (Figure4-25, stepZf). Tianslocationthus rerurnsthe ri-
bosome conformation to a state in which the A site is ooen
and able to accept another aminoacylatedIRNA complexed
with EFlcr.GTP,beginninganother cycleof chain elongation.
Repetition of the elongationcycle depictedin Figure 4-25
adds amino acids one at a time to the C-terminus of the

136 . cHAprER
4 B A s t cM o L E c u L AG
R E N E T tMc E c H A N I s M s
|
 (a) generaldomains of all tRNAs result in the movement of the
tnXRr betweenthe A, P' and E sitesas the ribosome translo-
catesalong the mRNA one three-nucleotidecodon at a time'

Translationls Terminatedby ReleaseFactors

W h e n a S t o p C o d o nl s R e a c h e d
The final stageof translation, like initiation and elongation,
requireshighly specificmolecular signalsthat decidethe fate
of the mRNA-ribosome-peptidyl-tRNA complex' Two

(b)

eukaryotic releasefactor, eRF3, is a GTP-binding protein'

The eRF3'GTP actsin concertwith eRFl to promote cleav-
age of the peptidyl-tRNA, thus releasingthe completed pro-
tJin chain (Figure 4-27). Bacteria have two releasefactors
(RF1 and RF2) that are functionally analogousto eRFl and

A FfGURE4-26 Low-resolutionmodel of E. coli 7OSribosome.

eRFl + eRF3.GTP
(a)Toppanels showcryoelectron microscoprc images oI E colil0S
ribosomes and5OSand30SsubunitsBottompanels showcomputer-
derived averages of manydozens of images in the sameorientation
(b)Modelof a 7OSribosome basedon thecomputer-derived images
andon chemical cross-linking studies Three tRNAs are superimposed
on theA (pink), P(green), andE(yellow) sitesThenascent polypeptide
c h a i ni sb u r i e di n a t u n n eiln t h el a r g er i b o s o msaul b u n ti th a tb e g i n s
closeto the acceptor stemof theIRNAin the Psite [See I S Gabashvili
et al, 2OOO, Cell1OO:537, courtesy of J Frank l

growing polypeptideas directedby the mRNA sequence,until

a stop codon is encountered.In subsequentcycles,the confor-
t
Peptidyl-tRNA \
mational changethat occurs in step f,l electsthe unacylated -+
cleavage | \ eRFl+eRF3oGDP+Pi
IRNA from the E site. As the nascent polypeptide chain
becomeslonger,it threadsthrough a channelin the large ribo-
somal subunit, exiting at a position opposite the side that
interactswith the small subunit (Figure4-26).
RNA-
In the absenceof the ribosome'the three-base-pair
RNA hybrid between the IRNA anticodons and the mRNA
codonsin the A and P siteswould not be stable;RNA-RNA
duplexesbetweenseparateRNA moleculesmust be consid-
erably longer to be stable under physiologicalconditions.
However, multiple interactions between the large and small A FIGURE4-27 Terminationof translationin eukaryotes'When
rRNAs and general domains of tRNAs (e.g., the D and a ribosome bearinga nascent a stopcodon(UAA'
proteinchainreaches
ribosomalcomplex, probably
TVCG loops, seeFigure 4-20) stabilizethe tRNAs in the A UGA,UAG),release factoreRFlentersthe
at or neartheA sitetogetherwith eRF3'GTP of
Hydrolysis the bound
and P sites,while other RNA-RNA interactionssensecorrect
bycleavage chainfromtheIRNAin
of the peptide
codon-anticodonbasepairing, assuringthat the geneticcode GTPisaccompanied
the Psiteandrelease of thetRNAsandthetwo ribosomal subunits
is read properly. Then, interactions between rRNAs and the

ON RIBOSOMES .
5 F PROTEINS
E Y N T H E S IO
S T E P W I SS
137
 a GTP-binding factor (RF3) that is analosousto eRF3. Once
again,the eRF3 GTPasemonirors the coriect recognition of a
stop codon by eRF1. The peptidyl-rRNA bond of the IRNA
We can now seethat one or more GTp-binding proteins
participate in each stage of translation. These proteins be-
long to the GTPasesuperfamily of switch proteins that cycle
between a GTP-bound active form and GDp-bound inaciive
form (seeFigure 3-32). Hydrolysisof the bound GTp causes
a conformational change in the GTpase itself and other as-
sociatedproteins that are critical to various complex molec-
ular processes.In translation initiation, for instance,hydrol-
ysis of eIF2.GTP to eIF2.GDp prevenrsfurther scanning of
the mRNA once the start site is encounrered and allJws
binding of the large ribosomal subunit to the small subunit
(seeFigure 4-24, step p). Similarly, hydrolysis of EF2.GTp
to EF2.GDP during chain elongation leads to correct
translocation of the ribosome along the mRNA (seeFigure
4-25, step Zl), and hydrolysisof eRF3.GTp to eRF3.GDp
assurescorrect termination of translation. Since hvdrolvsis
of the high-energy B-1 phosphoesterbond of GTi, is irre-
versible, coupling of these stepsin protein synthesisto GTp
hydrolysis preventsthem from going in rhe reversedirection.
One kind of mutation that can inactivate a gene in any
organism is a base-pairchange that converts a codon nor_
mally encoding an amino acid into a stop codon, e.g., UAC
(encodingtyrosine) -+ UAG (stop). When this occurs early in
the reading frame, the resulting truncated protein usually is
nonfunctional. Such mutations are called nonsense mvta_
tions becausewhen the genetic code equating each triplet
codon sequencewith a single amino *"i being dlci_
phered, the three stop codons were found ".id
,rot to .rr.od. ,.ry
amino acid-they did not ,,make sense.',
In geneticstudieswith the bacterium E. coli, it was dis_
138 . c H A p r E4R | B A S tM
c oLEcuLA
GRE N E TM
t cE c H A N t s M s
the protein encoded by the original gene with the nonsense
mutation is produced to provide its essentialfunctions, the ef-
fect of the first mutation is said to be suppressedby the second
mutation in the anticodon of the IRNA gene.
This mechanism of nonsensesuppressionis a powerful
tool in geneticstudiesin bacteria.For example, mutant bac-
terial virusescan be isolatedthat cannot grow in normal cells,
but can grow in cellsexpressinga nonsense-suppressing IRNA
becausethe mutant virus has a nonsensemutation in an es-
sential gene. Such mutant viruses grown on the nonsense-sup-
pressingcells can then be used in experimentsto analyzethe
function of the mutant geneby infecting normal cellsthat do
not suppressthe mutation and analyzing what step in the vi-
ral life cycle is defectivein the absenceof the mutant protein.
Polysomesand RapidRibosomeRecycling
Increasethe Efficiencyof Translation
an mRNA. Simultaneoustranslation of an mRNA bv multiole
ribosomesis readily observablein electronmicrographsandty
sedimentation analysis,revealing mRNA attached to multiple
ribosomes bearing nascentgrowing polypeptide chains. These
structures, referred to as polyribosomes or polysomes, were
seen to be circular in electron micrographs of some tissues.
Subsequentstudieswith yeastcellsexplained the circular shape
of polyribosomes and suggestedthe mechanism by which
ribosomes recycleefficiently.
These studies revealed that multiple copies of a cytosolic
protein found in all eukaryotic cells,poly(A)-binding prroteinI
(PABPI), can interacr with both an mRNA poly(A) tail and the
4G subunit of yeast eIF4. Recall that the 4E subunit of yeast
eIF4 binds to the 5' end of an nRNA. As a result of thesein-
cap. The circular pathway depictedin Figure 4-Z9b,which may
operate in many eukaryotic cells, would enhanceribosome re_
cycling and thus increasethe efficiency of protein synthesis.
Stepwise Synthesisof proteins on Ribosomes
prokaryoric and eukaryotic ribosomes-the large
.Both
bonucleoproteincomplexeson which translation o..urr-
consistof a small and a large subunit (seeFigure4-22).Each
subunit contains numerous different proteins and one ma-
jor rRNA molecule (small or large). The large subunit also
contains one accessory55 rRNA in bacteriaand two acces,
sory rRNAs in eukaryores(5S and 5.8S in vertebrates).
 ing GTP-binding proteins that hydrolyze their bound GTP
TJGDP whe.t aitip has been completed successfully'
During initiation' the ribosomal subunits assemblenear
e translation start site in an mRNA molecule with the
IRNA carrying the amino-terminal methionine (Met-
tRNAiM"t) base-pairedwith the start codon (Figute 4-24)'
ain elongation entails a repetitive four-step cycle: (1)
binding of an incoming aminoacyl-tRNA to the A site
e ribosome, (2) tight binding of the correct aminoacyl-
IRNA to the A site accompanied by releaseof the previ-

to the E site (seeFigure4-25).

(b)

E' leads to translocation.

r Termination of translation is carried out by two types of
termination factors: those that recognizestop codons and
those that promote hydrolysis of peptidyl-tRNA (see
Figure 4-27). Once again, correct recognition of a stop
codon is monitored by a GTPase(eRF3)'
The efficiency of protein synthesis is increased by the si-
ultaneoustranslation of a singlemRNA by multiple ribo-

a EXPERIMENTAL FIGURE 4-28 The circularstructureof mRNA

increasestranslationefficiency.Eukaryotic mRNAformsa circular
structure owingto interactions of threeproteins(a)In the presence
of purifiedpoly(A)-bindingprotein| (PABPI),elF4E,andelF4G, eukaryotic
mRNAs formcircularstructures,visiblein thisatomicforcemicrograph
Inthesestructures, protein-protein andprotein-mRNA interactlons !f,l DNAReplication
forma bridgebetween the 5' and3' endsof the mRNA.(b)Model Now that we haveseenhow geneticinformationencodedin
of proteinsynthesis on circular polysomes andrecycling of ribosomal
subunits. Multipleindividualribosomes cansimultaneously translate
a eukaryotic mRNA,shownherein circular formstabilized by
interactions betweenproteins boundat the 3' and5' ends.Whena
ribosome completestranslation anddissociates fromthe 3' end,the
separated subunitscan rapidlyfind the nearby 5' cap(m7G) and
initrateanotherroundof svnthesrs. [Part(a)courtesy
of A Sachs ]

r Analogous rRNAs from many different speciesfold into

quite similar three-dimensionalstructures containing nu-
merous stem-loopsand binding sites for proteins, mRNA,
and tRNAs. Much smaller ribosomal proteins are associ-
ated with the periphery of the rRNAs. (duplex\DNA
strandswould form a new double-stranded
r Of the two methionine tRNAs found in all cells,only one molecule,and the parentalduplexwould remain intact' In a
(tRNAiM't) functions in initiation of translation. semiconservativemechanism,the parental strands are per-
manentlyseparated, and eachforms a duplex molecule with
r Each stage of translation-initiation, chain elongation,
the daughterstrand base-paired to it. Definitive evidence
and termination-requires specific protein factors, includ-

DNA REPLICATION 139

 (a) Predicted results (b) Actual results
C o n s e r v a t i vm
e echanism S e m i c o n s e r v a t i vmee c h a n r s m Density- Density-
Generation
P a r e n t asl t r a n d s 0
s y n t h e s i z e idn 1 5 N

HH HH 0.7

1.0

1.1
After first
d o u b l i n gi n t a N 1.5

1.9

A
HH LL LH
2.5

3.0
/\ /\ /\ /\
4.1
After second
d o u b l i n gi n t r N 0 and 1.9
mrxed

0 and4.1
mrxed
HHLL LLLL LLHL LHLL L-L H-L H-H L-L H-L H-H
IGURE4-29 The Meselson_Stahl experiment of L-Lduplexes with eithermechanism(b)Actualbandingpatternsof
be semiconservative.Thrsexperimentshowed DNAsubjected to equilibrium density-gradientcentrifugationbefore
a semiconseryative mechanism. E colicells and aftershiftinglsN-labeled E colicellsto laN-containing medium.
initially
weregrown in a mediumcontaining ammoniumsaltsprepared DNA bandswerevisualized underUV lightand photographedThe
wlth "heavy"nitrogen(1sN)untilallthe cellularDNAwas labeledAfter traceson the left are a measureof the densityof the photographic
the cellsweretransferred to a mediumcontaining the normal,,light,, signal,and hencethe DNAconcentratlon, alongthe lengthof the
isotope(raN),samples were removedperiodically from the cultures and centrifugecellsfrom left to right.The numberof generations(far left)
the DNA in eachsamplewas analyzed by equilibrium density_gradient followingthe shiftto 1aN-containing mediumwas determined by
centrifugation,a procedure that separatesmacromolecules on the basis countrngthe concentration of E colicellsin the cultureThisvalue
of theirdensity.Thistechniquecan separate heavy_heavy (H_H),light_ corresponds to the numberof DNA replicatron cyclesthat had occurred
light(L-L),and heavy-light (H-L)duplexesinto distinctbands. at the time eachsamplewas taken.After one generation of growth,all
(a) Exp_ectedcompositionof daughterduplexmoleculessynthesized the extractedDNA had the densityof H-LDNA.After '19 generations,
from 1sN-labeled DNAafterF. colicellsareshiftedto raN_containing approximately halfthe DNA hadthe densityof H-LDNA;the otherhalf
medium if DNA replicationoccursby a conservative or semtconseryative had the densityof L-LDNA.With additionalgenerations, a largerand
mechanismParental heavy(H)strandsarein red;light(L)strands largerfractionof the extractedDNA consistedof L-Lduplexes;H_H
synthesized aftershiftto laN-containingmediumarein brue.Notethat duplexesneverappearedTheseresultsmatchthe predictedpatternfor
the conservativemechanism nevergenerates H_LDNAand that the the semiconservative replicationmechanismdepictedin (a) The bottom
semiconservative mechanism nevergenerates H_HDNA but does two centrifuge cellscontainedmixtures of H-HDNAand DNA isolated
generateH-LDNA duringthe secondand subsequent doublingsWith at 1 9 and 4 1 generations in orderto clearlyshowthe positions of H_H,
additionalreplicationcycles,the lsN-labeled(H)strandsfrom the H-1,and L-LDNA in the densitygradient.[part(b)fromM Meselson
originalDNAarediluted,so that the vastbulkof the DNAwould consist andF.
W Stahl, 1958,Proc Nat'lAcadSciUSAM:6711

that duplex DNA is replicated by a semiconservarive mech_

DNA strands. However, the vast preponderance of RNA and
DNA in cells is synthesized from preexisting duplex DNA.

D N A P o l y m e r a s eRs e q u i r ea p r i m e r
t o I n i t i a t eR e p l i c a t i o n
Analogousto RNA, DNA is synthesized from deoxynucleoside
5'-triphosphateprecursors(dNTps). Also like RNA synthesis,
DNA synthesisalwaysproceedsin the 5,+3, direction because
chain growth resultsfrom formation of a phosphoesterbond

140 . cHAprER
4 B A s t cM o L E c u L A G
R E N E I cM E C H A N t s M s
|
beftveenthe 3' oxygen of a growing strand and the o phos-
phate of a dNTP (seeFigure 4-10a). As discussedearlier, an
RNA polymerasecan find an appropriate transcription start
site on duplex DNA and initiate the synthesisof an RNA com-
plementaryto the template DNA strand (seeFigure 4-11). In
contrast, DNA polymerasescannot initiate chain synthesisde
novo; instead, they require a short, preexisting RNA or DNA
strand, called a primer, to begin chain growth. \7ith a primer
base-pairedto the template strand, a DNA polymeraseadds
deoxynucleotidesto the free hydroxyl group at the 3' end of
the primer as directed by the sequenceof the template strand:

Primer

lVhen RNA is the primer, the daughter strand that is formed

is RNA at the 5' end and DNA at the 3' end.

D u p l e xD N A l s U n w o u n da n d D a u g h t e rS t r a n d s
A r e F o r m e da t t h e D N A R e p l i c a t i o nF o r k
In order for duplex DNA to function as a template during
replication,the two intertwined strandsmust be unwound' or
melted, to make the basesavailablefor basepairing with the
basesof the dNTPs that are polymerizedinto the newly syn-
thesized daughter strands. This unwinding of the parental
DNA strandsis by specifichelicases,beginningat unique seg-
ments in a DNA moleculecalledreplication origins' or simply
origins. The nucleotide sequencesof origins from different
organisms vary gready,although they usually contain A'T- cent fragments'
rich sequences.Once helicaseshave unwound the parental
DNA at an origin, a specializedRNA polymerasecalled pri-
SeveralProteinsParticipatein DNA Replication
mase forms a short RNA primer complementaryto the un-
wound template strands. The primer, still base-pairedto its Detailed understanding of the eukaryotic proteins that par-
complementary DNA strand, is then elongated by a DNA ticipate in DNA replication has come largely from studies
polymerase,thereby forming a new daughter strand. witlh small viral DNAs, particularly SV40 DNA, the circular

llil+ FocusAnimation:NucleotidePolymerization
by DNA Polyrngra:g
DNA 5',
> FIGURE 4-30 leading-strandand lagging-strand
synthesis. Nucleotides areaddedby a DNApolymerase to each
(indicated by P o i n to f j o i n i n g
growingdaughter strandin the 5'-+3' direction
arrowheads) Theleading strandissynthesized continuously f roma L a g g i n gs t r a n d
singleRNA primer (red)
at its 5' end.
The lagging strand is
discontinuously frommultipleRNAprimers thatare Okazakifragment
synthesized
formedperiodically aseachnewregionof the parental duplexis l NAduPlex
P a r e n t aD S h o r tR N A p r i m e r
unwoundElongation of theseprimers initiallyproduces Okazaki
As each growing fragment approaches the prevtous 5',
fragments,
primer, the primerisremoved andthefragments areligated
of the entire Leadingstrand
Repetition of thisprocess eventuallyresults in synthesis
l a g g i nsgt r a n d .

DNA REPLICATION 14'l

 FocusAnimation:Coordinationof Leading- and flltt
strandsynthesis
(a) SV40DNA replicationfork

31
5',
E::i1,""'; L a g g i n gs t r a n d pPol E
Primase E Rfc
Primer PCNA

(b}PCNA
RPA
a Double-
stranded
DNA

Leadingstrand

(c)RPA

FIGURE 4-31 Model of an SV40DNA replicationfork. (a)A upperleftshowsthe iconrepresenting pCNAboundto DNAin
hexamer of largeT-antigen([), a viralprotein.
functionsasa parta. (c)Thelargesubunitof RPAcontains two domains thatbind
helicaseto unwindthe parental DNAstrands. Single-strandregions single-strandedDNA.Onthe left,thestructure determined for the
of the parentaltemplate unwoundby largeT-antigen areboundby two DNA-binding domains of the largesubunitboundto single_
multiplecopies of the heterotrimeric
proteinRpA(Z). Theleading stranded DNAisshownwith the DNAbackbone (whitebackbone
strandissynthesized by a complex of DNApolymerase S (polS), with bluebases)parallelto the planeof the page Notethatthe
singleDNAstrandisextended withthe bases exposed, an optimal
conformation for replication
by a DNApolymerase Onthe right,the
viewisdownthe lengthof thesingleDNAstrand,revealing how RpA
B strandswraparoundthe DNA.Thediagram at bottomcenter
showsthe iconrepresenting heterotrimeric RpAboundto single-
fragment (E). (b)Thethreesubunitsof pCNA,shownin different stranded DNAin part(a).[part (a)adaptedfromS J Flint
etal, 2000,
colors,
forma circularstructure
with a centralholethroughwhich Virology:
Molecular pathogenesis,
Biology, andControl,ASMpress; part(b)
double-stranded DNApasses. A diagram of DNAisshownjn the afterJ M Gulbis
et al, 1996,Cetl87:297;
andpart(c)afterA Bochkarev
centerof a ribbonmodelof the pCNAtrimer.Thediaqram at the etal, 1997,Nature385:176l

Figure 4-31 depicts the multiple proteins that coordinate

copying of SV40 DNA at a replication fork. The assembled
proteins at a replication fork further illustrate the concept of
molecular machines introduced in Chapter 3. These multi_
component complexes permit the cell to carry out an ordered
sequenceof eventsthat accomplishessentialcell functions.
The molecular machine that replicatesSV40 DNA con_
tains only one viral protein. All other proteins involved in

142 o c H A p r E R4 I B A s t cM o L E c u L A R
G E N E T tM
c ECHANtsMs
llll+ FocusAnimation:Bidirectional of DNA
Reptication
EcoRl
SV40 DNA replication are provided by the host cell. This vi-
ral protein, large T-antigen, forms a hexamer that unwinds
the parental strandsat a replication fork. Primers for leading
and lagging daughter-strandDNA are synthesizedby a com-
plex of primase, which synthesizesa short RNA primer, and C i r c u l a vr i r a l
DNA polymerased (Pol a), which extendsthe RNA primer cnromosome
with deoxynucleotides,forming a mixed RNA-DNA primer'
The primer is extended into daughter-strand DNA by
DNA polymerase6 (Pol 6), which is lesslikely to make errors
during copying of the templatestrand than is Pol ct becauseof o
its proofreading mechanism (see Section 4.6 below). Pol E
forms a complex with R/c (replication factor C) and PCNA
q)
(proliferating cell ntclear antigen), which displaces the pri-
mase-Pol crcomplex following primer synthesis.As illustrated o)
in Figure 4-31,b,PCNA is a homotrimeric protein that has a E
central hole through which the daughterduplex DNA passes,
thereby preventing the PCNA-Rfc-Pol 6 complex from disso-
ciating from the template.Pol E is the main polymeraseused
by eukaryotes for elongating DNA strands during replication.
After parental DNA is separatedinto single-strandedtem-
platesat the replication fork, it is bound by multiple copiesof
RPA (replicationprotein A), a heterotrimericprotein (Figure
4-31,c).Binding of RPA maintains the template in a uniform
conformation optimal for copying by DNA polymerases.
Bound RPA proteins are dislodgedfrom the parental strands
by Pol cr and Pol 6 as they synthesizethe complementary
strandsbase-pairedwith the parental strands.
Severaleukaryotic proteins that function in DNA replica-
tion are not depictedin Figure 4-31. DNA polymerasee also
contributes to the synthesisof cellular chromosomal DNA,
though its exact role is uncertain. Still other specializedDNA a EXPERTME;I r;,;*; o g;-ri.r-" .,*.,."0, o";";
polymerasesare involved in repair of mismatchesand dam- bidirectionalreplicationof SV40DNA.Electron microscopy of
agedlesionsin DNA (seeSection4.6). A topoisomeraseasso- replicatingSV40DNAindicates bidirectionalgrowthof DNAstrands
ciateswith the parental DNA aheadof the helicaseto remove froman origin.Thereplicating viralDNAfromSV4O-infected cells
torsional stressintroduced by the unwinding of the parental wascut by the restrictionenzyme EcoRl, which recognizes one site
strands. Ribonuclease H and FEN I remove the ribonu- in thecircularDNA.Thiswasdoneto provide a landmark for a
cleotidesat the 5' ends of Okazaki fragments;these are re- specificsequence in the 5V40genome: the FcoRl recognition
placedby deoxynucleotidesadded by DNA polymerase6 as it sequence isnoweasily recognized asthe endsof linearDNA
Okazaki molecules vrsualized by electronmicroscopy. Electron micrographs
extendsthe upstream Okazaki fragment. Successive
of EcoRl-cutreplicating SV40 DNA molecules showed a collection
fragments are coupled by DNA ligase through standard
of cut molecules with increasingly longerreplication "bubbles,"
5'-+3' phosphoesterbonds.Replicationof a linear DNA mol-
whosecentersarea constantdistance from eachendof the cut
ecule presentsa specialproblem at the ends of the molecule
molecules Thisfindingisconsistent chaingrowthin two
with
sincethe 5'-most RNA primers of the lagging strandscannot at thecenterof a bubble,
directionsfroma commonoriginlocated
be replacedby DNA by this mechanism.In most eukaryotes' diagrams' [See G C Fareed etal,
asillustratedin thecorresponding
this problem is solved by the RNA-protein complex called photographs courtesyof N P Salzman l
1972,J Virol10:484;
telomerasethat carriesits own templateas discussedin Chap-
ter 6, Genes,Genomics,and Chromosomes.
that moves in one direction. Alternatively, two replication
forks might assembleat a single origin and then move in op-
D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l l y posite directions, leading to bidirectional growth of both
f r o m E a c hO r i g i n i",rght., strands.Severaltypes of experiments,including the
As indicatedin Figures4-30 and 4-31, both parental DNA orr. sho*tt in Figure 4-32, provided early evidencein sup-
strands that are exposedby local unwinding at a replication port of bidirectional strand growth.
fork are copied into a daughter strand. In theory, DNA repli- The general consensusis that all prokaryotic and eu-
cation from a singleorigin could involve one replication fork karyoticlels employ a bidirectional mechanism of DNA

DNA REpL1CAT;ON o 143

 FocusAnimation:Coordination
of Leading-and Lagging-strand flltt
Synthesis
Helicases < FIGURE 4-33 Bidirectional mechanism of DNAreplication.
Theleftreplication fork hereiscomparable to the replication fork
diagrammed in Figure 4-31, whichalsoshowsproteins otherthan
largeT-antigen lop:TwolargeT-antigen hexameric helicases first
EJrn*'no'nn bindat the replication originin opposite orientations. Step[: Using
energyprovided fromATPhydrolysis, the helicases movein opposite
directions,unwinding the parental DNAandgenerating single-strand
templates thatareboundby RPAproteinsStepE: primase_pol o
complexes synthesize shortprimers (red)base-paired to eachof the
E t".o,nn-.trand primersynthesis separated parental strands. Stepg: pCNA-Rfc-pol 6 complexes
J replacethe primase-Pol crcomplexes andextendtheshortprimers,
generating the leading strands (darkgreen)at eachreplication fork
Step@: Thehelicases furtherunwindthe parental strands, andRpA
proteins bindto the newlyexposed single-strand regions. Stepg:
I
PCNA-Rfc-Pol 6 complexes extend the leading strands further Step6:
! | Leading-strand
extension
Primase-Pol ctcomplexes synthesize primers
t for lagging-strand
synthesis at eachreplication fork Stepfl: pCNA-Rfc-pol 6 complexes
displace the primase-Pol o complexes andextendthe lagging-strand
Okazaki fragments (lightgreen), whicheventually areligatedto the
5' endsof the leading strandsTheposition whereligation occursis
represented by a circleReplication continues byfurtherunwinding of
theparental strands andsynthesis of leading andlagging strands asin
Steps4-Z Althoughdepicted asindividual stepsfor clarity, unwinding
andsynthesis of leading andlaggingstrands occurconcunentlv

I
Unlike SV40 DNA, eukaryotic chromosomal DNA mol-
El I Leaoing-strand
extension eculescontain multiple replication origins separatedby tens
+ to hundreds of kilobases.A six-subunit protein called ORC,
for origin recognition complex, binds to each origin and as-
sociateswith other proteins required to load cellular hexam-
eric helicasescomposed of six homologous MCM proteins
(for minichromosome maintenance. the genetic screen ini-
tially_used to identify the genes encoding them). Two op-
E t.nn,"n-strand
primersynrhesis posed MCM helicases
separatethe parental strands at an
J

Z I a.nn,nn-strand
extension
+
transcription of most genes,control of the initiation step is the
primary mechanismfor regulatingcellular DNA replication. Ac_

S t r a n dl i g a t i o n

replication. In the caseof SV40 DNA, replication is initiated

by binding of two large T-antigen hexamiric helicasesto the into two daughtercells.We discussthe various regulatory mech_
anismsthat determinethe rate of cell division in Chapter 20.

DNA Replication
Each strand in a parental duplex DNA acts as a template
r synthesisof a daughter strand and remains base-paired
the new strand, forming a daughter duplex (using a

144 C H A P T E R4 | B A S T CM O L E C U L A RG E N E T T M
C ECHANTSMS
semiconservativemechanism).New strands are formed
the 5'--+3'direction.
r Replication begins at a sequencecalled an origin. Each
eukaryotic chromosomal DNA molecule contains multiple
replication origins.
r DNA polymerases,unlike RNA polymerases,cannot un-
wind the strandsof duplex DNA and cannot initiate synthe-
sis of new strandscomplementaryto the templatestrands.
r At a replication fork, one daughter strand (the leading
strand) is elongated continuously. The other daughter
strand (the lagging strand) is formed as a seriesof discon-
tinuous Okazaki fragments from primers synthesizedevery
few hundred nucleotides(Figure 4-30).
r The ribonucleotidesat the 5' end of each Okazaki frag-
ment are removed and replacedby elongation of the 3' end
of the next Okazaki fragment. Finally adjacent Okazaki
fragments are joined by DNA ligase.
r Helicasesuse energyfrom ATP hydrolysis to separatethe
parental (template)DNA strands. Primase synthesizesa
short RNA primer, which remains base-pairedto the tem-
plate DNA. This initially is extendedat the 3' end by DNA
polymerase a (Pol c), resulting in a short (S')RNA-
(3' )DNA daughterstrand.
r Most of the DNA in eukaryotic cells is synthesizedby
Pol 6, which takes over from Pol ct and continues elonga-
tion of the daughterstrandin the 5'+3' direction.Pol 6 re-
mains stably associatedwith the template by binding to Rfc
protein, which in turn binds to PCNA, a trimeric protein
that encirclesthe daughter duplex DNA (seeFigure 4-31).
r DNA replication generally occurs by a bidirectional
mechanism in which two replication forks form at an ori-
gin and move in opposite directions, with both template
strandsbeing copied at eachfork (seeFigure4-33)'
r Synthesisof eukaryotic DNA in vivo is regulatedby con-
trolling the activity of the MCM helicasesthat initiate
DNA replication at multiple origins spacedalong chromo-
somal DNA.
![ DNARepairand Recombination
Damage to DNA is unavoidable and arises in many ways.
DNA damage can be caused by spontaneouscleavageof
chemicalbonds in DNA, by environmental agentssuch as ul-
traviolet and ionizing radiation, and by reaction with geno-
toxic chemicalsthat are by-products of normal cellular me-
tabolism or occur in the environment' A mutation in the
normal DNA sequencecan occur during replication when a
DNA polymerase inserts the wrong nucleotide as it reads a
damagedtemplate. Mutations also occur at a low frequency
as the result of copying errors introduced by DNA poly-
meraseswhen they replicate an undamagedtemplate. If such
mutations were left uncorrected, cells might accumulate so
many mutations that they could no longer function properly,
In addition, the DNA in germ cells might incur too many
mutations for viable offspring to be formed' Thus the preven-
tion of DNA sequenceerrors in all types of cellsis important
for survival, and several cellular mechanismsfor repairing
damagedDNA and correcting sequenceerrors have evolved'
One mechanismfor repairing double-strandedDNA breaks'
by a processcalled recombination, is also used by eukaryotic
cells to generatenew combinations of maternal and paternal
geneson each chromosome through the exchangeof segments
of the chromosomesduring the production of germ cells (e'g',
sperm and eggs).
Significantln defects in DNA repair mechanisms and
cancerare closelyrelated. !7hen repair mechanismsare com-
promised, mutations accumulate in the cell's DNA. If these
mutations affect genes that are normally involved in the
careful regulation of cell division, cells can begin to divide
uncontrollably, leading to tumor formation, and cancer'
Chapter 25 outlines in detail how cancer arisesfrom defects
in DNA repair.We will encountera few examplesin this sec-
tion, as well, as we first consider the ways in which DNA in-
tegrity can be compromised' and then discussthe repair
mechanismsthat cells have evolved to ensurethe fidelity of
this very important molecule'
DNA Polymerases IntroduceCopyingErrors
and Also CorrectThem
The first line of defensein preventingmutations is DNA poly-
merase itself. Occasionally, when replicative DNA poly-
merasesprogressalong the template DNA, an incorrect nu-
cleotideit to the growing 3' end of the daughterstrand
(seeFigure"da.a
4-31,).E. coll DNA polymerases., for instance,in-
troduce about 1 incorrect nucleotideper 104 polymerizednu-
cleotides.Yet the measuredmutation rate in bacterial cells is
much lower: about 1 mistake in 10e nucleotidesincorporated
into a growing strand. This remarkable accuracy is largely
due to proofreading by E. coli DNA polymerases'
Pro-ofreadingdependson a 3'-+5' exonucleaseactiuity of
some DNA polymerases.Sfhen an incorrect baseis incorpo-
rated during DNA synthesis,base-pairingbetweenthe 3' nu-
cleotideof the nascentstrand and the templatestrand doesnot
occur.As a result,the polymerasepauses'then transfersthe 3'
end of the growing chain to its exonucleasesite, where the
incorrectmispairedbaseis removed (Figure4-34)' Then the 3'
end is transferredback to the polymerasesite' where this re-
gion is copied correctly. Like the E. coli DNA polymerases'
I*o enkaryotic DNA polymerases,6 and e, used for replica-
tion of most chromosomal DNA in animal cells' also have
proofreading activity. It seemslikely that proofreading is in-
dispensablefor all cellsto avoid excessivemutatlons'
C h e m i c aal n d R a d i a t i o nD a m a g et o D N A
Can Leadto Mutations
DNA is continually subiectedto a baffage of damaging
chemical reactions; estimatesof the number of DNA dam-
in a singlehuman cell range from 10a to 105 per
"g..,r..t,,
day! Even if DNA were not exposedto damaging chemicals,
..it"in aspectsof DNA structure are inherently unstable'
D N A R E P A I RA N D R E C O M B I N A T I O N O 145
 Frngers Thumb Fingers Thumb

t^ urowtng
strand Pol

Template
Exo strand

FIGURE 4-34 Proofreading by DNApolymerase. All DNA baseat the3' endcausesmeltingof thenewlyformedendof the
polymerases havea similar three-dimensional
structure,
which duplexAsa result,thepolymerase pauses,andthe3,endof the
resembles a half-opened righthandThe,,fingers,,
bindthesingle- growingstrandistransferred
to the 3,-+5,exonucleasesite(Exo)
stranded segment of thetemplatestrand,andthe polymerase catalytic about3 nm away,wherethe mtspaired baseandprobably otherbases
(Pol)liesin thejunctionbetween
activity thefingersandpalm.As long areremoved. Subsequently,
the 3' endflipsbackintothe polymerase
asthecorrect nucleotides areaddedto the3, endof thegrowing siteandelongationresumes.
[Adapted fromC M JoyceandT.T.Steltz,
strand,it remains in the polymerasesite Incorporation
of an incorrect 1995,1 Bacteriol
177:6321,and
S Bellandl Baker,
1998,Ceil92:2951

For example, the bond connecting a purine baseto deoxyri-
bose is prone ro hydrolysis at low rate under physiological
conditions, leaving a sugar without an attached base.Thus
coding information is lost, and this can lead to a mutarion
during DNA replication. Normal cellular reacrions,including
the movement of electronsalong the electron-transportchain
in mitochondria and lipid oxidation in peroxisomei, produce
severalchemicalsthat react with and damage DNA, includ-
ing hydroxyl radicals and superoxide (O2 ). These too can
causemutations, including those that lead to cancers.
Many spontaneous mutations are point mutations,
which involve a changein a single basepair in the DNA se-
quence. One of the most frequent point mutations comes
ftom deamination of a cytosine (C) base,which converts it
into a uracil (U) base. In addition, the common modified
baseS-methylcytosineforms thymine when it is deaminated.
If thesealterationsare not correctedbefore the DNA is repli-
cated,the cell will usethe strandcontainingU or T as template
to form a U.A or T.A basepair, thus creating a permanent
changeto the DNA sequence(Figure 4-35).
Radiation from the environment can also have dramatic
consequences for DNA. High-energy ionizing radiation such
as x-rays and gamma rays causedouble-strandedbreaks in
DNA. Uy radiation found in sunlight causesdistortions in

NH. U
l'
(.tl
N'c-c-cr. Deamination HNz"\c-cH"
ttl trl
ozc'-r-cH o-rctN.c
I
2-Deoxyribose 2-Deoxyribose
5-Methylcytosine Thymine

5', ?21
5', J

Deamination Replication
--+

.*
E z
Base-excision
repatr
3',
3', 5',
Witd-type Mutant Wild-type
DNA DNA DNA
FIGURE 4-35 Deamination leadsto point mutations.A mechanisms (step[), it willleadto a permanent
changeIn sequence
spontaneous pointmutation canformbydeamination of (r.e.,
a mutation)
following (stepZ). Afteroneround
DNAreplication
5-methylcytosine(C)to formthymine(T).lf the resulting
T.Gbasepair of replication,
onedaughter DNAmolecule willhavethe mutantT.A
isnot restored
to the normalC.Gbasepairby baseexcision_repair basepairandtheotherwillhavethewild-type C.Gbasepair.

146 . c H A p r E4R | B A s t cM o L E c u L AGRE N E TM

t cE c H A N t s M s
 5' 3',
the DNA double helix that interfere with proper replication .*"'J.-r1.!!?sif..'-"1*.r
i{:iT,iil
and transcription.
_|j*l-_l-e -L*l-i-J-
5'
''
High-FidelitDy N A E x c i s i o n - R e p aSi ry s t e m s ,)a
' -lDNAglycosYlase
Recognizea n d R e p a i rD a m a g e
In addition to proofreading,cellshaveother repair systemsfor T*ril*rTl*r
preventingmutations due to copying errors, spontaneousmu- -ji*j*|J*t-Li*L
tation, and exposureto chemicalsand radiation. SeveralDNA
excision-repair systemsthat normally operate with a high de-
ls
A P EeI n d o n u c l e a s e
I
gree of accvracy have been well studied. These systemswere
first elucidated through a combination of genetic and bio- * *T-r -i*rj""r t
chemicalstudiesin E. coli. Homologs of the key bacterialpro- I liG:!"; fJ-

teins exist in eukaryotes from yeast to humans, indicating that

!-r-.{e.+ rr*

theseerror-freemechanismsaroseearly in evolution to protect

DNA integrity. Each of these systems functions in a similar
manner-a segmentof the damagedDNA strand is excised,
and the gap is filled by DNA polymeraseand ligaseusing the
::*T-l- Tl*l]-
complementary DNA strand as template.
i-J-l-Lgl-l-i-J"-
I7e will now turn to a closer look at some of the mecha-
nisms of DNA repair, ranging from repair of single basemu-
tations to repair of DNA broken acrossboth strands. Some
of theseaccomplish their repairs with great accuracy;others
are lessprecise.
BaseExcisionRepairsT.G Mismatchesand
DamagedBases
In humans, the most common type of point mutation is a C
to T, which is causedby deamination of S-methyl C to T (see
Figure 4-35). The conceptual problem with base excision
repair in this case is determining which is the normal and
which is the mutant DNA strand, and repairing the latter so
that it is properly base-pairedwith the normal strand. Sincea
G.T mismatch is almost invariably causedby chemical con-
version of C to U or S-methyl C to T, the repair system
evolvedto removethe T and replaceit with a C (Figure4-36).
The G'T mismatch is recognized by a DNA glycosylase
that flips the thymine base out of the helix and then hy-
drolyzes the bond that connects it to the sugar-phosphate
DNA backbone. Following this initial incision, an apurinic
(AP) endonucleasecuts the DNA strand near the abasicsite.
The deoxyribose phosphate lacking the base is then re-
moved and replaced with a C by a specializedrepair DNA
polymerasethat reads the G in the template strand. As men-
tioned earlier, this repair must take place prior to DNA
replication becausethe incorrect base in this pair, T, occurs
naturally in normal DNA. Consequently,it would be able to
engagein normal \Tatson-Crick basepairing during replica-
tion, generatinga stablepoint mutation that is now unable
to be recognizedby repair mechanisms(seeFigure 4-35,
s t e pZ ) .
Human cells contain a battery of glycosylases'each of
which is specific for a different set of chemically modified
DNA bases.For example,one removesS-oxyguanine,an oxi-
dized form of guanine, allowing its replacementby an un-
damagedG, and others remove basesmodified by alkylating
agents.The resultingnucleotidelacking a baseis then replaced
-^. )-
ls
AP lyase
(partof DNAPol0)
I
PorF,
DNA
risase
DNA
E
A FIGURE 4-36 Baseexcisionrepair of a T'G mismatch.A DNA
glycosylase
specificfor G'Tmismatches, usuallyformedby
(see 4-35),flipsthethymine
deamination of 5-mC residues Figure
baseout of the helixandthencutsit awayfromthesugar-phosphate
DNAbackbone (stepn), leaving justthe deoxyribose (blackdot).An
endonuclease specificfor the resultant baselesssite[apurinic
(stepE), and
endonuclease | (APE1)lthencutsthe DNAbackbone
thedeoxyribose phosphate is removed by an endonuclease, apurinic
lyase(APlyase), associatedwith DNApolymerase B, a specialized
DNApolymerase usedin repair(stepB). Thegapisthenfilledin by
DNAPolB andsealed by DNAligase (stepZl),restoring theoriginal
G.Cbase pair [AfterO Schdrer, 2003,Angewandte Chemie 42:2946]
by the repair mechanismjust discussed.A similar mechanism
functions in the repair of lesions resulting from depwrination,
the loss of a guanine or adenine base from DNA resulting
from hydrolysis of the glycosylic bond between deoxyribose
and the base.Depurination occursspontaneouslyand is fairly
common in mammals. The resulting abasicsites,if left unre-
paired, generatemutations during DNA replication because
they cannot specifythe appropriatepaired base.
MismatchExcisionRepairsOther Mismatches
a n d S m a l lI n s e r t i o n sa n d D e l e t i o n s
Another process,also conservedfrom bacteria to man' prin-
cipally eliminates base-pair mismatches and insertions or
deletionsof one or a few nucleotidesthat are accidentallyin-
troduced by DNA polymerasesduring replication. As with
base excision repair of a T in a T'G mismatch, the concep-
tual problem with mismatch excision repair is determining
whic^his the normal and which is the mutant DNA strand,
and repairing the latter. How this happensin human cellsis not
known with certainty. It is thought that the proteins that bind
to the mismatchedsegmentof DNA distinguishthe template

AND RECOMBINATION .
DNA REPAIR 147
 -iil
Templatestrand

!ll;l
z'lttsuz
+A
lvtsxo\
t:l
N u c l e o t i d eE x c i s i o nR e p a i r sC h e m i c aAl d d u c t s
T h a t D i s t o r tN o r m a lD N A S h a p e
Cells use nucleotide excision repair to fix DNA regions con-
taining chemically modified bases,often called chemical
addwcts,that distort the normal shapeof DNA locally. A key
iilli c 1ii
to this type of repair is the ability of certain proteins to slide
Newly synthesized along the surfaceof a double-strandedDNA molecule look-
d a u g h t e rs t r a n d \ ing for bulgesor other irregularities in the shapeof the dou-
ble helix. For example, this mechanism repairs thymine-
MLHl endonuclease, thymine dimers, a common type of damage caused by UV
PMS2
a light (Figure 4-38); thesedimers interfere with both replica-
DNAhelicase tion and transcription of DNA.
DNAexonuclease
Figure 4-39 illustrates how the nucleotide excision-
repair system repairs damaged DNA. Some 30 pro-
teins are involved in this repair process,the first of which
j
3' .-i- .,',,,"*.],--r,-'--;-"i,- 5, 3'd. **5' were identified through a study of the defectsin DNA re-
pair in cultured cellsfrom individuals with xeroderma pig-
mentosum,a hereditarydiseaseassociatedwith a predispo-
G a p r e p a i rb y D N A
p o l y m e r a s ea n d l i g a s e
sition to cancer.Individuals with this diseasefrequently
develop the skin cancers called melanomas and squdmous
cell carcinomasif their skin is exposedto the UV rays in
5', 3', sunlight. Cells of affected patients lack a functional nu-
l!. :liilai cleotide excision-repair system. Mutations in any of at
iri
3,h ill||-fr lllti: least seven different genes, called XP-A through Xp-G,
FIGURE 4-37 Mismatchexcisionrepairin humancells.The
mismatch excision-repairpathway correctserrorsintroduced during

",x"
replication
A complex of the MSH2andMSH6proteins (bacterial
MutShomologs 2 and6) bindsto a mispaired segment of DNAin
sucha wayasto distinguish between thetemplate anonewty Deo
synthesized
daughter strands (steptr) Thistriggers bindingof MLH.j
andPMS2(bothhomologs of bacterial
MutL)Theresulting DNA-
proteincomplexthenbindsan endonuclease thatcutsthe newly
synthesized
daughter strandNexta DNAhelicase
@
unwinds the helix,
andan exonuclease removes severalnucleotides fromthecutendof
the daughter
strand,including the mismatched base(stepf,l) Finally, Oeo"yriU\e
aswith baseexcision repair,thegapisthenfilledin by a DNA
polymerase(Pol6, in thiscase) andsealed by DNAligase (stepS)

Two thymine residues

and daughter strands; then the mispaired segmentof the
daughter strand-the one with the replication error-is
excisedand repaired to becomean exact;omplement of the
J,""Y,1,,""
templatestrand (Figure4-37).In contrastto baseexcisionre- o.. H
pair, mismatch excision repair occurs after DNA replication.
ouo*u.x"
Predispositionto a colon cancerknown as bereditary
nonpolyposiscolorectalcancerresultsfrom an inher-
@
ited loss-of-function mutation in one copy of either the
MLHL or the MSH2 gene. The MSH2 and MLH1 proteins
are essentialfor DNA mismatch repair (seeFigure 4-37).
Cells with ar least one functional copy of each of these
o"o,.rx"
1..t
" CH.

Thymine-thyminedimer residue
FIGURE4-38 Formation of thymine-thymine dimers. The
most commontype of DNA damagecausedby UV irradiation,
thymine-thymine
drmerscan be repairedby an excision-repair
mon in noninherited forms of colon cancer.I mechanism

148 CHAPTER
4 | BASTC
M O L E C U L AG
R E N E T TM
CE C H A N T S M S
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seua811e;o uorldrrcsueJtrot perrnberroloe; uortdrJ)suerl
'HIIJI, 'dlqelreruaA
le.raue8e Jo strunqns aprrdeddlodaar;
a ' G t - V e . r n 8 r ga e s )p o o l s
-Jepun
lle,t^.lAouare rreder uorsrJxeaprloelrnu ur surelord
'seruanbrsuoJaIUES eql o^Eq
dX esoqrJo tsoru Jo solor aq1
pue edltouaqd erueseqt ernpo.rd11elunsoluau8rd erurep
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 Double-strandbreak vast majority of women with inherited susceptibility to
TTTTTTTTTT TTTTTTTTTT breast cancer have a mutation in one allele of either the
rttttttl
BRCA-1 or the BRCA-2 genesrhat encodeproteins partic-
ipating in this repair process.Loss or inactivation of the
second allele inhibits the homologous recombination re-
pair pathway and thus tends to induce cancerin mammary
or ovarian epithelialcells,although at presentit is not clear
why these estrogen-responsive tissuesare favored sites of
carcinogenesis. Yeastscan repair double-strandbreaks in-
duced by 1-irradiation. Isolation and analysisof radiation-
sensitive(RAD) mutants that are deficient in this reoair
a proteins system facilitated study of the process. Virtually all the
yeast Rad proteins have homologs in the human genome,
Jo,n". and the human and yeastproteins function in an essentially
identical fashion.
A variety of DNA lesions not repaired by mechanisms
discussedearlier can be repaired by mechanismsin which the
damagedsequenceis replaced by a segmentcopied from the
same or a highly homologous DNA sequenceon the homol-
ogous chromosome of diploid organisms,or the sister chro-
mosome following DNA replication in all organisms.These
mechanismsinvolve an exchange of strands between sepa-
rate DNA moleculesand henceare collectivelyreferred to as
FIGURE 4-40 Nonhomologous end-joining.Whensister D N A r ecombination mechanisms.
chromatids arenot available to helprepairdouble-strand breaks, In addition to providing a mechanism for DNA repair,
nucleotide sequences arebuttedtogetherthatwerenot apposed in similar recombination mechanismsgenerategeneticdiversity
the unbroken DNA TheseDNAendsareusually fromthesame among the individuals of a speciesby causing the exchange
chromosome locus,andwhenlinkedtogether, several
basepairsare of large regions of chromosomesbetween the maternal and
lost Occasionally, endsfromdifferent chromosomes areaccidentally
joinedtogetherA complex paternal pair of homologous chromosomesduring the spe-
of two proteins, KuandDNA-dependent
protern kinase, bindsto theendsof a double-strand cial type of cellular division that generatesgerm cells (sperm
break(steptr)
Afterformation of a synapse, the endsarefurtherprocessed and eggs),meiosis (Figure 5-3). In fact the exchange of re-
by
nucleases, resulting in removal of a few bases (stepE), andthetwo gions of homologous chromosomes,called crossing over, is
double-stranded molecules areligatedtogether(stepB) Asa result, required for proper segregationof chromosomesduring the
the double-strand breakis repaired, but several basepairsat thesite first meiotic cell division. Meiosis and the consequencesof
of the breakareremoved[Adapted fromG Chu,1997, J Biot Chem generating new combinations of maternal and paternal
272:24097; M Lieber et al, 1997,Curr. OpinGenet. Devel.
T:99;andD van geneson one chromosome by recombination are discussed
G a n t e t a l , 2 O O 1 ,N a t u r eR e v .G e n e t 2 : 1 9 6 I further in Chapter 5. The mechanismsleading to proper seg-
regation of chromosomes during meiosis are discussedin
Chapter 20. Here we will focus on the molecular mecha-
nisms of DNA recombination, highlighting the exchangeof
DNA strands betweentwo recombining DNA molecules.

Repair of a Collapsed Replication Fork An exampleof re-

chromosome to another. Such translocations may generare
chimeric genesthat can have drastic effects on normal cell
function, such as uncontrollable cell growth, which is the
hallmark of cancer.The devastatingeffectsof double-strand
breaks make this the "most unkindest cut of all," to borrow
a phrase from Shakespeare'sJulius Caesar.
H o m o l o g o u sR e c o m b i n a t i oC
n a n R e p a i rD N A
Damageand GenerateGeneticDiversity
combinationalDNA repair is the repair of a "collapsed,'repli-
cation fork. If a break in the phosphodiesterbackboneof one
DNA strand is not repaired before a replication fork passes,
the replicatedportions of the daughterchromosomesbecome
separatedwhen the replication helicasereachesthe ,,nick,, in
the parental DNA strand becausethere are no covalent bonds
between the two fragments of the parental strand on either
side of the nick. This processis called replication fork collapse
(Figure4-41, stepn). If it is not repaired,it is generallylethal
to at leastone daughtercell following cell division becauseof
the loss of genetic information between the nick and the end
of the chromosome.The recombination processthat repairs
the resultingdouble-strandedbreak and regenerat.s r.pli."-
tion fork involves multiple enzymesand other proteins, "
only
some of which are mentionedhere.

150 . cHAprER
4 B A s t cM o L E c u L A G
R E N E T tMc E C H A N t s M s
|
 DNAnick (Figure 4-41.,steptr ). The lagging nascentstrand (pink) cre-
ated on the unnicked, homologous' parent strand is ligated
to the unreplicated portion of the parent chromosome, as
shown in Figure 4-41, step Z). A critical protein required
for the next step is RecA in bacteria, or the homologous
Rad51 in S. cereuisiae and other eukaryotes. Multiple
RecA/RadS1 molecules bind to the single-stranded DNA
5',
(now considered the invading strand) and catalyze its hy-
3', bridization to a perfectly or nearly perfectly complementary
I
s'-e*onrcleaseacts on sequencein another, homologous, double-strandedDNA
I ll
I brokenend. Other daughter molecule, either the ligated molecule created after fork col-
Y strand (pink)ligatedto lapse (as shown in the figure) or the other homologous chro-
repairedparentalstrand
( l i g h tb l u e )i n u n b r o k e n mosome in diploid organisms.The other strand (dark blue)
chromosome. of this target double-stranded DNA (the strand not base-
5',
pairing with the invading strand) is displaced as a single-
3',
stranded loop of DNA along the region of hybridization

Jo RecA-or Rad51-mediated
strand invasion
between its complement and the invading strand (refer to
Figure 4-4L, step B). The RecA/RadS1-catalyzedinvasion
of a duplex DNA by a single-strandedcomplement of one of
the strands is key to the recombination process. Since no
Hollidaystructure
basepairs are lost or gained in this process,calledstrand in-
uasion,it doesnot requirean input of energy.
\--*
Next, the hybrid region between target DNA (pink) and
the invading strand (dark red) is extended in the direction
away from the break by proteins that use energy from ATP
hydrolysis. This process is called branch migration (Ftg-
ure 4-41,, step 4) becausethe point at which the target
DNA strand (pink) crossesfrom one complementary strand
(dark blue) to its complement in the broken DNA molecule
3' (dark red), is called a branch in the DNA structure. In this
diagram, the diagonal lines represent only one phosphodi-
Ligateends esterbond. Molecular modeling and other studiesshow that
the first baseon either side of the branch is base-pairedto a
complementary nucleotide. As this branch miSrates to the
J
Ieft, the number of base pairs remains constant; one new
base pair formed with the (red) invading strand is matched
R e b u i l dr e p l i c a t i o fno r k a n d by the loss of one base pair with the parental (dark blue)
c o n t i n u er e p l i c a t i o n
strand.
When the region of hybridization extends beyond the 5'
3', end of the broken strand (light blue), this broken parental
DNA strand becomes increasingly single-stranded, as its
A FIGURE 4-41 Recombinational repairof a collapsed complement, the invading (dark red) strand' base-pairsin-
replication fork. Parental strandsarelightanddarkblueTheleading steadwith the target (pink) DNA strand. This single-stranded
daughter strand is darkred, and the lagging daughter strandpink. (light blue) parcntal strand then base-pairswith the comple-
Diagonal linesin stepB andbeyond represent a singlephosphodiester mentary region of the other parental strand (dark blue) that
bondfromtheDNAstrandof theconesponding colorSmallblack has likewise become single-strandedas the branch migrates
arrowsfollowingstep@ represent cleavage of the phosphodiester to the left (Figure 4-41., step 4). The resulting structure is
bondsat the crossover of DNAstrands in the Holliday See
structure.
called a Hotliday strwcture, after the geneticist who first pro-
http://www. sdscedu/jou rnalVm htmI f or an animationof
bb/ruva.
posed it as an intermediatein Seneticrecombination. Again,
branchmigration catalyzed by E coliproteinsRuvAandRuvB. See
genetics.wisc.edu/Holliday/holliday3D htmlfor an the diagonal lines in the diagram following step 4 represent
http://engels
animation of theHolliday structureanditsresolution Seethetextfor a single phosphodiesterbonds (not a stretch of DNA), and all
discussion. fromD L Nelson andM M Cox,2005, Lehninger basesin the Holliday structure are base-pairedto comple-
[Adapted
of Biochemistry
Principles 4thed, W H Freeman andCompany.l mentary basesin the parental strands. Cleavageof the phos-
phodiesterbonds that crossover from one parental strand to
'
The first step in the repair of the double-strand break is the other and ligation of the 5' and 3 ends base-pairedto the
exonucleolytic digestionof the strand (light blue) that has its same parental strands (stepsE and 6) result in the genera-
5' end at the break, leaving a portion of the other (dark red) tion of a structure similar to a replication fork. Rebinding of
strand (the one with the 3' end at the break) single-stranded replication fork proteins results in extension of the leading

D N A R E P A I RA N D R E C O M B I N A T I O N 151

A FIGURE 4-42 Double-strand DNAbreakrepairby
homologousrecombination. Forsimplicity,
eachDNAdoublehelix
is represented
by two parallel
lineswith the polarities
of the strands
indicatedby arrowheadsat their3, endsTheuppermolecule
double-strandbreakNotethat in the diagram of the upperDNA
strand past the point of the original strand break and re-
initiation of lagging-strandsynthesis(step Z), thus regen-
erating a replication fork. The overall processallows the
undamagedupper strand in the lower molecule following
step E (pink/light blue) to serveas template for extensionof
the leading (dark red) strand in step Z.
Double-Stranded DNA Break Repair by Homologous
Recombination A similar mechanism called bomolopous
152 o c H A p r E4R | B A s t cM o L E c u L AGRE N E TM
Jn Ends digestedby exonucleases,leaving
3 ' s i n g l e - s t r a n d eedn d s
RecA-or Rads1-mediated
strand invasion
! 3 ' e n d o f i n v a d i n gs t r a n di s e x t e n d e db y
D N A p o l y m e r a s eu n t i lt h e d i s p l a c e d
single-strand(dark blue) base-pairswith
the other 3' single strand generated
i n i t i a l l y( p i n k )
A a e n di s e x t e n d ebdy D N Ap o l y m e r a s e
J
Ends are ligated
I
I
Jo Cleavephosphodiesterbonds indicated
with arrows and ligatealternativeends
molecule
the strandwith its3' endat the rightison thetop,whilein
thediagramof the lowerDNAmolecule thisstrandisdrawnon the
bottom Seethetextfordiscussion. [AfterT.L Orr-WeaverandJ
W
hasa Szostak,
1985,MicrobiolRev.49:33.1
recombination can repafua double-strand break in a chro-
mosome and can also exchangelarge segmentsof two dou-
ble-strandedDNA molecules (Figure 4-42). Homologous
recombination is also dependenton strand invasion catalyzed
principally by RecA in bacteriaand Rad51 in eukaryotes(steps
Il and tr). The 3' end of the invading DNA strand is then ex-
tended by a DNA polymerase,displacingthe parental strand as
an enlargingsingle-stranded loop of DNA (dark blue, step E).
Vhen DNA synthesis extends sufficiently far, the displaced
t cE c H A N t s M s
parentalstrand (dark blue)that is complementaryto the 3' sin-
gle-strandedregion generatedat the other broken end of DNA
(the pink single-stranded region on the left following step n),
the complementarysequences base-pair(step B). This 3' end
(pink) is then extendedby a DNA polymerase,using the dis-
placed single-strandedloop of parental DNA (dark blue) as
template(step4).
Next, the two 3' ends generatedby DNA synthesisare
ligated(stepE)to the 5'ends generatedin step I by 5'-ex-
onucleasedigestion of the broken ends. This generatestwo
Holliday structuresin the paired molecules(stepE).Branch
migration of theseHolliday structurescan occur in either di-
rection (not diagrammed).Finally, cleavageof the strands at
the positions shown by the arrows, and ligation of the alter-
native 5' and 3' ends at each cleavedHolliday structure gen-
eratestwo recombinant chromosomesthat each contain the
DNA of one parental DNA molecule (pink and red strands)
on one side of the break point and the DNA of the other
parental DNA molecule (light and dark blue) on the other
sideof the break point (step6). Eachchromosomecontains
a third region, located in the immediate vicinity of the initial
break point, that forms a heterodwplex;here one strand
from one parent is base-pairedto the complementary strand
of the other parent (pink or red base-pairedto dark or light
blue). Base-pair mismatches between the two parental
strandsare usually repairedby repair mechanismsdiscussed
above to generatea complementary basepair. In the process,
sequencedifferencesbetween the two parents are lost, a
processreferredto as geneconuersion.
Figure 4-43 diagrams how cleavageof one or the other
pair of strandsat the four-way strand junction in the Holliday
structure generatesparental or recombinant molecules.This
process,calledresolution of the Holliday structure,separates
DNA molecules initially joined by RecA/RadS1-catalyzed
strand invasion. Each Holliday structure in the intermediate
following step El of Figure 4-42 can be cleavedand religated
in the two possibleways shown by the two setsof small black
arrows. Consequently,there are four possibleproducts of the
5', 3' 5',
5', 3',
.- ---
: |
I
t
AI
FIGURE 4-43 Alternativeresolutionof a Hollidaystru€ture.
Diagonal a singlephosphodiester
linesrepresent
andvertical bond.lt
to diagram
issimplest the process
by rotating of the
the diagram
bottommolecule 180'sothatthetop andbottommolecules have
the samestrandorientationsCuttinqthe bondsasshownin Il and
recombination process.Two of theseregeneratethe parental
chromosomes(with the exceptionof the heteroduplexregion
at the break point that is repaired into the sequenceof one
parent or the other (geneconversion),and t'wo generatere-
combinant chromosomesas shown inFigure 4-42.
Meiotic Recombination Meiosis is the specializedform of
cell division in eukaryotesthat generateshaploid germ cells
(e.g.,sperm and eggs)from a diploid cell (Figure 20-38). At
least one recombination occurs between the paternal and
maternal homologous chromosomesbefore the first meiotic
cell division. Recombination is initiated by an enzyme that
makes a double-strandedbreak in the DNA of one chromo-
some at any one of a very large number of sites.The process
diagrammed in Figure 4-42 is then followed. The entire
process from cleavageof the DNA of one chromosome
through resolution of the Holliday structuresis repeatedun-
til at least one recombination, also called a crossouer'occurs
between one pair of each of the homologous chromosomes.
As mentioned earlier,the resulting link betweenhomologous
chromosomesis required for their proper segregationduring
the first meiotic cell division (seeChapter 20). As a conse-
quence,every germ cell contains multiple recombinant chro-
mosomes made of large segmentsof either the maternal or
the oaternal chromosome.
DNA Repair and Recombination
Changesin the DNA sequenceresult from copying er-
rs and the effects of various physical and chemical
agents.
r Many copying errors that occur during DNA replication
are corrected by the proofreading function of DNA poly-
merasesthat can recognize incorrect (mispaired) basesat
the 3' end of the growing strand and then remove them by
an inherent3'-+5' exonuclease activity (seeFigure4-34).
theendsasindicated
ligating theoriginal
regenerates chromosomes
Cuttingthe strandsasshownin E andreligating asshownat the
bottomgenerates recombtnantchromosomes Seehttp://engels
for a three-dimensional
genetics.wisc.edu/Holliday/holliday3D.html
animation anditsresolution
structure
of the Holliday
AND RECOMBINATION O
DNA REPAIR 153
 r Eukaryotic cells have three excision-repair systemsfor
correcting mispaired basesand for removing W-induced
thymine-thymine dimers or large chemical adducts from
DNA. Base excision repair, mismatch repair, and nu-
cleotide excision repair operate with high accuracy and
generally do not introduce errors.
r Repair of double-strand breaks by the nonhomologous
end-joining pathway can link segmentsof DNA from dif-
ferent chromosomes, possibly forming an oncogenic
translocation. The repair mechanismalso producesa small
deletion, even when segmentsfrom the same chromosome
are joined.
r Inherited defectsin the nucleotideexcision-repairpath-
way, as in individuals with xeroderma pigmentosum,
predisposethem to skin cancer.Inherited colon cancer
frequently is associatedwith mutant forms of proteins
essentialfor the mismatch repair pathway. Defects in re-
pair by homologous recombination are associatedwith
inheritanceof one murant alleleof the BRCA-1 or BRCA-
2 gene and result in predispositionto breast and uterine
cancer.
r Error-free repair of double-strand breaks in DNA is ac-
complished by homologous recombination using the un-
damaged sister chromatid as a template. This processcan
lead to recombination of parental chromosomesand is
exploited by eukaryotes to generate genetic diversity by
recombination of paternal and maternal chromosomes in
developinggerm cells.
Wl Viruses:Parasites
of the Cellutar
GeneticSystem
Virusesare obligate,intracellularparasites.They cannot re-
produce by themselvesand must commandeera host cell's
machineryto synthesizeviral proteins and in some casesto
replicate the viral genome. RNA viruses, which usually
replicate in the host-cell cytoplasm, have an RNA genome,
and DNA viruses,which commonly replicate in the host-
cell nucleus, have a DNA genome (seeFigure 4-1). Viral
genomesmay be single-or double-stranded,dependingon
the specific type of virus. The entire infectious virus parti-
cle, called a virion, consistsof the nucleicacid and an outer
shell of protein that both protects the viral nucleic acid and
functions in the processof host-cell infection. The simplest
viruses contain only enough RNA or DNA to encode four
proteins; the most complex can encode=200 proteins. In ad-
dition to their obvious importanceas causesof disease,viruses
are extremely useful as researchtools in the study of basic
biological processes,such as thosediscussedin this chaoter.
Most Viral Host RangesAre Narrow
The surfaceof a virion contains many copies of one type of
protein that binds specificallyto multiple copies of a recep-
tor protein on a host cell. This interaction determines the
154 CHAPTER
4 | BASTC
M O L E C U L AG
R E N E T TM
CE C H A N T S M S
host range-the group of cell types that a virus can infect-
and beginsthe infection process.Most viruses have a rather
limited host range.
A virus that infects only bacteria is called a bacterio-
phage, or simply a phage. Viruses that infect animal or
plant cells are referred to generally as animal uiruses or
plant uiruses.A few viruses can grow in both plants or an-
imals and the insects that feed on rhem. The highly mobile
insects serve as vectors for transferring such viruses be-
tween susceptibleplant or animal hosts. Wide host ranges
are also characteristicof some strictly animal viruses,such
as vesicularstomatitis virus, which grows in insect vectors
and in many different types of mammals. Most animal
viruses,however, do not cross phyla, and some (e.g.,po-
liovirus) infect only closely related speciessuch as pri-
mates. The host-cell range of some animal viruses is further
restricted to a limited number of cell types because only
these cells have appropriare surface receptors to which the
virions can attach.
V i r a l C a p s i d sA r e R e g u l a rA r r a y so f O n e o r a
Few Typesof Protein
The nucleic acid of a virion is enclosedwithin a protein coat,
or capsid, composed of multiple copies of one prorein or a
few different proteins, each of which is encoded by a single
viral gene.Becauseof this structure, a virus is able to encode
all the information for making a relatively large capsid in a
small number of genes.This efficient use of geneticinforma-
tion is important, since only a limited amount of DNA or
RNA, and therefore a limited number of genes,can fit into a
virion capsid. A capsid plus the enclosednucleic acid is
called a nucleocapsid.
Nature has found two basic ways of arranging the multi-
ple capsid protein subunits and the viral genome into a nu-
cleocapsid.In some viruses,multiple copies of a single coat
protein form a helical structure that enclosesand protects
the viral RNA or DNA, which runs in a helical groove
within the protein tube. Viruses with such a helical nucleo-
capsid, such as tobacco mosaic virus, have a rodlike shape
(Figure 4-44a). The other major structural type is based on
the icosahedron, a solid, approximately spherical object
built of 20 identical faces,each of which is an equilateral tri-
angle (Figure 4-44b). During infection, some icosahedral
viruses interact with host cell-surfacereceptors via clefts in
between the capsid subunits; others interact via long fiber-
like proteins extending from the nucleocapsid.
In many DNA bacteriophages,the viral DNA is lo-
cated within an icosahedral "head" that is attached to a
rodlike "tail." During infection, viral proteins at the tip of
the tail bind to host-cellreceptors,and then the viral DNA
passesdown the tail into the cytoplasm of the host cell
(Figure 4-44c).
In someviruses,the symmetricallyarrangednucleocapsid
is coveredby an external membrane,or envelope,which con-
sistsmainly of a phospholipid bilayer but also conrains one
or two types of virus-encodedglycoproteins (Figure 4-44d).
 Poliovirus

Tobaccomosaicvirus

(c) 50 nm (d) 50 nm

A v i a n i n f l u e n z av i r u s

< FIGURE 4-44 Virionstructures. (a)Helical tobaccomosaic vlrus

i rlu sT h ee x a m p lseh o w ni sp o l i o v i r u s .
( b )S m a li lc o s a h e dvr a
(c)Bacteriophage Ta (d)Influenza virus,an example of an enveloped
virus[Part (a)O Bradfute, Peter Arnold/Science Photo Library; part(b)
courtesy of T S Baker; part (c)Department of Microbiology, Biozentrum/
t oe s e a r c h e rl sn,c l
S c l e n c eP h o t oL i b r a r yp; a r t ( d ) J a m e sC a v a l l i n i / P h o R

T4
Bacteriophage

The phospholipidsin the viral envelopeare similar to thosein
the plasmamembraneof an infectedhost cell.The virai enve-
lope is, in fact, derivedby budding from that membrane,but
contains mainly viral glycoproteins,as we discussshortly.
V i r u s e sC a n B e C l o n e da n d C o u n t e d
i n P l a q u eA s s a y s
The number of infectiousviral particlesin a samplecan be
quantifiedby a plaque assay.This assayis performedby cul-
turing a dilute sample of viral particleson a plate covered
with host cells and then counting the number of local Ie-
sions,called plaques,that develop (Figure 4-45). A plaque
developson the plate wherevera singlevirion initially infects
a singlecell. The virus replicatesin this initial host cell and
then lyses(ruptures)the cell, releasingmany progenyvirions
that infect the neighboring cells on the plate. After a few
such cyclesof infection, enough cells are lysed to produce a
visibleclear area,the plaque,in the layer of remainingunin-
fectedcells.
Sinceall the progeny virions in a plaque are derived from
a single parental virus, they constitute a virus clone. This
type of plaque assayis in standardusefor bacterialand ani-
mal viruses.Plant virusescan be assayedsimilarly by count-
ing local lesions on plant leavesinoculated with viruses.
Analysis of viral mutants' which are commonly isolated by
plaque assays,has contributedextensivelyto current under-
standingof molecularcellularprocesses.

V I R U 5 E SP: A R A S I T EOSF T H E C E L L U L A G SYSTEM

RENETIC 155
 (a) Confluentlayer of susceptiblehost cells 3. Replication-Yiral mRNAs are produced with the aid
growing on surfaceof a plate
of the host-cell transcription machinery (DNA viruses)or
by viral enzymes(RNA viruses).For both types of viruses,
viral mRNAs are translated by the host-cell translation ma-
chinery. Production of multiple copies of the viral genome
is carried out either by viral proteins alone or with the help
A d d d i l u t es u s p e n s i o nc o n t a i n i n gv i r u s ; of host-cell proteins.
after infection,cover layer of cells
with agar; incubate 4. Assembly-Yiral proteins and replicated genomesasso-
ciate to form progeny virions.
Plaoue
5. Release-Infected cell either ruptures suddenly (lysis),
releasingall the newly formed virions at once, or disinte-
gratesgraduallS with slow releaseof virions. Both cases
lead to the death of the infected cell.
Eachplaque representscell lysis initiatedby one viral
particle(agar restrictsmovement so that virus can
infectonly contiguouscells) Figure 4-46 rllustratesthe lytic cycle forT4 bacteriophage,a
nonenveloped DNA virus that infects E. coli. Viral capsid
proteins generally are made in large amounts becausemany

Freevirion

E. coli
Xffi;ffi["-E chromosome
a
a nn
d d iinni aj nef ic
n nt i o n
,
/
/.. ,,
EsE *? l

l.:
'll

.T4DNA

Plaque
A EXPERIMENTAL FTGURE 4-45 The plaqueassaydetermines
the numberof infectiousparticlesin a viral suspension. (a)Each
lesion,
or plaque, whichdevelops wherea singlevirioninitially
infected a pureviralclone.(b)plaques
a singlecell,constitutes on a
lawnof Pseudomonasfluorescens bacteriamadeby bacteriophage
$S1 [Part(b)Courtesyof Dr Pierre
Rossi,
Ecolepolytechnique
F6d€ralede
Lausanne(LBE-EPFL)
l
Replicationof viralDNA
Expression
of virallateproteins
A FIGURE 4-46 tytic replicationcycleof a nonenveloped,
LyticViral Growth CyclesLeadto the
bacterialvirus.E.colibacteriophage T4 hasa double-stranded DNA
Death of Host Cells genomeandlacksa membrane envelope Afterviralcoatproteins at
Although details vary among different types of viruses,those thetip of thetailin T4 interactwith specific
receptor proteinson the
that exhibit a lytic cycle of growth proceed through the fol- exteriorof the hostcell,theviralgenomeisinjected intothe host
lowing general stages: (stepIl). Host-cellenzymes thentranscribe viral"early"genesinto
mRNAs andsubsequently theseintoviral"early"proteins
translate
(stepZ) Theearlyproteins replicatetheviralDNAandinduce
l, Adsorption-Virion interacts with a host cell by bind-
expression of viral"late"proteinsby host-cellenzymes (stepS) The
ing of multiple copies of capsid protein to specificreceptors
virallateproteins include capsidandassembly proteins andenzymes
on the cell surface.
thatdegrade the host-cell DNA,supplying nucleotides for synthesis
of moreviralDNA Progeny virions
areassembled in thecell(stepZl)
2. Penetration-Yiral genome crossesthe plasma mem-
andreleased (step5) whenviralproteins lysethecell.Newly
brane. For some viruses,viral proteins packagedinside the
liberatedviruses initiateanothercycleof infectionin otherhost
capsid also enter the host cell.
ceils

155 C H A P T E R4 | B A S t CM O L E C U L A RG E N E T T M
C ECHANTSMS
 Rabiesvirus
* N u c l e o c a p s i dp r o t e i n
Matrixprotein
't R e c e p t o r - b i n d i nggl y c o p r o t e i n
o V i r a lR N A p o l y m e r a s e

arOOinS
$
;{b V i r u s r e c e p t o r C e l lm e m b r a n e
:' I' i ': ..,/
Cytosol

\enoo"vtori.

I M a t r i xa n d
Transnort n u c l e o c pa s i d
fl synthesrs
A
{
Replication
h' l.
t
ER
.{
Transcription E#
Nucleus Release

FIGURE4-47 Lytic replication cycle of an enveloped, animal synthesized on ER-bound ribosomes (stepZ). Carbohydrate isadded
v i r u s . R a b i evsi r u si s a n e n v e l o p evdi r u sw i t h a s i n g l e - s t r a n d R
e dN A i n s i d e E Rl u m e na n di sm o d i f i eads
t o t h el a r g ef o l d e dd o m a i n t h e
genome The structuralcomponentsof this virusare depictedat the the membrane andtheassociated glycoprotein passthroughthe
top After a virionadsorbsto multiplecopiesof a specifichost Golgiapparatus (stepE) Vesicles with matureglycoprotern fuse
membraneprotein(steptr), the cellengulfsit in an endosome with the hostplasma membrane, depositing viralglycoprotein on the
( s t e pE ) A c e l l u l apr r o t e i ni n t h e e n d o s o m em e m b r a n ep u m p s cellsurface withthe largereceptor-binding domainoutside the cell
H* ionsfrom the cytosolinto the endosomeinterior.The resulting (step9) Meanwhile, otherviralmRNAs aretranslated on host-cell
d e c r e a sien e n d o s o m apl H i n d u c e a s c o n f o r m a t i o n cahl a n g ei n t h e ribosomes intonucleocapsid protein, matrixprotein, andviralRNA
viralglycoprotein, leadingto fusionof the viralenvelopewith the polymerase GtepIE). Theseproteins areassembled with replicated
e n d o s o m al il p i db i l a y em r e m b r a n ea n d r e l e a soef t h e n u c l e o c a p s i d viralgenomic RNA(brightred)intoprogeny nucleocapsids GtepI[),
into the cytosol(stepsB and 4) ViralRNApolymerase uses whichthenassociate withthecytosolic domainof viraltransmembrane
ribonucleoside triphosphates in the cytosolto replicatethe viralRNA glycoproteins in the plasma membrane (stepIE) Theplasma
genome(stepE) and to synthesize viralmRNAs(step6) One of the m e m b r a ni sef o l d e da r o u n d t h en u c l e o c a p sf oi dr ,m i n ga " b u d "t h a t
viralmRNAsencodesthe viraltransmembrane glycoprotein, which is eventually isreleased (stePIE)
r n s e r t e idn t o t h e m e m b r a n eo f t h e e n d o p l a s m ri ce t i c u l u m ( E R a) s i t i s

copiesof them are requiredfor the assemblyof eachprogeny teins by host-cellribosomes,tRNA, and translationfactors.
virion. In eachinfectedcell, about 100-200 T4 progenyviri- The viral proteins are then transported back into the nu-
ons are producedand releasedby lysis. cleus,where some of them either replicate the viral DNA di-
The lytic cycle is somewhatmore complicatedfor DNA rectly or direct cellular proteinsto replicatethe viral DNA'
virusesthat infect eukaryoticcells.In most suchviruses,the as in the case of SV40 discussedearlier.Assembly of the
DNA genome is transported (with some associated capsid proteins with the newly replicated viral DNA occurs
proteins)into the cell nucleus.Once inside the nucleus,the in the nucleus,yieldingthousandsto hundredsof thousands
viral DNA is transcribedinto RNA by the host'stranscrip- of progenyvirions.
tion machinery.Processingof the viral RNA primary tran- Most plant and animal viruseswith an RNA genome do
script by host-cell enzymesyields viral mRNA, which is not require nuclear functions for lytic replication. In some of
transportedto the cytoplasmand translatedinto viral pro- these viruses, a virus-encoded enzyme that enters the host

G E N E T I CS Y S T E M
SF T H E C E L L U L A R
V I R U S E SP: A R A S I T EO 157
 d u r i n g p e n e t r a t i o n t r a n s c r i b e st h e g e n o m i c R N A i n t o
mRNAs in the cell cytoplasm. The mRNA is directly trans-
lated into viral proteins by the host-cell translation machin-
ery. One or more of theseproteins then produces additional
copies of the viral RNA genome. Finally, progeny genomes
are assembledwith newly synthesizedcapsid proteins into
p r o g e n yv i r i o n si n t h e c y t o p l a s m .
After the synthesisof hundreds to hundreds of thou-
sandsof new virions has beencompleted,dependingon the
type of virus and host cell, most infecredbacterialcellsand
some infectedplant and animal cellsare lysed,releasingall
the virions at once. In many plant and animal viral infec-
tions, however, no discretelytic event occurs; rather, the
dead host cell releasesthe virions as it gradually disinte-
grates.
As noted previously,envelopedanimal virusesare sur-
rounded by an outer phospholipid layer derived from the
plasma membrane of host cells and containing abundant
viral glycoproteins. The processesof adsorption and re-
lease of envelopedviruses differ substantially from these
p r o c e s s e sf o r n o n e n v e l o p e dv i r u s e s . T o i l l u s t r a t e l y t i c
replication of enveloped viruses, we consider the rabies
v i r u s , w h o s e n u c l e o c a p s i dc o n s i s t so f a s i n g l e - s t r a n d e d A EXPERIMENTAL FIGURE 4-48 Progenyvirionsare released
RNA genome surrounded by multiple copies of nucleo- by budding.Progeny virionsof enveloped viruses arereleasedby
capsid protein. Like most other lytic RNA viruses,rabies buddingfrominfected cellsIn thistransmission electron
micrograph
virions are replicated in the cytoplasm and do not require of a cellinfected with measles virus,virionbudsareclearly visible
protruding fromthe cellsurfaceMeasles virusisan enveloped RNA
host-cell nuclear enzymes.As shown in Figure 4-47, a ra-
viruswith a helical nucleocapsid, likerabiesvirus,andreplicatesas
bies virion is adsorbedto a host cell by binding to a spe-
illustrated in Figure4-47 [From A Levine.1991, Viruses,
Scientific
cific cell-surfacereceptor moleculeand then entersthe cell p 221
American tibrary,
by endocytosis.Progeny virions are releasedfrom a host
cell by budding from the host-cellplasma membrane.Bud-
ding virions are clearly visible in electron micrographs of molecule-the opposite flow of geneticinformation com-
infected cells, as illustrated in Figure 4-48. Many tens of pared with the more common transcription of DNA into
thousands of progeny virions bud from an infected host RNA. In the retroviral life cycle (Figure 4-49), a viral en-
cell before it dies. zyme called reversetranscriptaseinitially copies the viral
RNA genome into single-strandedDNA complementary
V i r a l D N A l s I n t e g r a t e di n t o t h e H o s t - C e l l to the virion RNA; the same enzyme then catalyzessyn-
thesisof a complementaryDNA strand. (This complex re-
G e n o m ei n S o m eN o n l y t i cV i r a l G r o w t h C y c l e s
action is detailed in Chapter 6 when we consider closely
Some bacterial viruses, called temperate phages,can estab- r e l a t e d i n t r a c e l l u l a r p a r a s i t e sc a l l e d r e t r o t r a n s p o s o n s . )
lish a nonlytic associationwith their host cellsthat doesnot The resulting double-strandedDNA is integrated into the
kill the cell. For example,when bacteriophageinfectsE. coli, chromosomal DNA of the infected cell. Finally, the inte-
the viral DNA may be integrated into the host-cell chromo- grated DNA, called a provirus, is transcribed by the cell's
some rather than being replicated. The integrated viral own machinery into RNA, which either is translated into
DNA, called a prophage, is replicated as part of the cell's viral proteins or is packagedwithin virion coat proteins to
DNA from one host-cell generation to the next. This phe- form progeny virions that are releasedby budding from
nomenon is referred to as lysogeny. Under certain condi- the host-cell membrane. Becausemost retrovirusesdo not
tions, the prophage DNA is activated,leading to its excision kill their host cells, infected cells can replicate,producing
from the host-cell chromosome and entrance into the lytic daughter cells with integrated proviral DNA. These
cycle, with subsequentproduction and releaseof progeny daughter cells continue to rranscribe the proviral DNA
vlrtons. and bud progeny virions.
The genomesof a number of animal viruses also can Some retrovirusescontain cancer-causing genes (onco-
rntegrateinto the host-cell genome. One of the most im- genes),and cellsinfectedby suchretrovirusesare oncogenically
portant are the retroviruses,which are envelopedviruses transformedinto tumor cells.Studiesof oncogenicretroviruses
with a genomeconsistingof two identical srrandsof RNA. (mostly virusesof birds and mice) have revealeda great deal
These viruses are known as retrouiruses because their about the processes that leadto transformationof a normal cell
RNA genome acts as a template for formation of a DNA into a cancercell (Chapter25).

158 CHAPTER
4 I BASTC
MOLECULAG
R E N E T TM
CE C H A N T S M S
llll| overview Animation: Life Cycle of a Retrovirus
Genomic
ssRNA
Retrovirus
Host-cell

Transportto
Reverse
transcription
I \
n u c l e u sa n d
integration
*Y

FIGURE4-49 Retrovirallife cycle.Retroviruses havea genome nucleusandintegratedintooneof manypossible sitesin the host-
of two identical
copies RNA
of single-stranded and an outer cellchromosomal DNA.Forsimplicity, chromosome
onlyonehost-cell
envelopeStep[: Afterviralglycoproteins in the envelopeinteract isdepictedStepZl: Theintegrated viralDNA(provirus)
istranscribed
host-cell
with a specific membrane protein,the retroviral
envelope RNApolymerase,
bythe host-cell generatingmRNAs (darkred)and
with the plasma
fusesdirectly membrane, allowing entryof the genomicRNAmolecules (brightred).Thehost-cell
machinery
nucleocapsidintothe cytoplasmof the cell StepE: Viralreverse theviralmRNAs
translates andnucleocapsid
intoglycoproteins
andotherproteins
transcriptase copytheviralssRNA genomeintoa StepEt: Progeny
proteins. thenassemble
virions andarereleased by
double-stranded DNA StepB: TheviraldsDNAis imported intothe in Figure
buddingasillustrated 4-47.

Among the known human retrovirusesare human T-

c e l l l y m p h o t r o p h i c v i r u s ( H T L V ) , w h i c h c a u s e sa
form of leukemia, and human immunodeficiency virus
( H I V ) , w h i c h c a u s e sa c q u i r e d i m m u n e d e f i c i e n c y s y n -
drome (AIDS). Both of these viruses can infect only spe-
cific cell types, primarily certain cells of the immune sys-
tem and, in the caseof HIV, some central nervous system
neurons and glial cells. Only these cells have cell-surface
receptors that interact with viral envelope proteins, ac-
counting for the host-cell specificity of these viruses.Un-
like most other retroviruses,HIV eventually kills its host
cells. The eventual death of large numbers of immune-
system cells results in the defectiveimmune response
characteristicof AIDS.
Some DNA viruses also can integrate into a host-cell
chromosome. One example is the human papillomaviruses
(HPVs), which most commonly cause warts and other be-
nign skin lesions. The genomes of certain HPV serotypes,
however,occasionallyintegrate into the chromosomal DNA
of infected cervical epithelial cells, initiating developmentof
cervical cancer. Routine Pap smears can detect cells in the
early stagesof the transformation processinitiated by HPV
integration, permitting effectivetreatment. I
Viruses: Parasitesof the Cellular Genetic System
r Viruses are small parasitesthat can replicate only in
host cells. Viral genomes may be either DNA (DNA
viruses)or RNA (RNA viruses)and either single-or double-
stranded.
r The capsid, which surrounds the viral genome, is com-
posed of multiple copiesof one or a small number of virus-
encodedproteins. Somevirusesalso have an outer envelope'
which is similar to the plasma membrane but contains viral
transmembraneproteins.
r Most animal and plant DNA viruses require host-cell
nuclear enzymesto carry out transcription of the viral
genome into mRNA and production of progeny genomes'
In contrast, most RNA viruses encode enzymesthat can
transcribe the RNA genomeinto viral mRNA and produce
new copies of the RNA genome.
r Host-cell ribosomes, tRNAs, and translation factors
are used in the synthesisof all viral proteins in infected
cells.

G E N E T I CS Y S T E M
SF T H E C E L L U L A R
V I R U S E SP: A R A S I T EO 159
 r Lytic viral infection entails adsorption, penetration,
synthesisof viral proteins and progeny genomes(replica-
tion), assemblyof progeny virions, and releaseof hun-
dreds to thousandsof virions, leadingto death of the host
cell (seeFigure 4-46).Releaseofenveloped vrrusesoccurs
by budding through the host-cell plasma membrane (see
Figure 4-471.
r Nonlytic infection occurs when the viral genome is inte-
grated into the host-cell DNA and generally does not lead
to cell death.
r Retroviruses are enveloped animal viruses containing a
single-stranded RNA genome. After a host cell is pene-
trated, reversetranscriptase,a viral enzymecarried in the
virion, converts the viral RNA genome into double-
stranded DNA, which integratesinto chromosomal DNA
(seeFigure4-49).
r Unlike infection by other retroviruses,HIV infection
eventually kills host cells, causing the defects in the im-
mune responsecharacteristicof AIDS.
r Tumor viruses, which contain oncogenes,may have an
RNA genome (e.g.,human T:cell lymphotrophic virus) or a
DNA genome (e.g., human papillomaviruses).In the case
of theseviruses,integration of the viral genomeinto a host-
cell chromosome can causetransformation of the cell into
a tumor cell.
In this chapter we first reviewed the basic structure of
DNA and RNA and then describedfundamental aspectsof
the transcription of DNA by RNA polym.rur.t. RNA
polymerases are discussed in greater detail in Chapter 7,
along with additional factors required for transcriotion
i n i t i a t i o n i n e u k a r y o t i cc e l l sa n d i n t e r a c t i o n sw i t h . e g u l a -
tory transcription factors that control transcription initia-
tion in both bacterial and eukaryotic cells. Next, we dis-
cussedthe geneticcode and the participation of IRNA and
the protein-synthesizingmachine, the ribosome, in decod-
ing the information in mRNA to allow accurateassembly
of protein chains. Mechanisms that regulate protein syn-
thesis are consideredfurther in Chapter 8. Then, we con-
sideredthe molecular detailsunderlying the accuraterepli-
cation of DNA required for cell division. Chapter 20
coversthe mechanismsthat regulatewhen a cell replicates
its DNA and that coordinate DNA replication with the
complex process of mitosis that distributes the daughter
DNA molecules equally to each daughter cell. The next
section addressedmechanisms for repairing damage to
DNA, including recombination mechanismsthat also lead
to the generationof geneticdiversity among individuals of
a species.This genetic recombination contributes to the
diversity of traits subjectedro natural selectionduring the
evolution of contemporary species.In Chapter 20, we dis-
cuss the mechanisms that segregatechromosomes into
haploid germ cells, a processthat requires recombination
160 . c H A p r E4R | B A S tM
c o L E c u L AGRE N E TM
t cE c H A N t s M s
between maternal and paternal chromosomes.Finally, we
discussed viruses, parasites of the cellular molecular
genetic system and important model systemsand useful
tools for studying multiple aspects of molecular cell
biology.
The basicmolecular geneticprocessesdiscussedin this
chapter form the foundation of contemporary molecular
cell biology. Our current understandingof theseprocesses
is grounded in a wealth of experimentalresults and is not
likely to change.However, the depth of our understanding
will continue to increaseas additional details of the struc-
tures and interactionsof the macromolecularmachinesin-
volved are uncovered. The determination in recent years
of the three-dimensionalstructures of RNA polymerases,
ribosomal subunits, and DNA replication proteins has
allowed researchersto design ever more penerraung ex-
perimental approachesfor revealinghow thesemacromol-
eculesoperate at the molecular level. The detailed level of
understanding currently being developed may allow the
design of new and more effective drugs for treating
illnessesof humans, crops, and livestock.For example,the
recent high-resolutionstructuresof ribosomesare provid-
ing insights into the mechanism by which antibiotics
inhibit bacterial protein synthesiswithout affecting the
function of mammalian ribosomes. This new knowledge
may allow the design of even more effective antibiotics.
Similarln detailed understanding of the mechanisms
regulating transcription of specifichuman genesmay lead
to therapeutic strategies that can reduce or prevent
inappropriate immune responsesthat lead to multiple
sclerosisand arthritis, the inappropriate cell division that
is the hallmark of cancer, and other pathological
processes.
Much of current biological researchis focusedon dis-
covering how molecular interactions endow cells with de-
cision-making capacity and their special properties. For
this reason severalof the following chapters describecur-
rent knowledge about how such interactions regulate
transcription and protein synthesisin multicellular organ-
isms and how such regulation endows cells with the ca-
pacity to become specializedand grow into complicated
organs. Other chapters deal with how protein-protein
interactions underlie the construction of specialized
organellesin cells, and how they determine cell shapeand
movement. The rapid advancesin molecular cell biology
in recent years hold promise that in the not too distant fu-
ture we will understand how the regulation of specialized
cell function, shape,and mobility coupled wirh regulared
c e l l r e p l i c a t i o n a n d c e l l d e a t h ( a p o p t o s i s )l e a d t o t h e
growth of complex organisms like flowering plants and
human beings.
KeyTerms
anticodon127 crossingover 150
codons127 deamination 146
complementaryL14 depurination 147
 '$7hat
DNA end-joining 149 polyribosomes138 (Figure 4-1.3a).'What is an operon? advantagesare
DNA polymerases141 primary transcript 121 there to having genesarrangedin an operon' compared with
primer 141 the arrangementin eukaryotes?
double helix 114
8. Contrast how selectionof the translational start site oc-
envelope(viral) 154 promoter 1-21-
curs on bacterial,eukaryotic, and poliovirus mRNAs.
excision-repair systems147 reading frame 1.28
9. \fhat is the evidence that the 23S rRNA in the large
exons 123 recombination112
rRNA subunit has a peptidyltransferaseactivity?
geneconversion153 replicationfork 141
10. How would a mutation in the poly(A)-binding protein I
genetic code 127 retroviruses152 gene affect translation? How would an electron micrograph
Holliday structure154 reversetranscriptase152 of polyribosomesfrom such a mutant differ from the normal
homologousrecombination ribosomal RNA pattern?
repair L50 (rRNA) 112 L1. What characteristic of DNA results in the require-
introns 123 ribosomes127 ment that some DNA synthesis is discontinuous? How
lagging strand 141 RNA polymerase120 are Okazaki fragments and DNA ligase utilized by the
cell?
leading strand 141 thymine-thymine
dimers 148 L2. Eukaryotes have repair systems that prevent muta-
messengerRNA
(mRNA) 112 tions due to copying errors and exposure to mutagens.
transcription1 12 'What
are the three excision-repair systemsfound in eu-
mutation 138 transferRNA (IRNA) 112
karyotes, and which one is responsible for correcting
Okazaki fragments 141 translation 112 thymine-thymine dimers that form as a result of UV light
'Watson-Crick
phosphodiesterbond 1 14 basepairs 114 damageto DNA?
13. DNA-repair systemsare responsiblefor maintaining ge-
nomic fidelity in normal cellsdespitethe high frequencywith
which mutational eventsoccur.'Sfhattype of DNA mutation
Review the Concepts is generatedby (a) UV irradiation and (b) ionizing radiation?
Describe the system responsiblefor repairing each of these
l. \fhat are'Watson-Crickbasepairs? lWhy are they im- types of mutations in mammalian cells. Postulatewhy a loss
portant? of function in one or more DNA-repair systems typifies
2, TAIA box-binding protein binds to the minor groove of many cancers.
DNA, resulting in the bending of the DNA helix (seeFigure 14. lVhat is the name given to the processthat can repair
4-5). Vhat property of DNA allows the TAIA box-binding DNA damage and generate genetic diversity? Briefly
protein to recognizethe DNA helix? describe the similarities and differences of the two
3. Preparing plasmid (double-stranded,circular) DNA for processes.
sequencinginvolves annealing a complementary,short, sin- 15. The genome of a retrovirus can integrate into the
gle-strandedoligonucleotide DNA primer to one strand of host-cellgenome.What geneis unique to retroviruses,and
the plasmid template. This is routinely accomplishedby why is the protein encoded by this gene absolutely neces-
heating the plasmid DNA and primer to 90 "C and then sary for maintaining the retroviral life cycle?A number of
'C.
slowly bringing the temperature down to 25 \ilhy does retroviruses can infect certain human cells. List two of
this protocol work? them, briefly describe the medical implications resulting
4. \fhat difference between RNA and DNA helps to ex- from these infections, and describewhy only certain cells
plain the greater stability of DNA? What implications does are infected.
this have for the function of DNA?
5. What are the major differencesin the synthesisand Analyze the Data
structure of prokaryotic and eukaryotic mRNAs?
6, 'While investigatingthe function of a specificgrowth fac- Protein synthesisin eukaryotes normally begins at the first
tor receptor gene from humans, researchersfound that two AUG codon in the mRNA. Sometimes,however' the ribo-
types of proteins are synthesizedfrom this gene. A larger somes do not begin protein synthesisat this first AUG but
protein containing a membrane-spanningdomain functions scan past it (leaky scanning),and protein synthesisbegins
to recognizegrowth factors at the cell surface,stimulating a instead at an internal AUG. In order to understandwhat
specific downstream signaling pathway. In contrast, a re- features of an mRNA affect efficiency of initiation at the
lated, smaller protein is secretedfrom the cell and functions first AUG, studies have been undertaken in which the syn-
to bind availablegrowth factor circulating in the blood, thus thesisof chloramphenicolacetyltransferase was examined.
inhibiting the downstream signaling pathway. Speculateon Translation of its messagecan give rise to a protein referred
how the cell synthesizesthesedisparateproteins. to as preCM or give rise to a slightly smaller protein, CAT
7. The transcription of many bacterial genesrelieson func- (see M. Kozak. 2005. Gene 367:1'3). The two proteins
tional groups called operons,such as the tryptophan operon differ in that CAT lacks several amino acids found at the

A N A L Y Z ET H E D A T A T 161
N-terminus of preCAT. CAT is not derived by cleavageof
preCAT but, instead, by initiation of translation of the
mRNA at an internal AUG:
precAT CAT
Start Start
llvt
m 7 c p p p " , ' ' , ", A U G AUG
,:r [J[[*;,[[[[n
12
a. Resultsfrom a number of studieshave given rise to
the hypothesisthat the sequence(-3)ACCAUGG(+4), in
which the start codon AUG is shown in boldface,provides an
optimal context for initiation of protein synthesisand ensures
that ribosomesdo not scanpast this first AUG to begin initia-
tion insteadat a downstreamAUG. In the numbering scheme
used here, the A of the AUG initiation is designated(*1);
bases5' of this are given negarivenumbers [so that the first
b a s eo f t h i s s e q u e n cies ( - 3 ) ] , a n d b a s e s3 ' t o t h e ( + 1 ) A a r e
given positive numbers [so that the last baseof this sequence
is (+4)]. To test the hypothesisthat the start sire sequence
(-3)ACCAUGG(+4) preventsleaky scanning,the chloram-
phenicol acetyltransferase mRNA sequencewas modified and
the resulting effectson translation assessed.
figure, the sequence(red) surrounding the first AUG codon
(black) of the mRNA that gives rise to the synthesisof pre-
CAT is shown above lane 3. Modification of this messageis
shown above the other gel lanes (altered nucleotides are in
blue), and the completedproteins generatedfrom eachmod-
ified messageappear as bands on the SDS-polyacrylamide
gel below. The intensity of each band is an indication of the
amount of that protein synthesized.Analyzethe alterations
to the wild-type sequence,and describe how they affect
translation. Are the positions of some nucleotidesmore im-
portant than others? Do the data shown in this figure pro-
vide support for the hypothesisthat the context in which the
first AUG is presentaffectsefficiencyof translation from this
site? Is ACCAUGG an optimal contexr for initiation from
the first AUG?
CCCCA
-3UUAAC
UUCCC
UUCCA
(t
AUG1 I A A A A A
^-^^"-1U U U U U
PrtrLAr
IC G G G G
+4UGGAA
preCAT+ *
***{}
CAT+* s +
Lane12345
b. What are some additional alterations to this
message, other than those shown in the figure, that would
further elucidate the importance of the ACCAUGG
'162 . c H A p r E R4 | B A S t cM o L E c u L A R
G E N E I cM E c H A N t s M s
sequence as an optimal context for synthesis of preCAT
rather than CAT? How would you further examine
w h e t h e rA a t t h e ( - 3 ) p o s i t i o n a n d G a t t h e ( + 4 ) p o s i t i o n
are the most important nucleotidesto provide context for
the AUG start?
c. A mutation causing a severe blood diseasehas
Stop been found in a single family (seeT. Matthes et a1.,2004,
I
Blood 104:2181). The mutation, shown in red in the fig,
ure below, has been mapped to the 5' untranslatedregion
of the gene encoding hepcidin and has been found to alter
the gene'smRNA. The shadedregions indicate the coding
sequenceof the normal and mutant genes.No hepcidin is
produced from the altered mRNA, and lack of hepcidin
resultsin the disease.Can you provide a reasonableexpla-
nation for the lack of synthesisof hepcidin in the family
memberswho have inherited this mutation? $fhat can you
deduce about the importance of the context in which the
start site for initiation of protein synthesisoccurs in this
c a s e?
Starthepcidin
.....cCAGUGGGACAGCCAGACAGACGGcncCnricccncUG.....
Norma
Y
I
In the following .....GCAAUGGGACAGCCAGACAGACGGCACGAUGGCACU........
Mutant
References
Structure of Nucleic Acids
Arnott, S. 2006. Historical article: DNA polymorphism and the
earfy history of the double helix. TrendsBiochem. Sci.3l:349-354.
Berger,J. M., and J. C. Wang. 1996. Recentdevelopmentsin
DNA topoisomeraseII structureand mechanism.Curr. Opin. Struc.
Biol. 6:84-90.
Dickerson,R. E. 1983. The DNA helix and how it is read. Sci.
Am. 249:94-1.'1.1..
Dickerson,R. E., and H. L. Ng. 2001. DNA srructurefrom A to
B. Proc. Nat'l Acad. Sci.USA.98:6986-69888.
Doudna, J. A., and T. R. Cech.2002.The chemicalrepertoireof
natural ribozymes.N ature 418:222-228.
Kornberg, A., and T. A. Baker.2005. DNA Replication.Univer-
sity Science,chap. 1. A good summary of the principlesof DNA
structure.
Lilley, D. M. 2005. Structure,folding and mechanismsof ri-
bozymes.Curr. Opin. Struc.Biol. t5:313-323.
Vicens,Q., and T. R. Cech.2005. Atomic level architectureof
group I introns revealed.TrendsBiochem. Sci.3l:41-51.
'Wang,
J. C. 1980. SuperhelicalDNA. TrendsBiochem. Sci.
5:279-221.
Wigley,D.B. 1995. Srructureand mechanismof DNA topoiso-
merases.Ann. Reu.Biophys. Biomol. Struc.24:L85-208.
Transcription of Protein-Coding Genes and Formation of
Functional mRNA
Brenner,S., F. Jacob,and M. Meselson.1,96L.An unstableinter-
mediatecarrying information from genesto ribosomesfor protein
synthesis.Natur e 190:576- 5 8'1,.
Murakami, K. S., and S. A. Darst. 2003. BacterialRNA polv-
merases:the whole story.Curr. Opin. Strwc.Biol. t3:31-39.
 Okamoto K., Y. Sugino,and M. Nomura. 1962. Synthesisand
turnover of phagemessengerRNA in E. coli infectedwith bacterio-
phageT4 in the presenceof chloromycetin.J. Mol. Biol. 5:527-534.
Steitz,T. A. 2006. Visualizingpolynucleotidepolymerase
machinesat work. EMBO J.25:3458-3468.
The Decoding of mRNA by tRNAs
Alexander,R. !(., and P. Schimmel.2001. Domain-domain com-
munication in aminoacyl-tRNA synthetases. Prog. Nucl. Acid Res.
Mol. Biol. 69:31,7-349.
Hatfield, D. L., and V. N. Gladyshev.2002.How seleniumhas
altered our understanding of the genetic code.Mol. Cell Biol.
22:3565-3576.
Hoagland, M. B., et al. 1958. A solubleribonucleicacid inter-
mediatein protein synthesis./. Biol. Chem.23l:241-257.
Ibba, M., and D. Soll. 2004. Aminoacyl-tRNAs: settingthe
limits of the geneticcode.GenesDeu. 18:731-738.
Khorana, G. H., et al. 1966. Polynucleotidesynthesisand the
geneticcode. Cold Spring Harbor Symp. Quant. Biol.3l:3949.
Nakanishi, K., and O. Nureki. 2005. Recentprogressof struc-
tural biology of IRNA processingand modification. MoL Cells
l91.57-166.
Nirenberg, M., et al. 1966.The RNA code in protein synthesis.
Cold Spring Harbor Symp. Quant. Biol. 3t:ll-24.
Rich. A.. and S.-H. Kim. 1978. The three-dimensionalstructure
of transfer RNA. Scl.Am.240(1,1:52-62(offprint 1.377).
Stepwise Synthesis of Proteins on Ribosomes
Abbott, C. M., and C. G. Proud. 2004. Translationfactors:in
sicknessand in health. TrendsBiocbem.Sci.2925-131.
Auerbach,T., A. Bashan,and A. Yonath. 2004. Ribosomal
antibiotics:structural basisfor resistance,synergismand selectivity.
TrendsB iotech nol. 22:570- 576.
Frank, J., et al. 2005. The role of IRNA as a molecularspring in
decoding,accommodation,and peptidyl transfer.FEBSLett.
579:959-962.
Ganoza,M. C., M. C. Kiel, and H. Aoki. 2002. Evolutionary
conservationof reactionsin translation.Microbiol. Mol. Biol. Reu.
66:460485.
Gualerzi,C. O., et al. 2001. Initiation factorsin the early events
of mRNA translation in bacteria.Cold Spring Harbor Symp. Quant.
Biol. 66:363-376.
Hellen, C. U., and P. Sarnow.2001. Internal ribosomeentry
sitesin eukaryotic mRNA molecules.Genet.Deuel. 15:1593-161'2.
Kahveiian,A., G. Roy, and N. Sonenbery.200l. The mRNA
closed-loopmodel: the function of PABPand PABP-interactingpro-
teins in mRNA translation.Cold Spring Harbor Symp. Quant. Biol.
66:293-300.
Kapp, L. D., and J. R. Lorsch. 2004. The molecularmechanics
of eukaryotictranslation.Ann. Reu.Biochem. 73:657-704.
Mitra, K., and J. Frank. 2006. Ribosomedynamics:insights
from atomic structuremodeling into cryo-electronmicroscopy
maps.Ann. Reu.Biophys.Biomol. Struc.35:299-3t7.
Noller, H. F. 2005. RNA structure:readingthe ribosome.
Science309:1508-15 14.
Noller, H. F., et al. 2002. Translocationof IRNA during protein
synthesis.FEBSLett. 514i1.1.-16.
Polacek,N., and A. S. Mankin. 2005. The ribosomal peptidyl
transferasecenter:structure,function, evolution, inhibition' Cdr.
Reu.Biocbem.Mol. Biol. 4O:285-31.1.
Preiss,T., and M. W. Hentze.2003. Startingthe protein synthe-
sis machine:eukaryotic translation initiation. BioEssays
25:1201-1.211'.
Richter,J. D., and N. Sonenberg.2005. Regulationof cap-
dependenttranslation by eIF4E inhibitory proteins.Nature
433:477480.
Scheper,G. C., C. G. Proud, and M. S' van der Knaap. 2005'
Defectivetranslation initiation causesvanishingof cerebralwhite
matter.TrendsMol. Med. t2:1'59-1'66.
Sonenberg,N. 2006. TranslationalControl in Biology and
Medicine (CSH monograph).Cold SpringHarbor Press.
Sonenberg,N., and T. E. Dever.2003. Eukaryotic translation
initiation factors and regulators.Curr. Opin. Struc.Biol' 13:56-63'
Steitz,T. A. 2005. On the structural basisof peptide-bondfor-
mation and antibiotic resistancefrom atomic structuresof the large
ribosomal subunit. FEBSLett. 579:955-958'
Taylor, S. S., N. M. Haste' and G. Ghosh. 2005. PKR and
eIF2ct:integration of kinasedimerization,activation' and substrate
docking. Cell 122:823-82 5.
DNA Replication
Brautigam, C. A, and T. A. Steitz. 1998' Structural and func-
tional insights provided by crystal structures of DNA polymerases
and their snbtti"t. complexes.Curr. Opin. Struc. Biol. 8:54-63.
Bullock, P.A. 1997. The initiation of simian virus 40 DNA
replication in vitro. Crit. Reu.Biochem.Mol. Biol' 32:503'568.
DePamphilis,M. L., ed. 2006 DNA Replicationand Human
Disease.Cold SpringHarbor Laboratory Press.
Kornberg. A., and T. A. Baker.2005. DNA Replication'
University Science.
Langston,L. D., and M. O'Donnell. 2005. DNA replication:
keep moving and don't mind the gap.Mol. Cells23:155-160'
Mendez,J., and B. Stillman.2003. Perpetuatingthe double
helix: molecularmachinesat eukaryotic DNA replication origins'
BioEssays 25:11'58-I1'67.
O'Donnell, M.2006. Replisomearchitectureand dynamicsin
Eschericbia coli. l. B iol. Cbem. 281:106 5 3-106 56.
Sclafani,R. A., R. J. Fletcher,and X. S' Chen' 2004' Two heads
are better than one: regulation of DNA replication by hexameric
helicases.GenesDeu. 18:2039-2045.
DNA Repair and Recombination
Andressoo,J. O., and J. H. Hoeijmakers'2005. Transcription-
coupled repair and premature aging.Mutat. Res.577:179-1'94'
Barnes,D. 8., and T. Lindahl. 2004. Repair and geneticconse-
quencesof endogenousDNA basedamagein mammalian cells'Ann'
Reu.Genet.38:445476.
Bell, C. E. 2005. Structureand mechanismoI Escherichiacoli
RecA ATPase.Mol. Microbiol. 58:358-366.
Friedberg,E. C., et al' 2006. DNA repair: from molecularmech-
anism to human disease.DNA Repair 5:986-996.
Haber,J. E. 2000. Partnersand pathwaysrepairing a double-
strand break. Trends Genet. 16:259-264.
system'Na/'
Jiricny,1.2006. The multifacetedmismatch-repair
Reu.Mol. Cell Biol.7:335-346.
Khuu. P.A.. et al. 2005. The stacked-XDNA Holliday iunction
and protein recognition.l. Mol. Recog.192234-242.
Lilley, D. M., and R. M. Clegg' t993-The structureof the
four--ay jonction in DNA. Ann. Reu-Biophys. Biomol' Struc'
22:299-328.
Mirchandani, K. D., and A. D. D'Andrea- 2006. The Fanconi
anemia/BRCAp"lh*"yt a coordinator of cross-link repail Exp' Cell
Res.372:2647-2653'
Mitchell, J. R., J. H. Hoeijmakers,and L. J. Niedernhofer'2003'
Divide and.*qu.t, nucleotideexcisionrepair battlescancerand
aging.Curr. Opin. Cell Biol.l5:232-240.
Orr-Weaver,T. L., and J. I7. Szostak.1985. Fungal recombina-
tion. Microbiol. Reu. 49:33-58.
Shin, D. S., et al. 2004. Structureand function of the double
strand break repair machinery.DNA Repair 32863-873'
'!food,
R. D., M. Mitchell, and T. Lindahl. Human DNA repair
senes. Mutat. Res.577:275-283.
R E F E R E N C E 5o 16 3
 Yoshida,K., and Y. Miki. 2004. Role of BRCA1 and BRCA2 as Klug, A. 1999.The tobacco mosaic virus particle: structure and
re-gulatorsof DNA repair, transcription, and cell cycle in responseto assembly.Phil. Trans, R. Soc.Lond. B Biol. SZi.354:531-535.
DNA damage.CancerSci. 95,:865-87t.
Knipe, D. M., and P. M. Howley eds. 2001. Fields Virology.
Lippincott Villiams & Ifilkins.
Viruses:Parasites of the CellularGeneticSystem
Kornberg, A., and T. A. Baker. 1992. DNA Replication,2d, ed,.
Flint,S.J.,et al. 2000.Principles
of Virology:
Molecular
Biol- W. H. Freeman and Company. Good summary of bacteriophagemo-
ogy, Pathogenesis,and Control. ASM press. lecular biology.
Hull, R. 2002. Matheuts' Plant Virology. Academic press.

164 C H A P T E R4 I B A S T CM O L E C U L A RG E N E T T M
C ECHANTSMS
 CHAPTER

GENETIC
MOLECULAR
TECHNIQUES
RNAinterference (RNA|)canbe usedto silence mostgenesin the
C. elegansgenome Thetransgenic worm on the right (markedby
a GFPreporterin headneurons) expressesdsRNAto the muscle
geneunc-\5, resultingin the potentdegradationof the unc-/5
mRNAand leadingto completeparalysis of the worm ln contrast,
body
the typicalsinusoidal
the wild-typeworm on the leftexhibits
movement[Courtesy of JohnKim]

I n previous chapters, we were introduced to the variety of encoded protein to its function in the context of a living
I tasks that proteins perform in biological systems.Indeed' cell or multicellular organism.
I the very field of molecular cell biology seeksto understand An important component in both strategiesfor studying a
the molecular mechanismsof individual proteins and how protein and its biological function is isolation of the corre-
groups of proteins work together to perform their biological sponding gene. Thus we discussvarious techniques by which
functions. In studying a newly discoveredprotein' cell biolo- res.archits can isolate, sequence'and manipulate specific re-
gistsusually begin by asking three questionsabout it: what is
its function, where is it located, and what is its structure?To
answer thesequestions,investigatorsemploy three tools: the
genethat encodesthe protein, a mutant cell line or organism
that lacks the function of the protein, and a sourceof the pu-
rified protein for biochemicalstudies.In this chapter we con-
sider various aspectsof two basic experimentalstrategiesfor
obtaining all three tools (Figure5-1). we discusstechniquesthat abolish normal protein function in
The first strategy,often referred to as classicalgenetics, order to analyzethe role of the protein in the cell.
beginswith isolation of a mutant that appearsto be defec-
tive in some process of interest. Genetic methods then are OUTLIN
used to identify and isolate the affected gene. The isolated
genecan be manipulatedto produce large quantitiesof the 5.1 GeneticAnalysisof Mutationsto
protein for biochemical experiments and to design probes ldentify and StudYGenes 166
for studies of where and when the encoded protein is ex-
5.2 DNA Cloningand Characterization 176
pressedin an organism.The secondstrategyfollows essen-
tially the same stepsas the classicalapproach but in reverse
5,3 UsingClonedDNA Fragmentsto
order, beginning with isolation of an interesting protein or
StudyGene ExPression
its identification based on analysis of an organism's ge-
nomic sequence.Once the corresponding gene has been 5.4 ldentifying and LocatingHuman
isolated, the gene can be altered and then reinsertedinto DiseaseGenes
an organism. In both strategies,by examining the pheno-
typic consequences of mutations that inactivate a particu- 5.5 Inactivatingthe Functionof Specific
lar gene, geneticistsare able to connect knowledge about Genesin EukarYotes
the sequence,structure, and biochemical activity of the

165
 > FIGURE 5-1 Overviewof two strategies
for relatingthe function,location,and
structureof gene products,A mutant
organism isthestarting pointfor the classical
geneticstrategy(greenarrows)Thereverse G e n e t i ca n a l y s i s
strategy(orange arrows)usuallybeginswith Screeningof DNA library
identificationof a protein-codrngsequence by
analysisof genomesequence databases In
bothstrategies, the actualgeneisisolated
eitherfroma DNAlibrary or by specific
amplification of the genesequence from
genomicDNA Oncea clonedgeneis isolated,
it canbe usedto produce theencoded protein
in bacterialor eukaryoticexpression systems Expression
in cultured
Alternatively, cells
a clonedgenecanbe rnactivated
by oneof various techniques andusedto
generate mutantcellsor orqanisms
ff,t Genetic
Analysis
of Mutations
to ldentifyand StudyGenes
As describedin Chapter 4, the information encoded in the
DNA sequenceof genesspecifiesthe sequence-and there-
fore the structure and function-of every protein moleculein
a cell. The power of geneticsas a tool foi studying cells and
organismslies in the ability of researchersto selectivelyalter
every copy of just one type of protein in a cell by making a
change in the gene for that protein. Genetic analysesof mu-
tants defectivein a particular processcan reveal (a) new
genes required for the process to occur, (b) the order in
which gene products act in the process,and (c) whether the
proteins encoded by different genes interact with one an-
other. Before seeinghow geneticstudiesof this rype can pro-
vide insights into the mechanism of complicated cellular or
developmental process,we first explain some basic genetic
terms used throughout our discussion.
The different forms, or variants, of a geneare referred to
as alleles.Geneticistscommonly refer to the numerous natu-
rally occurring genetic variants that exist in populations,
particularly human populations, as alleles.The term muta-
tion usually is reserved for instances in which an allele is
known to have been newly formed, such as after treatment
of an experimental organism with a muragen, an agent that
causesa heritable changein the DNA sequence.
Stricdyspeaking,the particular serof alielesfor all the genes
carried by an individual is its genotype.However, this term also
is usedin a more restrictedsenseto denoteiust the allelesof the
type usually denoresan allele that is present at a much higher
frequency than any of the other possible alternatives.
166 . cHAprER
5 | M o L E c u L AG
R E N E T tr cE c H N t e u E s
Mutant organism/cell
Comparisonof mutant and
wild-type function
Gene inactivation
Clonedgene
DNA sequencing
Databasesearchto identify
protein-codingsequence
PCRisolationof corresponding
gene
Protein
Localization
Biochemical studies
Determ i nati o n of structu re
Geneticistsdraw an important distinction between the
genotype and the phenotype of an organism. The phenotype
refers to all the physical attributes or traits of an individual
that are the consequenceof a given genotype. In practice,
however, the term phenotype often is used to denote the phys-
ical consequencesthat result from just the alleles that are
under experimental study. Readily observable phenotypic
characteristicsare critical in the geneticanalysisof mutations.
R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e s
GenerallyHave OppositeEffectson
G e n eF u n c t i o n
A fundamental genetic difference between experimental
organismsis whether their cells ca:i::ya single set of chromo-
somes or two copies of each chromosome. The former are
referred to as haploid; the latter, as diploid. Complex multi-
cellular organisms (e.g., fruit flies, mice, humans) are
diploid, whereasmany simple unicellular organismsare hap-
loid. Someorganisms,notably the yeastSaccharomycescere-
uisiae, can exist in either haploid or diploid states. Many
cancer cells and rhe normal cells of some organisms, both
plants and animals) caffy more than two copies of each
chromosome.However, our discussionof genetictechniques
and analysis relates to diploid organisms, including dipioid
yeasts.
Although many different allelesof a gene might occur in
different organisms in a population, any individual diploid
organism will carry two copiesof eachgeneand thus at most
can have two different alleles.An individual with two differ-
ent allelesis heterozygousfor a gene, whereas an individual
that carriestwo identical allelesis homozygousfor a gene.A
recessivemutant allele is defined as one in which both alleles
must be mutant in order for the mutant phenotype to be ob-
served;that is, the individual must be homozygous for the
mutant allele to show the mutant phenotype.In contrast, the
phenotypic consequences of a dominant mutant allelecan be
 ;;-;-'*i
GENOTYPE I l-----t i
= Dominant =

DIPLOID M uta nt
Wild type
PHENOTYPE

mutant alleles allelemustbe present

of a recessive to causea mutantphenotype
FIGURE 5-2 Effectsof dominantand recessive
Recessivemutations causea lossof function;dominant
usually
on phenotypein diploidorganisms.A singlecopyof a dominant
to produce
alleleissufficient a mutantphenotype,
whereas bothcopies mutationsusuallycausea gainof functionor an alteredfunction

observedin a heterozygousindividual carrying one mutant

and one wild-type allele(Figure5-2).
'Whether
a mutant allele is recessiveor dominant provides
valuable information about the function of the affectedgene
and the nature of the causativemutation' Recessivealleles
usually result from a mutation that inactivates the affected
gene,leadingto a partial or completelossof fwnctioz. Suchre-
cessivemutations may remove part of the gene or the entire
genefrom the chromosome,disrupt expressionof the gene,or
alter the structure of the encoded protein, thereby altering its
function. Conversely,dominant alleles are often the conse-
quenceof a mutation that causessome kind of gain of func- mutationsthan dominant mutations.
tion. Such dominant mutations may increase the activity of
the encodedprotein, confer a new function on it, or lead to its Segregationof Mutations in Breeding
inappropriate spatial or temporal pattern of expression. ExperimentsRevealsTheir Dominance
Dominant mutations in certain genes,however,are associ-
or RecessivitY
ated with a lossof function. For instance,somegenesatehaplo-
inswfficient, meaning that both allelesare required for normal Geneticistsexploit the normal life cycle of an organism to
function. Removing or inactivating a singleallele in such a gene test for the dominance or recessivityof alleles' To see how
leadsto a mutant phenotype.In other rare instancesa domi- this is done, we need first to review the type of cell division
nant mutation in one allelemay lead to a structuralchangein that gives rise to gametes (sperm and egg cells in higher
the protein that interferes with the function of the wild-rype plants and animals). l7hereas the body (somatic)cells of
protein encoded by the other allele. This type of mutation, -ort *.tl,i.ellular organisms divide by mitosis, the germ
referred to as a dominant-negatiue,producesa phenotype sim-
ilar to that obtainedfrom a loss-of-functionmutation.

Someallelescan exhibit both recessiveand dominant

properties.In such cases,statementsabout whether
an allele is dominant or recessivemust specify the pheno- their genesmay exist in different allelic forms' Figure 5-3
type. For example, the allele of the hemoglobin gene in depicts the major eventsin mitotic and meiotic cell division'
humans designatedHb'has more than one phenotypiccon- InLitosis DNA replication is always followed by cell divi-
sequence.Individuals who are homozygousfor this allele sion, yielding two diploid daughter cells' In meiosis oze
(Hb'/Hb') have the debilitating diseasesickle-cellanemia, round'of DNe replication is followed by two separatecell
but heterozygousindividuals (Hb'/Hb") do not have the divisions, yieldingfour haploid (12) cells that contain only
disease.Therefore, Hb' is recessiuefor the trait of sickle- one chromosome of each homologous pair' The apportion-
cell disease.On the other hand, heterozygous(Hb'/Hb')
individuals are more resistantto malaria than homozygous
(Hb"/Hb") individuals, revealing that Hb' is dominant for
the trait of malaria resistance.I

A commonly used agent for inducing mutations (muta-

genesis) in experimental organisms is ethylmethane
sulfonate (EMS). Although this mutagen can alter DNA se-
quencesin severalways, one of its most common effectsis to
chemically modify guanine basesin DNA, ultimately leading
to the conversionof a G'C basepair into an A T basepair.
Such an alteration in the sequenceof a gene,which involves
only a singlebasepair, is known as a point mutation. A silent

A N D s T U D YG E N E S
TO IDENTIFY
OF MUTATIONS
ANALYSIS
167
GENETIC
 Focus Mitosisftttt
Animation:
Focus Meiosis
MEIOTICCELL Paternal
DIVISION homolog

Maternal
homolog
S o m a t i cc e l l( 2 n ) Premeioticcell l2nl

I DNA reptication DNA reptication

v vI
Replicated Replicated
cnromosomes cnromosomes
(4nl

v
I I Homologous chromosomes
+ align; synapsisand crossing over
Mitotic
Mitotic appara
apparatus
; MetaphaseI
o
o
o

v
I I
v

/\
/ Ceil division \
++

D a u g h t e cr e l l s( 2 n )

o
o
o
o
E

FIGURE 5-3 Comparison of mitosisand meiosis.Both chromosomes is evidentat this stage One replicatedchromosomeof
somatrccellsandpremeiotic germcellshavetwo copies of each eachmorphological type then goesinto eachdaughtercell The
chromosome (2n),onematernal andonepaternal. In mitosis, resultingcellsundergoa seconddivisionwithout interveningDNA
the replicated
chromosomes, eachcomposed of two sister replication,with the srsterchromatidsof eachmorphological type
chromatids,alignat the cellcenterin sucha waythatboth beingapportionedto the daughtercells.In the secondmeiotic
daughter cellsreceivea maternal andpaternal fromolog of each divisionthe alignmentof chromatidsand their equalsegregation into
morphological typeof chromosome. Durinqthefirstmerctrc daughtercellsis the sameas in mttoticdivision.The alignmentof
division,
however, eachreplicated chromosome pairswith its pairsof homologouschromosomein metaphaseI is randomwith
nomotogous partnerat the cellcenter;thispairingoff isreferred respectto other chromosomepairs,resultingln a mix of paternally
Io assynapsis,andcrossing overbetweenhomoloqous and maternallyderivedchromosomes in eachdaughtercell.

168 CHAPTER
5 | M O L E C U L AG
R E N E T TTCE C H N T O U E S
(a) Segregationof dominant mutation (Figure 5-4). If the F1 progeny exhibit the mutant trait' then
ih."r.r.rr"n, allele is dominant; if the F1 progeny exhibit the
Mutant Wild-tYPe
wild-type trait, then the mutant is recessive'Further crossing
b"t*.Ln F1 individualswill also revealdifferent patternsof in-
heritanceaccording to whether the mutation is dominant or
recessive.When F1 individuals that are heterozygousfor a
dominant allele are crossedamong themselves,three-fourths
Gametes
of the resulting F2 progeny will exhibit the mutant trait' In
contrast, when F1 individuals that are heterozygous for a re-
F i r s tf i l i a l cessiveallele are crossedamong themselves,only one-fourth
g e n e r a t i o nF, . : of the resultingF2 progeny will exhibit the mutant tratt'
all offspringhave As noted earlier,the yeast S. cereuisiae,an lmportant ex-
mutant phenotype
perimental organism' can exist in either a haploid or a
iiotoid state.1n these unicellular eukaryotes' crossesbe-
tween haploid cells can determinewhether a mutant allele is
Gametes dominani or recessive.Haploid yeast cells' which carry one
copy of each chromosome' can be of two different mating
S e c o n df i l i a l types k.town as a and ct. Haploid cells of opposite mating
g e n e r a t i o nF, r :
3/aof offspringhave
type can mate to produce a/ct diploids, which carry two
mutant phenotype .*i., of each chromosome. If a new mutation with an ob-
Normal servablephenotypeis isolatedin a haploid strain, the mutant
strain can be mated to a wild-type strain of the opposite mat-
ing type to produce a/o diploids that are heterozygousfor
thl mutant ull.t.. ff these diploids exhibit the mutant trait,
then the mutant allele is dominant, but if the diploids appear
as wild-type, then the mutant allele is recessive'I7hen a/ct
diploids aie placed under starvation conditions, the cells un-
(b) Segregationof recessivemutation dergo -eiosfu, giving rise to a tetrad of four haploid spores'
t*Jof type a and two of type ct. Sporulation of a heterozy-
Mutanl
go.rsdipiiid cell yields two sporescalryils the mutant allele
ina t*o carrying the wild-type allele (Figure 5-5)' Under

Wild type Mutant

(type a) (type o)
Gametes

F i r s tf i l i a l
g e n e r a t i o nF, r .
no offspringhave
mutant phenotype
Diploid cells:
w i l l n o t e x h i b i tm u t a n t
phenotypeif mutation
Gametes is recessive

S e c o n df i l i a l
g e n e r a t i o nF, r :
1/+of offspringhave
mutant phenotype
Haploid spores in tetrad:
2 will be mutant
2 will be wild tYPe
A FIGURE 5-4 Segregationpatternsof dominantand
FIGURE 5-5 Segregation of allelesin yeast'Haploid
recessivemutationsin crossesbetweentrue-breedingstrains
Saccharomyces cellsof oppositematingtype(i e , oneof matingtype
o f d i p l o i do r g a n i s m sA. l l t h e o f f s p r i nign t h e f i r s t( F r ) an a/ctdiploidlf
generation areheterozygous. lf the mutantalleleisdominant, the F1 ctandoneof matingtypea) canmateto produce
p a r t( a ) l f t he onehaploid a
carries dominant wild-type alleleand theothercarries
o f f s p r i nwg ill e x h i b itth e m u t a n p
t h e n o t y p a
e s
, i n
a recessivemutant alleleof the same gene, the resulting
m u t a na t l l e l ei s r e c e s s i vt h
ee , F 1o f f s p r i nw g i l le x h i b itth e
heterozygous diploidwillexpress the dominant trait Undercertaln
w i l d - t y ppeh e n o t y p e a ,si n p a r t( b ) C r o s s i nogf t h e F 1
conditions,a diploidcellwillforma tetradof four haploidspores'
heterozygotes amongthemselves alsoproduces different
Twoof the sporesin the tetrad will the
express trait and
recessive
f
s e g r e g a t i or ant i o so r d o m i n a nat n d r e c e s s i m
v e u t a nat lleles
two will expressthe dominant tratt
intheF,qeneration

A N D S T U D YG E N E S 169
G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F Y
 appropriate conditions, yeastsporeswill germinate,produc_ (a)
rng vegetativehaploid srrains of both mating at 23 .C for 5 n
p Incubate
rypes.
-+ E
C o n d i t i o n aM
l u t a t i o n sC a n B e U s e dt o S t u d y Add mutagen;
distributeinto
E s s e n t i aGl e n e si n y e a s t s m a l l e ra l i q u o t s
00001000
[000t[00
The proceduresusedto identify and isolate mutants, referred VVVV rI V V V
to as genettc screens,depend on whether the experimental
organism is haploid or diploid and, if the latter, whether the
mutation is recessiveor dominant. Genes that encode Colonies
proteins essentialfor life are among the most interestingand
important ones ro study. Since phenotypic expressionof
mutationsin essentialgenesleadsto death of the individual,
Incubate
ingeniousgeneticscreensare neededto isolateand maintain at23'C
organismswith a lethal mutation.
ff5fi:JJ:ru Temperature-sensitive
for growth; growth at 23.,
no growth at 36.

(b)
Wild type

cdc28 mutants

l
t

tagenizedyeastcellsthat could grow normally at23 "C but that

o
could not form a colony when placedat 36;C (Figure5_6a).
Once temperature-sensitive murants were isolated,fu.th.,
analysisrevealedthat some indeed were defectivein cell divi- cdcZmutants

Cg
6
l{
t..
v
,G
o'
t-
ao

170 o c H A p r E5R | MoLEcuLA

GRE N E TrtEcc H N t e u E S
< EXPERIMENTAL FIGURE 5-6 Haploidyeastscarrying ComplementationTestsDetermineWhether
temperature-sensitive lethalmutationsare maintainedat Mutations Are in the
Different Recessive
permissive temperatureand analyzedat nonpermissive
cell-
S a m eG e n e
temperature.(a)Genetic screen for temperature-sensttive
divisioncycle (cdc) mutants in yeast Yeasts that grow and form In the genetic approach to studying a particular cellular
colonresat 23 'C (permissive temperature) but not at 36 "C process, researchersoften isolate multiple recessivemuta-
(nonpermissive temperature) maycarrya lethalmutation thatblocks iion, that produce the same phenotype. A common test for
celldivision.(b)Assay of temperature-sensitive colonies for blocksat
specificstages in thecellcycle.Shownherearemicrographs of wild-
type yeastand two differenttemperature-sensitive mutants after
incubationat the nonpermissive temperature for 6 h Wild-type cells,
whichcontinue to grow,canbe seenwith alldifferent sizes of buds,
different
reflecting stages of the cellcycleIn contrast, cellsin the
ganism heterozygousfor both mutations (i'e', carrying one a
lowertwo micrographs exhibit a block at a specificstage in thecell
Ill.le one b allele)will exhibit the mutant phenotype be-
cycleThecdc28mutantsarrestat a pointbeforeemergence of a "nd
cellsThecdcZmutants, causeneither allele provides a functional copy of the gene' In
newbudandtherefore appearasunbudded
justbeforeseparation of the mothercellandbud contrast, if mutation a and b arein separategenes,then het-
whicharrest
(emerging daughter cell),appearascellswith largebudslPart (a)see erozygotescarrying a single copy of each mutant allele will
L H H a r t w e l l ,1 9 6 -,l J B a c t e r i o9l 3 j 6 6 2 ; p a r t ( b ) f r o m L M H e r e f o r d a n d not;;hibit the mutant phenotype becausea wild-type allele
L H Hartwell.1914,J Mol Biol 84.4451

theseyeastmutants did not simply fail to grow, as they might

if they carried a mutation affecting generalcellular metabo-
lism. Rather,at the nonpermissivetemperature,the mutants of
interest grew normally for part of the cell cycle but then ar-
restedat a particular stageof the cell cycle,so that many cells
at this stagewere seen(Figure 5-6b). Most cdc mutations in
yeastare recessive; that is, when haploid cdc strainsare mated
to wild-type haploids,the resulting heterozygousdiploids are
neither temperature-sensitive nor defectivein cell division.

R e c e s s i vLee t h a lM u t a t i o n si n D i p l o i d sC a n
B e l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e d
in Heterozygotes
In diploid organisms,phenotypesresulting from recessive
lar characterization of the CDC genes and their encoded
mutations can be observedonly in individuals homozygous
proteins, as describedin detail in Chapter 20, has provided a
for the mutant alleles.Sincemutagenesisin a diploid organ-
?r"-.*ork for understanding how cell division is regulated
ism typically changesonly one allele of a gene' yielding het-
in organismsranging from yeast to humans'
erozygousmutants, geneticscreensmust include inbreeding
stepsto generateprogeny that are homozygous for the mu-
tanl alleles. The geneticist H. Muller developed a general Double Mutants Are Useful in Assessing
and efficient procedure for carrying out such inbreeding ex-
t h e O r d e ri n W h i c h P r o t e i n sF u n c t i o n
periments in the fruit fly Drosophila. Recessivelethal muta-
iions in Drosophila and other diploid organisms can be Based on careful analysis of mutant phenotypes associated
maintained in heterozygousindividuals and their phenotypic with a particular cellular process' researchersoften can de-
consequencesanalyzedin homozygotes. duce the order in which a set of genesand their protein prod-
The Muller approach was used to great effect by C' ucts function. Two general types of processesare amenable
Niisslein-Volhard and E. \Tieschaus, who systematically to such analysis: (a) biosynthetic pathways in which a pre-
screenedfor recessivelethal mutations affectingembryogenesis cursor material is convertedvia one or more intermediatesto
in Drosophila. Dead homozygousembryos carrying recessive a final product and (b) signaling pathways that regulate
lethal mutations identifiedby this screenwere examinedunder other processesand involve the flow of information rather
the microscope for specific morphological defects in the than chemical intermediates.
embryos.Current understandingof the molecularmechanisms
underlyingdevelopmentof multicellularorganismsis based,in
large part, on the detailedpicture of embryonic development
rWe
revealedby characterizationof theseDrosoprila mutants.
will discusssome of the fundamental discoveriesbased on
thesegeneticstudies\n Chapter 22.

A N D S T U D YG E N E S
TO IDENTIFY
OF MUTATIONS
ANALYSIS
171
GENETIC
 > EXPERIMENTA FLI G U R E5 - 7
Mutant Mutant Mutant Mutant
Complementation analysisdetermines (type a) (type cr) (typea)
M a t e h a p l o i d so f (typeo)
whether recessivemutations are in the
oppositemating types (cdcX) (cdcY\
same or different genes. Complementation and carryingdifferent \cdcx) (cdcz)
testsin yeastare performedby matinghaploida recessivetemperature-
\_./ \_-/ \_-./ \_-,i

and o cellscarryingdifferentrecessrve mutations sensitivecdc mutations \/ \/

to producediploidcells In the analysis of cdc \r' \r'
mutations,pairsof differenthaploid cdcXlcdcY cdcXlcdcZ
(type a/o) (type a/o)
temperature-sensitive cdc strainswere
systematically matedand the resultingdiploids P l a t ea n d i n c u b a t e
at permissive
testedfor growth at the permissive and temperature
nonpermrsstve temperaturesIn this hypothetical
example,the cdcX and cdcy mutants
c o m p l e m e net a c ho t h e ra n d t h u s h a v e
mutationstn differentgenes,whereasthe cdcX T e s tr e s u l t i n gd i p l o i d s
for a temperature-
and cdcZmutantshavemutationsin the
sensitivecdc phenotype
s a m eg e n e

2C aa
Growth 36'C No growth

PHENOTYPE: 6A
Wild type
Y9
M utant
INTERPRETATION: Growth indicatesthat Absenceof growth
mutations cdcX and cdcY indicatesthat mutations
are in differentgenes c d c Xa n d c d c Z a r e i n t h e
s a m eg e n e
-Ir-V----l-
...t-^_i-
a-
"'-l___LF

Respectivewild-typealleles B o t h a l l e l e sn o n f u n c t i o n a l
p r o v i d en o r m a lf u n c t i o n

next. In E. coLi,the genesencodingtheseenzymeslie adja_
cent to one another in the genome, constituting the trp
operon (seeFigure 4-73a). The order of action of the differ_
ent genesfor theseenzymes,hencethe order of the biochemi_
cal reactions in the pathway, initially was deducedfrom the
types of intermediatecompoundsthat accumulatedin each
In Chapter 14 we discussthe classicuse of the double_
mutant.strategyto help elucidatethe secretorypathway. In
this pathway proteins to be secreredfrom the cell rnou. i.o-
their site of synthesison the rough endoplasmicreticulum
(ER) to the Golgi complex. then to ,..r.toiy vesicles,
and fi_
n a l l y t o r h ec e l l s u r f a c e .
Ordering of Signaling pathways As we learn in later
chapters,expressionof many eukaryoticgenesis regulated
by signaling pathways that are initiateJ by extracellular
hormones, growth factors, or other signals. Such signaling
pathways may include numerouscomponents,and double-
mutant analysisofren can provide insight into the functions
and interactionsof thesecompon.nrr.th. only prerequisite
for obtaining useful information from this type of analysisis
that the two mutations must have opposite effects on the
output of the sameregulatedpathway. Most commonl5 one
mutatio_nrepressesexpressionof a particular reporrer gene
even when the signal is present, while another mutation
resultsin reporter geneexpressionevenwhen the signalis ab-
sent(i.e.,constirutiveexpression).As illustratedin Figure5_gb,
two simple regulatory mechanismsare consistentwith such
Note that this technique differs from complementation
analysisjust describedin that when testingtwo recessive
mu_
tations, the double mutant created is homozygolzsfor both
mutations.Furthermore,dominant mutantscan be subiected
t o d o u b l e - m u t a natn a l v s i s .

172 . c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

(a)Analysisof a biosyntheticpathway
1.
A m u t a t i o ni n A a c c u m u l a t e isn t e r m e d i a t e
A m u t a t i o ni n B a c c u m u l a t e isn t e r m e d i a t 2
e.

PHENOTYPE OF
A d o u b l em u t a t i o ni n A a n d B a c c u m u l a t e s
D O U B L EM U T A N T
: intermediate 1.
'
INTERPRETATION: The reaction catalYzedby A precedes the
I reaction catalYzedbY B.

Eo'Et'E
(b)Analysisof a signalingpathway
mutant allelewould have a mutant phenotype(Figure5-9a)'
A mutation in A gives repressedreporterexpresslon. The observation of genetic suppressionin yeast strains
A mutation in B gives constitutivereporterexpression. carrying a mutant actin allele (act1-1) and a secondmuta-
PHENOTYPE OF tion (sic5) in another gene provided early evidence for a
: D O U B L EM U T A N T : A d o u b l em u t a t i o ni n A a n d B g i v e s
I repressedreporterexPression.
, INTERPRETATION: A positively regulates repofter expression (a)Suppression
. and is negativelYregulated bY B'
GenotyPe AB aB Ab ab
Phenotype Wildtype Mutant Mutant SuPPressed

rNrERPRErAlo-
/ed\
P H E N O T Y PO
EF t I i/t l
D O U B L EM U T A N T A d o u b l em u t a t i o ni n A a n d B g i v e s \UU,/
constitutivereporterexpression.
INr ERPRErArIoN: pression
7:; i:';:;':, t""1 i7 f .x
i' fJ3i,,1,133 (b) Synthetic lethality 1
A--€--l B Genotype AB aB Ab ab
Phenotype Wild type Partial Partial Severe
defect defect defect

FIGURE 5-8 Analysisof doublemutantsoften canorderthe rNrERPRErArto- ,fii\

stepsin biosyntheticor signalingpathways'Whenmutations in ( | 11I )
two different genesaffectthe samecellular process but have \U U,/
i
distinctly different phenotypes, the phenotype of the doublemutant
canoftenreveal the order in which the two genes mustfunction(a)
Inthe caseof mutations thataffectthe samebiosynthetic pathway, a (cl Synthetic lethality 2
t i l la c c u m u l attheei n t e r m e d i ai m
d o u b l em u t a nw t em e d t a t e l y
ab
AB aB Ab
preceding the stepcatalyzed by the proteinthatactsearlier in the Genotype
wild-type organism(b)Double-mutant analysis of a signaling Phenotype Wildtype Wild type Wild tYPe
pathwayispossible if two mutations haveopposite effects on
expression of a reporter gene In thiscase,the observed phenotype
of the doublemutantprovides information aboutthe orderin which
the proteins actandwhethertheyarepositive or negative regulators

G e n e t i cS u p p r e s s i o an n d S y n t h e t i cL e t h a l i t y or
A FIGURE 5-9 Mutationsthat resultin geneticsuppression
C a n R e v e a Il n t e r a c t i n go r R e d u n d a n P t roteins
syntheticlethalityreveal interacting or redundant proteins'
Two other types of genetic analysis can provide additional (a)Observationthatdoublemutants with two defective proteins
clues about how proteinsthat function in the samecellular (A andB)havea wild-type phenotype butthatsinglemutantsgive
processmay interactwith one anotherin the living cell. Both a mutantphenotype indicatesthatthefunctionof eachprotein
depends on interactionwith the other.(b)Observation thatdouble
of thesemethods,which are applicablein many experimen-
mutantshave a more severephenotypic than
defect single mutants
tal organisms,involve the use of double mutants in which (e g , subunits
alsoisevidence thattwo proteins of a heterodimer)
the phenotypic effects of one mutation are changed by the (c)Observation thata double
mustinteractto functionnormally.
presenceof a secondmutation.
mUtantisnonviab|ebutthatthecorrespondingsing|emutants
phenotype indicatesthattwo proteins f unctionin
Suppressor Mutations The first type of analysisis basedon thewild-type
redundant pathways to produce an essential product
geneticsuppression.To understandthis phenomenon,suppose

A N D S T U D YG E N E S . 173
O F M U T A T I O N sT O I D E N T I F Y
ANALYSIS
GENETIC
 direct interaction in vivo between the proteins encoded by
the two genes.Later biochemical studies showed that these
two proteins-Act1 and Sac6-do indeed inreract in the
construction of functional actin structureswithin the cell.
Synthetic Lethal Mutations Another phenomenon,called
synthetic lethality, produces a phenotypic effect opposrte to
that of suppression.In this case,the deleteriouseffectof one
mutation is greatly exacerbated(rather than suppressed)by
a secondmutation in a relatedgene.One situation in which
such synthetic lethal mutations can occur is illustrated in
Figure 5-9b. In this example,a heterodimericprotein is par-
tiall5 but not completely,inactivatedby mutations in eiiher
one of the nonidentical subunits.However, in double mu-
tants carrying specificmurationsin the genesencodingboth
subunits,little interaction betweensubunits occurs,iesult-
ing in severephenotypic effects. Synthetic lethal mutations
product cannor be synthesizedand the double mutanrs will
be nonviable.
G e n e sC a nB e l d e n t i f i e db y T h e i r M a p p o s i t i o n
on the Chromosome
The preceding discussionof genetic analysisillustrates how
a geneticistcan gain insight into gene function by observing
the phenotypic effectsproduced by joining together different
combinations of mutant allelesin the samecell or organism.
For example, combinations of different alleles of the same
gene in a diploid can be used to determine whether a muta-
tion is dominant or recessiveor whether two different reces-
sive mutations are in the same gene.Furthermore, combina-
tions of mutations in different genes can be used to
determine the order of gene function in a pathway or to
identify functional relationships between genessuch as sup-
pressionand synthetic enhancement.Generally speaking,all
these methods can be viewed as analytical tests baseJ on
gene functioz. \7e will now consider a fundamentally differ-
ent type of genetic analysis based on gene positioz. Studies
designedto determine the position of a gene on a chromo-
some, often referred to as genetic mapping studies, can be
usedto identify the geneaffected by a particular mutation or
to determine whether two mutations are in the samegene.
In many organisms generic mapping studies rely on
exchangesof genetic information that occur during meiosis.
As discussedin Chapter 4 and as shown in Figure 5-10a,

(a) (b)Consider
-'-lf m2
Replicated two linkedgenesA and Bwith recessive
allelesa and b.
chromosomes
(4nl Crossof two mutantsto construct
a doublyheterozygous
strain:
and
AlAblb x alaBlB
over

MetaphaseI I
A

i A n a p h a s eI Crossof double heterozygoteto test strain:

A b .. a b
aBab
I
v
*\ / "; A n a p h a s el l
AbaBABab
abuA"brb
I I v I \___________-Y_

Parentaltypes
_________YJ

Recombinanttypes
m1
GeneticdistancebetweenA and B can be determinedfrom
1n 1n tn tn frequencyof parentaland recombinantgametes:

Parental Recombinantgametes parental gametes

gamete gamete
A FIGURE5-10 Recombinationduring meiosis can be used to
map the position of genes. (a)Shown is an individualthat carries
two mutations,designatedm/ (yellow)andm2 (green),that are on
the maternaland paternalversionsof the samechromosomelf
crosslngoveroccursat an intervalbetweenml andm2 beforethe
first meloticdivision,then two recombinantgametesare produced;
one carriesboth m / and m2, whereasthe other carriesneither
mutation The longerthe distancebetweentwo mutationson a
Geneticdistancein cM = 100, Itotbi.nant
total gametes
chromatid,the more likelythey are to be separatedby recombination
and the greaterthe proportionof recombinantgametesproduced
(b) In a typicalmappingexperiment,a strainthat is heterozygous for
two differentgenesis constructedThe frequencyof parentalor
recombinantgametesproducedby thls straincan be determjned
from the phenotypesof the progenyin a testcross to a homozygous
recessive strain The geneticmap distancein centimorgans(cM) is
givenas the percentof the gametesthat are recombinant

174 . cHAprER
s M o l E c u L A RG E N E T tTcE C H N t o u E S
I
genetic recombination takes place before the first meiotic
cell division in germ cells,when the replicatedchromosomes
of each homologous pair align with each other. At this time,
homologous DNA sequenceson maternally and paternally
derived chromatids can exchangewith each other, a process
'We
known as crossing over. now know that the resulting
crossovers between homologous chromosomes provide
structural links that are important for the proper segregation
of pairs of homologous chromatids to opposite poles during
the first meiotic cell division (for discussionseeChapter 20).
Considertwo different mutations,one inheritedfrom each
parent, that arc located close to one another on the same
chromosome.Two different typesof gametescan be produced
according to whether a crossoveroccurs betweenthe muta-
tions during meiosis. If no crossoveroccurs between them,
gametesknown as parental types,which contain either one or
the other mutation, will be produced. In contrast' if a
crossoveroccurs betweenthe two mutations' gametesknown
as recombinant types will be produced. In this example
recombinant chromosomeswould contain either both muta-
tions, or neither of them. The sites of recombination occur
more or lessat random along the length of chromosomes;thus
the closertogethertwo genesare, the lesslikely that recombi-
nation will occur between them during meiosis. In other
words, the lessfrequently recombination occursbetweentwo
genes on the same chromosome, the more tightly they are
Iinked and tbe closer together they are. Two genesthat are suf-
ficiently close together such that there are significantly fewer
recombinant gametes produced than parental gametes are
consideredto be geneticallylinked.
The technique of recombinational mapping was devised
in 1911 by A. Sturtevantwhile he was an undergraduate
working in the laboratory of T. H. Morgan at Columbia
University. Originally used in studies on Drosophila, this
technique is still used today to assessthe distance between
two genetic loci on the same chromosome in many experl-
mental organisms. A typical experiment designedto deter-
mine the map distancebetweentwo geneticpositions would
involve two steps. In the first step' a strain is constructed
that carriesa different mutation at eachposition, or locus. In
the secondstep, the progeny of this strain are assessed to de-
termine the relative frequency of inheritance of parental or
recombinant types.A typical way to determinethe frequency
of recombination between two genesis to cross one diploid
parent heterozygousat each of the genetic loci to another
parent homozygousfor each gene.For such a cross,the pro-
portion of recombinant progeny is readily determined be-
causerecombinant phenotypeswill differ from the parental
phenotypes.By convention, one genetic map unit is defined
as the distance between two positions along a chromosome
that results in one recombinant individual in 100 total prog-
eny. The distance corresponding to this 1 percent recombi-
nation frequency is called a centimorgan (cM) in honor of
Sturtevant'smentor,Morgan (seeFigure 5-10b).
A complete discussion of the methods of genetic map-
ping experiments is beyond the scope of this introductory
discussion;however,two featuresof measuringdistancesby
recombination mapping need particular emphasis.First, the
G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F YA N D S T U D Y
frequency of genetic exchange between two loci is strictly
proportional to the physical distancein basepairs separating
ih.- o.tly for loci that are relatively close together (sa5 less
than about 10 cM). For loci that are farther apart than this'
a distance measured by the frequency of genetic exchange
tends to underestimatethe physical distance becauseof the
possibility of two or more crossoversoccurring within an in-
ie.ual. In the limiting casein which the number of recombi-
nant types will equal the number of parental types, the two
loci under considerationcould be far apart on the same
chromosome or they could be on different chromosomes,
and in such casesthe loci are consideredto be unlinked'
A secondimportant concept neededfor interpretation of
recombination frequency (i.e.' a genetic distance of 1 cM)
representsa physical distanceof about 2.8 kilobasesin yeast
compared *lth distance of about 400 kilobases in
"
Drosophila and about 780 kilobasesin humans'
One of the chief uses of genetic mapping studies is to
human diseasescan be identified using such methods' A sec-
ond general use of mapping experiments is to determine
*hethe. two different mutations are in the samegene' If two
mutations are in the same gene, they will exhibit tight
linkage in mapping experiments,but if they are in different
g..r.{ they will usually be unlinked or exhibit weak linkage'
Genetic Analysis of Mutations to ldentify
and Study Genes
r Diploid organisms carry two copies (alleles)of each
gene,whereashaploid organismscarry only one copy'
r Recessivemutations lead to a loss of function, which is
masked if a normal allele of the gene is present' For the
mutant phenotype to occur' both alleles must carry the
mutatl0n.
r Dominant mutations lead to a mutant phenotype in the
presenceof a normal allele of the gene.The phenotypesas-
sociatedwith dominant mutations often representa gain of
function but in the caseof some genesresult from a loss of
function.
r In meiosis,a diploid cell undergoesone DNA replication
and two cell divisions, yielding four haploid cells in which
maternal and paternal alleles are randomly assorted (see
Figure5-3).
GENES 175
 r Dominant and recessivemutations exhibit characteristic
segregatlonpatternsin geneticcrosses(seeFigure5_4).
r I n h a p l o i d y e a s t .t e m p e r a t u r e - s e n s i t i vmeu t a t i o n s a r e
particularly useful for identifying and studying genesessen-
tial to survival.
r The number of functionally related genesinvolved in a
process can be defined by complemenration analysis (see
Figure-5-7).
r The order in which genesfunction in a signaling path-
way can be deducedfrom the phenotype of double mutants
defectivein two stepsin the affectedprocess.
r Functionally significant interactions between Drorelns
can be deduced from the phenotypic effects o? allele-
specificsuppressormutations or syntheticlethal mutations.
r Genetic mapping experimentsmake use of crossingover
between homologous chromosomes during meiosis to
measurethe genetic distance between two different muta_
tions on the samechromosome.
f[ DNACloningand Characterization
is simply any DNA molecule composed of sequencesde_
rived from different sources.
The key to cloning a DNA fragment of interest is to link
it to a vector DNA molecule that can replicate within a host
cell. After a singlerecombinant DNA molecule,composedof
a vector plus an insertedDNA fragmenr, is introdu.id irrto
"
host cell, the inserted DNA is replicated along with the vec_
tor, generating a large number of identical DNA molecules.
The basic schemecan be summarizedas follows:
Vector + DNA fragment
J
RecombinantDNA
J J'
Replication of recombinant DNA within host cells
J
Isolation, sequencing,and manipulation
of purified DNA fragment
Although investigatorshave devisednumerous experimental
variations, this flow diagram indicatesthe essentialsteDsin
DNA cloning. In this section,we first describemethods for
isolating a specificsequenceof DNA from a seaof other DNA
sequences. This processoften involvescutting the genomernto
fragmentsand then placing each fragment ii a vector so that
the entire collection can be propagatedas recombinantmole_
cules in separatehost cells. While many different types of
'176 .
c H A p r E5R I M o L E c u L AGRE N E Tr tEcc H N t e u E s
vectorsexist, our discussionwill mainly focus on plasmid vec-
tors in E. coli host cells,which are commonly used. Various
techniquescan then be employed to identify the sequenceo{
interest from this collection of DNA fragments,known as a
DNA library. Once a specificDNA fragment is isolated,it is
typically characterizedby determining the exact sequenceof
nucleotidesin the molecule. We end with a discussionof the
polymerasechain reaction (PCR).This powerful and versatile
techniquecan be usedin many ways to generatelarge quanti-
ties of a specificsequenceand otherwisemanipulate ONR m
the laboratory.The various usesof cloned DNA frasmentsare
discussed in subsequent sections.
RestrictionEnzymesand DNA Ligases
A l l o w I n s e r t i o no f D N A F r a g m e n t si n t o
CloningVectors
A major objective of DNA cloning is to obtain discrete,
small regions of an organism'sDNA that constitute specific
genes.In addition, only relatively small DNA moleculescan
be cloned in any of the availablevectors.For thesereasons,
the very long DNA molecules that compose an organism's
genome must be cleavedinto fragments that can be inserted
into the vector DNA. Two types of enzymes-restriction en-
zymes and DNA ligases-facilitate production of such re-
combinantDNA molecules.
Cutting DNA Molecules into Small Fragments Restric_
tion enzymesare endonucleasesproduced by bacteria that typi-
cally recognizespecific 4- to 8-bp sequences,calledrestrictin
sites, and then cleaveboth DNA strandsat this site. Restriction
sites commonly are short palindromic sequences;that is, the
restriction-sitesequenceis the sameon each DNA strand when
read in the 5'-+3' direction (Figure5-11).
For each restriction enzyme) bacteria also produce a
modification enzyme, which protects a bacterium's own
DNA from cleavageby modifying it at or near eachpotenrial
cleavagesite. The modification enzymeadds a methyl group
to one or two bases,usually within the restriction site. When
EcoRl
+
r(- | |
ti
Cleavage EcoRl
I
+
Stickyends
-------r
s',-
.), G c ----r 3'
. TTAA \l 5
FIGURE 5-11 Cleavage of DNAby the restrictionenzyme
EcoRf.Thisrestriction
enzymefromE colimakesstaqqered cutsat
the specific
6-bppalindromicsequenceshown,yielding fragments
withsingle-stranded,
complementary"sticky,,
ends.Manyother
restriction
enzymes alsoproducefragmentswith stickyends
a methyl group is presentthere, the restriction endonuclease
is prevented from cutting the DNA. Together with the
restriction endonuclease,the methylating enzyme forms a
restriction-modification system that protects the host DNA
while it destroysincomingforeign DNA (e.g.,bacteriophage
DNA or DNA taken up during transformation) by cleaving
it at all the restriction sitesin the DNA.
Many restriction enzymesmake staggeredcuts in the two
DNA strands at their recognition site, generatingfragments
that have a single-stranded"tail" at both ends,sticky ends
(seeFigure 5-11). The tails on the fragmentsgeneratedat a
given restriction site are complementaryto those on all other
fragments generated by the same restriction enzyme. At
room temperature, these single-strandedregions can tran-
siently base-pairwith thoseon other DNA fragmentsgener-
ated with the same restriction enzyme. A few restriction
enzymes,such as Alul and SmaI, cleaveboth DNA strandsat
the same point within the restriction site, generating frag-
mentswith "blunt" (flush)endsin which all the nucleotides
at the fragment ends are base-pairedto nucleotides in the
complementarystrand.
The DNA isolated from an individual organism has a
specificsequence,which purely by chancewill contain a spe-
cific set of restriction sites' Thus a given restriction enzyme
will cut the DNA from a particular source into a repro-
ducible set of fragmentscalled restriction fragments.The fre-
quency with which a restriction enzymecuts DNA, and thus
the average size of the resulting restriction fragments, de-
pends largely on the length of the recognition site. For ex-
ample, a restriction enzyme that recognizesa 4-bp site will
cleaveDNA an averageof once every4", or 256, basepairs,
whereas an enzyme that recognizesan 8-bp sequencewill
cleaveDNA an averageof onceevery4E basepairs (=65 kb)'
Restriction enzymes have been purified from several hun-
dred different speciesof bacteria, allowing DNA molecules
to be cut at a large number of different sequencescorrespon-
ding to the recognitionsitesof theseenzymes(seeTable5-1)'
Inserting DNA Fragments into Vectors DNA fragments
with either sticky endsor blunt endscan be insertedinto vector
DNA with the aid of DNA ligases.During normal DNA repli-
cation, DNA ligase catalyzestheend-to-endioining (ligation) of

ENZYME MICRO()RGANISM
SOURCE SITE-
RECOGNITION PBODUCED
ENOS

J
BamHl B a cillu s amy lo liq u efaciens -G-G-A-T-C.C. Sticky
-C-C-T-A-G-G.
1

J
Sau3A aureus
Staphylococcus -G-A-T-C- Sticky
.C-T-A-G-
t
J
EcoRl Escherichia coli -G-A-A-T-T-C- Sticky
-C.T-T-A-A-G
t

J
Hindlll Haemophilus influenzae -A,A-G.C-T-T. Sticky
-T-T-C-G-A-4.
T
J
Serratia mdrcescens -C-C-C-G-G-G- Blunt
Smal
-G-G-G-C-C-C-
1

Notl No cardia otiti di s-cauiarum -G-C_G-G.C-C-G-C- Sticky

-C-G-C-C-G-G-C-G-
t

Many of these recognition sequencesare included in a common polylinker sequence(seeFigure 5-13).

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N 177
 short fragments of DNA called Okazaki fragments. For pur-
posesof DNA cloning, purified DNA ligaseis usedto covalently
join the ends of a restriction fragment and vector DNA that
have complementaryends (Figure5-12). The vector DNA and
restriction fragment are covalently ligated rogetherthrough the
standard3'-+5' phosphodiesterbonds of DNA. In addition to
Iigating complementary sticky ends, the DNA ligasefrom bac-
teriophage T4 can ligate any two blunt DNA ends. However,
blunt-end ligation is inherently inefficient and requires a higher
concentration of both DNA and DNA ligasethan doesligation
of sticky ends. Polylinker Plasmid
cloning vector

E. coliPlasmidVectorsAre Suitablefor Cloning FIGURE 5-13 Basiccomponentsof a plasmidcloningvector

that can replicatewithin an E. coli cell. plasmid vectorscontaina
lsolatedDNA Fragments selectable genesuchasamp',whichencodes the enzyme
Plasmidsare circular, double-strandedDNA (dsDNA) mole- P-lactamase andconfers resistanceto ampicillin.
Exogenous DNAcan
cules that are separatefrom a cell's chromosomal DNA. be inserted intothe bracketed regionwithoutdisturbing theabilityof
These extrachromosomal DNAs, which occur naturally in the plasmidto replicate or expresstheamp,gene plasmid vectors
bacteria and in lower eukaryotic cells (e.g.,yeast),exist in a alsocontaina replication origin(ORl) sequence whereDNA
replication isinitiatedby host-cell
enzymes. Inclusionof a synthetic
polylinker containing the recognition sequences for severaldifferent
Genomic DNA fragments
restriction enzymes increases the versatility
of a plasmidvectorThe
vectorisdesigned sothateachsitein the polvlinker is unioueon
(a)
t h ep l a s m i d .
P-AATT 3'
VectorDNA oH__r-----::-.---r
5,
(a') (b) parasitic or symbiotic relationship with their host cell. Like
s', oH + P-C G- 3'
3'r---------r-TTAA-p the host-cell chromosomal DNA, plasmid DNA is dupli-
HO-:5'
cated before every cell division. During cell division, copies
(c)
P-A G CT 3' of the plasmid DNA segregateto each daughtercell, assuring
HOj-:-:r 5, continued propagation of the plasmid through successive
generationsof the host cell.
I The plasmidsmost commonly usedin recombinant DNA
I Complementary
I endsbase-pair technology are those that replicate in E. col/. Investigators
I
v have engineeredtheseplasmids to optimize their use as vec-
OHP tors in DNA cloning. For instance, removal of unneeded
portions from naturally occurring E. coli plasmids yields
lltr plasmid vectors, (=1.2-3 kb in circumferential length, that
3'r------]-TTAA
contain three regions essentialfor DNA cloning: a replica-
/\ tion origin; a marker that permits selection,usually a drug-
PHO
resistancegene;and a region in which exogenousDNA frag-
2 ATP ments can be inserted (Figure 5-13). Host-cell enzymes
T4 DNA ligase replicate a plasmid beginning at the replication origin (ORI),
a specific DNA sequenceof 50-100 basepairs. Once DNA
2AMP+2PPi replication is initiated at the ORI, it continues around the
circular plasmid regardlessof its nucleotide sequence.Thus
(a') (a) any DNA sequenceinsertedinto such a plasmid is replicated
5'I-AATT 3' along with the rest of the plasmid DNA.
3',---------F++ii s, Figure 5-14 outlines the general procedure for cloning a
FIGURE 5-12 Ligationof restrictionfragmentswith DNA fragment using E. coli plasmid vectors. \lhen E. coli
complementary stickyends.In thisexample, cells are mixed with recombinant vector DNA under certain
vectorDNAcutwith
EcoRl ismixedwith a sample containingrestriction conditions, a small fraction of the cells will take up the
fragments
produced by cleavinggenomicDNAwith several differentrestriction plasmid DNA, a process known as transformation. Typi-
enzymes Theshortbasesequences composing the stickyendsof cally, 1, cell in about 10,000 incorporates a single plasmld
eachfragment typeareshownThestickyendon the cutvectorDNA DNA molecule and thus becomes transformed. After
(a')base-pairs
onlywith thecomplementary stickyendson theEcoR/ plasmid vectors are incubated with E. coli, those cells that
fragment(a)in the genomic sample.Theadjacent 3, hydroxyl
and5, take up the plasmid can be easily selectedfrom the much
phosphate groups(red)on the base-pairedfragments thenare larger number of cells. For instance,if the plasmid carries a
joined(ligated)
covalently byT4 DNAligase. gene that confers resistance to the antibiotic ampicillin,

178 . c H A p r E R5 MoLEcuLAR
G E N E T trcE c H N t o u E s
|
> EXPERIMENTAL FIGURE 5-14 DNAcloningin a plasmid
vector permitsamplificationof a DNAfragment.A fragmentof
intoa plasmid
DNAto be clonedisfirstinserted vectorcontaining an
DNA fragment
gene(amp'),suchasthatshownin Figure
ampicillin-resistance 5-13 to be cloned
Onlythefew cellstransformed of a plasmid
by incorporation molecule
mediumIntransformed
on ampicillin-containing
willsurvive cells,
the
plasmid intodaughter cells,resulting
in Enzymaticallyinsert
DNAreplicatesandsegregates
DNA into plasmid vector
formationof an ampicillin-resistant
colonv

Recombinant
transformed cells can be selected by growing them in an plasmid
ampicillin-containing medium.
DNA fragments from a few base pairs up to =10 kb
commonly are insertedinto plasmid vectors. When a recom- Mix E. coliwith olasmids
binant plasmid with an inserted DNA fragment transforms in presenceol CaCl2;heat-pulse
an E. coli cell, all the antibiotic-resistantprogeny cells that C u l t u r eo n n u t r i e n ta g a r
arise from the initial transformed cell will contain plasmids p l a t e sc o n t a i n i n ga m p i c i l l i n
E. coli
with the sameinsertedDNA. The insertedDNA is replicated cnromosome
along with the rest of the plasmid DNA and segregatesto
daughter cells as the colony grows. In this way, the initial
fragment of DNA is replicated in the colony of cells into a
large number of identical copies. Since all the cells in a
Transformedcell Cellsthat do not
colony arise from a single transformed parental cell, they
SUTVIVES t a k e u p p l a s m i dd i e
constitute a clone of cells, and the initial fragment of DNA o n a m p i c i l l i np l a t e s
inserted into the parental plasmid is referred to as cloned I P l a s m i replication
d
I
DNA or a DNA clone.
The versatility of an E. coli plasmid vector is increased +
by the addition of a polylinker, a synthetically generated
sequencecontaining one copy of severaldifferent restric-
tion sitesthat are not presentelsewherein the plasmid se-
quence (seeFigure 5-13). \fhen such a vector is treated
with a restriction enzyme that recognizesa restriction site certmurtiotication
in the polylinker, the vector is cut only once within the
polylinker. Subsequentlyany DNA fragment of appropri- I
ate length produced with the same restriction enzymecan
be insertedinto the cut plasmid with DNA ligase.Plasmids
containing a polylinker permit a researcher to use the
same plasmid vector when cloning DNA fragmentsgener-
ated with different restriction enzymes,which simplifies
experimentalprocedures.
For some purposes,such as the isolation and manipula-
tion of large segmentsof the human genome, it is desirable
g
to clone DNA segments as large as several megabases Colony of cells,each containingcopies
of the same recombinantPlasmid
[L megabase(Mb) : 1 million nucleotides).For this purpose
specializedplasmid vectors known as BACs (bacterialartifi-
cial chromosomes)have been developed.One type of BAC
uses a replication origin derived from an endogenousplas-
mid of E. coli known as the F factor. The F factor and
cloning vectors derived from it can be stably maintained at a
GDNALibrariesRepresentthe Sequences
single copy per E. coli cell even when they contain inserted
sequencesof up to about 2 Mb. Production of BAC libraries
of Protein-CodingGenes
requires special methods for the isolation, ligation, and A collection of DNA molecules each cloned into a vector
transformation of large segmentsof DNA becausesegments molecule is known as a DNA library. !7hen genomic DNA
of DNA larger than about 20 kb are highly vulnerable to from a particular organism is the source of the starting
mechanical breakage by even standard manipulations such DNA, the set of clones that collectively represent all the
as prpetung. DNA sequencesin the genome is known as a genomic

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N . 179
 < FIGURE 5-15 A cDNAlibrary containsrepresentative
copiesof cellularmRNAsequences. A mixtureof mRNAsis
pointfor preparing
thestarting recombinant plasmid clones
eachcontaining a cDNA.TransformingE coliwith the
recombinantplasmids generates
a setof cDNAclones
representing
allthecellular
mRNAs. Seethetextfor a step-by-
+F*ry65' steodiscussion.
I
lJ TranscriUe
RNAintocDNA

g I n " . o u " R N Aw i t ha t k a t i
E J A d dp o t y ( d c ) t a i l

Single-stranded 3' G GGGT-___--I T T T T 5'

cDNA
t,,,.,.
EI lil$:fl6"#lli",
5'I Y
3'GGGGI_---__--lTTTT5'

- | Synttresize
complementary
sJ strand

Double-stranded 3',
cDNA r (Jbbba - i ^ F - - r
I fTT5

oRl sites

5',
3'GGGG
I

E C T T A A G E G G G G I _ _ _ _ _ _ - _ _T_ IT T T E C T TAAGE

lf .,*"" withEcoRl

Gf]GGGGI-.-_lT T T TECTTAA
nJ.,,with EcoRl
Stickyend
I

Individual
rJl:ru:HT:;,0?,,
clones

180 . c H A p r E Rs I MoLEcuLAR
G E N E I Cr E c H N t e u E s
library. Such genomic libraries are ideal for representingthe
geneticcontent of relatively simple organismssuch as bacte-
ria or yeast, but presentcertain experimental difficulties for
higher eukaryotes.First, the genesfrom such organismsusu-
ally contain extensiveintron sequencesand therefore can be
too large to be inserted intact into plasmid vectors. As a re-
sult, the sequencesof individual genesare broken apart and
carried in more than one clone. Moreover, the presenceof in-
trons and long intergenic regions in genomic DNA often
makes it difficult to identify the important parts of a gene
that actually encode protein sequences.For example, only
about 1.5 percentof the hunran genomeactually represents
protein-coding genesequences. Thus for many studies,cellu-
lar mRNAs, which lack the noncoding regionspresentin ge-
nomic DNA, are a more useful starting material for generat-
ing a DNA library. In this approach, DNA copies of
mRNAs, called complementaryDNAs (cDNAs), are synthe-
sized and cloned into plasmid vectors. A large collection of
the resulting cDNA clones, representingall the mRNAs
expressedin a cell type, is called a cDNA library.
cDNAsPreparedby ReverseTranscription
o f C e l l u l a rm R N A sC a n B e C l o n e dt o
G e n e r a t ec D N AL i b r a r i e s
The first stepin preparing a cDNA library is to isolatethe to-
tal mRNA from the cell type or tissueof interest.Becauseof
their poly(A) tails, mRNAs are easily separated from the
much more prevalent rRNAs and tRNAs presentin a cell ex-
tract by use of a column to which short strings of thymidy-
late (oligo-dTs) are linked to the matrix. The generalproce-
dure for preparing a cDNA library from a mixture of cellular
mRNAs is outlined in Figure 5-15. The enzymereversetran-
scriptase,which is found in retroviruses,is usedto synthesize
a strand of DNA complementary to each mRNA molecule,
starting from an oligo-dT primer (steps1 and2). The result-
ing cDNA-mRNA hybrid moleculesare converted in several
stepsto double-strandedcDNA moleculescorrespondingto
all the mRNA molecules in the original preparation (steps
3-5). Each double-stranded cDNA contains an oligo-
dC.oligo-dG double-strandedregion at one end and an
oligo-dT.oligo-dA double-strandedregion at the other end.
Methylation of the cDNA protects it from subsequent
restrictionenzymecleavage(step6).
To prepare double-stranded cDNAs for cloning, short
double-strandedDNA moleculescontaining the recognition
site for a particular restriction enzymeare ligatedto both ends
of the cDNAs using DNA ligasefrom bacteriophageT4 (Fig-
ure 5-15, step 7). As noted earlier,this ligasecan join "blunt-
ended" double-strandedDNA moleculeslacking sticky ends.
The resulting moleculesare then treated with the restriction
enzymespecificfor the attachedlinker, generatingcDNA mol-
eculeswith sticky endsat eachend (step8a). In a separatepro-
cedure,plasmid DNA first is treatedwith the samerestriction
enzymeto produce the appropriate sticky ends (step8b).
The vector and the collection of cDNAs, all containing
complementary sticky ends, then are mixed and joined
covalently by DNA ligase(Figure 5-15, step 9). The resulting
DNA moleculesare transformed into E. coli cellsto generate
individual clones;each clone carrying a cDNA derived from
a singlemRNA.
Becausedifferent genesare transcribed at very different
rates,cDNA clonescorrespondingto abundantly transcribed
genes will be representedmany times in a cDNA library,
whereas cDNAs corresponding to infrequently transcribed
geneswill be extremely rare or not presentat all. This prop-
erty is advantageousif an investigatoris interestedin a gene
that is transcribedat a high rate in a particular cell type' In this
case,a cDNA library preparedfrom mRNAs expressedin that
cell type will be enriched in the cDNA of interest, facilitating
isolation of clonescarrying that cDNA from the library. How-
eve! to have a reasonablechance of including clones corre-
sponding to slowly transcribedgenes'mammalian cDNA li-
biaries must contain 1.06-107individual recombinantclones.
D N A L i b r a r i e sC a n B e S c r e e n e db y H y b r i d i z a t i o n
to an OligonucleotidP e robe
Both genomic and cDNA libraries of various organisms
contain hundredsof thousandsto upwards of a million in-
dividual clones in the case of higher eukaryotes. Two gen-
eral approaches are available for screening libraries to
identify clonescarrying a geneor other DNA region of in-
terest: (1) detectionwith oligonucleotideprobes that bind
to the clone of interest and (2) detection basedon expres-
sion of the encoded protein. Here we describe the first
method; an example of the secondmethod is presentedin
the next section.
The basis for screeningwith oligonucleotideprobes is
hybridization, the ability of complementary single-
strandedDNA or RNA moleculesto associate(hybridize)
specificallywith each other via basepairing. As discussed
in Chapter 4, double-stranded(duplex) DNA can be dena-
tured (melted) into single strands by heating in a dilute
salt solution. If the temperature then is lowered and the
ion concentration raised, complementary single strands
will reassociate(hybridize) into duplexes.In a mixture of
nucleic acids, only complementary single strands (or
strands containing complementary regions) will reassoci-
ate; moreover,the extent of their reassociationis virtually
unaffectedby the presenceof noncomplementarystrands.
As we will seelater in this chapter,the ability to identify a
particular DNA or RNA sequencewithin a highly com-
plex mixture of moleculesthrough nucleic acid hybridiza-
tion is the basis for many techniquesemployed to study
gene expresslon.
The stepsinvolved in screeningan E. coli plasmid cDNA
library are depicted in Figure 5-16. First' the DNA to be
screenedmust be attachedto a solid support. A replica of the
petri dish containing a large number of individual E. coli
clones is reproduced on the surfaceof a nitrocellulose mem-
brane. The DNA on the membrane is denatured, and the
membrane is then incubated in a solution containing a ra-
dioactively labeled probe specificfor the recombinant DNA
containing the fragment of interest. Under hybridization
conditions (near neutral pH, 40-65 "C' 0.3-0'6 M NaCl),
D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N . 181
 I n d i v i d u acl o l o n i e s B o u n ds i n g l e - s t r a n d eDdN A

*'** Master plate of Filter

5eu E. coli colonies

P l a c en i t r o c e l l u l o sfei l t e ro n D l a t e
t o p i c ku p c e l l sf r o m e a c hc o l o n y

Hvbridized
N i t r o c e l l u l o sfei l t e r
c o m p l e m e n t a rD
y NAs

Wash away labeledDNA that does

not hybridizeto DNA bound to filter

f
r"uorr autoradiography I
Performaut o r a d i o g r a p h y

S i g n a la p p e a r so v e r
p l a s m i dD N A t h a t i s
o)
complementary
to probe

A EXPERIMENTAL FIGURE 5-16 cDNAlibrariescan be screened i m a g eo f t h ep o s i t i oonf t h a tp a r t i c u l a

c lro n eo n t h eo r i g i n aple t r i
with a radiolabeledprobeto identifya cloneof interest.The dish(although for easeof comparison, it isnot shownreversed
a p p e a r a noc fea s p o to n t h ea u t o r a d i o g r ai nmd i c a t et hs ep r e s e n c e h e r e )A. l i g n i ntgh ea u t o r a d i o g r awmi t ht h eo r i g i n aple t r d
i i s hw i l l
o f a r e c o m b i n acnl to n ec o n t a i n i nDgN Ac o m p l e m e n t a t or yt h e locatethe corresponding clonefromwhichE colicellscanbe
probeTheposition of the spoton the autoradioqram isthe mirror recovered

this labeledprobe hybridizesro any complemenrarynucleic
acid strandsbound to the membrane.Any excessprobe that
doesnot hybridize is washedaway, and the labeledhybrids
are detectedby autoradiography of the filter. This technique
can be usedto screenboth genomicand cDNA libraries,but
is most commonly usedto isolatespecificcDNAs.
ClearlS identification of specific clones by the
membrane-hybridizationtechnique depends on the avail-
a b i l i t y o f c o m p l e m e n r a r yr a d i o l a b e l e d p r o b e s . F o r a n
oligonucleotideto be useful as a probe, ir must be long
enough for its sequenceto occur uniquely in the clone of
interest and not in any other clones. For most purposes,
this condition is satisfied by oligonucleotidescontaining
a b o u t 2 0 n u c l e o t i d e s .T h i s i s b e c a u s ea s p e c i f i c2 0 - n u -
c l e o t i d e s e q u e n c eo c c u r s o n c e i n e v e r y 4 2 0 ( = 1 0 1 2 )n u -
cleotides.Since all genomesare much smaller (=3 x t0e
nucleotidesfor humans),a specific20-nucleotidesequence
in a genomeusually occurs only once. With automated in-
s t r u m e n t s n o w a v a i l a b l e , r e s e a r c h e r sc a n p r o g r a m t h e
chemicalsynthesisof oligonucleotidesof specificsequence
up to about 100 nucleotideslong. Longer probes can be
preparedby the polymerasechain reaction (pCR), a widely
usedtechniquefor amplifying specificDNA sequences rhat
is describedlater.
How might an investigator design an oligonucleotide
probe to identify a clone encoding a particular protein? It
helps if all or a portion of the amino acid sequenceof the
protein is known. Thanks to the availability of the complete
genomic sequencesfor humans and some important model
organisms such as the mouse, Drosophila, and the round-
worm Caenorbabditis elegans,a researchercan use an ap-
propriate computer program to searchthe genomic sequence
database for the coding sequencethat corresponds to the
amino acid sequenceof the protein under study. If a match is
found, then a single, unique DNA probe based on this
known genomic sequencewill hybridize perfectly with the
clone encoding the protein of interest.
YeastGenomicLibrariesCan Be Constructed
with Shuttle Vectorsand Screenedby
F u n c t i o n aC
l omplementation
In somecasesa DNA library can be screenedfor the ability to
express a functional protein that complements a recessive

182 . cHAprER
s M o l E c u L A RG E N E T rI cE c H N t e u E S
I
 Polylinker < EXPERIMENTAL FIGURE 5-17 A yeastgenomiclibrarycan be
(a) plasmid shuttle vector that can replicatein
constructed in a
yeastand E.coli.(a)Components of a typicalplasmid shuttlevector
for cloningSaccharomyces genesThepresence of a yeastoriginof
DNAreplication (ARS) anda yeastcentromere (CEN) allowsstable
replicationandsegregation in yeast Also included isa yeast
Shuttlevector
selectablemarkersuchasURA3,whichallowsa ura3 mutantto
growon mediumlackinguracilFinally, thevectorcontains sequences
andselection
for replication in E.coli(ORlandamp')anda polylinker
for easyinsertion of yeastDNAfragments. (b)Typical protocol for
constructinga yeast genomic libraryPartialdigestion of totalyeast
genomic DNAwith5au3Aisadjusted to generate fragments with an
CEN average sizeof about10 kb Thevectorisprepared to acceptthe
genomic fragments by digestion with BamHl, whichproduces the
(b) transformed clone of E. colithat
samestickyendsas5au3A.Each
growsafterselection for ampicillinresistancecontains a singletype
l^ dNos mil$ mmrs of yeastDNAfragment

Yeastgenomic DNA in a genomic DNA fragment inserted into a plasmid vector.

To construct a plasmid genomic library that is to be
screenedby functional complementationin yeast cells,the
plasmid vector must be capable of replication in both E.
coli cells and yeast cells. This type of vector' capable of
propagation in two different hosts, is called a shuttle vec-
tor. The structure of a typical yeast shuttle vector is shown
in Figure 5-L7a. This vector contains the basic elements
that permit cloning of DNA fragmentsin E- coli.In addi-
tion, the shuttle vector contains an autonomouslyreplicat-
ing sequence(ARS), which functions as an origin for DNA
replication in yeast; a yeast centromere (called CEN),
which allows faithful segregationof the plasmid during
yeastcell division; and a yeastgeneencodingan enzymefor
uracil synthesis (URA3), which serves as a selectable
marker in an approprlate yeastmutant.
To increasethe probability that all regions of the yeast
Transform E. coli genome are successfullycloned and represented in the
Screenfor amoicillinresistance
plasmid library, the genomic DNA usually is only partially
digest.d to yield overlapping restriction fragments of
'as\
=tb t U. These fragments are then ligated into the shuttle
* ***
+
vector in which the polylinker has been cleavedwith a re-
striction enzymethat produces sticky ends complementary
I lsolateandpoolrecombinant to those on the yeast DNA fragments (Figure 5-17b). Be-
I plasmidsfrom 105transformed causethe 10-kb restriction fragmentsof yeastDNA are in-
{ F. colicolonies corporated into the shuttle u.ito., randomly, at least 10s
Assayyeastgenomiclibraryby functionalcomplementation E. coli colonies,each containing a particular recombinant
shuttle vector, are necessaryto assurethat each region of
yeast DNA has a high probability of being representedin
mutation. Sucha screeningstrategywould be an efficientway the library at least once.
to isolate a cloned genethat correspondsto an interestingre- Figure 5-18 outlines how such a yeast genomic library
cessivemutation identified in an experimental organism. To can be screenedto isolate the wild-type gene corresponding
illustrate this method, referredto as functional complementa- to one of the temperature-sensitivecdc mutations mentioned
tion, we describehow yeast genescloned in special E. coli earlier in this chapter. The starting yeast strain is a double
plasmidscan be introduced into mutant yeastcells to identify mutant that requires uracil for growth due to a ura3 mtta-
the wild-type genethat is defective in the mutant strain. tion and is temperature-sensitivedue to a cdc28 mutation
Libraries constructed for the purpose of screening identified by its phenotype (see Figure 5-6). Recombinant
among yeast gene sequencesusually are constructed from plasmids isolated from the yeast genomic library are mixed
genomic DNA rather than cDNA. BecauseSaccharomyces with yeast cells under conditions that promote transforma-
genesdo not contain multiple introns, they are sufficiently tion of the cells with foreign DNA. Sincetransformed yeast
compact that the entire sequenceof a genecan be included cells carry a plasmid-borne copy of the wild-type URA3

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N 183

Temperature-sensitive
cdc-mutant yeast;
ura3 (requiresuracil) Transformyeast by treatmentwith
LiOAC,PEG.and heat shocr
Plateand incubateat
permissivetemperature
o n m e d i u m l a c k i n gu r a c i l
Only colonies
carrYtng a
URA3 marker
are able to
grow
23'C
EXPERIMENTAL FIGURE 5-18 Screening of a yeastgenomic
libraryby functionalcomplementation can identifyclones
carryingthe normalform of a mutantyeastgene.Inthis
example, a wild-type
CDCgeneis isolatedby complementationof a
cdcyeastmutant TheSaccharomyces strainusedfor screentng
rne
yeastlibrary
carriesura3 anda temperature-sensitive
cdcmutation
Thismutantstrainisgrownandmaintained at a permisstve
temperature (23"C) Pooledrecombinantplasmids prepared
as
gene,they can be selectedby their ability to grow in the ab-
senceof uracil. Typically, about 20 petri dishes, each con-
taining about 500 yeast transformants, are sufficient to
represent the entire yeast genome. This collection of yeast
transformants can be maintained at 23"C, a temperature
permissivefor growth of the cdc28 mutant. The entire col-
Iection on 20 plates is then transferredto replica plates,
which are placed at 36 "C, a nonpermissivetemperature for
cdc mutants. Yeastcoloniesthat carry recombinant plasmids
expressinga wild-type copy of the CDC28 genewill be able
to grow at 36'C. Once temperature-resistant yeastcolonies
have been identified, plasmid DNA can be extracted from
the cultured yeast cells and analyzed by subcloning and
DNA sequencing,topics we take up next.
Gel Electrophoresis
Allows Separationof Vector
DNA from ClonedFragments
In order to manipulate or sequencea cloned DNA fragment, it
sometimesmust first be separatedfrom the vector DNA. This
can be accomplished by cutting the recombinant DNA clone
with the samerestriction enzymeusedto produce the recombi-
nant vectors originally. The cloned DNA and vector DNA then
are subjected to gel electrophoresis, a powerful method for
separatingDNA moleculesof different size(Figure5-19).
Near neutral pH, DNA molecules carry a large negative
charge and therefore move toward the positive electrode
during gel electrophoresis.Becausethe gel marrix restricrs
random diffusion of the molecules, molecules of the same
length migrate together as a band whose width equalsthat of
'184 .
c H A p r E5
R | M o L E c u L AGRE N E TrtEc c H N t e u E s
Libraryof yeast genomic DNA
carrying URA3selectivemarker
Only colonies carrying
a wild-type CDC gene
are able to grow
R e p l i c a - p l a taen d
incubateat nonpermissive
te m perature 36'C
shownin Figure5-17arerncubated with the mutantyeastcellsunder
thatpromotetransformation
conditions Therelatively
few
transformedyeastcells,
whichcontainrecombinant plasmid
DNA.
cangrowin theabsence of uracilat 23 'C Whentransformed yeast
colonies
arereplica-plated
andplacedat 36'C (a nonpermissrve
temperature),
onlyclones carrying a libraryplasmid
thatcontainsthe
wild-type
copyof the CDCgenewillsurviveL|OAC= lithiumacetate;
PEG= polyethyleneglycol
the well into which the original DNA mixture was placed at
the start of the electrophoreticrun. Smaller moleculesmove
through the gel matrix more readily than larger molecules,
so that molecules of different length migrate as distinct
bands. Smaller DNA moleculesfrom about 10 to 2000 nu-
cleotides can be separatedelectrophoretically on polyacry-
lamide gels, and larger molecules from about 200 nu-
cleotidesto more than 20 kb on agarose gels.
A common method for visualizingseparatedDNA bands
on a gel is to incubatethe gel in a solution containing the flu-
orescentdye ethidium bromide. This planar moleculebinds to
DNA by intercalating berweenthe base pairs. Binding con-
centratesethidium in the DNA and also increasesits intrinsic
fluorescence.As a result, when the gel is illuminated with ul-
traviolet light, the regions of the gel containing DNA fluoresce
much more brightly than the regionsof the gel without DNA.
Once a cloned DNA fragment, especiallya long one, has
beenseparatedfrom vector DNA, it often is treated with var-
ious restriction enzymesto yield smallerfragments.After sep-
aration by gel electrophoresis,all or some of these smaller
fragments can be ligated individually into a plasmid vector
and cloned in E. coli by the usual procedure. This process,
known as subcloning,is an important step in rearranging
parts of genesinto useful new configurations. For instance,
an investigator who wants to change the conditions under
which a geneis expressedmight usesubcloningto replacethe
normal promoter associatedwith a cloned genewith a DNA
segmentcontaining a different promoter. Subcloningalso can
be used to obtain cloned DNA fragments that are of an ap-
propriate length for determining the nucleotidesequence.
(a) DNA restrictionfragments

dKs

I n , u . " m i x t u r ei n t h ew e l lo f
I a n a g a r o soer p o l y a c r y l a m i d e
A n n l ve l e c t r ifci e l d
I sel.
v < EXPERIMENTAL FIGURE 5-19 Gel electrophoresis separates
DNA moleculesof different lengths.(a)A gel is prepared by
Well p o u r i n ga l i q u i dc o n t a i n i negi t h e rm e l t e da g a r o soer
__l - (Negativeelectrode) u n p o l y m e r i zaecdr y l a m i dbee t w e e n t w o g l a s sp l a t e sa f e w
Gelparticle m i l l i m e t ear sp a r t A s t h e a g a r o sseo l i d i f i eosr t h e a c r y l a m i d e
p o l y m e r i z ienst op o l y a c r y l a m i ad eg ,e lm a t r i x( o r a n g o e v a l sf)o r m s
c o n s i s t i nogf l o n g ,t a n g l e dc h a i n o
s f p o l y m e r s
T h ed i m e n s i o nosf
t h e i n t e r c o n n e c t icnhga n n e l o s ,r p o r e sd, e p e n do n t h e
concentration f t h e a g a r o soer a c r y l a m i dues e dt o f o r mt h e g e l
T h es e p a r a t ebda n d sc a nb e v i s u a l i z ebdy a u t o r a d i o g r a p(hi fyt h e
Pores f r a g m e n tasr er a d i o l a b e l eodr)b y a d d i t i o no f a f l u o r e s c e d n yt e
( e g , e t h i d i u mb r o m i d et )h a t b i n d st o D N A ( b )A p h o t o g r a pohf a
g e ls t a i n e wd i t h e t h i d i u mb r o m i d e ( E t B r )E t B rb i n d st o D N Aa n d
f l u o r e s c eusn d e rU Vl i g h t ,T h eb a n d si n t h ef a r l e f ta n df a r r i g h t
+ (Positive lectrode) l a n e sa r ek n o w na s D N Al a d d e r s - D N A f r a g m e n tosf k n o w ns i z e
t h a ts e r v ea sa r e f e r e n c e f o r d e t e r m i n h e l e n g t ho f t h e D N A
i nt g
M o l e c u l e sm o v e t h r o u g hp o r e s f r a g m e n tisn t h e e x p e r i m e n tsaal m p l eI P a r(tb )S c i e n cPeh o t o
in gel at a rate inversely L i b r alr y
p r o p o r t i o n atlo t h e i r c h a i nl e n g t h

o-
-O-P:O - o - PI : o
I I
o o
- O - P I: O - O - P I: O
I I
o
-O-P:O - O - PI : O
Subjectto autoradiography
or incubatewith fluorescentdye l I
o ase O
I
CH, QH,

HH
Deoxyribonucleoside Dideoxyribonucleoside
Signals
triphosPhate triphosPhate
corresponding (dNTP) (ddNTP)
to DNA bands
FIGURE 5-20 Structures of deoxyribonucleoside
triphosphate(dNTP) and dideoxyribonucleoside triphosphate
(ddNTP). of a ddNTP
Incorporation residueintoa growingDNA
elongation
strandterminates at thatpoint

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N 185
 Technique of DNA{tttt
Animation:DideoxySequencing
(b) P r i m e r5 ' -
T e m p l a t e3 ' - 5'

(a)

5'TAGCTGACTC3'
3' A T C G A C T G A G T C A A G A A C T A T T G G G C T T A A
I DNApolymerase
I + ddGTP
| + dATB dGTB dCTB dTTp
| + ddGTPin low concentration
I
v
5'TAGCTGACTCAG3' -A -G
3' ATCGACTGAGTCAAGAACTATTGGGCTTAA
5 ' T A G C T G A CT C A GT T C + T T G 3 ' _A EG
3' ATCGACTGAGTCAAGAACTATTGGGCTTAA
5' TAGCTGACTCAGTTJTTCNTNACCCG3' -G -T
3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

T
etc. etc.

I I
Denatureand separatedaughterstrandsby electrophoresis
I
I N N N N A A T G A A TA G A T A T A T A G G G G A A T T G A G T GGTA GGGGAT T TAGAGT GA TG AGG AIG AAG TTGAGTATT I
20 30 40 80 90-

AAATAG TTGG GTAA ATGGT ATAG TGTTT GTGAAATTGTTATGTAAA A

110 120 A AA
'180 AT
130 140 150 160 170

a EXPERIMENTAL FTGURE 5-21 ClonedDNAscan be fragments endingat everyoccurrence of ddGTp(b)Toobtainthe

sequencedby the Sangermethod, usingfluorescent-tagged complete sequence of a templateDNA,four separate reactions are
dideoxyribonucleoside triphosphates (ddNTps). (a)A single performed, eachwith a different dideoxyribonucleoside triphosphate
(template)
strandof the DNAto be sequenced (blueletters)is (ddNTP) TheddNTP thatterminates eachtruncated fragment canbe
hybridized
to a syntheticdeoxyribonucleotide primer(blackletters) identified
by useof ddNTPs taggedwith four differentfluorescent
Theprimeriselongated in a reactionmixture containing thefour dyes(indicatedbycolored highlights). (c)In an automated
normaldeoxyribonucleoside triphosphatesplusa relativelysmall sequencing machine, thefourreaction mixtures aresubjected to gel
amountof oneof thefourdideoxyribonucleoside triphosphates. ln electrophoresis,
andtheorderof appearance of eachof thefour
thisexample,ddGTP (yellow) ispresentBecause of the relatively drfferent
fluorescentdyesat the endof the gelis recordedShown
low concentrationof ddGTP, incorporation
of a ddGTBandthus hereisa sample printoutfroman automated sequencer fromwhich
chaintermination,occurs at a givenpositionin the sequence only thesequence of theoriginal template DNAcanbe deduced fromthe
about1 percent of thetime Eventually the reaction mixturewill sequence of thesynthesized strandN = nucleotide thatcannotbe
containa mixtureof prematurely terminated(truncated) dauqhter assigned[Part(c)fromGriffithset al, Figure14-27I

186 CHAPTER
5 I MOLECULAG
RENETIC
TECHNIQUES
 lsolation of a genomic library spanning the genome of interest

A l i g n i n gl i b r a r yc l o n e s
Sequencing of
by hybridizationor
r a n d o ml i b r a r yc l o n e s
restriction-site mapping

Sequenceof unordered fragments,

Ordered set of clones
for about lO-fold coverageof
spanningthe genome
eachgenomic segment

Sequencing of Aligning
sequenced
orderedclones clonesby comPuter

Genomicsequence Assembled
genomlcsequence
> FIGURE 5-22 Two Strategiesfor Assembling WholeGenome methoddepends
alternative on the relativeeaseof automated DNA
Sequences. Onemethoddepends on isolatingandassembling a set sequencingandbypasses the laborrousstepof ordering the library
of clonedDNAsegments thatspanthe genomeThiscanbe doneby Bysequencingenoughrandomlibrary clonessothateachsegment
matching clonedsegmentsby hybridizationor by alignmentof of the genomeis from
represented 3 to 10times it ispossibleto
restriction
sitemapsTheDNAsequence of the ordered clones can thegenomrc
reconstruct sequence by computer alignment of the
thenbe assembledintoa completegenomic sequence The verylargenumberof sequence fragments

guished by their corresponding fluorescent label (Figure 5-

C l o n e dD N A M o l e c u l e sA r e S e q u e n c e d Rapidly
21b). For example,all truncated fragmentsthat end with a G
b y t h e D i d e o x yC h a i n - T e r m i n a t i oMne t h o d
would fluoresce one color (e.g., yellow), and those ending
The complete characterizationof any cloned DNA fragment with an A would fluoresceanother color (e.g',red), regardless
requires determination of its nucleotide sequence.F. Sanger of their lengths. The mixtures of truncated daughter frag-
and his colleaguesdeveloped the method now most com- ments from each of the four reactionsare subjectedto elec-
monly used to determine the exact nucleotide sequenceof trophoresison specialpolyacrylamidegels that can separate
DNA fragmentsup to =500 nucleotideslong. The basic idea single-strandedDNA moleculesdiffering in length by only 1
behindthis method is to synthesizefromthe DNA fragmentto nucleotide.A fluorescencedetector that can distinguish the
be sequenceda set of daughterstrandsthat are labeledat one four fluorescenttags is located at the end of the gel. The se-
end and differ in length by one nucleotide.Separationof the quence of the original DNA template strand can be deter-
truncated daughterstrandsby gel electrophoresiscan then es- mined from the order in which different labeledfragmentsmi-
tablish the nucleotidesequenceof the original DNA fragment. grate past the fluorescencedetector (Figure 5-21'c).
Synthesisof truncated daughterstandsis accomplishedby In order to sequencea long continuousregion of genomic
use of 2',3'-dideoxyribonucleoside triphosphates(ddNTPs). DNA or even the entire genomeof an organism, researchers
These molecules,in contrast to normal deoxyribonucleotides usually employ one of the strategiesoutlined in Figure 5-22.
(dNTPs), lack a 3' hydroxyl group (Figure 5-20). Although The first method requiresthe isolation of a collection of cloned
ddNTPs can be incorporated into a growing DNA chain by DNA fragmentswhose sequences overlap. Once the sequence
DNA polymerase,once incorporated they cannot form a of one of these fragments is determined, oligonucleotidesbased
phosphodiesterbond with the next incoming nucleotide on that sequence can be chemically synthesized for use as
triphosphate.Thus incorporation of a ddNTP terminates primers in sequencing the adjacent overlapping fragments. In
chain synthesis, resulting in a daughterstrand truncated at this way, the sequence of a long stretch of DNA is determined
specific positions correspondingto the basecomplementary incrementally by sequencingof the overlapping cloned DNA
to the added ddNTP on the template strand. fragments that compose it. A second method, which is called
Sequencingusing the Sangerdideoxy chain-termination whole genomeshotgunseqwencing, bypasses the time-consum-
method is usually carried out using an automated DNA se- ing step of isolating an ordered collection of DNA segments
quencing machine. The reaction begins by denaturing a that span the genome.This method involves simply sequencing
double-strandedDNA fragment to generatetemplate strands random clones from a genomic library. A total number of
for in vitro DNA synthesis.A syntheticoligodeoxynucleotide clonesare chosenfor sequencingso that on averageeach seg-
is usedas the primer for the polymerizationreactionthat con- ment of the genomeis sequenced about 10 times.This degreeof
tains a low concentrationof eachof the four ddNTPs in addi- coverageensuresthat eachsegmentof the genomeis sequenced
tion to higher concentrationsof the normal dNTPs. The more than once.The entiregenomicsequenceis then assembled
ddNTPs are randomly incorporated at the positions of the using a computeralgorithm that alignsall the sequences, using
correspondingdNTP, causingtermination of polymerization their regionsof overlap.\7hole genomeshotgunsequencingis
at thosepositionsin the sequence(Figure5-21,a).Inclusion of the fastestand most cost-effectivemethod for sequencinglong
fluorescenttagsof different colors on eachof the four ddNTPs stretchesof DNA, and most genomes'including the human
allows each set of truncated daughterfragmentsto be distin- genome,have beensequencedby this method.

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N 187
 TechniqueAnimation: PolymeraseChain Reaction{tttt

< EXPERIMENTAL FIGURE 5-23 The polymerase chainreaction(PCR) is widely used

, Denaturationof DNA to amplifyDNAregionsof known sequences. Toamplifya specific regionof DNA,an
c Y c l e1 I A n n e a l i n go f p r i m e r s investigatorwillchemicallysynthesizetwo different
oligonucleotrde primerscomplementary to
sequences of approximately 18 basesflankingtheregionof interest (designated aslightblue
anddarkbluebars)Thecomplete reaction iscomposed of a complex mixture of double-
strandedDNA(usually genomic DNAcontaining thetargetsequence of interest),a
E l o n g a t i o no f p r i m e r s stoichiometricexcessof bothprimers, thefourdeoxynucleoside triphosphates, anda heat-
I
stableDNApolymerase knownasTaqpolymerase. DuringeachPCRcycle, the reaction
mixtureisfirstheatedto separate thestrands andthencooledto allowtheprimers to bindto
complementary sequences flankingthe regionto beamplified. Iaq polymerase thenextends
, Denaturationof DNA
eachprimerfromits3' end,generating newlysynthesized strands thatextendin the3'
c Y c l e2 I A n n e a l i n go f p r i m e r s direction
to the 5' endof thetemplate strand.Duringthethirdcycle, two double-stranded
DNAmolecules aregenerated equalin lengthto thesequence of theregionto beamplified
Ineachsuccessive cyclethetargetsegment, whichwillannealto theprimers, isduplicated,
andwilleventually vastlyoutnumber allotherDNAsegments in thereaction mixture
SuccessivePCRcycles canbeautomated bycycling
the reaction for timedintervals at high
temperature for DNAmeltingandat a definedlowertemperature for theannealing and
elongationportions of thecycleA reaction thatcycles20 timeswillamplifythespecific target
{ Elongationof primers sequence 1-million-fold

The PCR dependson the ability to alternately denature

(melt) double-strandedDNA moleculesand hybridizecomple-
mentary singlestrandsin a controlled fashion. As outlined in
Figure5-23, a typical PCR procedurebeginsby heat-denatura-
, Denaturationof DNA
G Y c l e3 I n n n e a t i n so f p r i m e r s tion of a DNA sampleinto singlestrands.Next, two synthetic
oligonucleotidescomplementaryto the 3' ends of the target
DNA segmentof interestare addedin greatexcessto the dena-
tured DNA, and the temperarure is lowered to 50-60 'C.
Thesespecificoligonucleotides,which are at a very high con-
centration,will hybridizewith their complementarysequences
in the DNA sample, whereas the long strands of the sample
DNA remain apart becauseof their low concentration.The hy-
bridized oligonucleotidesthen serveas primers for DNA chain
synthesisin the presenceof deoxynucleotides(dNTPs) and a
E l o n g a t i o no f p r i m e r s temperature-resistantDNA polymerase such as that from
I
Thermusaquaticus(a bacteriumthat livesin hot springs).This
enzymq called Taq polymerase, can remain active even after
being heatedto 95 oC and can extend the primers at tempera-
turesup to72"C. When synthesisis complete,the whole mix-
ture is then heatedto 95 "C to denaturethe newly formed DNA
duplexes.After the temperatureis lowered again, another cycle
of synthesistakes place becauseexcessprimer is still present.
Repeated cycles of denaturation (heating) followed by hy-
bridization and synthesis(cooling) quickly amplify the se-
L quence of interest. At each cycle, the number of copies of the
Cycles4, 5, 6, etc. sequencebetweenthe primer sitesis doubled; therefore, the de-
sired sequenceincreasesexponentially-about a million-fold
after 20 cycles-whereas all other sequencesin the original
DNA sampleremain unamplified.
T h e P o l y m e r a sC
e h a i nR e a c t i o nA m p l i f i e sa
Direct lsolation of a Specific Segment of Genomic DNA
SpecificDNA Sequencefrom a ComplexMixture
For organismsin which all or most of the genome has been
If the nucleotide sequencesat the ends of a parricular DNA sequenced,PCR amplification starting with the total genomic
region are known, the intervening fragment can be amplified DNA often is the easiestway to obtain a specificDNA region
directly by the polymerase chain reaction (PCR). Here we of interest for cloning. In this application, the two oligonu-
describethe basic PCR technique and three situations in cleotideprimers are designedto hybridize to sequences flank-
which it is used. ing the genomic region of interest and to include sequences

188 . cHAprER
s I M o L E c u L AG
R E N E I cr E c H N t e u E s
 Regionto be amplified
5'
D N As y n t h e s i s
P r i m e r1
s'
P r i m e r2
Prime1
r method, known as reuersetranscriptase-PcR /R?PCR/, be-
C o n t i n u ef o r = 2 0
PCRcycles
Cut with restriction
enzymes
-Stickyend
Stickyend
Ligatewith plasmidvector
with stickyends
that are recognizedby specificrestriction enzymes(Figure 5-
24). After amplification of the desired target sequencefor
about 20 PCR cycles,cleavagewith the appropriate restric-
tion enzymesproduces sticky ends that allow efficient liga-
tion of the fragment into a plasmid vector cleaved by the
same restriction enzymesin the polylinker. The resulting re-
combinant plasmids,all carrying the identical genomic DNA
segment,can then be cloned in E. coli cells.\fith certain re-
finements of the PCR, even DNA segmentsgreater than 10
kb in length can be amplified and cloned in this way.
Note that this method does not involve cloning of large
numbers of restriction fragments derived from genomic DNA
and their subsequentscreeningto identify the specificfragment
of interest. In effect, the PCR method inverts this traditional
approach and thus avoids its most tedious aspects.The PCR
method is useful for isolating genesequencesto be manipulated
in a variety of useful ways describedlater. In addition the PCR
method can be used to isolate gene sequencesfrom mutant or-
ganismsto determinehow they differ from the wild type.
A variation on the PCR method allows PCR amplifica-
tion of a specificcDNA sequencefrom cellular mRNAs. This
< EXPERIMENTAL FIGURE 5-24 A specifictarget region in total
genomicDNAcan be amplifiedby PCRfor usein cloning.Each
3'
primerfor PCRiscomplementary to oneendof thetargetsequence
andincludes the recognition
sequence for a restriction
enzyme that
doesnot havea sitewithinthetargetregion.In thisexample, primer
1 containsa BamHl sequence, whereas primer2 contains a Hindlll
sequence (Notethatfor clarity,
in anyround,amplif icationof only
5'
oneof thetwo strands isshown,the onein brackets ) After
thetargetsegments
amplification, aretreatedwith appropriate
restriction
enzymes,generating fragments with stickyendsThese
canbe incorporated intocomplementary plasmid vectorsandcloned
in E colibythe usualprocedure (seeFigure 5-13).
gins with the same procedure describedpreviously for isola-
tion of cDNA from a collection of cellular mRNAs' Typically'
an oligo-dT primer, which will hybridize to the 3' poly(A) tail
of the mRNA, is used as the primer for the first strand of
cDNA synthesisby reversetranscriptase.A specific cDNA
can then be isolatedfrom this complex mixture of cDNAs by
PCR amplification using two oligonucleotide primers de-
signedto match sequencesat the 5' and 3' ends of the corre-
sponding mRNA. As described previously, these primers
could be designedto include restriction sitesto facilitate the
insertion of amplified cDNA into a suitableplasmid vector.
Preparation of Probes Earlier we discussedhow oligonu-
cleotide probes for hybridization assayscan be chemically
synthesized.Preparation of such probes by PCR amplifica-
tion requires chemical synthesisof only two relatively short
primers corresponding to the two ends of the target se-
quence. The starting sample for PCR amplification of the
target sequencecan be a preparation of genomic DNA, or a
preparation of cDNA synthesizedfrom the total cellular
32P-
mRNA. To generatea radiolabeled product from PCR,
labeled dNTPs are included during the last several amplifi-
cation cycles.Becauseprobes prepared by PCR are relatively
3'P atoms incorporated into
long and have many radioactive
them, theseprobes usually give a stronger and more specific
signal than chemically synthesizedprobes.
Tagging of Genes by Insertion Mutations Another useful
application of the PCR is to amplify a "tagged" gene from
the genomic DNA of a mutant strain. This approach is a
simpler method for identifying genesassociatedwith a par-
ticular mutant phenotype than screening of a library by
functionalcomplementation(seeFigure5-18).
The key to this use of the PCR is the ability to produce
mutations by insertion of a known DNA sequenceinto the
genome of an experimental organism. Such insertion muta-
tions can be generated by use of mobile DNA elements,
which can move (or transpose)from one chromosomal site
to another. As discussedin more detail in Chapter 5, these
DNA sequencesoccur naturally in the genomesof most or-
ganisms and may give rise to loss-of-function mutations if
they transposeinto a protein-coding region.
For example, researchershave modified a Drosophila
mobile DNA element, known as the P element, to optimize
D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N r89
> EXPERIMENTAL FIGURE 5-25 The genomic I ransDoson
sequenceat the insertionsite of a transposon
is revealedby PCRamplificationand
sequencing. Toobtainthe DNAsequence of the Restriction
sites:t
insertionsiteof a P-elementtransooson it is
necessary to PCR-amplify thejunctionbetween I cr, *it,
knowntransposon sequences andunknown restriction
enzyme
I
flankingchromosomal sequences Onemethodto
achievethisisto cleave genomic DNAwith a
restriction
enzyme thatcleaves oncewithinthe
transposon sequence Ligation
of the resulting
restriction
fragments willgeneratecircular DNA
molecules. Byusingappropriately designed DNA
primersthatmatchtransposon sequences it is
possibleto PCR-amplify the desiredjunction
Ligate
fragmentFinally, a DNAsequencing reaction (see to circularize
Figure5-21)is performed usrngthe PCR-amplified
fragmentasa template andan oligonucleotide
primerthat matches sequences neartheendof the
transposon, to obtainthesequence of thejunction PCRprimers
between thetransposon andchromosome

I PCRamplification
with Orimers
to transposon
Sequencing I
pflmer
-+

its use in the experimental generation of insertron muta-

tions. Once it has been demonstratedthat insertion of a P
element causesa mutation with an interestingphenotype,
the genomic sequencesadjacentto the insertion site can be
amplified by a variation of the standardPCR protocol that
usessyntheticprimers complementaryto the known P-ele-
ment sequencebut that allows unknown neighboring se-
quencesto be amplified. One such method, depicted in
Figure 5-25, beginsby cleavingDrosophila genomic DNA
containing a P-elementinsertion with a restriction enzyme
that cleavesonce within the P-elementDNA. The collec-
tion of cleaved DNA fragmenrs treated with DNA ligase
yields circular molecules,some of which will contain P-ele-
ment DNA. The chromosomal region flanking the P ele-
ment can then be amplified by PCR using primers that
match P-elementsequencesand are elongatedin opposite
directions. The sequenceof the resulting amplified frag-
ment can then be determined using a third DNA primer.
The crucial sequencefor identifying the site of P-element
insertion is the junction between the end of the P-element
and genomic sequences.Overall, this approach avoids the
cloning of large numbers of DNA fragments and their
screeningto detect a cloned DNA correspondingto a mu-
tated geneof interest.
Similar methods have been applied to other organrsms
for which insertion mutations can be generatedusing either
mobile DNA elements or viruses with sequencedgenomes
that can insertrandomly into the genome.
DNA Cloning and Characterization
r In DNA cloning, recombinant DNA molecules are
formed in vitro by inserting DNA fragments into vecror
DNA molecules.The recombinant DNA molecules are
then introduced into host cells, where they replicate, pro-
ducing large numbers of recombinant DNA molecules.
r Restriction enzymes (endonucleases)typically cut DNA
at specific4- to 8-bp palindromic sequences,producing de-
fined fragments that often have self-complementarysingle-
strandedtails (stickyends).
I Two restriction fragments with complementary ends can
be joined with DNA ligase to form a recombinant DNA
molecule(seeFigure 5-12).
t E. coli cloning vectors are small circular DNA molecules
(plasmids)that include three functional regions:an origin of
replication, a drug-resistancegene, and a site where a DNA
fragmentcan be inserted.Transformedcellscarrying a vector
grow into colonieson the selectionmedium (seeFigure5-13).
r A cDNA library is a set of cDNA clones prepared from
the mRNAs isolated from a particular type of tissue. A
genomic library is a set of clones carrying restriction frag-
ments produced by cleavageof the entire genome.
r In cDNA cloning, expressed mRNAs are reverse-
transcribedinto complementaryDNAs, or cDNAs. By a se-
ries of reactions,single-strandedcDNAs are converted into

190 CHAPTER
5 | MOLECULAG
R E N E T TTCE C H N T Q U E S
 double-strandedDNAs, which can then be ligated into a
plasmidvector (seeFigure5-15).
r A particular cloned DNA fragment within a library can
be detected by hybridization to a radiolabeledoligonu-
cleotidewhose sequenceis complementaryto a portion of
the fragment(seeFigure5-16).
r Shuttle vectors that replicate in both yeast and E. coli can
be used to construct a yeastgenomic library. Specificgenes
can be isolated by their ability to complementthe corre-
spondingmutant genesin yeastcells(seeFigure5-17).
r Long cloned DNA fragments often are cleavedwith re-
striction enzymes,producing smallerfragmentsthat are then
separatedby gel electrophoresisand subclonedin plasmid
vectorsprior to sequencingor experimentalmanipulation.
r DNA fragmentsup to about 500 nucleotideslong are se-
quenced in automated instruments based on the Sanger
(dideoxychain-termination)method (seeFigure5-21).
r lil/hole genome sequencescan be assembledfrom the se-
quencesof a large number of overlappingclones from a
genomiclibrary (seeFigure 5-22).
r The polymerasechain reaction(PCR)permitsexponential
amplification of a specificsegmentof DNA from just a sin-
gle initial template DNA molecule if the sequenceflanking
the DNA regionto be amplifiedis known (seeFigure5-23).
r PCR is a highly versatilemethod that can be programmed
to amplify a specificgenomicDNA sequence,a cDNA, or
a sequenceat the junction betweena transposableelement
and flanking chromosomalsequences.
EE UsingClonedDNAFragments
to
StudyGeneExpression
In the last sectionwe describedthe basictechniquesfor using
recombinantDNA technologyto isolatespecificDNA clones,
and ways in which the clones can be further characterized.
DNA
I
I Cleavewith
restriction
enzymes
I Ni t r o c e l l luo s e
Gel V
Ni t r o c e l l luo s e
Gel
----.f----
---r---T---T
A l k a l i n es o l u t i o n
C a p i l l a r ya c t i o nt r a n s f e r s
DNAfrom gelto nitrocellulose
A EXPERIMENTAL FIGURE 5-25 Southernblot techniquecan
detecta specificDNAfragmentin a complexmixtureof
restrictionfragments.Thediagram depictsthreedifferent
fragments
restriction in the gel,butthe procedure
canbe applied
to
U S I N GC L O N E DD N A F R A G M E N TTSO S T U D YG E N EE X P R E S S I O N
Now we considerhow an isolatedDNA clone can be usedto
study geneexpression.We discussseveralwidely usedgeneral
techniquesthat rely on nucleic acid hybridization to elucidate
when and where genesare expressed,as well as methods for
generatinglarge quantitiesof protein and otherwisemanipu-
lating amino acid sequencesto determinetheir expressionpat-
terns,structure,and function. More specificapplicationsof all
thesebasictechniquesare examinedin the following sections.
H y b r i d i z a t i o nT e c h n i q u ePs e r m i tD e t e c t i o no f
S p e c i f i cD N A F r a g m e n t s
a n d mRNAs
Two very sensitivemethodsfor detectinga particular DNA or
RNA sequencewithin a complex mixture combine separation
and hybridizationwith a complementary
by gel electrophoresis
radiolabeled DNA probe. A third method involveshybridizing
labeledprobesdirectly onto a preparedtissuesample.\Wewill
encounter referencesto all three of these techniques,which
have numerousapplications,in other chapters.
Southern Blotting The first hybridizationtechniqueto detect
DNA fragments of a specific sequenceis known as Southern
blotting after its originator E. M. Southern.This techniqueis ca-
pable of detecting a single specific restriction fragment in the
highly complex mixture of fragments produced by cleavageof
the entire human genomewith a restrictionenzyme.When sucha
complex mixture is subjectedto gel electrophoresis,so many dif-
ferent fragmentsof nearly the same length are presentit is not
possibleto resolveany particular DNA fragmentsas a discrete
band on the gel. Neverthelessit is possibleto identify a particular
fragmentmigrating as a band on the gel by its ability to hybridize
to a specificDNA probe. To accomplish this, the restriction
fragments present in the gel are denatured with alkali and
transferredonto a nitrocellulosefilter or nylon membraneby
blotting (Figure 5-26). This procedurepreservesthe distribution
of the fragmentsin the gel, creatinga replica of the gel on the fil-
ter. (The blot is usedbecauseprobesdo not readily diffuseinto the
original gel.)The filter then is incubatedunder hybridization con-
ditions with a specificradiolabeledDNA probe, which usually is
Autoradiogram
Hybridizewith
l a b e l e dD N A o r
R N Ap r o b e
a mixture of DNAfragmentsOnlyfragments
of millions that
to a labeled
hybridize probewillgivea signalon an autoradiogram
technique
A similar blottingdetects
calledtVorthern mRNAs
specifrc
withina mixturelseeE M Southern, 1975,J Mol Blol98:508
l
191
generatedfrom a cloned restriction fragment. The DNA restric-
tion fragmentthat is complementaryto the probe hybridizes,and
its location on the filter can be revealedby autoradiography.
Northern Blotting One of the most basicways to character-
ize a cloned geneis to determinewhen and where rn an organ-
ism the geneis expressed.Expressionof a particular genecan be
followed by assayingfor the corresponding mRNA by North-
ern blotting, named, in a play on words, after the related
method of Southern blotting. An RNA sample, often the total
cellular RNA, is denatured by treatment with an agent such as
formaldehyde that disrupts the hydrogen bonds between base
pairs, ensuring that all the RNA moleculeshave an unfolded,
linear conformation. The individual RNAs are separated
according to size by gel electrophoresisand transferred to a
nitrocellulose filter to which the extendeddenatured RNAs ad-
here.As in Southernbloning, the filter then is exposedto a la-
beled DNA probe that is complementaryto the geneof interest;
finally, the labeled filter is subjected to autoradiography. Be-
causethe amount of a specificRNA in a samplecan be esti-
mated from a Norrhern blot, the procedureis widely used ro
compare the amounts of a particular mRNA in cells under dif-
ferentconditions(Figure5-27).
In Situ Hybridization Northern blotting requiresextracring
the mRNA from a cell or mixture of cells,which meansthat the
cellsare removedfrom their normal location within an orsan-
UN
{k
5kb-
2kb-
1kb-
-0.6s kb -
w
t-flHl,.'l'"
A EXPERIMENTAL FIcURE5-27 Northernblot analysisreveals
increased expression of p-globinmRNAin differentiated
erythroleukemia
cells.ThetotalmRNAin extracts of
erythroleukemiacellsthatweregrowingbut uninduced
andin cells
induced to stopgrowingandallowedto differentiate for 4g hoursor
96 hourswasanalyzed by Northern blottingfor B-globinmRNAThe
densityof a bandisproportionalto theamountof mRNApresent
TheB-globin mRNAisbarelydetectable in uninduced cells(UNlane)
but increasesmorethan1000-fold by 96 hoursafterdifferentiationis
induced[Courtesyof L Kole]
192 . cHAprER
5 | M o L E c u L AG
R E N E T tr cE c H N t e u E s
ism or tissue.As a result, the location of a cell and its relation to
its neighbors is lost. To retain such positional information in
precisestudiesof geneexpression,a whole or sectionedtissueor
evena whole permeabilizedembryo may be subjectedto in situ
hybridization to detect the mRNA encoded by a particular
gene.This technique allows genetranscription to be monitored
in both time and space(Figure5-28).
D N A M i c r o a r r a y sC a nB e U s e dt o E v a l u a t et h e
E x p r e s s i o on f M a n y G e n e sa t O n e T i m e
Monitoring the expressionof thousandsof genessimultane-
ously is possiblewith DNA microarray analysis,another
technique based on the concept of nucleic acid hybridiza-
tion. A DNA microarray consists of an organized array of
thousands of individual, closely packed gene-specific
sequencesattached to the surfaceof a glassmicroscopeslide.
By coupling microarray analysis with the results from
genome sequencing projects, researchers can analyze the
global patterns of gene expression of an organism during
specificphysiological responsesor developmentalprocesses.
Preparation of DNA Microarrays In one methodfor prepar-
ing microarrays,an =1-kb portion of the coding region of each
gene analyzedis individually amplified by the PCR. A robotic
deviceis used to apply each amplified DNA sample to the sur-
face of a glass microscope slide, which then is chemically
processedto permanently attach the DNA sequencesto the
glasssurfaceand to denature them. A typical array might con-
tain =6000 spotsof DNA in a2 x 2- cm grid.
In an alternative merhod, multiple DNA oligonu-
cleotides, usually at least 20 nucleotides in length, are syn-
thesizedfrom an initial nucleotide that is covalently bound
to the surface of a glass slide. The synthesisof an oligonu-
cleotide of specific sequencecan be programmed in a small
region on the surfaceof the slide. Severaloligonucleotidese-
quencesfrom a singlegeneare thus synthesizedin neighbor-
ing regions of the slide to analyzeexpressionof that gene.
Vith this method, oligonucleotidesrepresentingthousands
of genescan be produced on a singleglassslide. Becausethe
methods for constructing these arrays of synthetic oligonu-
cleotideswere adapted from methods for manufacturing mi-
croscopic integrated circuits used in computers, these types
of oligonucleotidemicroarrays are often called DNA chips.
Using Microarrays to Compare Gene Expression under
Different Conditions The initial stepin a microarrayexpres-
sion study is to preparefluorescentlylabeledcDNAs correspon-
ding to the mRNAs expressedby the cellsunder study.When the
cDNA preparation is applied to a microarray,sporsrepresenting
genesthat are expressedwill hybridize under appropriate condi-
tions to their complementarycDNAs in the labeledprobe mix,
and can subsequentlybe detectedin a scanninglasermicroscope.
Figure 5-29 depicts how this method can be applied to
examine the changes in gene expression observed after
starved human fibroblasts are transferred to a rich, serum-
containing, growth medium. In this type of experiment, the
separatecDNA preparations from starvedand serum-grown
 (c)

DorsaI
Head

NT

Notochord

A EXPERIMENTAL FIGURE 5-28 In situ hybridizationcandetect o r t h e r e p o r t eern z y m iesa d d e dA c o l o r e p

s u b s t r a tf e drecipitate
activityof specificgenesin whole and sectionedembryos.The f o r m sw h e r et h e p r o b eh a sh y b r i d i z et od t h e m R N Ab e i n g
s p e c i m ei n s p e r m e a b i l i zbeydt r e a t m e nwt i t h d e t e r g e natn da d e t e c t e d( a )A w h o l em o u s e m b r y oa t a b o u t1 0 d a y so f
protease to expose the mRNAto the probeA DNAor RNAprobe, development probedfor Sonichedgehog mRNAThestainmarks
s p e c i f ifco r t h e m R N Ao f i n t e r e sits, m a d ew i t h n u c l e o t i daen a l o g s t h en o t o c h o r d ( r e da r r o w )a, r o d o f m e s o d e r rmu n n i n g a l o n gt h e
c o n t a i n i ncgh e m i c aglr o u p tsh a tc a nb e r e c o g n i z ebdy a n t i b o d i e s f u t u r es p i n acl o r d ( b )A s e c t i oonf a m o u s e m b r y os i m i l atro t h a t
A f t e rt h e p e r m e a b i l i zsepde c i m ehna sb e e ni n c u b a t ewdi t ht h e i n o a r t( a ) T h ed o r s a l / v e n tar xailso f t h e n e u r atlu b e( N T c) a nb e
p r o b eu n d e cr o n d i t i o nt hs a tp r o m o t e hybrid z a t i o nt,h ee x c e s s seen,with the Sonichedgehog-expressing notochord (redarrow)
p r o b ei s r e m o v ew d i t ha s e r i eosf w a s h e sT h es p e c i m ei n st h e n b e l o wi t a n dt h e e n d o d e r (
m b l u e
a r r o w s
) t i l f
l a r t h e v
r e n t r a (l c )A
i n c u b a t ei d n a s o l u t i ocno n t a i n i nagn a n t i b o dtyh a tb i n d st o t h e wholeDrosophila embryoprobedfor an mRNAproduced during
p r o b eT hs a n t i b o diysc o v a i e n tl loyi n e dt o a r e p o r t eern z y m (ee g , tracheadevelopment Therepeating patternof bodysegments is
h o r s e r asdh p e r o x i d a o se
r a l k a l i npeh o s p h a t a st hea) tp r o d u c eas visibleAnterior(head)is up,ventralisto the left Icourtesy of L
coloredreaction productAfterexcess antibodyhasbeenremoved, Milenkovic andN/ P Scott l

fibroblastsare labeledwith differently colored fluorescent the many different changesin cell physiology that occur
dyes.A DNA array comprising8600 mammaliangenesthen when cells are transferred from one medium to another.
is incubatedwith a mixture containingequal amountsof the I n o t h e r w o r d s , g e n e st h a t a p p e a rt o b e c o - r e g u l a t e di n a
t w o c D N A p r e p a r a t i o n su n d e r h y b r i d i z a t i o nc o n d i t i o n s . s i n g l e m i c r o a r r a y e x p r e s s i o ne x p e r i m e n t m a y u n d e r g o
After unhybridizedcDNA is washed away, the intensity of changesin expressionfor very different reasonsand may
green and red fluorescenceat each DNA spot is measured a c t u a l l y h a v e v e r y d i f f e r e n t b i o l o g i c a l f u n c t i o n s .A s o l u -
using a fluorescence microscopeand storedin computerfiles t i o n t o t h i s p r o b l e m i s t o c o m b i n et h e i n f o r m a t i o n f r o m a
under the name of eachgeneaccordingto its known position set of expressionarray experimentsto find genesthat are
on the slide.The relativeintensitiesof red and greenfluores- similarly regulated under a variety of conditions or over a
cencesignalsat eachspot are a ffreasureof the relativelevel oeriod of time.
of expressionof that genein resp()nseto serum.Genesthat This more informative use of multiple expressionarray
are not transcribedunder thesegrowth conditions give no experiments is illustrated by examining the relative expres-
detectablesignal. Genesthat are transcribed at the same sion of the 8600 genesat different times after serum addi-
level under both conditions will hybridize equally to both tion, generatingmore than 104 individual piecesof data. A
r e d a n d g r e e n - l a b e l e dc D N A p r e p a r a t i o n s .M i c r o a r r a y computer program, relatedto the one usedto determinethe
analysisof geneexpressionin fibroblastsshowedthat tran- relatedness of differentprotein sequences' can organizethese
scriptionof about 500 of the 8600 genesexaminedchanged data and cluster genes that show similar expression over the
substantiallyafter addition of serum. time course after serum addition. Remarkably, such cluster
analysis groups sets of genes whose encoded proteins partic-
ipate in a common cellular process, such as cholesterol
C l u s t e rA n a l y s i so f M u l t i p l e E x p r e s s i o n
biosynthesisor the cell cycle (Figure5-30).
E x p e r i m e n t sl d e n t i f i e sC o - r e g u l a t e G
d enes
analysiswill be a powerful
F i r m c o n c l u s i c l n sr a r e l y c a n b e d r a w n f r o m a s i n g l e m i - F.i In the future, microarray
c r o a r r a y e x p e r i m e n t a b o u t w h e t h e r g e n e st h a t e x h i b i t fiil di"enustic tool in medicine. For instance,particular
s i m i l a r c h a n g e si n e x p r e s s i o na r e c o - r e g u l a t e da n d h e n c e setsof .RXRt have beenfound to distinguishtumors with a
likely to be closely related functionally. For example, poor prognosisfrom those with a good prognosis.Previously
m a n y o f t h e o b s e r v e dd i f f e r e n c e si n g e n e e x p r e s s i o nj u s t indistinguishablediseasevariations are now detectable'
d e s c r i b e di n f i b r o b l a s t sc o u l d b e i n d i r e c t c o n s e < r u e n c o
e fs Analysisof tumor biopsiesfor thesedistinguishingmRNAs

U S I N GC L O N E DD N A F R A G M E N TTSO S T U D YG E N EE X P R E S S I O N . 193
 Technique
Animation:Synthesizing Array flllt
an Oligonucleotide
TechniqueAnimation: Screeningfor Patternsof Gene Expression
Fibroblasts < EXPERIMENTAL FIGURE 5-29 DNAmicroarrayanalysiscan
w i t h o u ts e r u m revealdifferencesin gene expressionin fibroblastsunder
differentexperimentalconditions.(a)In thisexample, cDNA
prepared frommRNAisolated fromfibroblasts eitherstarved for
serumor afterserumadditionislabeled with differentfluorescent
dyesA microarray composed of DNAspotsrepresenting 8600

Green
dye--J
I t s o t a tteo t a tm R N A I

Reverse-transcribe I
+
mammalian
preparations
intensities
genesisexposed to an equalmixture
underhybridization conditions
of redandgreenfluorescence
of thetwo cDNA
Theratioof the
overeachspot,detected
to cDNA labeledwith lr/ with a scanning confocal lasermicroscope, indicates the relative
a fluorescentdye { expression of eachgenein response to serum.(b)A micrograph of a
smallsegment of an actualDNAmicroarray. Eachspotin this 16 x
16arraycontains DNAfroma different genehybridized to control
andexperimental cDNAsamples labeled with redandgreen
fluorescentdyes(A yellowspotindicates equalhybridization of

cDNAs hvbridized to J Hybridizeto DNA

mrcroarray
greenandredfluorescence,
expression) (b)Alfred
[Part
indicating
Pasieka/Photo
no changein gene
Researchers,
Inc]
DNAs for a single gene
I Wash

I Measuregreen and red

fluorescenceover eachspot
will help physiciansto selectthe most appropriate treat-
ment. As more patternsof geneexpressioncharacteristicof
various diseasedtissuesare recognized,the diagnostic use
of DNA microarrays will be extendedto other conditions.I

E. coliExpressionSystemsCan ProduceLarge
Quantitiesof Proteinsfrom ClonedGenes

Many protein hormones and other signalingor regu-

latory proteins are normally expressedat very low
A lf a spot is green,expressionof that gene decreasesin concentrations,precluding their isolation and purification
cells after serum addition in large quantities by standard biochemical techniques.
.E l f a s p o t i s r e d ,e x p r e s s i o no f t h a t g e n e i n c r e a s e isn c e l l s Widespread therapeutic use of such proteins, as well as
after,seru
m,addition basicresearchon their structureand functions, dependson
efficient proceduresfor producing them in large amounts
at reasonablecost. RecombinantDNA techniquesthat turn
E. coli cells into factories for synthesizinglow-abundance
proteins now are used to commercially produce granulo-
cyte colony-stimulating factor (G-CSF), insulin, growth
hormone, and other human proteins with therapeuticuses.
For example, G-CSF stimulatesthe production of granulo-
cytes, the phagocytic white blood cells critical to defense
against bacterial infections. Administration of G-CSF to
cancer patients helps offset the reduction in granulocyte
production caused by chemotherapeuticagents, thereby
protecting patients againstseriousinfection while they are
receiving chemotherapy.I

The first step in producing large amounts of a low-

abundanceprotein is to obtain a cDNA clone encoding the
full-length protein by methods discussedpreviously.The sec-
ond step is to engineerplasmid vectorsthat will expresslarge
amounts of the encoded protein when it is inserted into
E. coli cells.The key to designingsuch expressionvectors is
inclusion of a promoter, a DNA sequencefrom which tran-
scription of the cDNA can begin. Consider,for example, the

194 c H A P T E R5 | M O L E C U L AG
R E N E T TTCE C H N T Q U E S
 Eachcolumn representsa differentgene at
times after addition of serum

0)
E
-

-r-
A BCD

A EXPERIMENTAL FIGURE 5-30 Clusteranalysisof data from icantchangein expression.

signif The"tree"diagram at thetop
multiple microarrayexpressionexperimentscan identify co- showshowthe expression patterns genescanbe
for individual
regulatedgenes.Theexpression of 8600mammalian geneswas organized fashion
in a hierarchical to grouptogetherthe geneswith
detected by microarray
analysisat timeintervalsovera 24-hour the greatest in theirpatterns
similarity overtime'Five
of expression
periodafterserum-starvedfibroblasts wereprovidedwith serum.The clustersof coordinately regulated geneswereidentified in this
diagram
cluster shownhereisbasedon a computer algorithm that experiment, asindicated by the barsat the bottom.Eachcluster
groupsgenesshowingsimilar changes compared
in expression with a contains multiple geneswhoseencoded proteins
functionin a
serum-starvedcontrolsample overtime.Eachcolumnof colored particularcellularprocess: cholesterol (A),thecellcycle
biosynthesis
boxesrepresentsa singlegene,andeachrow represents a time (B),the immediate-early response (C),signalingandangiogenesis
point.A redboxindicates an increase relative
in expression to the (D),andwoundhealing andtissueremodeling (E).[Courtesyof Michael
control;a greenbox,a decrease in expression;anda blackbox,no B Eisen,Lawrence BerkeleyNationalLaboratoryl

relatively simple system for expressing G-CSF shown in

Figure 5-31. In this case,G-CSF is expressedin E. coli trans-
formed with plasmid vectors that contain the lac promoter
adjacentto the cloned cDNA encoding G-CSF.Transcription
from the lac promoter occurs at high rates only when lac-
tose, or a lactose analog such as isopropylthiogalactoside
(IPTG), is added to the culture medium. Even larger quanti-
(- -*R^*fu
\/
ties of a desired protein can be produced in more compli- , p-galactosidase.
cated E. coli expressionsystems.
To aid in purification of a eukaryotic protein produced in - IPTG
an E. coli expression system, researchersoften modify the
cDNA encodingthe recombinant protein to facilitate its sep- (b)
aration from endogenousE. coli proteins. A commonly used
modification of this type is to add a short nucleotide
sequenceto the end of the cDNA, so that the expressedpro-
tein will have six histidine residuesat the C-terminus. Pro-
teins modified in this way bind tightly to an affinity matrix

> EXPERIMENTAL FIGURE 5-31 Someeukaryoticproteinscan

be producedin E coli cellsfrom plasmidvectorscontainingthe
lac promoter.(a)Theplasmidexpression vectorcontainsa fragment
of the E colichromosome containingthe /acpromoterandthe Transform
E. coli
neighboring lacZgene.Inthe presence analogIPTG,
of the lactose
RNApolymerase normallytranscribesthe iacZgene,producing /acZ
mRNA,whichistranslated intotheencoded protein,B-galactosidase
(b)The/acZgenecanbe cut out of the expression vectorwith
restriction
enzymes andreplaced by a clonedcDNA,in thiscaseone
encoding granulocyte colony-stimulatingfactor(G-CSF).Whenthe
resultingplasmid istransformed intoE.collcells,additionof IPTG
andsubsequent fromthe/acpromoter
transcription produce G-CSF
mRNA.whichistranslated intoG-CSF protein - IPTG + IPTG

G E N EE X P R E S S I O N
TO STUDY
U S I N GC L O N E DD N A F R A G M E N T S 195
that contains chelated nickel atoms, whereas most E. coli {a) Transient transfection
proteins will not bind to such a matrix. The bound proteins cDNA
can be releasedfrom the nickel atoms by decreasingthe pH
of the surrounding medium. In most cases,this procedure
yields a pure recombinant protein that is functional, since
addition of short amino acid sequencesto either the C-
terminusor the N-terminus of a protein usually doesnot in-
terfere with the protein's biochemicalactivity.
I transtectcultured
I c e l l sb y l i p i dt r e a t m e n t
PlasmidExpressionVectorsCan Be Designedfor or electroporation
J
U s ei n A n i m a lC e l l s
\Vhile bacterial expressionsystemscan be used successfully
to createlarge quantitiesof someproteins, bacteriacannot be
used in all cases.Many experimenrsto examine the function
of a protein in an appropriate cellular context requlre expres- f r o m c D N A i n p l a s m i dD N A
P r o t e i ni s e x p r e s s e d
sion of a geneticallymodified protein in cultured animal cells.
Genesare cloned into specializedeukaryotic expressionvec, (b) Stable transfection (transformation)
tors and are introduced into cultured animal cells by a
processcalled transfection.Two common methods for trans- cDNA
Promoter
fecting animal cells differ in whether the recombinant vecor
DNA is or is not integratedinto the host-cellgenomic DNA.
In both methods,culturedanimal cellsmust be treatedto
facilitatetheir initial uptake of the recombinantplasmidvec-
tor. This can be done by exposingcells to a preparationof
lipids that penetrarethe plasma membrane, increasingits
permeability to DNA. Alternatively, subjecting cells to a I Transfect cultured
I c e l l sb y l i p i dt r e a t m e n t
brief electric shock of severalthousand volts, a technique or electroporation
known as electroporation, makes them transiently perme- {
ableto DNA. Usuallythe plasmidDNA is addedin sufficient
concentrationto ensurethat a large proportion of the cul-
tured cellswill receiveat leastone copy of the plasmidDNA.
Researchers have also harnessedvirusesfor their use in the
laboratory; virusescan be modified ro contain DNA of in-
terest, which is then introduced into host cells by simply
infecting them with the recombinant virus.

Transient Transfection The simplestof the two expression

methods, calledtransient transfectioz, employs a vector similar
to the yeast shuttle vectors describedpreviously. For use in
mammalian cells,plasmid vectorsare engineeredalso to carry
an origin of replicationderivedfrom a virus that infecrsmam-
malian cells, a strong promoter recognized by mammalian
RNA polymerase,and the cloned cDNA encodingthe protein
to be expressedadjacentto the promoter (Figure5-32a). Once
such a plasmid vector entersa mammalian cell, the viral origin
of replication allows it to replicate efficientl5 generating
numerousplasmidsfrom which the protein is expressed.How-
ever,duringcell divisionsuchplasmidsare nor faithfullysegre-
gatedinto both daughtercellsand in time a substantialfraction
of the cells in a culture will not contain a plasmid, hencethe
name t ransi ent t ransfecti on.
Stable Transfection (Transformation) If an introduced vec-
tor integratesinto the genomeof the host cell, the genomeis
permanentlyalteredand the cell is said to be transformed.ln-
tegration most likely is accomplishedby mammalian enzymes
that normally function in DNA repair and recombination.
Proteinis expressedfrom cDNA integrated
i n t o h o s tc h r o m o s o m e
A EXPERIMENTAL FIGURE5-32 Transientand stable transfection
with specially designed plasmid vectors permit expression of
cloned genes in cultured animal cells.Bothmethodsemployplasmid
vectorsthat containthe usualelements-ORl,selectablemarker(e.g ,
amp'), and polylinker-that permitpropagationin E. coli and inserlionof
a clonedcDNAwith an adjacentanimalpromoter.Forsimplicity, these
elementsarenot depicted(a)In transient transfection,the plasmid
vectorcontainsan originof replication for a virusthat can replicatern the
culturedanimalcells Sincethe vectoris not incorporated into the genome
of the culturedcells,production of the cDNA-encoded proteincontinues
onlyfor a limitedtime (b) In stabletransfection, the vectorcarriesa
selectable markersuchas neo',whichconfersresistance to G-418,The
relatively
few transfectedanimalcellsthat integratethe exogenousDNA
into theirgenomesareselected on mediumcontainingG-418 Because
the vectoris integratedinto the genome,thesestablytransfected,or
transformed, cellswill continueto producethe cDNA-encoded protein
as longasthe cultureis maintainedSeethe textfor discussion

196 c H A P T E R5 | MOLECULAG
R E N E T TTCE C H N T Q U E S
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AUTOSOMAL RECESSIVE

Sickle-cellanemia Abnormal hemoelobin causesdeformation of 11625of sub-SaharanAfrican origin

red blood cells, which can become lodged in
capillaries; also confers resistanceto malaria'

Cysric fibrosis Defective chloride channel (CFTR) in epithelial cells 1'12500of European origin
leads to excessivemucus in lungs.

Phenylketonuria (PKU) Defective enzyme in phenylalanine metabolism 1/10,000 of European origin

(tyrosine hydroxylase) results in excessphenylalanine,
leading to mental retardation, unless restricted by diet.

Tay-Sachsdisease Defective hexosaminidaseenzyme leads to accumulation 1/1000 easternEuropean Jews

of excesssphingolipids in the lysosomesof neurons,
impairing neural development.

AUTOSOMAL DOMINANT

Huntington's disease Defective neural protein (huntingtin) may assembleinto 1/10,000of Europeanorigin
aggregatescausing damage to neural ttssue.

Hypercholesterolemia Defective LDL receptor leads to excessivecholesterol in tlI22French Canadians

blood and early heart attacks.

X-LINKED RECESSIVE

Duchenne muscular Defective cytoskeletal protein dystrophin leads to 1/3500 males

dystrophy (DMD) impaired muscle function.

Hemophilia A Defective blood clotting factor VIII leads to 1-2110,000 males

uncontrolled bleedine.

provide clues to the molecular and cellular causeof the dis-
ease.Historically, researchershave usedwhatever phenotypic
clues might be relevant to make guessesabout the molecular
basis of inherited diseases.An early example of successful
guessworkwas the hypothesisthat sickle-cellanemia,known
to be a diseaseof blood cells,might be causedby defectivehe-
moglobin. This idea led to identification of a specificamino
acid substitution in hemoglobin that causespolymerization
of the defectivehemoglobin molecules,causingthe sickle-like
deformation of red blood cellsin individuals who have inher-
ited two copiesof the Hb' allele for sickle-cellhemoglobin.
Most often, however,the genesresponsiblefor inherited
diseasesmust be found without any prior knowledge or rea-
sonablehypothesesabout the nature of the affectedgeneor its
encodedprotein. In this section,we will seehow human ge-
neticistscan find the generesponsiblefor an inherited disease
by following the segregationof the diseasein families' The
segregationof the diseasecan be correlatedwith the segrega-
tion of many other genetic markers, eventually leading to
identification of the chromosomalposition of the affected
gene.This information, along with knowledgeof the sequence
of th. hn-".t genome,can ultimately allow the affectedgene
and the disease-causingmutations to be pinpointed.
s howOneof Three
M a n y I n h e r i t e dD i s e a s e S
Major Patternsof Inheritance
Human genetic diseasesthat result from mutation in one
specific gene exhibit severalinheritance patterns depend-
ing on the nature and chromosomal location of the alleles
that causethem. One characteristicpattern is that exhib-
ited by a dominant allele in an autosome (that is, one of
the 22 human chromosomes that is not a sex chromo-
some). Becausean autosomal dominant allele is expressed
in the heterozygote,usually at least one of the parents of
an affected individual will also have the disease'It is of-
ten the case that the diseasescaused by dominant alleles
appear later in life after the reproductive age. If this were
n o i t h e c a s e , n a t u r a l s e l e c t i o nw o u l d h a v e e l i m i n a t e d
the allele during human evolution. An example of an

H U M A N D I S E A SG
EENES . 199
I D E N T I F Y I NAGN D L O C A T I N G
 (al Autosomal AHD/A* x
probability of being carriers for the same recessivealleles.
d A+/A+ Q
dominant: Affected Thus children born to related parents are much more likely
Huntington's
disease I
Males and females
than those born to unrelated parents to be homozygousfor,
and thereforeaffectedby, an autosomalrecessivedisorder.
The third common pattern of inheritanceis that of an X-
AHD/A+ A-/A-
Linked recessiueallele. A recessiveallele on the X chromo-
Affected Not affected
some will most often be expressedin males,who receiveonly
(b) Autosomal one X chromosome from their mother, but not in females,
d AcFrR/A+ x AcFrR/A+ ?
recessive: Carrier Carrier
who receivean X chromosome from both their mother and
,
Cystic fibrosis I their father. This leadsto a distinctive sex-linked segregation
* pattern where the diseaseis exhibited much more frequently
Males and females
in males than in females.For example, Duchenne muscular
ACFTR /ACFTR ACFTR/A+ A+/AcFrR A-/A- dystrophy(DMD), a muscledegenerative
Affected Carrier Carrier Noncarrier diseasethat specif-
ically affects males, is causedby a recessiveallele on the X
(c) X-finked recessive:
chromosome. DMD exhibits the typical sex-linked segrega-
d X* ly x XDMD/X+ Q tion pattern in which mothers who are heterozygous and
Duchennemuscular Carrier
dystrophy I therefore phenotypically normal can act as carriers, rrans-
v mitting the DMD allele, and therefore the disease,to 50 per-
Males Females
cent of their male progeny (Figure5-35c).
PMDIY x+ly YDMD/x+ x+/x+
Affected Unaffected Carrier Noncarrier
D N A P o l y m o r p h i s mAs r e U s e di n L i n k a g e -
FIGURE 5-35 Threecommoninheritancepatternsfor human M a p p i n gH u m a nM u t a t i o n s
geneticdiseases. Wild-type autosomal (A)andsexchromosomes (X Once the mode of inheritancehas beendetermined,the next
andY)areindicated by superscript plussigns(a)In an autosomal
d o m i n a ndti s o r d es ru c ha sH u n t i n g t osnd i s e a soen, l yo n em u t a n t step in determining the position of a diseaseallele is to ge-
alleleisneeded to conferthe diseaself eitherparentis heterozygous netically map its position with respect to known genetic
for the mutantHDallele,hisor herchildren markers using the basic principle of genetic linkage as de-
havea 50 percent
c h a n coef i n h e r i t i nt h
g em u t a nat l l e l e
a n dg e t t i n g
t h ed i s e a s(eb )l n scribed in Section5.1. The presenceof many different al-
an autosomal recessive disorder suchascystic fibrosis, two mutant ready mapped genetic rraits, or markers, distributed along
alleles mustbe present to conferthe diseaseBothparents mustbe the length of a chromosome facilitatesthe mapping of a new
heterozygous carriers of the mutantCFIRgenefor theirchildren to mutation by assessingits possiblelinkage to these marker
be at riskof beingaffected or beingcarriers(c)An X-linked recessive genesln appropriate crosses.The more markers that are
disease suchasDuchenne muscular dystrophy iscauseo oy a available,the more preciselya mutarion can be mapped.The
recessive mutation on theX chromosome andexhibits thetypicalsex_ density of genetic markers neededfor a high-resolution hu-
linkedsegregatron patternMalesbornto mothers heterozygous for man genetic map is about one marker every 5 centimorgans
a mutantDMDallelehavea 50 percent chance of inheriting the (cM) (as discussedpreviously,one geneticmap unir, or cen-
mutantalleleandbeingaffectedFemales bornto heterozvqous timorgan, is defined as the distance between two positions
mothers havea 50 percent chance of beinqcarriers along a chromosome that results in one recombinant indi-
vidual in 100 progeny).Thus a high-resolutiongenericmap
requires25 or so geneticmarkersof known position spread
a u t o s o m a l d o m i n a n t d i s e a s ei s H u n t i n g t o n ' s d i s e a s e ,a along the length of each human chromosome.
neural degenerativediseasethat generally strikes in mid- In the experimentalorganismscommonly used in genetic
to late life. If either parenr carries a murant HD allele, studies,numerous markers with easily detectablephenotypes
e a c h o f h i s o r h e r c h i l d r e n ( r e g a r d l e s so f s e x ) h a s a 5 0 are readily availablefor geneticmapping of mutations.This is
percent chance of inheriting rhe mutanr allele and being not the casefor mapping geneswhose mutant allelesare asso-
a f f e c t e d( F i g u r e5 - 3 5 a ) . ciated with inherited diseasesin humans. However" recombi-
A recessiveallelein an autosomeexhibits a quite different nant DNA technologyhas made availablea wealth of useful
segregatronpattern. For an autosomal recessiueallele, both DNA-based molecular markers. Becausemost of the human
parentsmust be hererozygouscarriersof the allelein order for genomedoesnot code for protein, alarge amount of sequence
their children to be at risk of being affectedwith the disease. variation exists betweenindividuals. Indeed, it has been esti-
Each child of heterozygousparenrshas a 25 percentchanceof mated that nucleotidedifferencesbetweenunrelatedindividu-
receiving both recessiveallelesand thus being affected,a 50 als can be detectedon an averageof every 103 nucleotides.If
percentchanceof receivingone normal and one mutant allele thesevariations in DNA sequence,referredto as DNA poly-
and thus being a carrier, and a 25 percentchanceof receiving morphisms,can be followed from one generationto the next,
two normal alleles.A clear exampleof an autosomalrecessive they can serve as genetic markers for linkage studies. Cur-
diseaseis cystic fibrosis, which results from a defectivechlo- rently, a panel of as many as 104 different known polymor-
ride-channelgeneknown as CFTR (Figure5-35b). Relatedin- phisms whose locations have been mapped in the human
dividuals (e.g.,first or secondcousins)have a relatively high genomeis usedfor geneticlinkage studiesin humans.

200 . c H A p r EsR I M o L E c u L AGRE N E Tr tEcc H N l o u E s

 Restriction fragment length polymorphisms (RFLPs)
were the first type of molecular markers used in linkage
studies.RFLPsarisebecausemutationscan createor destroy
the sitesrecognizedby specificrestriction enzymesthat hap-
pen to lie in human DNA, leadingto variationsbetweenin-
dividuals in the length of restriction fragments produced
from identical regionsof the genome.Differencesin the sizes
of restriction fragments betweenindividuals can be detected
by Southern blotting with a probe specific for a region of
DNA known to contain an RFLP (Figure5-36a).The segre-
gation and meiotic recombination of such DNA polymor-
phisms can be followed like typical genetic markers. Figure
5-36b illustrates how RFLP analysis of a family can detect
the segregaticlnof an RFLP that can be used to test for sta-
tistically significant linkage to the allele for an inherited dis-
easeor some other human trait of interest.
The amassedgenomic sequenceinformation from differ-
ent humans has led to identification of other useful DNA
polymorphisms in recent years. Single-nwcleotidepolymor-
phisms (SNPs) constitute the most abundant type and are
therefore useful for constructing geneticmaps of maximum
resolution. Another useful type of DNA polymorphism con-
sistsof a variable number of repetitionsof a one- two-, or
three-basesequence. Suchpolymorphisms,known as simple
sequencerepeats (SSRs)or microsatellites, presumably are
formed by recombinationor a slippagemechanismof either
the template or newly synthesizedstrands during DNA
replication. A useful property of SSRsis that different indi-
viduals will often have different numbers of repeats.The ex-
istenceof multiple versionsof an SSRmakes it more likely to
produce an informative segregationpattern in a given pedi-
greeand therefore be of more generalusein mapping the po-
sitionsof diseasegenes.If an SNP or SSRaltersa restriction
site, it can be detectedby RFLP analysis. More commonly'
however, these polymorphisms do not alter restriction frag-
ments and must be detectedby PCR amplification and DNA
sequenclng.
LinkageStudiesCanMap Disease Geneswith a
Resolutionof About 1 Centimorgan
lVithout going into all the technical considerations,let's see
how the allele conferring a particular dominant trait (e.9.,
familial hypercholesterolemia)might be mapped. The first
step is to obtain DNA samplesfrom all the members of a
family containing individuals that exhibit the disease.The
DNA from each affected and unaffected individual then is
analyzed to determine the identity of a large number of
known DNA polymorphisms (either SSR or SNP markers
can be used). The segregationpattern of each DNA poly-
morphism within the family is then compared with the
segrigation of the diseaseunder study to find those poly-
morphisms that tend to segregatealong with the disease'
Finaily, computer analysisof the segregationdata is used to

lat Hybridization (b)

banding pattern G randparents
Grandparents
from individual
with both allele 1
Chromosomalarrangement and allele2

't'iri
Enzyme Enzynre
; i't 't B
g
Mutation at site a2
: : Preventscleavage
t 'ti i+ .1, Alleles Fragment
lengths
I
tr F-okb-l
R e s t r i c t i o ne n d o n u c l e a s A
e
,f r,,*, Ilikb-l
As
j
v
B
R e s t r i c t i o ne n d o n u c l e a s e

E
tr .!!, Fs kb-l
[ - - - l P r o b e ds i n g l e - c o p yr e g i o n

A EXPERIMENTAL FIGURE5-36 Restrictionfragment length (a1-a2anda1-a3),and two bandsare seen,indicatingthat a

polymorphisms (RFLPs) can be followed like genetic markers. mutationhascausedthe lossof one of the a sitesin one of the two
chromosomes(b) Pedigreebasedon RFLP analysis of the DNA from a
(a) In the two homologouschromosomes shown,DNA is treatedwith
two differentrestrictionenzymes(A and B),which cut DNA at regionknown to be presenton chromosome5. The DNA samples
differentsequences (a and b) The resultingf ragmentsare subjected were cut with the restriction enzymeIaql and analyzedby Southern
(seeFigure5-26)with a radioactive probe blotting In this family, this region of the genomeexistsin threeallelic
to Southernblot analysis
that bindsto the indicatedDNA region(green)to detectthe forms characterized by Iaql sitesspaced10, 7 '7, or 6 5 kb apart
fragments Sinceno differences betweenthe two homologous Eachindividualhastwo alleles;somecontainallele2 (7 7 kb) on both
recoqnizedby the B enzyme, chromosomes, and othersare heterozygous at this site Circles
chromosomes occurin the sequences
indicatefemales;squares indicate males The gel lanesare in the
only one fragmentis recognized by the probe,as indicatedby a
same order as the subjects in the pedigree above and are generally
singlehybridizationband However,treatmentwith enzymeA
r D o n i s - K ee
a l i g n e db e l o wt h e m l A f t e H , e l5l 1 : 3 1 ]9
l l tearl , 1 9 8 7C
producesradiographically distinctf ragmentsof two differentlengths

. 201
I D E N T I F Y IA
NNGD L O C A T I NHGU M A ND I S E A SGEE N E S
 calculatethe likelihood of linkage between each DNA poly-
morphism and the disease-causing f f l cr"ototot"
allele. Generation Newmu+6+:^h
In practice, segregation data are collected from different 1 inp.,.ti",l?i'.":
\ | | F Yitl dirrerent
families exhibiting the same diseaseand pooled. The more
huptotvp?'' mf I J nantotvoe
families exhibiting a parricular diseasethaican be examined, fVeioti"recombination
the greater the statistical significance of evidence for linkage I

ilI tl ff
that can be obtained and the grearerthe precisionwith which
Generation
the distancecan be measuredbetween a linked DNA oolvmor- 2
phism and a diseaseallele.Most family studieshave a maxi-
mum of about 100 individualsin which linkage betweena dis-
easegene and a panel of DNA polymorphismscan be tested.
This number of individuals setsthe practical upper limit on the
resolutionof sucha mapping study to about 1 cenrimorgan,or
a physicaldistanceof about 7.5 x 10s basepairs.
I
A phenomenoncalledlinkagedisequilibriumisthe basisfor
I
an alternative strategy,which in some casescan afford a higher
'""tt''""
ll iltl ililililt|
degreeof resolution in mapping studies.This approach de-
pendson the particular circumstancein which a genericdisease
commonly found in a particular population resultsfrom a sin-
gle mutation that occurredmany generationsin the past. The
A FIGURE 5-37 Linkagedisequilibrium studiesof human
DNA polymorphismscarriedby this ancestralchromosomeare populationscan be usedto map genesat high resolution.A
collectively known as the haplotype of that chromosome. As newdisease mutationwillarisein the contextof an ancestral
the diseasealleleis passedfrom one generationto the next, only chromosome amonga setof polymorphisms known asthehaplotype
the polymorphisms that are closestto the diseasegenewill not (indicated
by pinkshading).Aftermanygenerations, chromosomes
be separatedfrom it by recombination. After many generarlons thatcarrythe diseasemutation willalsocarrysegments of the
the region that contains the diseasegenewill be evident because ancestral
haplotype thathavenot beenseparated fromthe disease
this will be the only region of the chromosome thar will carry mutationby recombination Thebluesegments of these
the haplotype of the ancestralchromosome conservedthrough chromosomes general
represent haplotypes derived fromthegeneral
many generations(Figure5-37).By assessing the distributionof population andnotfromtheancestral haplotype in whichthe
specificmarkersin all the affectedindividualsin a population, mutatronoriginallyaroseThisphenomenon is knownaslinkage
geneticistscan identify DNA markers tightly associatedwith disequilibriumThepositionof the diseasemutationcanbe located
by scanning chromosomes containing thedisease mutation for highly
conserved polymorphismscorresponding to theancestral haplotype

from normal and affected individuals may suggesttissuesin

appearedon the ancestralchromosome-in somecasesthis can
amount to finding markersthat are so closelylinked to the dis-
easegenethat even after hundredsof meiosesthey have never
beenseparatedby recombinarion.
FurtherAnalysisls Neededto Locatea Disease
G e n ei n C l o n e dD N A
Although linkage mapping can usually locare a human dis-
easegene to a region containing about 105 base pairs, as
many as 10 different genesmay be located in a region of this
size. The ultimate objective of a mapping study is to locate
the genewithin a cloned segmenrof DNA and then to deter-
mine the nucleotide sequenceof this fragment. The relative
scalesof a chromosomalgenetic-ap anJphysicalmaps cor-
responding to ordered sers of plasmid clones and the nu-
cleotidesequenceare shown in Figure5-38.
One strategyfor further localizinga diseasegenewithin the
genome is to identify mRNA encoded by DNA in the region of
the geneunder study. Comparison of geneexpressionin tissues
which a particular diseasegenenormally is expressed.For in-
stance,a mutation that phenotypically affects muscle, but no
other tissue,might be in a genethat is expressedonly in muscle
tissue. The expressionof mRNA in both normal and affected
individuals generally is determined by Northern blotting or in
situ hybridization of labeledDNA or RNA to tissuesections.
Northern blots, in situ hybridization, or microarray experi-
ments permit comparison of both the level of expression and
the size of mRNAs in mutant and wild-type tissues.Although
the sensitivity of in situ hybridization is lower than that of
Northern blot analysis,it can be very helpful in identifying an
mRNA that is expressedat low levels in a given tissue but at
very high levels in a subclassof cells within that tissue.An
mRNA that is altered or missing in various individuals affected
with a diseasecomparedwith wild-type individualswould be
an excellent candidate for encoding the protein whose dis-
rupted function causesthat disease.
In many cases,point mutations that give rise to disease-
causing allelesmay result in no detectablechange in the
level of expression or electrophoretic mobility of mRNAs.
Thus if comparison of the mRNAs expressedin normal and
affected individuals reveals no detectabledifferencesin the
candidate mRNAs, a search for point mutarions in the

2O2 . c H A p r E 5R | M o l E c u L AG
R E N E TrtEc c H N t e u E s
 Polymorphic
or BAC

(l

A
T
U
T
T
A
tr
A
A
T
(-
A
750 kb
T

A
c
u
T
A
T
T

'l
a
u
T
A
u
U
\T

LEVELOF
RESOLUTION:Cytogenetic Linkage Physical Sequence
map map map map

M E T H O DO F
DETECTION:Chromosome Linkageto restriction Hybridization Sanger
to plasmid (dideoxY)
b a n d i n gp a t t e r n f r a g m e n tl e n g t hp o l y -
Fluorescence in morPhismsRFLPS, clones sequenclng
s i t u h y b r i d i z a t i o n s i n g l en u c l e o t i d ep o l y -
(FISH) m o r P h i s m sS N P s ,a n d
s l m p l es e q u e n c e
repeatsSSRs

A FIGURE 5-38 The relationshipbetweenthe geneticand

physicalmapsof a humanchromosome' Thediagram a
deptcts
humanchromosome analyzed levels
at different of detailThe
chromosome asa wholecanbeviewedin the lightmicroscope when
it isin a condensed statethatoccurs at metaphase, andthe
FromGenes
et al, 2003,Genetics:
fromL Hartwell 2d
to Genomes'
approximate ic sequences
locationof specif canbe determined by lAdapted
fluorescence in sltuhybridization(FISH)At the nextlevelof detail, Hill
ed, McGraw I

DNA regions encoding the mRNAs is undertaken. Now s e s u l tf r o m M u l t i p l e

that highly efficientmethodsfor sequencingDNA are avail-
able, researchers frequently determinethe sequenceof can-
didate regions of DNA isolatedfrom affectedindividualsto
identify point mutations. The overall strategyis to search
for a coding sequencethat consistentlyshowspossiblydele-
reriousalterationsin DNA from individualsthat exhibit the
disease.A limitation of this approachis that the region near
the affectedgene may carry naturally occurring polymor-
phisms unrelated to the gene of interest. Such polymor-
phisms,not functionally related to the disease,can lead to
misidentificationof the DNA fragmentcarrying the geneof
interest.For this reason,the more mutant allelesavailable
for analysis,the more likely that a gene will be correctly
identified.
M a n y I n h e r i t e dD i s e a s e R
GeneticDefects
Most of the inherited human diseasesthat are now un-
derstood at the molecular level are monogenetic traits;
that is, a clearly discerniblediseasestate is produced by a
defect in a single gene.Monogenic diseasescausedby mu-
tation in one specific gene exhibit one of the characteris-
tic inheritance patterns shown in Figure 5-35' The genes
associatedwith most of the common monogenic diseases
have already been mapped using DNA-based markers as
describedpreviouslY.
However, many other inherited diseasesshow more
complicatedpatterns of inheritance,making the identifica-
tion of the underlying genetic causemuch more difficult'

H U M A N D I S E A SG
EENES O 203
I D E N T I F Y I NAGN D L O C A T I N G
 One type of added complexity that is frequently encoun-
tered is genetic heterogeneity.In such cases,mutations in
any one of multiple different genes can cause the same
disease.For example, retinitis pigmentosa,which is char-
acterizedby degenerationof the retina usually leading to
blindness,can be causedby mutations in any one of more
than 60 different genes. In human linkage studies, data
from multiple families usually must be combined to deter_
mine whether a statisricallysignificant linkage exists be_
t w e e n a d i s e a s eg e n e a n d k n o w n m o l e c u l a r m a r k e r s .
Genetic heterogeneitysuch as rhar exhibited by retinitis
pigmentosa can confound such an approach becauseany
statisticaltrend in the mapping data from one family tends
to be canceledout by the data obtained from another fam_
i l y w i t h a n u n r e l a t e dc a u s a t i v eg e n e .
Human geneticisrsused two different approachesto
_
identify the many genesassociatedwith retinitii pigmenrosa.
The first approach relied on mapping studies in &ception_
ally large single families rhar contained a sufficient number
of affectedindividuals to provide statisticallysignificant evi-
dencefor linkage betweenknown DNA polymolphisms and
a single causativegene. The genesidentified in such studies
retinitis pigmentosa.This approach of using additional in_
formation to focus screeningefforts on a s.rbiet of candidate
genesled to identificationof additional rare causativemura_
ldentifying and Locating Human DiseaseGenes
r Inherited diseasesand other traits in humans show
t h r e e m a j o r p a t t e r n s o f i n h e r i t a n c e :a u t o s o m a l d o m i -
nant, autosomal recessive,and X-linked recessive(see
F i g u r e5 - 3 5 ) .
r Genesfor human diseasesand other trairs can be mapped
by determining their cosegregation during meiosis with
markers whose locations in the genome are known. The
closer a geneis to a particular marker, the more likelv thev
are to cosegregate,
of human geneswith great precision requires
of molecular markers distributed along the
es. The most useful markers are differencesin
the DNA sequence(polymorphisms) between individuals
in noncoding regions of the genome.
r DNA polymorphisms useful in mapping human genesin-
clude restriction fragment length polymorphisms (RFLps),
single-nucleotidepolymorphisms (SNps), and simple se-
quencerepeats(SSRs).
inkage mapping often can locate a human diseasegene
a chromosomal region that includes as many as l0
genes.To identify the geneof interestwithin this candidate
region_typicallyrequires expressionanalysis and compari_
son of DNA sequencesbetween wild-type and disiase-
affectedindividuals.
r Some inherited diseasescan result from mutations in dif_
ferent genesin different individuals (geneticheterogeneity).
The occurrence and severity of other diseasesdepend on
the presenceof mutant allelesof multiple genesin the same
individuals (polygenictraits). Mapping of the genesassoci-
ated with such diseasesis particularly difficult becausethe
occurrenceof the diseasecannot readily be correlated to a
singlechromosomallocus.

ff,l Inactivatingthe Functionof

SpecificGenesin Eukaryotes
The elucidation of DNA and protein sequencesrn recent
y e a r s h a s l e d t o i d e n t i f i c a t i o no f m a n y g e n e s .u s i n g s e -
quencepatternsin genomicDNA and the sequencesimilar_
ity of the encodedproteins with proteins oi known func-
tion. As discussedin Chapter 6, the general functions of
proteins identified by sequencesearchesmay be predicted
by analogy with known proteins. However, the precisein
vivo roles of such "new" proteins may be lrnclearin the ab_
senceof mutant forms of the correspondinggenes.In this

CHAPTER
5 J MOLECULAG
RENETIC
TECHNIQUES
 Three basic approachesunderlie these gene-inactivation (a) 20-nt flanking
sequence
techniques:(1) replacinga normal genewith other sequences,
(2) introducing an allelewhose encodedprotein inhibits func-
tioning of the expressednormal protein, and (3) promoting
destructionof the mRNA expressedfrom a gene.The normal .-f-hi"_l
s y n r h sr i s
DNJA
*Fri-"|.
Z
endogenousgeneis modified in techniquesbasedon the first - -
approach but is not modified in the other approaches' - -

NormalYeastGenesCan Be Replacedwith
M u t a n t A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n Disruption
Modifying the genomeof the yeastS. cereuisiaeis particularly construct
easy for two reasons:yeast cells readily take up exogenous
DNA under certain conditions, and the introduced DNA is
efficiently exchangedfor the homologous chromosomal site (b)
in the recipient cell. This specific,targeted recombination of
identical stretchesof DNA allows any genein yeastchromo-
somesto be replacedwith a mutant allele. (As we discussin
Section5.1, recombination between homologous chromo-
somesalso occurs naturally during meiosis.)
In one popular method for disrupting yeast genesin this
fashion, the PCR is used to generatea disruption construct
containing a selectablemarker that subsequentlyis trans-
fectedinto yeastcells.As shown in Figure 5-39a,primersfor
PCR amplification of the selectablemarker are designedto
include about 20 nucleotidesidenticalwith sequences flank-
ing the yeast gene to be replaced. The resulting amplified
constructcomprisesthe selectablemarker (e.g.,the kdnMX
gene,which llke neo' confers resistanceto G-41 8 ) flanked by
about 20 basepairs that match the ends of the target yeast
gene. Transformeddiploid yeast cells in which one of the
two copiesof the target endogenousgenehas beenreplaced
by the disruption construct are identified by their resistance
to G-418 or other selectable phenotype.Theseheterozygous
diploid yeastcellsgenerallygrow normally regardlessof the Four
haploid
function of the target gene,but half the haploid sporesde- spores
rived from thesecellswill carry only the disruptedallele(Fig-
ure 5-39b). If a geneis essentialfor viability,then sporescar-
rying a disruptedallelewill not survive.
Disruption of yeastgenesby this method is proving par-
ticularly usefulin assessing the role of proteinsidentifiedby a EXPERIMENTA FLI G U R E5 - 3 9 H o m o l o g o u s r e c o m b i n a t i o n
analysisof the entire genomic DNA sequence(seeChapter with transfected disruption constructs can inactivate specific
g e n e s i n y e a s t . ( a ) A s u i t a b l ec o n s t r u cfto r d i s r u p t i n ga
6). A large consortiumof scientistshas replacedeach of the target
t a r g e tg e n ec a n b e p r e p a r e db y t h e P C R T . h et w o p r i m e r s
approximately 6000 genesidentified by this analysiswith
d e s i g n e df o r t h i s p u r p o s ee a c hc o n t a i na s e q u e n c eo f a b o u t 2 0
the kanMX disruption construct and determinedwhich gene yeast
n u c l e o t i d e(sn t )t h a t i s h o m o l o g o u st o o n e e n d o f t h e t a r g e t
disruptionslead to nonviablehaploid spores.Theseanalyses n e e d e dt o a m p l i f ya s e g m e n o
t f D NA
g e n ea s w e l l a s s e q u e n c e s
have shown that about 4500 of the 6000 yeastgenesare not
carryinga selectablemarkergene such as kanMX, which confers
resistance to G-418. (b) When recipientdiploid Saccharomyces
c e l l sa r et r a n s f o r m e dw i t h t h e g e n e d i s r u p t i o nc o n s t r u c t ,
h o m o l o g o u sr e c o m b i n a t i obne t w e e nt h e e n d so f t h e c o n s t r u c t
a n d t h e c o r r e s p o n d i ncgh r o m o s o m asl e q u e n c ew s i l l i n t e g r a t et h e

gene may be viable becauseof operation of backup or

compensatorypathways.To investigatethis possibility,yeast
geneticistscurrently are searchingfor syntheticlethal muta-
tions that might reveal nonessentialgeneswith redundant
functions(seeFigure 5-9c). nonvrable

O F S P E C I F IGCE N E SI N E U K A R Y O T E S
I N A C T I V A T I NTGH E F U N C T I O N
205
 (a) Formation of ES cells carrying a knockout mutation < EXPERIMENTAL FIGURE 5-40 lsolationof mouseEScellswith
neor fuHSV a gene-targeteddisruption is the first stage in productionof
knockoutmice.(a)Whenexogenous DNAisintroduced rnto
embryonic stem(ES) cells,randominsertion vianonhomologous
G e n eX r e p l a c e m e nct o n s t r u c t
recombrnation occursmuchmorefrequently thangene-targeted
Homologous ,/ r. insertion viahomologous recombination Recombinant cellsin which
recombination,? onealleleof geneX (orange
"il" andwhite)isdisrupted canbe obtained
./
r' by usinga recombinant
neo'(green), whichconfers
vectorthatcarries
resistance
geneX disrupted
to G-418,and,outside
with
the
regionof homology, tkHsv(yellow), thethymidine kinase genefrom
herpes simplex vrrusTheviralthymidlne kinase, unlikethe
; ES-ceilDNA endogenous mouseenzyme, canconvert the nucleotide analog
G e n eX O t h e rg e n e s ganciclovir intothe monophosphate form;thisisthenmodified to
I I t h et r i p h o s p h af o
t er m ,w h i c hi n h i b i tcse l l u l aDrN Ar e p l i c a t i o
i nnE S
I Gene-targeted II R a n d o m cellsThusganciclovir iscytotoxic for recombinant EScellscarrying
linsertion ltnserilon the tkHsv gene.Nonhomologous insertion includes the tkHsv gene,
v v whereas homologous insertion doesnot;therefore, onlycellswith
nonhomologous insertion aresensitive to ganciclovir
M u t a t i o ni n g e n eX N o m u t a t i o ni n g e n eX (b) Recombinant cellsareselected by treatment with G-41g,since
Cellsare resistantto C e l l sa r e r e s i s t a ntto cellsthatfailto pickup DNAor integrate it intotheirgenomeare
G - 4 1 8a n d g a n c i c l o v i r G - 4 1 8b u t s e n s i t i v e sensitive to thiscytotoxic compoundThesurvivrng recombinant cells
to ganciclovir aretreatedwith ganciclovir Onlycellswith a targeted disruption in
geneX, andtherefore lacklng the fkHsv geneanditsaccompanying
(b) Positive and negative selection of recombinant ES cells cytotoxicity, willsurvive[See S L Mansour etal, 1988,Nature336.348l

O O- N o n r e c o m b i n a ncte l l s

O 6+ Recombinantswith
gene-targetedinsertion A useful promoter for this purpose is the yeast GAL1
promoter, which is active in cells grown on galactose but
completely inactive in cells grown on glucose. In this ap-
| - r " . , w i t hG - 4 1 8 proach, the coding sequenceof an essentialgene (X) ligated
( p o s i t i vsee l e c t i o n )
J to the GALl promoter is inserted into a yeast shuttle u..to.
o.',oo (seeFigure 5-17a).The recombinant vector then is intro-
duced into haploid yeastcellsin which geneX has beendis-
o-oo rupted. Haploid cells that are transformed will grow on
Ag galactosemedium, since the normal copy of gene X on the
vector is expressedin the presenceof galactose.\fhen the
I fr"u, with ganciclovir
( n e g a t i v es e l e c t i o n ) cells are transferredto a glucose-containingmedium, geneX
|
* no longer is transcribed;as the cells divide, the amount of
o,
-o
rro c'l
o o-
E S c e l l sw i t h t a r g e t e dd i s r u p t i o ni n g e n eX

In an early application of this method, researchersex,

Transcriptionof GenesLigatedto a Regulated
P r o m o t e rC a n B e C o n t r o l l e dE x p e r i m e n t a l l y
Although disruption of an essenrialgene required for cell
g r o w t h w i l l y i e l dn o n v i a h l es p o r e sr.h i i m e r h o dp r o v i d e sl i m l e
infclrmation about what the encodedprotein actually does in
cells.To learn more about how a specificgenecontributesto
cell growth and viability investigarorsmust be able to selec_
tively inactivatethe genein a population of growing cells.One
method for doing rhis empkrysa regularedpro-oi.. to selec_
tively shut off transcription of an essentialgene.
plored the function of cytosolic Hsc70 genes ln yeasr.
Haploid cells with a disruption in all four redundant
Hsc70 geneswere nonviable, unlessthe cells carried a vec-
tor containing a copy of the Hsc70 gene that could be ex_
pressedfrom the GALI promoter on galactosemedium.
On transfer to glucose,the vector-carryingcells eventually
stopped growing becauseof insufficient Hsc70 acivity.
C a r e f u l e x a m i n a r i o n o f t h e s e d y i n g c e l l s r e v e a l e dt h a t
their secretory proteins could no longer enter the endo-
plasmic rericulum (ER). This study provided the first evi_
dencefor the unexpectedrole of Hsc70 protein in translo-
cation of secretory proteins into the ER, a process
e x a m i n e di n d e t a i l i n C h a p t e r 1 3 .

206 o c H A p r E5R I M o L E c u L AGRE N E Tr tEcc H N t o u E S

# uia"o: Microinjectionof ESCellsinto a Blastocyst

E gffl'"?
> EXPERIMENTAL FIGURE 5-41 EScellsheterozygous for a
disruptedgene are usedto producegene-targetedknockout ::l[T;"0?]X'J"'""'
mice.Step1: Embryonic stem(ES) cellsheterozygous for a knockout Brown mouse
(X andhomozygous for a dominant (NA, X-lx+)
mutationin a geneof interest
alleleof a markergene(here,browncoatcolor,A) aretransplanted B l a c km o u s e
(ala, X+lX+l
intothe blastocoel cavityof 4 5-dayembryos thatarehomozygous
for a recessivealleleof the marker(here,blackcoatcolor,a) Step2: 4.5-dayblastocyst
Theearlyembryos thenareimplanted intoa pseudopregnant female I
Thoseprogeny containing ES-derived cellsarechimeras, indicated
micethenare
by EIl"[';:Xl'.ffi
['Ji:;i]:il:;
theirmixedblackandbrowncoats.Step3: Chimeric *
backcrossed to blackmice;brownprogeny fromthismatinghaveE5-
derived cellsin theirgermline Steps 4-6: Analysis of DNAisolated A
froma smallamountof tailtissue canidentify brownmice
heterozygous for the knockout alleleIntercrossing of thesemice F o s t e rm o t h e r
produces someindividuals homozygous for the disrupted that
allele,
is,knockout mice.lAdapted fromM R.Capecchi, 1989,Trends
Genet5:70.1

SpecificGenesCan Be PermanentlyInactivated
Black
i n t h e G e r m L i n eo f M i c e
I S.t""t chimericmicefor
Many of the methods for disrupting genesin yeast can be ap- to wild-typeblackmice
I crosses
plied to genesof higher eukaryotes.Thesealteredgenescan be v
introduced into the germ line via homologous recombination
to produce animals with a gene knockout, or simply "knock-
out." Knockout mice in which a specificgeneis disrupted are a
powerful experimental systemfor studying mammalian devel- germcells: All germcells:
Possible
opment, behavior, and physiology. They also are useful in A/X+tA/X-: a/X* a/X+
studying the molecular basisof certain human geneticdiseases. =r I tt cell-deriveo
ProgenY
Gene-targetedknockout mice are generatedby a two-stage s
I will be brown
procedure.In the first stage,a DNA construct containing a dis- v
rupted allele of a particular target gene is introduced into em-
bryonic stem (ES)cells.Thesecells,which are derived from the
a/a, X+/X*
blastocyst,can be grown in culture through many generations
(seeFigure 21'-7).In a small fraction of transfectedcells, the
Progenyfrom ES cell-derived
introduced DNA undergoeshomologousrecombinationwith germcells
the target gene, although recombination at nonhomologous
chromosomal sitesoccurs much more frequently' To selectfor !| | s.re"n brown ProgenYDNA
-*
to identify X'lX+ heterozygotes
cells in which homologous gene-targetedinsertion occurs' the
al
recombinant DNA construct introduced into ES cells needsto EI Mate X-lX* heterozYgotes
include two selectablemarker genes (Figure 5-40). One of
thesegenes(neo'),which confersG-418 resistance,is inserted lil Screen Progeny DNA to identifY
- I
within the target gene (X), thereby disrupting it. The other se- l, X tx- homozYgotes
lectable gene, the thymidine kinase gene from herpes simplex
virus (lAHsv),confers sensitivity to ganciclovir, a cytotoxic nu-
cleotide analog; it is inserted into the construct outside the tar- Knockoutmouse
get-genesequence.Only ES cells that undergo homologous re-
Io-bitr"tiott, and therefore do not incorporate tkHsv, can
survivein the presenceof both G-418 and ganciclovir.In these
cellsone alleleof geneX will be disrupted. mozygous for a visible marker trait (e.g., coat color)' then
In the second stage in production of knockout mice' ES chimeric progeny in which the ES cells survived and prolifer-
cellsheterozygousfor a knockout mutation in geneX are in- ated can te identified easily.Chimeric mice are then mated
jected into a recipient wild-type mouse blastocyst,which with mice homozygous for another allele of the marker
subsequentlyis transferred into a surrogatepseudopregnant trait to determine if the knockout mutation is incorporated
female mouse (Figure 5-41). The resultingprogeny will be into the germ line. Finally, mating of mice' each heterozy-
chimeras, containing tissues derived from both the trans- gous for the knockout allele, will produce progeny ho-
plantedES cellsand the host cells.If the ES cellsalso are ho- mozygous for the knockout mutation.

C E N E SI N E U K A R Y O T E S
T H E F U N C T I O NO F S P E C I F I G
INACTIVATING
207
 loxP Cre
mouse mouse
A l l c e l l sc a r r ye n d o g e n o u sg e n e Heterozygousfor gene X knock-
Xwith /oxPsitesflankingexon 2 out; all cellscarry cre gene

Cell{ype-specif
ic
promoter

Cellsnot expressingCre CellsexpressingCre

-4lJ@

G e n ef u n c t i o ni s n o r m a r

IoxP-Cre
mouse
All cells carry one copy of loxp-
modified gene X, one copy of
g e n eX k n o c k o u ta, n d c r e g e n e s
-
FE
G e n ef u n c t i o n
isdisrupted

EXPERIMENTAL FTGURE 5-42 The loxp-Crerecombination functionof othergenesln the/oxp-Cre mtcethat resultfrom
system can knock out genes in specificcell types. A /oxpsjte is crossing, Creproteinisproduced onlyin thosecellsin whichthe
insertedon eachsideof the essential exon2 of the targetgeneX promoter isactiveThusthesearethe onlycellsin which
(blue)by homologousrecombination, producinga /oxpmouse.Since recombrnation between the/oxPsites catalyzed by Creoccurs,
the /oxPsitesare in introns,they do not disruptthe functionof X leading to deletion of exon2 Since the otheralleleisa constitutive
T h e C r e m o u s ec a r r i e o
s n e g e n eX k n o c k o u at l l e l ea n d a n i n t r o d u c e d geneX knockout, deletion between the/oxpsitesresults in complete
cre gene (orange)from bacteriophage p'l linkedto a cell-type_specific l o s so f f u n c t i o on f g e n e X i na l lc e l l se x p r e s s iC
ngr e B yu s i n g
promoter(yellow)The cre gene is incorporatedinto the mouse different promoters, researchers canstudytheeffects of knockinq
genomeby nonhomologousrecombination and doesnot affectthe out geneX in various typesof cells

Development of knockout mice that mimic certain
human diseasescan be illustrated by cystic fibrosis.
By methodsdiscussedin Section5.4, the recessrvemurarion
that causesthis diseaseeventuallywas shown to be located
in a geneknown as CFTR, which encodesa chloride chan_
rnice are currently being used as a model systemfor stud
ing this geneticdiseaseand developingeffectivetherapies
S o m a t i cC e l lR e c o m b i n a t i o C
n a nI n a c t i v a t e
G e n e si n S p e c i f i cT i s s u e s
Investigatorsoften are interestedin examining the effects
o f k n o c k o u t m u t a t i o n si n a p a r t i c u l a rt i s s u eo i t h e m o u s e ,
at a specificstagein development,or both. However, mice
carrying a germ-line knockout may have defectsin numer_
ous tissuesor die before the developmentalstageof inter_
est. To addressthis problem, mouse geneticistshave de_
v i s e d a c l e v e r t e c h n i q u et o i n a c t i v a l er a r g e r g e n e s l n
specifictypes of somatic cells or at particular times during
development
This techniqueemploys site-specificDNA recombination
sites(calledloxP sires)and the enzymeCre that catalyzesre-
combination between them. The loxP-Cre recombination
system is derived from bacteriophageP1, but this site-
specificrecombination sysremalso functions when placed in
mouse cells. An essentialfeature of this technique is that
expression of Cre is controlled by a cell-type-specificpro-
moter. In loxP-Cre mice generatedby the procedure depicted
in Figure 5-42,inactivation of the geneof interest (X) occurs
only in cells in which the promoter controlling the cre gene
is active.
An early application of this techniqueprovided strong
evidencethat a particular neurotransmitterreceptor is im-
portant for learning and memory. Previouspharmacological
and physiologicalstudieshad indicated that normal learn-
ing requiresthe NMDA classof glutamatereceptorsin the
hippocampus,a region of the brain. But mice in which the
gene encoding an NMDA receptor subunit was knocked
out died neonatally,precluding analysis of the receptor's
role in learning. Following the protocol in Figure 5-42, re-
searchersgeneratedmice in which the receptorsubunit gene
was inactivatedin the hippocampusbut expressedin other
tissues. These mice survived to adulthood and showed

208 . c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

llll+ Technique Mouse
Animation:creatinga Transgenic
z/ar\
\\"2 DNA injected into a Pronucleusof a Mouse

> EXPERIMENTAL FIGURE 5-43 Transgenic miceare produced Injectforeign DNA

intooneofthe pronuclei
by randomintegrationof a foreigngeneinto the mousegerm
line.Foreign DNAinjected intooneof thetwo pronuclei (themale
andfemalehaploidnucleicontributed bythe parents) hasa good
chance of beingrandomly integrated intothe chromosomes of the Pronuclei
diploidzygoteBecause a transgene is integrated intothe recipient
genomeby nonhomologous recombination, it doesnot disrupt
e n d o g e n o u s g e n[ S L B r i n s t e r e, t1a9l 8 1C
e se e R , e2l l7 . 2 7 3 1 Fertilizedmouse egg Prlor
t o f u s i o no f m a l e a n d
f e m a l ep r o n u c l e i

II Transferinjectedeggs
learning and memory defects,confirming a role for thesere- I into foster mother
I
ceptors in the ability of mice to encode their experiences +
rnto memory.

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y
l n h i b i t S o m eG e n e s
In diploid organisms,as noted in Section5.1, the phenotypic
effect of a recessiveallele is expressedonly in homozygous
individuals,whereasdominant allelesare expressedin het-
o
erozygotes.Thus an individual must carry two copies of a
About 10-30% of offspringwill contain
recessiveallele but only one copy of a dominant allele to ex- foreign DNA in chromosomesof
hibit the corresponding phenotypes. Sfe have seen how a l l t h e i r t i s s u e sa n d g e r m l i n e
strains of mice that are homozygous for a given recessive
knockout mutation can be produced by crossingindividuals Breed mice expressing
that are heterozygousfor the same knockout mutation (see foreign DNA to proPagate
DNA in germline
Figure 5-41). For experimentswith cultured animal cells,
however, it is usually difficult to disrupt both copies of a
genein order to produce a mutant phenotype.Moreover, the @ @
difficulty in producing strainswith both copiesof a genemu-
tated is often compounded by the presenceof related genes
of similar function that must also be inactivated in order to
reveal an observablephenotype.
For certain genes,the difficulties in producing homozy-
gous knockout mutants can be avoided by use of an allele
carrying a dominant-negativemutation. Theseallelesare ge- a much higher frequency than insertion via homologous re-
netically dominant; that is, they produce a mutant pheno- combination. Becauseof this phenomenon' the production
type even in cells carrying a wild-type copy of the gene. of transgenicmice is an efficient and straightforward process
However, unlike other types of dominant alleles,dominant-
negativeallelesproduce a phenotypeequivalentto that of a
loss-of-function mutatton.
Useful dominant-negativealleleshave been identified for
a variety of genesand can be introduced into cultured cells
by transfection or into the germ line of mice or other organ-
isms. In both cases,the introduced geneis integratedinto the
genome by nonhomologous recombination. Such randomly GTPasesfrom an inactive GDP-bound state to an active
inserted genesare called transgenes;the cells or organisms GTP-bound state dependson their interacting with a cor-
carrying them are referred to as transgenic.Transgenescar- responding guanine nucleotide exchangefactor (GEF)' A
rying a dominant-negative allele usually are engineeredso ,rr,.riu.r,small GTPase that permanently binds to the GEF
that the allele is controlled by a regulatedpromoter, allowing orotein will block conversion of endogenous wild-type
expressionof the mutant protein in different tissuesat dif- imall GTPasesto the active GTP-bound state' thereby in-
ferent times. As noted above, the random integration of ex- hibiting them from performing their switching function
ogenousDNA via nonhomologousrecombinationoccursat ( F i g u r e5 - 4 4 ) .

C E N E SI N E U K A R Y O T E S
T H E F U N C T I O NO F S P E C I F I G
INACTIVATING
 (a) Cellsexpressingonly Inactive permanently destroyed by nucleolytic degradation. The
wild-type allelesof a normal function of both Dicer and RISC is to allow for
small GTPase
gene regulation by small endogenous RNA molecules
j known as micro RNAs (miRNAs).
fGo'
\-/ Researchersexploit the micro RNA pathway for inten-
Wildtype tional silencing of a gene of interest by using either of two
generalmethods for generatingsiRNAs of defined sequence.
(b) Cellsexpressingboth
In the first method a double-strandedRNA corresponding to
wild-type allelesand a the target genesequenceis produced by in vitro transcription of
dominant-negativeallele both senseand antisensecopiesofthis sequence(Figure5-45a).
This dsRNA is injected into the gonad of an adult worm.
where it is converted to siRNA by Dicer in the developing
D o m i n a n t - n e guve
a embryos. In conjunction with the RISC complex, the siRNA
mutant
molecules causethe corresponding mRNA molecules to be
destroyedrapidly. The resulting worms display a pheno-
FIGURE 5-44 Inactivationof the functionof a wild-type type similar to the one that would result from disruption
GTPase by the actionof a dominant-negative mutantallele. of the corresponding gene itself. In some cases,entry of
(a)Small(monomeric) GTPases (purple)areactivatedby their just a few moleculesof a particular dsRNA into a cell is
interaction
with a guaninenucleotide exchange factor(GEF),which sufficient to inactivate many copies of the corresponding
catalyzes
the exchange of GDpfor GTp(b)Introduction of a
dominant-negative mRNA. Figure 5-45b illustrates the ability of an injected
alleleof a smallGTpase geneintocultured cellsor
transgenic
animals leadsto expression dsRNA to interfere with production of the corresponding en-
of a mutantGTpase that binds
to andinactivatesthe GEFAsa result, endogenous dogenousmRNA in C. elegansembryos.In this experiment, the
wild-typecopies
of thesamesmallGTPase aretrappedin the inactiveGDp-bound mRNA levels in embryos were determined by incubating
stateA singledominant-negative allelethuscauses a loss-of- the embryos with a fluorescently labeled probe specific for
functionphenotype in heterozygotessimilarto thatseenin the mRNA of interest. This technique,in situ hybridization,
homozygotes carryingtwo recessiveloss-of-f
unctionalleles is useful in assayingexpressionof a particular mRNA in cells
and tissuesections.
The second method is to produce a specific double-
stranded RNA in vivo. An efficient way ro do this is to
expressa synthetic gene thar is designedto contain tandem
R N AI n t e r f e r e n c e
C a u s e sG e n eI n a c t i v a t i o nb y segmentsof both senseand anti-sensesequencescorresDon-
Destroyingthe CorrespondingmRNA ding to the target gene (Figure 5-45c). \X/henthis gene is
transcribed, a double-strandedRNA "hairpin" srructure
A recently discoveredphenomenon known as RNA inter- forms, known as small hairpin RNA, or shRNA. The
ference (RNAi) is perhaps the mosr straightforward shRNA will then be cleaved by Dicer to form siRNA mole-
method to inhibir the function of specificgenes.This ap- cules. The lentiviral expressionvectors are particularly use-
proach is technically simpler than the methods described ful for introducing synrhericgenes for the expressionof
above for disrupting genes.First observed in the round- shRNA constructsinto animal cells.
worm C. elegans,RNAi refers to rhe ability of double- Both RNAi methods lend themselves to systematic
stranded RNA to block expression of its corresponding studiesto inactivateeach of the known genesin an organ-
single-strandedmRNA but not that of mRNAs with a difl ism and to observewhat goeswrong. For example, in ini-
ferent sequence. tial studies with C. elegans,RNA interferencewith 16,700
As describedin Chapter 8, the phenomenon of RNAi genes(about 86 percent of the genome)yielded 1722 visi-
rests on the general ability of eukaryotic cells to cleave bly abnormal phenotypes.The geneswhose functional in-
double-strandedRNA inro short (23-nt) double-stranded activation causesparticular abnormal phenotypescan be
segmentsknown as small inhibitory RNA (siRNA). The grouped into sets;each member of a set presumably con-
RNA endonuclease that catalyzesthis reaction, known as trols the same signals or events.The regulatory relations
between the genes in the set-for example, the genes that
control muscledevelopment-can then be worked out.
Other organismsin which RNAi-mediated geneinactiva-
tion has been successfulinclude Drosophila, many kinds of
plants, zebra fish, the frog Xenopus, and mice, and are now
tween one strand of the siRNA and its complementaryse_ the subjects of large-scaleRNAi screens.For example,
quenceon the target nRNA; subsequently,specificnucle_ lentiviral vectors have been designedto inactivate by RNAi
asesin the RISC complex then cleave the mRNA/siRNA more than 10,000 different genes expressedin cultured
hybrid. This model accounts for the specificity of RNAi, mammalian cells. The function of the inactivated genescan
since it dependson basepairing, and for its potency in si_ be inferred from defects in growth or morphology of cell
lencing gene function, since the complem entary mRNA is clones transfectedwith lentiviral vectors.

21O . cHAprER
5 M o L E c u L AG
R E N E T tr cE c H N t e u E s
|
fi| ,oo."rt: RNAInterference
(a) In vitro production of double-stranded RNA < EXPERIMENTAL FIGURE 5-45 RNAinterference (RNA|)can
functionallyinactivategenesin C.elegansand other
Antisensetranscript organisms.(a)Invitroproduction of double-stranded RNA(dsRNA) for
RNA|of a specific target gene The coding sequence of the gene,
--\(-
derived fromeithera cDNAcloneor a segment of genomic DNA,is
placedin two orientations in a plasmid vectoradjacent to a strong
promoterTranscription of bothconstructs in vitrousingRNA
polymerase andribonucleoside triphosphates yieldsmanyRNAcoptes in
thesense orientation (identicalwith the mRNA sequence) or
complementary antisense orientationUndersuitable conditions, these
complementary RNAmolecules willhybridize to formdsRNAWhenthe
dsRNA isinjected intocells,it iscleaved by DicerintosiRNAs(b)
lnhibition of mex3RNAexpression in worm embryos by RNA|(seethe
text for the mechanism) (Left)Expression of mex3 RNA in embryos was
assayed by in situhybridization with a probespecific for thismRNA,that
is,linkedto an enzyme thatproduces a colored(purple) product(Rtgtht)
Theembryoderived froma worminjected with double-stranded mex3
mRNA produces littleor no endogenous mex3 mRNA, as indicated by
theabsence of color.Eachfour-cell-stage embryois=50 Fm in length
(c)Invivoproduction of double-stranded RNAoccurs viaan engineered
plasmid introduced directlyintocellsThesynthetic geneconstruct isa
tandemarrangement of both sense and antisense sequences of the
Noninjected Injected targetgeneWhenit istranscribed, double-stranded smallhairpin RNA
forms(shRNA) TheshRNA iscleaved by Dicerto formsiRNAlPart (b)
(c) In vivo production of double-stranded RNA fromA Fire etal. 1998, Nature 391:806 l

Sensetranscript

r The /oxP-Cre recombination system permits production

siRNA
2+
Inactivating the Function of SpecificGenes in
Eukaryotes
r Once a gene has beencloned, important clues about its
normal function in vivo can be deducedfrom the observed
phenotypic effectsof mutating the gene.
r Genescan be disrupted in yeast by inserting a selectable
marker geneinto one allele of a wild-rype genevia homolo-
gous recombination, producing a heterozygousmutant. \7hen
such a heterozygoteis sporulated, disruption of an essential
genewill producetwo nonviablehaploid spores(Figure5-39).
r A yeastgenecan be inactivatedin a controlledmannerby
using the GALI promoter to shut off transcription of a
genewhen cellsare transferredto glucosemedium.
r In mice, modified genescan be incorporated into the
germ line at their original genomic location by homologous
recombination,producingknockouts (seeFigures5-40 and
5-41).Mouse knockoutscan providemodelsfor human ge-
netic diseases such as cysticfibrosis.
of mice in which a geneis knocked out in a specifictissue.
r In the production of transgeniccells or organisms,exoge-
nous DNA is integrated into the host genome by
nonhomologous recombination (seeFigure 5-43). Intro-
duction of a dominant-negativeallele in this way can func-
tionally inactivate a genewithout altering its sequence.
r In many organisms,including the roundworm C. elegans,
double-stranded RNA triggers destruction of the all the
mRNA moleculeswith the samesequence(seeFigure 5-45).
This phenomenon, known as RNAI (RNA interference),
provides a specificand potent means of functionally inacti-
vating geneswithout altering their structure.
As the examplesin this chapter and throughout the book il-
lustrate, genetic analysis is the foundation of our under-
standing of many fundamental processesin cell biology. By
examining the phenotypic consequencesof mutations that
inactivate a particular gene' geneticistsare able to connect
knowledge about the sequence'structure' and biochemical

P E R S P E C T I VF
EOS RT H E F U T U R E 211
 Although scientistscontinue to use this classicalgenetic
approachto dissectfundamentalcellular processes and bio-
chemical pathways, the availability of iomplete genomic
sequenceinformation for most of the common experimental
organisms has fundamentally changed the way genetic ex-
periments are conducted. Using various computational
methods, scientistshave identified the protein-coding gene
sequences in most experimentalorganismsincludingE. coli,
yeast, C. elegans,Drosophila, Arabidopsis, mouse, and hu-
mans.The genesequences, in turn, revealthe primary amino
acid sequenceof the encodedprotein products, providing us
with a nearly complete list of the proteins found in each of
the major experimentalorganisms.
The approach taken by most researchershas thus shifted
from discoveringnew genesand proteins to discoveringthe
functions of genesand proteins whose sequencesare already
known. Once an interestinggenehas beenidentified,genomic
sequenceinformation greatly speedssubsequentgeneticma-
nipulations of the gene,including its designedinactivation, to
learn more about its function. Already sets of vectors for
RNAi inactivation of most defined senesin the nematode C.
elegansnow allow efficientgeneric,ir..n, to be performed in
this multicellular organism.Thesemethodsare now being ap-
plied to largecollectionsof genesin cultured mammalian cells
and in the near future either RNAi or knockout methodswill
have beenusedto inactivateeverygenein the mouse.
In the past, a scientistmight spend many years studying
only a single gene, but nowadays scientistscommonly study
whole setsof genesar once. For example,with DNA microar-
rays the level of expressionof all genesin an organismcan be
measuredalmost as easily as the expressionof a singlegene.
One of the great challengesfacing geneticistsin the twenty-
first century will be to exploit the vast amount of available
data on the function and regulation of individual genesto un-
derstandhow groups of genesare organizedto form complex
biochemicalpathways and regulatory nerworks.
KeyTerms
alleles
166 linkage175
ctone I /')
| 4 - ^
temperature-sensrtrve transgenes209
mutations 170 vector 1-76
transfection 196
transformationL78
Review the Concepts
1, Genetic mutations can provide insights into the mecha-
nisms of complex cellular or developmentalprocesses.How
might your analysisof a geneticmutation be different depend-
ing on whether a particular mutation is recessiveor dominant?
2. Give an example of how and why temperature-sensitive
mutations might be usedto study the function of essentialgenes.
3. Describehow complementation analysiscan be used to
reveal whether two mutations are in the sameor in different
genes.Explain why complementation analysiswill not work
with dominant mutations.
4, Compare the different usesof suppressorand synthetic
lethal mutations in geneticanalysis.
5. Restriction enzymesand DNA ligaseplay essentialroles
in DNA cloning. How is it that a bacterium that produces a
restriction enzyme does not cut its own DNA? Describe
some general features of restriction enzymesites.ril/hat are
the three types of DNA ends that can be generatedafter cut-
ting DNA with restriction enzymes?\{hat reaction is cat-
alyzedby DNA ligase?
6. Bacterial plasmids often serve as cloning vectors. De-
scribethe essentialfeaturesof a plasmid vector. Sfhat are the
advantagesand applicationsof plasmidsas cloning vectors?
7. A DNA library is a collectionof clones,eachcontaining
a different fragment of DNA, inserted into a cloning vector.
What is the differencebetweena cDNA and a genomic DNA
library? How can you use hybridization or expressionto
screen a library for a specific gene? How many different
oligonucleotideprimers would need to be synthesizedas
probes to screena library for the gene encoding the peptide
Met-Pro-Glu-Phe-Tyr?
8. In 1993, Kerry Mullis won rhe Nobel prize in chemistry

tlntation 765 for his invention of the PCR process.Describethe three steps
complementary in each cycle of a PCR reaction. $fhy was the discoveryof a
Northern blotting 192
DNAs (cDNAs) 181 thermostableDNA polymerase(e.g.,Taq polymerase)so im-
phenotype155
portant for the developmentof PCR?
complementation183 plasmids178
9. Southernand Northern blotting are powerful tools in
DNA cloning 126 polymerasechain molecular biology basedon hybridization of nucleic acids.
DNA library 179 r e a c t i o n( P C R )1 8 8 How are these techniques the same? How do they differ?
DNA microarray 192 p r o b e s1 8 1 Give some specific applications for each blotting technique.
dominant L66 recessive165 10. A number of foreign proteins have been expressedin
geneknockoul'207 recombinantDNA 126 bacterial and mammalian cells. Describe the essential
genomics129 recombination175 featuresof a recombinantplasmid that are required for ex-
genotype166 pressionof a foreign gene.How can you modify the foreign
restriction enzymes176
protein to facilitate its purification? What is the advantage
heterozygous166 RNA interference
of expressinga protein in mammalian cellsversusbacteria?
homozygous165 (RNAi)210
11. What is a DNA microarray? How are DNA microarrays
hybridization 181 segregation167
usedfor studying geneexpression?How do experimentswith
in situ hybridizatton 192 Southern blotting 191 microarrays differ from Northern blotting experiments?

212 . c H A p r EsR MOLECULAG

RENETIC
TECHNIQUES
12. In determining the identity of the protein that corre- b. A wild-type yeastcDNA library, preparedin a plas-
sponds to a newly discoveredgene, it often helps to know mid that contains the wild-type URA3* gene' is used to
the pattern of tissueexpressionfor that gene.For example, transformX cells,which are then culturedas indicated.Each
researchers have found that a genecalledSERPINA6 is ex- black spot below representsa singleclone growing on a petri
'$7hat
pressedin the liver, kidney, and pancreasbut not in other plate. are the molecular differencesbetweenthe clones
tissues.!7hat techniques might researchersuse to find out growing on the two plates?How can theseresults be used to
which tissuesexpressa particular gene? identify the geneencodingX?
13. DNA polymorphisms can be used as DNA markers.
Describethe differencesbetweenRFLR SNR and SSRpoly-
morphisms.How can thesemarkersbe usedfor DNA map-
ping studies?
14. How can linkage disequilibrium mapping sometimcs G r o w t ha t 2 3 ' C o n G r o w t ha t 3 2 ' C o n
m e d i al a c k i n gu r a c i l m e d i al a c k i n gu r a c i l
provide a much higher resolutionof genelocation than clas-
sicallinkagemapping? c. DNA is extracted from the parental cells, from X
15. Genetic linkage studies can usually only roughly locate cells, and from Y cells and digested with a restriction
the chromosomalposition of a "disease"gene.How can ex- enzyme.The digests are analyzedby Southern blot analysis,
pression analysis and DNA sequenceanalysis help locate a shown at the left below, using a probe obtained from the
diseasegenewithin the region identified by linkage mapping? gene encoding X. In addition, PCR primers are used to
amplify the gene encoding X in both the parental and the X
16. Gene targetingby siRNA techniquesexploits a normal
cells.The primersare complementaryto regionsof DNA just
micro RNA pathway that is found in all metazoansbut not
external to the geneencoding X. The PCR results are shown
in simpler eukaryotessuch as yeast.\7hat are the roles of
in the gel at the right. What can be deducedabout the muta-
Dicer and RISC in this pathway?
tion in the X genefrom thesedata?
77. The ability to selectivelymodify the genome in the
mousehas revolutionizedmousegenetics.Outline the proce-
dure for generatinga knockout mouse at a specificgenetic
locus.How can the loxP-Cresystembe usedto conditionally
w
knock out a gene?\Vhat is an important medicalapplication
of knockout mice?
18. Two methods for functionally inactivating a gene with-
out alteringthe genesequenceare by dominant-negative mu-
tations and RNA interference(RNAi). Describehow each
method can inhibit expressionof a gene. Parental X Parental X
Southern PCR
Analyzethe Data
d. A construct of the wild-type geneX is engineeredto
encode a fusion protein in which the green fluorescentpro-
A culture of yeast that requiresuracil for growth (ura3-)
tein (GFP) is presentat the N-terminus (GFP-X) or the C-
was mutagenized,and two mutant colonies,X and Y, have
terminus (X-GFP) of protein X. Both constructs'presenton
been isolated.Mating type a cells of mutant X are mated
a URA3* plasmid, are used to transform X cells grown in
with mating type o cellsof mutant Y to form diploid cells.
the absenceof uracil. The transformants are then monitored
The parental (ura3 ), X, Y, and diploid cells are streaked
for growth at 32oC, shown below at the left. At the right are
onto agar platescontaininguracil and incubatedat 23 "C or
typical fluorescentimagesof X-GFP and GFP-X cells grown
32'C. Cell growth was monitored by the formation of
at 23 "C in which green denotesthe presenceof green fluo-
colonieson the culture platesas shown in the figure below.
rescentprotein. What is a reasonableexplanation for growth
Denotesgrowth of cells o f G F P - X b u t n o t X - G F P c e l l sa t 3 2 ' C ?
'/'
ura3 ura \X
\ \ G F P - Xc e l l

Diploid /, Diploid /,

Growthat 23'C G r o w t ha t 3 2 ' C

X-GFP
a. What can be deducedabout mutants X and Y from Growth at 32 "C G F Pl o c a l i z a t i oinn c e l l s
the data provided? grown at 23 'C

THE DATA
ANALYZE O 213
 e. Haploid offspring of the diploid cells from part (a)
above are generated.XY double mutants constitute 114 of
theseoffspring.Haploid X cells,Y cells,and XY cellsin liquid
culture are synchronizedat a stagejust prior to budding and
then are shifted from 23'C to 32 oC. Examination of the cells
24 hours later revealsthat X cellsare arresredwith small buds,
Y cellsare arrestedwith large buds, and XY cellsare arrested
with small buds.\Whatis the relationshipbetweenX and Y?
References
Genetic Analysis of Mutations to ldentify and Study Genes
Adams,A. E. M., D. Botstein,and D. B. Drubin. 1989.A yeast
actin-bindingprotein is encodedby sac6,a genefound by suppres-
sion of an actin muration. Science243:231.
Griffiths, A. G. F., et al. 2000. An Introduction to Genetic
Analysis,Tth ed. W. H. Freemanand Company.
Guarente,L.1993. Synthericenhancement in geneinteraction:a
genetictoof comesof age.TrendsCenet.g:362-3o6.
Hartwell, L. H. 1967.Macromolecularsynthesis of
temperature-sensitive mutanrs of yeast.J. Bacteriol. 93:1662.
Hartwell, L.H. 1974. Geneticcontrol of the cell divisioncycle
in yeast.Science183:46.
Niisslein-Volhard, C., and E.'Wieschaus. 1980.Mutations affectins
segmentnumber and polariryin Drosophila.Nature 287:795-801.
Simon,M. A., er al. 1991. Rasl and a putativeguaninenucleotide
exchangefactor perform crucialstepsin signalingby the sevenless
protein tyrosinekinase.Cell 67:701-776.
Tong,A. H., et al. 2001. Systematicgeneticanalysiswith ordered
arraysof yeastdeletionmurants.Science294:2364)368.
DNA Cloning and Characterization
Ausubel,F. M., et al.2002. Current Protocolsin Molecular Bi-
ology.Vlley.
Gubler,U., and B. J. Hoffman. 1983.A simpleand very efficient
method for generatingcDNA libraries. Gene 25:263-289.
Han, J. H., C. Stratowa,and W. I. Rutter.1987.Isolationof full-
lengthputativerat lysophospholipase cDNA usingimprovedmethods
for mRNA isolationand cDNA cloning.Biochem.26:.161.7-1632.
Itakura,K., J.J. Rossi,and R. B. Wallace.1984.Synthesis and
use of syntheticoligonucleotides.Ann. Reu.Biochem. 53:323-356.
Maniatis, T., et al. 1978.The isolation of structural senesfrom
libraries of eucaryoticDNA. Cel/ 15:687-701.
Nasmyth,K. A., and S. I. Reed.1980.Isolationof senesby
complementationin yeast:molecularcloning of a cell-Jyclegene.
Proc. Nat'l Acad. Sci.USA 77:21.19-2123.
Nathans,D., and H. O. Smith. 1975.Restrictionendonucleases
in
the analysisand restructuringof DNA molecules.Ann. Reu.Biochem.
44:273193.
Roberts,R. J., and D. Macelis.1997.REBAsE-restricrion
enzymesand methylases.Nacl. Acids Res.25:248-262. Information
on accessinga continuouslyupdateddatabaseon restrictionand
modification enzymesat http://www.neb.com/rebase.
214 c H A P T E R5 | M O L E C U L AG
R E N E T TTCE C H N T Q U E S
Using Cloned DNA Fragments to Study Gene Expression
Andrews, A.T. 1986. Electrophoresis,2d ed. Oxford University
Press.
Erlich, H., ed. 1.992.PCR Technology:Principlesand
Applicationsfor DNA Amplification. W. H. Freemanand Company.
Pellicer,A., M. Wigler, R. Axel, and S. Silverstein.1978. The
transfer and stableintegration of rhe HSV thymidine kinasegene
into mousecells.Cel/ 4t:1,33-1{1,.
Saiki,R. K., et al. 1988. Primer-directedenzymaticamplification
of DNA with a thermostableDNA polymerase.Science239:487491.
Sanger,F. 1981. Determinationof nucleotidesequences in DNA.
Science214:720 5-'1210.
Souza,L. M., et al. 1986.Recombinanthuman granulocyte-colony
stimulating factor: effectson normal and leukemic myeloid cells.
Science232:61-65.
'Wahl,
G. M., J. L. Meinkoth, and A. R. Kimmel. 1987. Northern
and Southern6lots. Metb. Enzymol. 152:572-581.
Wallace,R. B., et al. 1,987.The useof syntheticoligonucleotides
as hybridizationprobes.II: Hybridization of oiigonucleotides of
mixed sequence to rabbit B-globinDNA. Nzcl. Acids Res.9:879-887.
ldentifying and Locating Human Disease Genes
Botstein,D., et al. 1980. Constructionof a geneticlinkage map
in man using restriction fragmentlength polymorphisms.Az. /.
Genet.32:31.4-331..
Donis-Keller,H., et al. 1,987.A geneticlinkage map of the
human genome.Cell 5l:319-337.
Hartwell, et al. 2000. Genetics:From Genesto Genomes.
McGraw-Hill.
Hastbacka,T., et al. 1994.The diastrophicdysplasiagene
encodesa novel sulfatetransporter:positional cloning by fine-
structurelinkage disequilibriummapping. Cell 781.073.
Orita, M., et al. 1989. Rapid and sensitivedetecrionof point
mutations and DNA polymorphismsusing the polymerasechain
reaction. Genomics5:874.
Tabor,H. K., N.J. Risch,and R. M. Myers.2002. Opinion:
candidate-gene approachesfor studyingcomplex generictrairs:
practicalconsiderations.Nat. Reu.Genet.3:391,-397.
Inactivating the Function of Specific Genes in Eukaryotes
Capecchi,M. R. 1989. Altering the genomeby homologousre-
combination. Science 244 :'1,288-1,292.
Deshaies,R. J., et al. 1988. A subfamily of stressproteins facili,
tatestranslocationof secretoryand mitochondrial precursor
polypeptides.Nature 332:800-805.
Fire, A., et al. 1,998.Porentand specificgeneticinterferenceby
double-strandedRNA in Caenorhabditiselegans.Nature
391:806-8'1.1.
Gu, H., et al. 1994. Delerion of a DNA polymerasebeta gene
segmentin T cellsusing cell type-specificgenetargeting.Science
265:L03-106.
Zamore, P. D., T. Tuschl,P.A. Sharp,and D. P. Bartel.2000.
RNAi: double-strandedRNA directsthe ATP-dependentcleavageof
mRNA at 21 to 23 nucleotideinrervals.Cell 101:25-33.
Zimmer, A. 1992. Manipulating the genomeby homorogous
recombinationin embryonic stem cells.Ann. Reu.Neurosci. 15:115
 CHAPTER

GENES,
AND
GENOMICS,
CHROMOSOMES
ThesebrightlycoloredRxFISH-painted chromosomes
areboth
beautifuland usefulin revealingchromosome and in
anomalies
comparingkaryotypes of differentspecies[@Departmentof Clinical
Cytogenetics,Addenbrookes Researchers,
Hospital/Photo Incl

I n previous chapterswe learnedhow the structure and com- of human chromosomal DNA! The noncoding DNA in mul-
I position of proteins allow them to perform a wide variety ticellular organisms contains many regions that are similar
I of cellular functions. We also examined another vital com- but not identical. Variations within some stretchesof this
ponent of cells, the nucleic acids, and the processby which repetitious DNA between individuals are so great that every
information encoded in the sequenceof DNA is translated person can be distinguishedby a DNA "fingerprint" based
into protein. In this chapter,our focus again is on DNA and on these sequencevariations. Moreover, some repetitious
proteins as we consider the characteristicsof eukaryotic nu- DNA sequencesare not found in the same positions in the
clear and organellar genomes:the features of genesand the genomesof different individuals of the same species.At one
other DNA sequencethat comprise the genome, and how time, all noncoding DNA was collectively termed "junk
'We
this DNA is structured and organized by proteins within the DNA" and was considered to serve no purpose. now
cell. understandthe evolutionary basisof all this extra DNA, and
By the beginning of the twenty-first century, molecular the variation in location of certain sequencesbetween
biologists had completed sequencingthe entire genomesof
hundredsof viruses,scoresof bacteria,and one unicellular OUTLINE
eukaryote, the budding yeast S. cereuisiae.In addition, the
vast majority of the genome sequenceis also known for the 6.1 EukaryoticGene Structure 217
fission yeast S. pombe, and severalmulticellular eukaryotes
6.2 C h r o m o s o m aOl r g a n i z a t i o no f G e n e s
including the roundworm C. elegans, the fruit fly D. 223
a n d N o n c o d i n gD N A
melanogastel,mice, and humans. Detailed analysis of these
sequencingdata has revealedinsights into genome organtza- 5.3 (Mobile) DNA Elements
Transposable 226
tion and genefunction. It has allowed researchersto identify
previously unknown genesand to estimatethe total number 6.4 O r g a n e l l eD N A s 236
of protein-coding genesencoded in each genome. Compar-
5.5 G e n o m i c sG: e n o m e - w i d eA n a l y s i so f G e n e
isons betweengenesequencesoften provide insight into pos-
Structureand ExPression
sible functions of newly identified genes. Comparisons of
genomesequenceand organizationbetweenspeciesalso help 6.6 StructuralOrganizationof Eukaryotic
us understand the evolution of organisms. Chromosomes
Surprisingly,DNA sequencingrevealedthat a large por-
tion of the genomesof higher eukaryotes does not encode 6.7 M o r p h o l o g ya n d F u n c t i o n aEl l e m e n t s
mRNAs or any other RNAs required by the organism. Re- of EukaryoticChromosomes
markably, such noncoding DNA constitutes :98.5 percent

215
individuals. Cellular genomesharbor symbiotic transposable
(mobile) DNA elements,sequencesthat can copy themselves
and move throughout the genome.Although transposable
DNA elementsseemro have little funcrion in the life cycleof
an individual organism, over evolutionary time they have
shapedour genomesand contributedto the rapid evolution
of multicellularorganisms.
In higher eukaryores,DNA regions encoding proteins or
functional RNAs-that is, genes-lie amidst this expanseof
apparently nonfunctional DNA. In addition ro the nonfunc-
tional DNA between genes,noncoding introns are common
within genesof multicellularplants and animals.Sequencing
of the same protein-coding gene in a variety of eukaryotic
specieshas shown that evolutionary pressureselectsfor
maintenanceof relativelysimilar sequences in the coding re-
glons,or exons.In contrast,wide sequence variation,evenin-
cludingtotal loss,occursamon€lintrons,suggesting that most
intron sequenceshave little functional significance.However,
as we shall see,although most of the DNA sequenceof in-
trons is not functional,the existenceof introns has favored
the evolution of multidomain proteins that are common ln
highereukaryotes.It also allowed the rapid evolutionof pro-
teinswith new combinationsof functionaldomains.
Mitochondria and chloroplastsalso contain DNA that
encodesproteins essentialto the function of thesevital or-
ganelles.!7e shall see that mitochondrial and chloroplast
DNAs are evolutionaryremnantsof the origins of theseor-
ganelles.Comparisonof DNA sequences betweendifferent
classes of bacteria and mitochondrial and chloroolast
genomeshas revealedthat theseorganellesevolvedfrom in-
tracellular bacteria that developed symbiotic relationships
with ancient eukaryotic cells.
The sheerlength of cellular DNA is a significant problem
with which cells must contend. The DNA in a single human
cell, which measuresabout 2 meters in total length, must be
contained within cells with diametersof less than 10 pm, a
compactionratio of greaterthan 105to 1.In relativeterms,if
a cell were 1 centimeter in diameter, the length of DNA
packed into its nucleuswould be about 2 kilometers! Special-
ized eukaryotic proteins associatedwith nuclear DNA ex-
quisitely fold and organizethe DNA so thar it fits into nuclei.
And yet at the same time, any given portion of this highly
compacted DNA can be accessedreadily for transcription,
DNA replication, and repair of DNA damage without the
long DNA moleculesbecoming tangled or broken. Further-
more, the integrity of DNA must be maintained during the
processof cell division when it is partitioned into daughter
cells. In eukaryotes,the complex of DNA and the proteins
that organize it, called chromatin, can be visualized as indi-
vidual chromosomesduring mitosis (seechapter opening fig-
ure). As we will seein this and the following chapter,the or-
ganization of DNA into chromatin allows a mechanism for
regulation of geneexpressionthat is not availablein bacteria.
In the first five sections of this chapter, we provide an
overview of the landscapeof eukaryotic genes and genomes.
First we discussthe structure of eukaryotic genesand the com-
plexities that arise in higher organismsfrom the processingof
mRNA precursorsinto alternatively spliced mRNAs. Next we
discussthe main classes of eukaryoticDNA includingthe special
properties of transposableDNA elementsand how they shaped

> FIGURE6-1 Overview of the structure of

genes and chromosomes.DNA of higher
eukaryotes consistsof uniqueand repeated
s e q u e n c eO s n l y: 1 5 p e r c e not f h u m a nD N A
encodesproteinsand functionalRNAsand
the regulatorysequences that controltheir
expression; the remainderis merelyintrons
within genesand intergenicDNA between
g e n e s M u c ho f t h e i n t e r g e n iD c N A ,- 4 5
percentin humans,is derivedf rom transposable
(mobile)DNA elements,geneticsymbiontsthat
havecontributedto the evolutionof
" B e a d so n a s t r i n g "
contemporary genomes Eachchromosome
consrsts of a single,long moleculeof DNA up
t o : 2 8 0 M b i n h u m a n so, r g a n i z e d into
increasing levelsof condensation by the histone
a n d n o n h i s t o n pe r o t e i n sw i t h w h i c h i t i s
intricately complexedMuch smallerDNA
m o l e c u l easr e l o c a l i z eidn m i t o c h o n d r iaan d
chloroplasts

Nucleosome

Major Types of DNA Sequence

S i n g l e - c o pg
yenes S i m p l e - s e q u e n cDeN A
G e n ef a m i l i e s T r a n s p o s a b lD
e N Ae l e m e n t s
T a n d e m l yr e p e a t e dg e n e s S p a c e rD N A
Introns

216 C H A P T E6R | GENES

G,E N O M t CASN, D C H R O M O S O M E S
contemporarygenomes.We then considerorganelleDNA and
how it differs from nuclear DNA. This background preparesus
to discussgenomics,computer-basedmethods for analyzingand
interpreting vast amounts of sequencedata. The final two sec-
tions of the chapter addresshow DNA is physically organizedin
eukaryoticcells.'Sfeconsiderthe packagingof DNA and histone
proteins into compact complexes(nucleosomes)that are the
fundamental building blocks of chromatin, the large-scalestruc-
ture of chromosomes,and the functional elementsrequired for
chromosomeduplication and segregation.Figure 6-1 provides
an overview of theseinterrelatedsubjects.The understandingof
genes,genomics,and chromosomesgained in this chapter will
prepareus to explore current knowledge about how the synthe-
sis and concentration of each protein and functional RNA in a
cell is regulatedin the following two chapters.
Eukaryotic
GeneStructure
lil
In molecular terms, a genecommonly is defined as tbe entire
nucleic acid seqwencethat is necessaryfor the synthesisof a
fwnctionalgeneproduct (polypeptideor RNA). According to
this definition, a geneincludesmore than the nucleotidesen-
coding an amino acid sequenceor a functional RNA, referred
to as the coding region. A genealso includesall the DNA se-
quencesrequired for synthesisof a particular RNA transcript,
no matter where those sequences are locatedin relation to the
coding region.For example,in eukaryoticgenes,transcription-
control regions known as enhancerscan lie 50 kb or more
from the coding region.As we learnedin Chapter4, other crit-
ical noncoding regions in eukaryotic genesinclude the pro-
moter, as well as sequencesthat specify 3' cleavage and
polyadenylation,known as poly(A) sites, and splicing of pri-
mary RNA transcripts,known as splicesiles(seeFigure4-15).
Mutations in thesesequences, which control transcriptionini-
tiation and RNA processing,affect the normal expressionand
function of RNAs, producing distinct phenotypesin mutant
organisms.We examine these various control elementsof
genesin greaterdetail in Chapters7 and 8.
Although most genes are transcribed into mRNAs,
which encode proteins, some DNA sequencesare tran-
scribedinto RNAs that do not encodeproteins (e.g.,tRNAs
and rRNAs described in Chapter 4 and micro RNAs that
regulatemRNA stability and translationdiscussedin Chap-
ter 8). Becausethe DNA that encodestRNAs, rRNAs and
micro RNAs can cause specific phenotypeswhen mutated,
theseDNA regions generallyare referred to as tRNA, rRNA
and micro RNA genes, even though the final products of
thesegenesare RNA moleculesand not proteins.
In this section,we will examine the structure of genesin
bacteria and eukaryotesand discusshow their respective
gene structuresinfluence geneexpressionand evolution.
M o s t E u k a r y o t i cG e n e sC o n t a i nI n t r o n s
a n d P r o d u c em R N A sE n c o d i n gS i n g l eP r o t e i n s
As discussedin Chapter4, many bacterialmRNAs (e.g.,the
mRNA encodedby the trp operon) include the coding region
for several proteins that function together in a biological
process.SuchmRNAs are said to be polycistronic. (A cistron
is a genetic unit encoding a single polypeptide.) In contrast'
most eukaryotic mRNAs are monocistronic; that is, each
mRNA molecule encodes a single protein. This difference
betweenpolycistronic and monocistronic mRNAs correlates
with a fundamental differencein their translatton.
Within a bacterial polycistronic mRNA a ribosome-binding
site is locatednear the start site for eachof the protein-coding
regions,or cistrons,in the mRNA. Translation initiation can
beginat any of thesemultiple internal sites,producing multiple
proteins (seeFigure 4-13a).In most eukaryoticmRNAs' how-
ever,the 5'-cap structuredirectsribosome binding, and trans-
lation beginsat the closestAUG start codon (seeFigure4-13b).
As a result, translation beginsonly at this site. In many cases,
the primary transcriptsof eukaryoticprotein-codinggenesare
processedinto a singletype of mRNA, which is translatedto
give a singletype of polypeptide(seeFigure4-15).
Unlike bacterialand yeastgenes,which generallylack in-
trons, most genesin multicellular animals and plants contain
introns, which are removedduring RNA processingin the nu-
cleus beforethe fully processedmRNA is exported to the cy-
tosol for translation. In many cases,the introns in a geneare
considerablylonger than the exons. Although many introns
are :90 bp long, the median intron length in human genesis
3.3 kb. Some,however,are much longer: the longestknown
human intron is 17,1.06bp, and lies within titan, a gene en-
coding a structural protein in muscle cells. In comparison,
most human exonscontain only 50-200 basepairs. The typi-
cal human geneencodingan average-size protein is :50,000
bp long, but more than 95 percentof that sequenceis present
in introns and flanking noncoding 5' and 3' regions.
Many large proteins in higher organisms that have re-
peated domains are encoded by genesconsisting of repeats
of similar exons separatedby introns of variable length. An
example of this is fibronectin, a component of the extracel-
lular matrix. The fibronectin gene contains multiple copies
of five types of exons (seeFigure 4-16). Suchgenesevolved
by tandem duplication of the DNA encoding the repeated
exon, probably by unequal crossingover during meiosisas
shown in Figure6-2a.
S i m p l ea n d C o m p l e xT r a n s c r i p t i o U
n nits
A r e F o u n di n E u k a r y o t i cG e n o m e s
The cluster of genesthat form a bacterial operon comprises
a single transcription unit that is transcribed from a specific
promoter in the DNA sequenceto a termination site. pro-
ducing a singleprimary transcript. In other words, genesand
transcription units often are distinguishablein prokaryotes
since a single transcription unit contains severalgeneswhen
they are part of an operon. In contrast' most eukaryotic
genes are expressedfrom separatetranscription units' and
each mRNA is translated into a single protein. Eukaryotic
transcription units, however, are classified into two types,
dependingon the fate of the primary transcript.
The primary transcript produced from a simple transcrip-
tion unit is processedto yield a singletype of mRNA' encoding
C E N Es T R U C T U R E .
E U K A R Y O T IG 217
(a) Exon duplication

L1 E x o n1 E x o n2 Exon 3

Parental
chromosomes
L1 Exon 1 Exon 2 Exon 3
=== ===
I
I R e c o m b i n a t i o(nu n e q u acl r o s s i n go v e r )
+
L1 Exon 1 Exon 2 Exon 3 Exon 3

Recombinant
cnromosomes -
Exon 1 Exon 2

(b) Gene duplication

B - g l o b i ng e n e

Parentat I
chromosomes
I

R e c o m b i n a t i o(nu n e q u acl r o s s i n g
I
* B - s l o b i ng e n e

Recombinant
cnromosomes

A FIGURE
t
5-2 Exonand geneduplication.(a)Exonduplication copies of exon3) andonechromosome in whichthe geneis missing
results fromunequal crossing overduringmeiosrs Eachparental exon3 (b)Unequal crossing overbetweenL'1sequences alsocan
chromosome contains oneancestral genecontaining threeexons generate duplications of entiregenesInthisexample, eachparental
( n u m b e r e1d- 3 )a n dt w o i n t r o n sH o m o l o g o n uos n c o d i nLg1 chromosome contains oneancestral B-globin gene,andoneof the
r e p e a t esde q u e n c lei es5 ' a n d3 ' o f t h eg e n ea, n da l s oi n t h ei n t r o n recombinant chromosomes contains two duplicated B-globin genes
between exons2 and3 As discussed in Section 6 3, L1sequences S u b s e q u ei n d t e p e n d em nut t a t i o ni ns t h ed u p l i c a t egde n e cs o u l d
havebeenrepeatedly transposed to newsitesin thegenomeoverrne l e a dt o s l i g hct h a n g ei sn s e q u e n ct hea tm i g h tr e s u litn s l i g h t l y
course of humanevolution, sothatallchromosomes arepeppered different functional properties of theencoded proteinsUnequal
withthem Theparental chromosomes areshowndisplaced relative crossing overalsocanresultfromrarerecombinations between
to eachother,sothatthe L1sequences arealignedHomologous unrelated sequences Notethatthe scalein part(b)is muchlarger
recombination betweenL1sequences asshownwouldgenerate one thanin part(a) [Part (b)seeD H A Fitch eral, 1991,ProcNat'|. AcadSci.
recombinant chromosome in whichthegenehasfourexons(two USA88:7396 I

a singleprotein.Mutations in exons,introns,and transcription- script in fibroblasts and hepatocytesdetermineswhether or

control regions all may influence expressionof the protein
encodedby a simpletranscriptionunit (Figure6-3a).
In the caseof complex rranscription units, which are quite
common in multicellular organisms,the primary RNA tran-
script can be processedin more than one way, leading to for-
mation of mRNAs containing different exons. Each alternate
mRNA, however, is monocistronic, being translated into a
single polypeptide, with translation usually initiating ar the
first AUG in the mRNA. Multiple mRNAs can arise from a
primary transcriptin threeways, as shown in Figure6-3b.
Examplesof all three types of alternariveRNA process-
ing occur in the genesthat regulate sexual differentiation in
Drosophila (see Figure 8-16). Commonly, one mRNA is
produced from a complex transcription unit in some cell
types, and a different mRNA is made in other cell types. For
example, alternative splicing of the primary fibronectin tran-
not the secretedprotein includes domains that adhereto cell
surfaces(seeFigure 4-16). The phenomenonof alternative
splicing greatly expands the number of proteins encoded in
the genomesof higher organisms. It is estimated that -60
percent of human genesare contained within complex tran-
scription units that give rise to alternatively spliced mRNAs
encoding proteins with distinct functions, as for the fibrob-
last and hepatocyteforms of fibronectin.
The relationshipbetweena mutation and a gene is not
always straightforward when it comes to complex tran-
scription units. A mutation in the control region or in an
exon shared by alternatively spliced mRNAs will affect all
the alternative proteins encoded by a given complex tran-
scription unit. On the other hand, mutations in an exon
present in only one of the alternative mRNAs will affect
only the protein encodedby that mRNA. As explained in

218 . cHAprER
6 | G E N E sG, E N o M t c sA,N Dc H R o M o s o M E s
ffi ,od."rt: Eukaryotic
Transcription
Units
> FIGURE 6-3 Simpleand complexeukaryotictrans€ription Simple transcription unit
units.(a)A simple transcription unitincludes a regionthatencodes , 50kb ,
oneprotein, extending fromthe 5' capsiteto the 3' poly(A) site, l-l
andassociated controlregionsIntrons liebetween exons(light CaPsite Poly(A)site
bluerectangles) andareremoved duringprocessing of the primary
transcripts (dashed redlines); thustheydo notoccurin thefunctional Gene
m o n o c i s t r o nmi cR N AM u t a t i o ni sn a t r a n s c r i p t i o n - c o nr et rgoilo n
(a b) mayreduce or prevent transcription, thusreducing or eliminatrng Control regions
synthesis of theencoded protein. A mutation withinan exon(c)may
resultin an abnormal proteinwith diminished activityA mutation nRNA 5'
withinan intron(d) thatintroduces a newsplicesiteresults in an
abnormally spliced mRNAencoding a nonfunctional protein(b)
Complex transcription unitsproduce primary transcripts thatcanbe (b) Complex transcription units
processed in alternative ways.(Iop)lf a primary transcript contains Cap site
alternative splicesites,it canbe processed intomRNAs with thesame
5' and 3' exonsbut differentinternalexons(Middle)lf a primary
transcript hastwo poly(A) sites,it canbe processed intomRNAs with Gene
alternative 3' exons(Botton)lf alternative promoters (f or g) are Exon1 Exon2 Exon3 Exon4
activein different celltypes,mRNA1, produced in a celltypein which
f isactivated, hasa different firstexon(1A)thanmRNA2 has,which mRNAT S'f----a
isproduced in a celltypein whichg isactivated (andwhereexon1B
(aandb) andthosedesignated or
isused)Mutations in controlregions -___---l3,
c withinexonssharedbythealternative mRNAs affectthe proteins nRNA2 5' f_l- T_
encoded by bothalternatively processed mRNAsIn contrast,
mutations (designated d ande)withinexonsuniqueto oneof the
alternatively processed mRNAs affectonlythe proteintranslated from Cap site Poly(A)
thatmRNAForgenesthataretranscribed fromdifferent promoters
in differentcelltypes(bottom),mutations in differentcontrolregions
(f andg) affectexpression onlyin thecelltypein whichthatcontrol Gene
regionisactive Exon1 E x o n3

mRNAT S'f J

or
Chapter 5, genetic complementationtests commonly are
used to determine if two mutations are in the same or dif- nRNA2 5'[---f .T
ferent genes(seeFigure5-7). However,in the complex tran-
scription unit shown in Figure 6-3b (middle), mutations d Cap site Cap site poty(A)
and e would complement each other in a genetic comple-
mentation test, even though they occur in the same gene.
This is becausea chromosomewith mutation d can express Gene
E x o n1 A ExonlB Exon2 E x o n3
a normal protein encoded by mRNA2 and a chromosome
with mutation e can express a normal protein encoded by
mRNA1. Both mRNAs produced from this gene would be nRNAI 5'
presentin a diploid cell carrying both mutations,generating or
both protein products and hence a wild-type phenotype. mRNA2
However, a chromosome with mutation c in an exon com-
mon to both mRNAs would not complement either muta-
tion d or e. In other words, mutation c would be in the same
complementation groups as mutations d and e, even though
P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g
d and e themselveswould not be in the same complementa-
tion group! Given these complications with the genetic def-
to a Gene F a m i ly
inition of a gene, the genomic definition outlined at the The nucleotidesequenceswithin chromosomal DNA can be
beginning of this section is commonly used. In the case of classified on the basis of structure and function' as shown in
'We
protein-coding genes, a gene is the DNA sequence tran- Thble 6-1. will examinethe propertiesof eachclass'begin-
scribed into a pre-mRNA precursor, equivalent to a tran- ning with protein-coding genes,which comprise two groups.
scription unit, plus any other regulatory elements required In multicellular organisms, roughly 25-50 percent of the
for synthesisof the primary transcript.The various proteins protein-codinggenesare representedonly once in the haploid
encoded by the alternatively spliced mRNAs expressed genome and thus are termed solitary genes.A well-studied
from one gene are called isoforms. example of a solitary protein-coding gene is the chicken

G E N ES T R U C T U R E .
EUKARYOTIC 219
 CLASS ITNGTH C0PY
NUMBEB
lNHUMAN
GEN0ME FRACTI0N
0FHUMAN (0/o)
GEN()ME

Protein-coding
genes 0.5-2200kb -25,000 :55" (1.8)1

Tandemlyrepeatedgenes
U2 snRNA 5 . 1k b + :20 <0.001
rRNAs 43 kb+ :300 0.4

RepetitiousDNA
Simple-sequenceDNA 1-500 bp Variable :$
Interspersed
repeats(mobileDNA elements)
DNA transposons 2-3 kb 300,000 3
LTR retrotransposons 5-11kb 440,000 8
Non-LIR retrotransposons
LINEs 5-8 kb 850,000 2T
SINEs 100-400 bp 1,600,000 13
Processedpseudogenes Variable 1- : 1 0 0 -0.4

Unclassified
spacerDNA$ Variable n.a. -25

"' Complete transcription units including introns.

t Transcription units not including introns.
Protein-coding regions (exons) total 1.1% ofthe genome.
+ Length of tandemly repeated
sequence.
s Sequencesbetween transcription
units that are not repeated in the genome; n.a. : not applicable.
SoURcE:International Human Genome SequencingConsortium, 200L, Nature 409860 and 2004, Nature 431:931.

lysozymegene. The 15-kb DNA sequenceencoding chicken
lysozyme constitutes a simple transcription unit containing
four exons and three introns. The flanking regions,extending
for about 20 kb upstreamand downstreamfrom the transcrip-
tion unit, do not encodeany detectablemRNAs. Lysozyme,an
enzymethat cleavesthe polysaccharidesin bacterial cell walls,
is an abundant component of chicken egg-whiteprotein and
also is found in human tears.Its activity helps to keep the sur-
face of the eye and the chicken egg sterile.
Duplicated genesconstitute the secondgroup of protein-
coding genes.These are geneswith close but nonidentical
sequencesthat often are located within 5-50 kb of one
another. A set of duplicated genesthat encodeproteins with
similar but nonidentical amino acid sequencesis called a
gene family; the encoded,closely related, homologous pro-
teins constitute a protein family. A few protein families, such
as protein kinases, vertebrate immunoglobulins, and olfac-
tory receptors include hundreds of members. Most protein
families, however, include from iust a few to 30 or so mem-
bers; common examples are cytoskeletal proteins, the
myosin heavy chain, and the o- and B-globinsin vertebrates.
The genesencodingthe B-like globins are a good exam-
ple of a gene family. As shown in Figure 6-4a, the B-like
globin gene family contains five functional genes desig-
nated B, E, A.y, G], and e; the encoded polypeptides are
similarly designated.Two identical B-like globin polypep-
tides combine with two identical o-globin polypeptides
(encoded by another gene family) and four small heme
groups to form a hemoglobin molecule (seeFigure 3-13).
AII the hemoglobins formed from the different B-like glo-
bins carry oxygen in the blood, but they exhibit somewhat
different properties that are suited to specific roles in
human physiology. For example, hemoglobins containing
either the A" or G, polypeptides are expressedonly during
fetal life. Becausethese fetal hemoglobins have a higher
affinity for oxygen than adult hemoglobins, they can effec-
tively extract oxygen from the maternal circulation in the
placenta. The lower oxygen affinrty of adult hemoglobins,
which are expressed after birth, permits better release of
oxygen to the tissues,especiallymuscles,which have a high
d e m a n df o r o x y g e nd u r i n g e x e r c i s e .
The different B-globin genesarose by duplication of an
ancestralgene, most likely as the result of an "unequal
crossover" during meiotic recombination in a developing
germ cell (egg or sperm) (Figure 6-2b). Over evolutionary
time the rwo copies of the gene that resulted accumulated
random mutations; beneficialmutations that conferred some
refinementin the basicoxygen-carryingfunction of hemoglo-
bin were retained by natural selection,resulting in sequence
drift. Repeatedgene duplications and subsequentsequence
drift are thought to have generatedthe contemporary globin-
like genesobservedin humans and other mammals today.
Two regionsin the human B-like globin genecluster con-
tain nonfunctional sequences,called pseudogenes,similar to
those of the functional B-like globin genes(seeFigure 6-4a).
Sequenceanalysis shows that these pseudogeneshave the
sameapparent exon-intron structure as the functional B-like
globin genes,suggestingthat they also arose by duplication
of the same ancestral gene. However, there was little selective
pressureto maintain the function of thesegenes.Consequently

220 . c H A p r E6R I G E N EG
s ,E N o M t cAsN
, Dc H R o M o s o M E s
 (a) Human B-globingene cluster (chromosome111

I t"on l--l Pseudogene I nlu site

lbl S. cerevisiae(chromosome lll)

l--l o p " n r e a d i n gf r a m e t R N Ag e n e

80 kb

A FIGURE 6-4 Structureof p-globingeneclusterand sequence, an :300-bpnoncoding repeated sequence thatis

comparisonof genedensityin higherand lower eukaryotes. abundant in the humangenome, (b)In the diagram of yeastDNA
(a)Inthediagram of the B-globingenecluster
on humanchromosome fromchromosome lll,the greenboxesindicate openreading frames
11,thegreenboxesrepresent genesExons
exonsof B-globin-related Mostof thesepotential protein-coding sequences arefunctional
splicedtogether
to formonemRNAareconnected bycaret-like
spikes geneswithoutintrons. Notethe muchhigherproportion of
ThehumanB-globin genecluster two pseudogenes
contains (white); noncoding-to-coding sequences in the humanDNAthanin theyeast
theseregionsarerelated to thefunctional genesbutare
globin-type DNA lPart(a),seeF 5 Collins andS M Weissman, 1984, ProgNuclAcid
nottranscribed.
Eachredarrowindicates thelocation
of anAlu R e sM o l B i o l . 3 1 : 3P1 a
5 r t ( b ) , s e e O
S l
G i v e r e t
, a
1 l9 9 2N, ature357'-78]l

sequencedrift during evolution generatedsequencesthat Severaldifferent genefamiliesencodethe various proteins

either terminate translation or block mRNA processing,
rendering such regions nonfunctional. Becausesuch pseudo-
genes are not deleterious, they remain in the genome and
mark the location of a geneduplication that occurred in one
of our ancestors.
Duplications of segmentsof a chromosome (called seg-
mentdl duplication) occurredfairly often during the evolution
of multicellular plants and animals. As a result, a large frac-
tion of the genesin these organismstoday have been dupli-
cated,allowing the processof sequencedrift to generategene
families and pseudogenes. The extent of sequencedivergence
betweenduplicatedcopiesof the genomeand characterization
of the homologousgenomesequences in relatedorganismsal-
low an estimateof the time in evolutionaryhistory when the
duplication occurred.For example,the human fetal 1-globin
genes(G" and A") evolvedfollowing the duplicationof a 5.5-kb
region in the B-globin locus that included the single1-globin
genein the common ancestorof caterrhineprimates(old world
monkeys, apes, and humans) and platyrrhine primates (new
world monkeys)about 50 million yearsago.
Although members of gene families that arose relatively
recently during evolution are often found near each other on
the samechromosome,as for genesof the human B-globin
locus, members of gene families may also be found on dif-
ferent chromosomesin the same organism.This is the case
for the human ct-globin genes,which were separatedfrom
the B-globin genesby an ancient chromosomal transloca-
tion. Both the a- and B-globin genesevolvedfrom a single
ancestralglobin genethat was duplicated(seeFigure 6-2b)
to generatethe predecessorsof the contemporary cr- and
B-globin genesin mammals.Both the primordial ct- and B-
globin genesthen underwent further duplicationsto gener-
ate the different genesof the cr- and B-globin gene clusters
found in mammalstoday.
that make up the cytoskeleton.Theseproteins are presentin
varying amounts in almost all cells. In vertebrates,the major
cytoskeletalproteins are the actins, tubulins, and intermedi-
ate filament proteins like the keratins discussedin Chapters
'We
17, 18 and 19. examine the origin of one such family, the
tubulin family, in Section6.5. Although the physiologicalra-
tionale for the cytoskeletalprotein families is not as obvious
as it is for the globins, the different members of a family
probably have similar but subtly different functions suited to
the particular type of cell in which they are expressed.
HeavilyUsedGene ProductsAre Encoded
b y M u l t i p l eC o p i e so f G e n e s
In vertebratesand invertebrates'the genesencoding riboso-
mal RNAs and some other nonprotein-coding RNAs such as
those involved in RNA splicing occur as tandemly repeated
arldys. These are distinguishedfrom the duplicated genesof
gene families in that the multiple tandemly repeated genes
encode identical or nearly identical proteins or functional
RNAs. Most often copiesof a sequenceappear one after the
other, in a head-to-tail fashion, over a long stretch of DNA.
Within a tandem array of rRNA genes,each copy is nearly
exactly like all the others. Although the transcribed portions
of rRNA genesare the same in a given individual, the non-
transcribed spacer regions between the transcribed regions
can vary.
These tandemly repeatedRNA genesare neededto meet
the great cellular demand for their transcripts. To under-
stand why, consider that a fixed maximal number of RNA
copies can be produced from a single gene during one cell
generation when the gene is fully loaded with RNA poly-
merase molecules.If more RNA is required than can be
transcribed from one gene, multiple copies of the gene are

G E N ES T R U C T U R E
EUKARYOTIC 221
necessary.For example, during early embryonic develop-
ment in humans, many embryonic cells have a doubling time
of :24 hours and contain 5-10 million ribosomes.To pro-
duce enough rRNA to form this many ribosomes,an embry-
onic human cell needs at least 100 copies of the large and
small subunit rRNA genes,and most of thesemust be close
to maximally active for rhe cell to divide every 24 hours.
That is, multiple RNA polymerasesmust be transcribing
each rRNA gene at the same time (seeFigure 8-33). Indeed,
all eukaryotes,includingyeasts,contain 100 or more copres
of the genesencoding 55 rRNA and the large and small
subunit rRNAs.
Multiple copies of IRNA genesand the genesencoding
the histone proteins also occur.As we'll seelater in this chap-
ter, histones bind and organize nuclear DNA. Just as the cell
requiresmultiple rRNA and IRNA genesto support efficient
translation, multiple copies of the histone genesare required
to produce sufficient histone protein to bind the large
amount of nuclear DNA. While IRNA and histone genesof-
ten occur in clusters,they generally do not occur in tandem
arrays in the human genome.
N o n p r o t e i n - C o d i nG
g e n e sE n c o d e
F u n c t i o n aRl NAs
In addition to rRNA and IRNA genes,there are hundreds of
additional genesthat are transcribedinto nonprotein-coding
RNAs, some with various known functions, and many
whose functions are not yet known. For example, small
nuclearRNAs (snRNAs) function in RNA splicing,and small
nucleolar RNAs (snoRNAs) function in rRNA processing
and basemodification in the nucleolus. The RNase P RNA
functionsin IRNA processing,and a largefamily (-1000) of
short micro RNAs (miRNAs) regulate the stability and
translation of specific mRNAs. The functions of these non-
protein-coding RNAs are discussedin Chapter 8. An RNA
found in telomerase(Chapter 4) functions in maintaining the
sequenceat the ends of chromosomes, and the 7SL RNA
functions in the import of secretedproteins and most mem-
brane proteins into the endoplasmicreticulum (Chapter 13).
These and other nonprotein-coding RNAs encoded in the
human genome and their functions, when known, are listed
inTable 6-2.

RNA (]FGENTS
NUMBER INI|UMAN
GTNI)MI FUNCTI()N

rRNAs -300 Protein synthesis

tRNAs :500 Protein synthesis

snRNAs :80 mRNA splicing

U7 snRNA 1 Histone mRNA 3' processing

snoRNAs :85 Pre-rRNA processingand rRNA modification

miRNAs :1 000 Regulation of gene expressron

Xist 1 X-chromosomeinactivation

7SK I Transcription control

RNAse P 1 IRNA 5' processing

7SL RNA Protein secretion (component of signal recognition particle, SRP)

RNAseMRP 1 rRNA processing

Telomerase
RNA 1 Template for addition of telomeres

Vault RNAs 3 Components of Vault ribonucleoproteins (RNPs), function unknown

hY1, hY3, hY4, hY5 :30 Components of Ro ribonucleoproteins (RNPs), function unknown

Hl9 I Unknown

souRcE: International Human Genome SequencingConsortium, 2001, Nature 409:860, and P. D. Zamore and B. Haley, 2005. Science 309':7519.

222 . c H A p T E R6 | G E N E 5G
, E N O M t C SA,N D C H R O M O S O M E S

Eukaryotic Gene Structure
r In molecular terms, a gene is the entire DNA sequence
required for synthesisof a functional protein or RNA mol-
ecule.In addition to the coding regions(exons),a genein-
cludescontrol regions and sometimesintrons.
r A simple eukaryotic transcription unit produces a single
monocistronic mRNA. which is translated into a single
proteln.
r A complex eukaryotic transcription unit is transcribed
into a primary transcript that can be processedinto two or
more different monocistronic mRNAs depending on the
choice of splice sites or polyadenylation sites. A complex
transcription unit with alternate promoters also generates
two or more different mRNAs (seeFigure 6-3b).
r Many complex transcription units (e.g., the fibronectin
gene) expressone mRNA in one cell type and an alternate
mRNA in a different cell type.
r About half the protein-coding genes in vertebrate ge-
nomic DNA are solitary genes,each occurring only once in
the haploid genome. The remainder are duplicated genes,
which arose by duplication of an ancestralgeneand subse-
quent independentmutations (seeFigure 6-2b). The pro-
teins encoded by a gene family have homologous but non-
identical amino acid sequencesand exhibit similar but
slightly di fferentproperties.
r In invertebratesand vertebrates,rRNAs are encoded by
multiple copies of genes located in tandem arrays in ge-
nomic DNA. Multiple copies of IRNA and histone genes
also occur, often in clusters, but not generally in tandem
arrays.
r Many genesalso encode functional RNAs that are not
translated into protein but nonethelessperform significant
functions, such as rRNA, IRNA and snRNAs. Among
these are micro RNAs, possibly up to 1000 in humans,
whose biological significancein regulating geneexpression
has only recentlybeenappreciated.
@ ChromosomalOrganization
of Genesand NoncodingDNA
Having reviewed the relationship between transcrlptlon
units and genes,we now consider the organization of genes
on chromosomesand the relationshipof noncodingDNA se-
quencesto coding sequences.
G e n o m e so f M a n y O r g a n i s m sC o n t a i nM u c h
N o n f u n c t i o n aD
l NA
Comparisons of the total chromosomal DNA per cell in var-
ious speciesfirst suggestedthat much of the DNA in certain
organismsdoes not encodeRNA or have any apparent regu-
latory function. For example, yeasts,fruit flies, chickens,
C H R O M O S O M AOL R G A N I Z A T I OO
and humans have successivelymore DNA in their haploid
c h r o m o s o m es e t s ( 1 2 ; 1 8 0 ; 1 3 0 0 ; a n d 3 3 0 0 M b , r e s p e c -
tively), in keepingwith what we perceiveto be the increasing
complexity of these organisms.Yet the vertebrateswith the
greatestamount of DNA per cell are amphibians, which are
surely less complex than humans in their structure and be-
havior. Even more surprising, the unicellular protozoan
speciesAmoeba dubia has 200 times more DNA per cell
than humans. Many plant speciesalso have considerably
more DNA per cell than humans have. For example, tulips
have 10 times as much DNA per cell as humans. The DNA
content per cell also varies considerably between closely re-
lated species.All insectsor all amphibians would appear to
be similarly complex, but the amount of haploid DNA in
specieswithin each of thesephylogenetic classesvaries by a
factor of 100.
Detailed sequencingand identification of exons in chro-
mosomal DNA have provided direct evidence that the
genomesof higher eukaryotescontain large amounts of non-
coding DNA. For instance, only a small portion of the B-
globin gene cluster of humans, about 80 kb long' encodes
protein (seeFigure 6-4a).In contrast,a typical 8O-kbstretch
of DNA from the yeast S. cereuisiae,a single-celledeukary-
ote, contains many closely spacedprotein-coding sequences
without introns and relatively much less noncoding DNA
(seeFigure 6-4b). Moreover, compared with other regions of
vertebrate DNA, the B-globin gene cluster is unusually rich
in protein-coding sequences,and the introns in globin genes
are considerablyshorter than those in many human genes.
The density of genesvaries greatly in different regions of
human chromosomal DNA, from "gene-rich" regions, such
as the B-globin cluster,to large gene-poor "gene deserts."Of
the 96 percent of human genomic DNA that has been se-
quenced,only :1.5 percentcorrespondsto protein-coding
sequences(exons). We learned in the previous section that
the intron sequencesof genesare often significantly longer
than the exon sequences. Approximately one-third of human
genomic DNA is thought to be transcribed into pre-mRNA
precursors or nonprotein-coding RNAs in one cell or an-
other, but some 95 percent of this sequenceis intronic, and
thus removed by RNA splicing. This amounts to a large frac-
tion of the total genome. The remaining two-thirds of
human DNA is noncoding DNA between genesas well as
regions of repeated DNA sequencesthat make up the
centromeresand telomeres of the human chromosomes'
Consequently,-98.5 percent of human DNA is noncoding'
Different selectivepressuresduring evolution may account,
at least in part, for the remarkable difference in the amount of
nonfunctionalDNA in unicellularand multicellularorganisms.
For example, microorganisms must compete for limited
amounts of nutrients in their environment, and metabolic
economythus is a critical characteristic.Sincesynthesisof non-
functional (i.e.,noncoding)DNA requirestime, nutrients and
energy,presumablythere was selectivepressureto lose non-
functional DNA during the evolution of microorganisms. On
the other hand, natural selectionin vertebratesdependslargely
on their behavior. The energy invested in DNA synthesis is
trivial compared with the metabolic energy required for the
DNA
NF G E N E SA N D N O N C O D I N G
223
movementof muscles;thus therewas little selectivepressureto (a) Normal replication
eliminatenonfunctionalDNA in vertebrates.

M o s t S i m p l e - S e q u e n cDeN A sA r e C o n c e n t r a t e d
i n S p e c i f i cC h r o m o s o m aLl o c a t i o n s
(b) Backwardslippage
Besidesduplicated protein-codinggenesand tandemly re-
peated genes,eukaryotic cells contain multiple copies of
other DNA sequences in the genome,generallyreferredto as
repetitiousDNA (seeTable 6-1). Of the two main types of
repetitiousDNA, the lessprevalentis simple-sequence DNA,
or satelliteDNA, which constiruresabout 6 percent of the
human genomeand is composedof perfector nearly perfect
repeats of relatively short sequences.The more common
type of repetitious DNA, collectively called interspersed
repeats,is composedof much longer sequences. These se-
quences,consisting of several types of transposableele,
ments,are discussedin Section6.3. a' u,
The length of each repeat in simple-sequence
DNA can
I
range from 1 to 500 basepairs. Simple-sequence DNAs in Second
replication
which the repearscontain 1-13 basepairs are often called I
microsatellites.Most microsateiliteDNA has a repeatlength (c)Secondreplication
ol 1-4 base pairs and usually occurs in tandem repeatsof
Dau
150 repeatsor fewer. Microsatellitesare rhought to have
53'
originatedby "backward slippage"of a daughrerstrand on
its templatestrand during DNA replicarionso that the same 3', 5'
short sequenceis copiedtwice (Figure6-5).
N o r m a ld a u g h t e rD N A
Microsatellitesoccasionallyoccur within transcription u' r,
units. Some individuals are born with a larser number 3', s,
of repeatsin specificgenesthan observedin the generalpopu-
lation, presumablybecauseof daughter-strandslippageduring FIGURE 6-5 Generationof microsatellite repeatsby
DNA replication in a germ cell from which they developed. backwardslippageof the nascentdaughterstrandduring DNA
Such expanded microsatelliteshave been found to cause at replication.lf duringreplication (a),the nascent daughter strand
least 14 different types of neuromusculardiseases,depending "slips"backward relative to thetemplate strandby onerepeat, one
on the genein which they occur.In some casesexpandedmi- newcopyof the repeatisaddedto the daughter strandwhenDNA
crosatellitesbehavelike a recessive replication continues (b) Thisextracopyof the repeatformsa single-
mutation becausethey in-
terferewith just the function or expressionof the encoded stranded loopin the daughter strandof thedaughter duplexDNA
moleculelf thissingle-stranded loopisnot removed by DNArepair
gene.But in the more common types of diseases associated proteins beforethe nextroundof DNAreplication (c),theextracopy
with expanded microsatelliterepeats,such as myotonic dys-
of the repeatisaddedto oneof the double-stranded daughter DNA
tropby and spinocerebelldrataxia, the expanded repeatsbe-
m o l e c u l et h
s ,eo t h e rd a u g h t emr o l e c u w
l ei l lb e n o r m a l
have like dominant mutarions becausethey interfere with all
RNA processingin the musclecellsand neuronswhere the af-
fectedgenesare expressed.For example,in patientswith my- studieswith metaphasechromosomeshave localizedthese
otonic dystrophy,transcripts of the DMPK gene contain be- simple-sequenceDNAs to specific chromosomal regions.
tween 100-4000 repeats of the sequenceCUG in the 3, Much of this DNA lies near centromeres,the discretechro-
untranslatedregion, compared to 50-100 repeatsin normal mosomal regions that attach to spindle microtubules dur-
individuals. The extendedstrerchof CUG reDeatsin affected ing mitosis and meiosis(Figure6-6). Experimentsin the fis-
individuals forms long RNA hairpins (seeFigure 4-9) that in- sion yeast Schizosaccharomyces pombe indicate that these
terferewith normal RNA processingand export of transcripts sequencesare required to form a specializedchromatin
from the nucleusto rhe cyrosol. The double-stranded(ds) structure called centromeric heterochromatin, necessary
regions of these long RNA hairpins bind nuclear dsRNA- for the proper segregationof chromosomes to daughter
binding proteins,interferingwith their normal function in reg- cellsduring mitosis. Simple-sequence DNA is also found in
ulating the alternative splicing of other specificpre-mRNAs l o n g t a n d e m r e p e a t s a t t h e e n d s o f c h r o m o s o m e s ,t h e
essentialfor normal muscleand nerve cell function. I telomeres,where they function to maintain chromosome
ends and prevent their joining ro the ends of other DNA
Most satelliteDNA is composedof repeatsof 14-500 base molecules,as discussedfurther in the last section of this
pairs in randem arrays of 20-700 kb. In situ hybridizarion chaoter.

224 CHAPTER
6 I G E N E SG
, E N O M I C SA, N D C H R O M O S O M E S
 DNA fingerprinting, which is superior to conventional fin-
gerprinting for identifying individuals (Figure 6-7). The use
of PCR methods allows analysis of minute amounts of
DNA, and individuals can be distinguished more precisely
and reliably than by conventional fingerprinting.

d p a c e rD N A O c c u p i e sa S i g n i f i c a n t
U n c l a s s i f i eS
Portion of the Genome
As Table5.1 shows,:25 percentof humanDNA liesbe-
tween transcription units and is not repeatedanywhere else
in the genome. Much of this DNA probably arose from
ancient transposable elements that accumulated so many
mutations over evolutionary time they can no longer be rec-
ognized as having arisen from this source (Section 6.3).

(a) Paternity (b) Criminal identification

determination
Ih
MCF1F2 6
-o
A EXPERIMENTAL FIGURE 6-6 Simple-sequence DNAis --!t<-

localizedat the centromerein mousechromosomes.Purified tr'^A

simple-sequence DNAfrommousecellswascopiedin vitrousingE + 6P
collDNApolymerase I andfluorescently
labeled dNTPs to generate a
fluorescently
labeled DNAprobefor mousesimple-sequence DNA
Chromosomes fromcultured mousecellswerefixedanddenatured e
on a microscope slide,andthenthechromosomal DNAwas
hybridizedin situto the labeledprobe(blue-green) Thesltdewas
alsostainedwith DAPI,a DNA-binding dye,to visualize thefull-
lengthof thechromosomes (darkblue)Fluorescence microscopy
showsthatthe simple-sequence probehybridizes primarily
to one
endof thetelocentric mousechromosomes (i e . chromosomes in
whichthe centromeres arelocatednearoneend) [Courtesy of Sabine
-=
M a l , P h D , M a n i t o b aI n s t i t u t eo f C e l lB i o l o g yC
, a n a d al
--E
=
-- --=
D N A F i n g e r p r i n t i n gD e p e n d so n D i f f e r e n c e s 3--
in Length of Simple-Sequence DNAs =-= I I--

---- E-=
\(ithin a species,the nucleotidesequencesof the repeat units
composing simple-sequenceDNA tandem arrays are highly A EXPERIMENTAL FIGURE 5-7 DNAfingerprintingis used
conservedamong individuals. In contrast, the nwmber of re- to identify individualsin paternity casesand criminal
peats, and thus the length of simple-sequence tandem arrays investigations. In DNAfingerprinting, a singlePCRreaction is
containing the same repeat unit, is quite variable among in- performed on template DNAfroman individual usingseveral setsof
dividuals. These differencesin length are thought to result primersto uniquesequences flanking repeat
the minisatellite
from unequal crossing over within regions of simple- sequences Gelelectrophoresis of the PCRproducts generates a DNA
sequenceDNA during meiosis.As a consequenceof this un- fingerprintfor the individual: thatis,a setof minisatellitesof
repeatlengths
different andhencemobility in the gel (a)Inthis
equal crossing over, the lengths of some tandem arrays are
of
analysis paternity, laneM shows the products usingthe mother's
unique in each individual.
DNAastemplate in the PCRreaction; C, usingthechild's DNA;and
In humans and other mammals, some of the simple-
F1andF2usingDNAfromtwo potential fathers.Thechildhas
sequenceDNA exists in relatively short 1- to 5-kb regions fromeitherthe motheror F1,
minisatelliterepeatlengths inherited
made up of 20-50 repeat units, each containing :14-1.00 thatF1isthe father Arrows indicate PCRproducts
demonstrating
basepairs. Theseregionsare called minisatellites.Even slight fromF1.but not F2,foundin the child's DNA.(b)In theseDNA
differencesin the total lengths of various minisatellitesfrom fingerprints of a specimen isolated froma rapevictimandthreemen
different individuals can be detectedby the polymerasechain susoected of the crime,it isclearthat minisatellite repeatlengths in
reaction (PCR), using a mixture of severalprimers that hy- the specimen matchthoseof suspect 1 Thevictim's DNAwas
bridize to unique sequencesflanking multiple minisatellites. includedin the analysis to ensure thatthe specimen DNAwasnot
TheseDNA polymorphisms (i.e., di{ferencesin sequencebe- contamtnated with DNAf romthevictimlFrom T.StrachanandA P
tween individuals of the same species)form the basis of Read,Human Molecular Genetics2, 1999,JohnWiley& Sons l

DNA
NF G E N E SA N D N O N C O D I N G
C H R O M O S O M AOL R G A N I Z A T I OO 225
Transcription-control regions on the order of 50-200 base
pairs in length that help to regulate transcription from dis-
tant promoters also occur in theselong stretchesof unclassi-
fied spacerDNA. In some cases,sequencesof this seemingly
nonfunctional DNA are nonethelessconservedduring evolu-
tion, indicating that they may perform a significant function
that is not yet understood.For example, they may contribute
to the structuresof chromosomesdiscussedin Section6.7.
Chromosomal Organization of Genes
a n d N o n c o d i n gD N A
r In the genomesof prokaryotes and most lower eukary-
otes, which contain few nonfunctional sequences,coding
regions are densely arrayedalong the genomic DNA.
r In contrast, vertebrateand higher plant genomescontain
many sequencesthat do not code for RNAs or have any
regulatory function. Much of this nonfunctional DNA is
composedof repeatedsequences. In humans, only about
1.5 percentof total DNA (the exons)actuallyencodespro-
teins or functional RNAs.
r Variation in the amount of nonfunctional DNA in the
genomes of various speciesis largely responsible for the
lack of a consistent relationship between the amount of
DNA in the haploid chromosomesof an animal or plant
and its phylogeneticcomplexity.
r Eukaryotic genomic DNA consistsof three major classes
of sequences:genes encoding proteins and functional
RNAs, repetitiousDNA, and spacerDNA (seeTable 6-1).
r Simple-sequence DNA, short sequencesrepeatedin long
tandem arrays, is preferentially located in centromeres,
telomeres,and specificlocations within the arms of partic-
ular chromosomes.
r The length of a particular simple-sequence tandem array
is quite variable betweenindividuals in a species,probably
becauseof unequal crossing over during meiosis. Differ-
encesin the lengthsof some simple-sequence tandem arrays
form the basisfor DNA fingerprinting (seeFigure 6-7).
!p (Mobile)
Transposable
DNAElements
Interspersedrepeats,the secondtype of repetitious DNA in
eukaryotic genomes,is composed of a very large number of
copies of relatively few sequencefamilies (seeTable 6-1).
AIso known as moderately repeatedDNA, or intermediate-
repeat DNA, these sequencesare interspersedthroughout
mammalian genomes and make up :25-50 percent of
mammalian DNA (-45 percentof human DNA).
Becauseinterspersedrepeatshave the unique ability to
"move" in the genome,they are collectivelyreferred to as
transposableDNA elementsor mobile DNA elements(we
use these terms interchangeably).Although transposable
DNA elementsoriginally were discoveredin eukaryotes.
226 CHAPTER
5 | c E N E S ,G E N O M t C SA, N D C H R O M O S O M E S
they also are found in prokaryotes.The processby which
these sequencesare copied and insertedinto a new site in
the genomeis called transposition.TransposableDNA ele-
ments are essentiallymolecular symbionts that in most
casesappear to have no specific function in the biology of
their host organisms, but exist only to maintain them-
selves.For this reason, Francis Crick referred to them as
"selfish DNA."
'When
transposition occurs in germ cells, the transposed
sequencesat their new sitesare passedon to succeedinggen-
erations. In this wag mobile elementshave multiplied and
slowly accumulated in eukaryotic genomesover evolution-
ary time. Sincemobile elementsare eliminated from eukary-
otic genomesvery slowly, they now constitute a significant
portion of the genomesof many eukaryotes.
Not only are mobile elementsthe source for much of the
DNA in our genomes,but they also provided a secondmech-
anism, in addition to meiotic recombination, for bringing
about chromosomal DNA rearrangementsduring evolution
(seeFigure 6-2). This is becauseduring transposition of a
particular mobile element, adjacent DNA sometimesalso is
mobilized. Transpositions occur rarely: in humans, about
one new germ-line transposition for every eight individuals.
Since98.5 percent of our DNA is noncoding, most transpo-
sitions have no deleteriouseffects.But over time they played
an essentialpart in the evolution of geneshaving multiple
exons and of geneswhose expressionis restrictedto specific
cell types or developmentalperiods. In other words, al-
though transposableelementsprobably evolved as cellular
symbionts,they have played a central role in the evolution of
complex, multicellularorganisms.
Transposition also may occur within a somatic cell; in
this casethe transposedsequenceis transmitted only to the
daughter cells derived from that cell. In rare cases,such so-
matic-cell transposition may lead to a somatic-cellmutation
with detrimental phenotypic effects,for example, the inacti-
vation of a tumor-suppressorgene (Chapter 25). In this sec-
tion, we first describethe structure and transposition mech-
anisms of the major types of transposable DNA elements
and then consider their likelv role in evolution.
M o v e m e n to f M o b i l e E l e m e n t sI n v o l v e sa D N A
o r a n R N AI n t e r m e d i a t e
Barbara McClintock discovered the first mobile elemenrs
while doing classical generic experimenrs in maize (corn)
during the 1940s. She characterizedgenetic entities that
could move into and back out of genes, changing the
phenotype of corn kernels. Her theories were very contro-
versial until similar mobile elementswere discoveredin
bacteria, where they were characterized as specific DNA
sequences, and the molecular basisof their transposition was
deciphered.
As researchon mobile elementsprogressed,they were found
to fall into two categories:(L) those that transposedirectly as
DNA and (2) those that transpose via an RNA intermediate
transcribed from the mobile element by an RNA polymerase
and then convertedback into double-strandedDNA bv a reverse
 (a)
DNA transposon Retrotransposon
,. -.:..:..:.)..:
,ir,-'ilil:ir:r:,
D o n o rD N A D o n o rD N A
- -
ansposed/
m o b i l ee l e m e n t s
transcriptase(Figure 5-8). Mobile elementsthat transpose di-
rectly as DNA are generallyreferredto as DNA transposons,or
simply transposons.Eukaryotic DNA transposonsexcisethem-
selvesfrom one place in the genome,leaving that site and mov-
ing to another.Mobile elementsthat transposeto new sitesin the
genomevia an RNA intermediateare called retrotransposons.
Retrotransposonsmake an RNA copy of themselvesand
introduce this new copy into another site in the genome,
while also remaining at their original location. The move-
ment of retrotransposons is analogous to the infectious
processof retroviruses.Indeed,retrovirusescan be thought of
as retrotransposonsthat evolved genesencoding viral coats,
thus allowing them to transpose between cells. Retrotrans-
posons can be further classifiedon the basis of their specific
mechanism of transposition. To summarize, DNA trans-
posonscan be thought ofas transposingby a "cut-and-paste"
mechanism, while retrotransposonsmove by a "copy-and-
paste" mechanismin which the copy is an RNA intermediate.
DNA TransposonsAre Presentin Prokaryotes
and Eukaryotes
Most mobile elementsin bacteriatransposedirectly as DNA.
In contrast, most mobile elementsin eukaryotes are retro-
transposons, but eukaryotic DNA transposons also occur.
Indeed, the original mobile elementsdiscoveredby Barbara
McClintock are DNA transposons.
Bacterial Insertion Sequences The first molecular un-
derstanding of mobile elementscame from the study of
(b) < FIGURE 6-8 Two maior classesof mobile
DNA elements.(a)Eukaryotic DNAtransposons
whichis
(orange)moveviaa DNAintermediate,
fromthe donorsite (b)Retrotransposons
excised
(green) intoan RNAmolecule,
arefirsttranscribed
whichthenis reverse-transcribedintodouble-
strandedDNA.In bothcases, thedouble-stranded
DNAintermediate intothetarget-
is integrated
siteDNAto complete movementThusDNA
transposonsmovebya cut-and-paste mechanism,
whereasretrotransposons moveby a copy-and-
oastemechantsm.
-
certain E. coli mutations causedby the spontaneousinser-
tion of a DNA sequence,-1-2 kb long, into the middle of
a gene. These inserted stretches of DNA ate called inser-
tiin seqwences,or 15 elements. So far, more than 20 dif-
ferent IS elementshave been found in E. coli and other
bacteria.
Transposition of an IS element is a very rare event'
occurring in only one in 105-107 cells per generation' de-
pending on the IS element. Often, transpositions inactivate
essentialgenes,killing the host cell and the IS elementsit car-
ries. Therefore,higher rates of transposition would probably
result in too great a mutation rate for the host organism to
survive, However, since IS elementstranspose more or less
randomly, some transposedsequencesenter nonessentialre-
gions of the genome (e.g.' regions between genes),allowing
the cell to survive. At a very low rate of transposition, most
host cells survive and therefore propagate the symbiotic IS
element. IS elementsalso can insert into plasmids or lyso-
genic viruses, and thus be transferred to other cells' In this
way, IS elementscan transposeinto the chromosomesof vir-
on the other strand, such as:
5' GAGC-GCTC 3',
s',
3 ' C T C G - C G A-G
T R A N S P O S A B L( E
M O B I L E )D N A E L E M E N T S . 227
 lS element {=1-2 kb) responsiblefor the second classdissociation (Ds) elements
becausethey also tended to be associatedwith chromosome
breaks.
Many years after McClintock's pioneering discoveries,
Inverted repeat Protein-coding Target-site
(=50bp) regron
cloning and sequencingrevealedthat Ac elementsare equiv-
direct repeal
(5-11bp) alent to bacterial IS elements.Like IS elements,they contain
inverted terminal repeat sequencesthat flank the coding re-
A FIGURE 6-9 Generalstructureof bacteriallS elements.The
relatively
largecentralregionof an lSelement, gion for a transposase,which recognizesthe terminal repeats
whichencodes oneor
two enzymes requiredfor transposition, isflankedby an inverted and catalyzestranspositionto a new site in DNA. Ds elements
repeatat eachend.Thesequences of the inverted repeats
arenearly
identical,
but theyareoriented in opposite directionsTheinverted- D o n o rD N A TargetDNA
repeatsequence of a particular
ischaracteristic lSelementThe5, and
5',
3' shortdlrect(asopposed to inverted) repeats arenottransposedwith
3', 5',
the insertion
element;rather, theyareinsertion-site sequences
that
become duplicated,withonecopyat eachend,duringinsertion of a 9-bp target site
mobileelement. Thelengthof thedirectrepeats isconstant
for a given I
lSelement,
therefore
buttheirsequence
varies
depends
witheachtransposition
on thesiteof insertion
and
of the lSelementArrows
EIllli?ffi'"xT,tsi,lx'.
cutsin targetDNA
:yffill;s.
indicatesequence orientation Theregions in thisdiagramarenotto I
scale;thecodingregionmakesup mostof thelenqthof an lSelement
5' 3',
3', s',

Betweenthe inverted repeatsis a region that encodestrans- s'-?,s-l .. 5'mil-+3',

posase,an enzyme required for transposition of the IS ele- 3'< _llulJlls' 3'+_ 5'
....r
ment to a new site. The transposaseis expressedvery rarely Unpaired
accounting for the very low frequency of transposition. An bases
important hallmark of IS elementsis the presenceof a short - | ,r.nroosase tigateslSTo
direct-repeatsequence,containing 5-1 1 basepairs, immedi- E I to b'singte-strfndedends
ately adjacent to both ends of the inserted element. The I of target DNA
length of the direct repeat is characteristicof each type of IS l.
element, but its sequencedependson the target site where a
particular copy of the IS element inserted. When the se-
quence of a mutated gene containing an IS element is com-
pared with the wild-type genesequence,only one copy of the
short direct-repeatsequenceis found in the wild-type gene.
Duplication of this target-sitesequencero createthe second
direct repeat adjacent to an IS elemenroccurs during the in-
sertlonprocess.
As depicted in Figure 6-10, transposition of an IS ele-

9-bptarget-site
directreoeats
A FIGURE 5-10 Modelfor transpositionof bacterialinsertion
sequences. Step[: Transposase, whichisencoded by the lS
donor DNA. Finally, a host-cell DNA polymerasefills in the element (lS/0 in thisexample), cleaves bothstrands of the donor
single-strandedgaps, generatingthe short direct repearsthat DNAnextto the inverted repeats (darkred),excising the lS/0
flank IS elements,and DNA ligasejoins the free ends. elementAt a largely randomtargetsite,transposase makes
staggered cutsin thetargetDNA.In thecaseof lSI0,thetwo cuts
Eukaryotic DNA Transposons McClintock's original dis- are9 bp apart.Step[: Ligation of the 3, endsof theexcised lS
covery of mobile elementscame from observation of sDonta- element to the staggered sitesin thetargetDNAalsoiscatalyzed by
transposase Stepf,l: The9-bpgapsof single-stranded DNAleftin
the resulting intermediate arefilledin by a cellular
DNApolymerase;
finallycellularDNAligase formsthe 3'-+5,phosphodiester bonds
between the 3' endsof theextended targetDNAstrands andthe 5,
endsof the lS70strandsThisprocess resultsin duplicationof the
target-sitesequence on eachsideof the inserted lSelement. Note
unlessthey occur in the presenceof the first class of muta-
thatthe lengthof thetargetsiteandlS/0 arenotto scale[See HW
tions. McClintock called the agent responsiblefor the first Benjamrnand N Kleckner,1989, Cell59:373, and 1992, proc Nat'1.Acad Sci.
class of mutations the actiuator (Ac) element and those USA 89:4648 I

228 CHAPTER
6 | G E N E SG, E N O M t C SA, N D C H R O M O S O M E S
are deletedforms of the Ac elementin which a portion of the
sequenceencoding transposaseis missing. Becauseit does
not encode a functional transposase,a Ds element cannot
move by itself. However, in plants that carry the Ac element
and thus expressa functional transposase,Ds elementscan
be transposed becausethey retain the inverted terminal re-
peats recognizedby the transposase.
Since McClintock's early work on mobile elements in
corn, transposonshave been identified in other eukaryotes.
For instance,approximately half of all the spontaneousmu-
tations observed in Drosophila are due to the insertion of
mobile elements.Although most of the mobile elementsin
Drosophila function as retrotransposons,at leastone-the P
element-functions as a DNA transposon, moving by a
mechanism similar to that used by bacterial insertion se-
quences. Current methods for constructing transgenic
Drosophila depend on engineered,high-level expressionof
the P-elementtransposaseand use of the P-elementinverted
terminal repeatsas targets for transposition, as discussedin
Chapter 5 (seeFigure5-25).
DNA transpositionby the cut-and-pastemechanismcan
result in an increasein the copy number of a transposon if
it occurs during the S phase of the cell cycle, when DNA
synthesisoccurs. An increasein the copy number happens
when the donor DNA is from one of the two daughter DNA
molecules in a region of a chromosome that has replicated
and the target DNA is in the region that has not yet repli-
'S7hen
cated. DNA replication is complete at the end of the
S phase, the target DNA in its new location is also repli-
cated, resulting in a net increasein the total number of these
transposonsin the cell (Figure6-11). Vhen such a transpo-
One copy of
transposon
before S phase
S phase:DNA
replicationand
DNA transposition
ili,lfiJ,^,ixi;3,1,?
ffirrrr ifJ#:J""JJff"
6-11 Mechanismfor increasing the copynumberof
A FIGURE
a DNA transposon thatmovesby a
DNAtransposons. In thismodel,
mechanism (seeFigure 6-10) transposes duringthe
cut-and-paste
S phasefroma regionof thechromosome that hasreplicatedto a
oneof thetwo
regronthat hasnotyetreplicated lf thisoccurs,
chromosomes will havea netincrease of onetransposon
daughter
is completed,
once
insertron chromosomal replication
sition occurs during the S phase preceding meiosis' one of
the four germ cells produced contains the extra copy of the
transposon. Repetition of this process over evolutionary
time has resulted in the accumulation of large numbers of
DNA transposons in the genomesof some organisms. Hu-
man DNA contains about 300,000 copies of full-length and
deletedDNA transposons,amounting to :3 percent of hu-
man DNA. As we will seeshortly, this mechanism can lead
to the transposition of genomic DNA as well as the trans-
poson itself.
LTRRetrotransposons BehaveLike
l n t r a c e l l u l aR
r etroviruses
The genomesof all eukaryotes studied from yeast to hu-
mans contain retrotransposons' mobile DNA elements
that transpose through an RNA intermediate utilizing a
reversetranscriptase(seeFigure 6-8b). These mobile ele-
ments are divided into two major categories,those con-
taining and those lacking long terminal repeats (LTRs).
LTR retrotransposons'which we discusshere, are common
in yeast (e.g.,Ty elements)and in Drosophila le.g., copia
elements).Although less abundant in mammals than non-
LTR retrotransposons, LTR retrotransposons nonetheless
constitute -8 percent of human genomic DNA. In mam-
mals, retrotransposonslacking LTRs are the most com-
mon type of mobile element; these are described in the
next sectlon.
The general structure of LTR retrotransposonsfound in
eukaryotesis depicted in Figure 6-12'ln addition to short 5'
and 3' direct repeatstypical of all transposons' these retro-
LTR retrotransposons encode all the proteins of the most
common type of retroviruses, except for the envelope pro-
teins. Lacking these envelope proteins' LIR retrotrans-
Dosons cannot bud from their host cell and infect other
cells; however, they can transpose to new sites in the DNA
LTRretrotransposon (=5;11 161
5'
LTR Protein-coding Target-site
(250-600 bp) regron direct repeat
(5-10bp)
FIGURE 6-12 Generalstructureof eukaryoticLTR
retrotransposons. Thecentralprotein-codingregionisflankedby
two longterminal repeats(LTRs),
whichareelement-specific direct
repeats.Likeothermobileelements, retrotransposons
integrated
directrepeatsat eachend Notethat the
haveshorttarget-site
different arenot drawnto scaleTheprotein-coding
regtons region
constitutes andencodes
80 percentor moreof a retrotransposon
reversetranscriptase, andotherretroviral
integrase, proteins
O 229
T R A N S P O S A B L( E
M O B I L E )D N A E L E M E N T S
 of their host cell. Becauseof their clear relationship with > FIGURE 6-14 Model for reversetranscriptionof retroviral
retroviruses,this classof retrotransposonsare often called genomicRNAinto DNA.Inthismodel,a complicated seriesof nine
r etr ouirus-like elements. eventsgenerates a double-stranded DNAcopyof the single-stranded
A key step in the retroviral life cycle is formation of RNAgenomeof a retrovrrus (top)Thegenomic RNAis packaged in
retroviral genomic RNA from integratedretroviral DNA (see thevirionwitha retrovirus-specific
cellularIRNAhybridized to a com-
Figure 4-49). This processservesas a model for generationof plementary sequence nearits 5' endcalledIheprimer-binding site
the RNA intermediate during transposition of LTR retro- (PBS)TheretroviralRNAhasa shortdirect-repeat terminal
sequence
transposons.As depictedin Figure 6-13, the leftward retrovi- (R)at eachend.Theoverall reaction iscarriedout by reverse
transcrip-
ral LTR functions as a promorer that directs host-cell RNA tase,whichcatalyzespolymerization of deoxyribonucleotides.RNaseH
digeststhe RNAstrandin a DNA-RNA hybridTheentireprocess yieldsa
polymeraseto initiate transcription at the 5, nucleotideof the
double-stranded
DNAmolecule thatislongerthanthetemplate RNA
R sequence.After the entire downstream retroviral DNA has
andhasa longterminalrepeat(LTR) at eachend Thedifferentregions
been transcribed, the RNA sequencecorresponding to the
arenotshownto scaleThePBS andRregions areactuallymuchshorter
rightward LTR direcrs host-cell RNA-processingenzymesto thantheU5andU3regions, andthecentral codingregionisverymuch
cleavethe primary transcript and add a poly(A) tail at the 3, longerthantheotherregions. E Gilboa
[See etal, 1979,
Ce//
18:93]
end of the R sequence.The resultingrerroviral RNA genome,
which lacks a completeLIR, exits the nucleusand is packaged is complex, it is a critical aspect of the retrovirus life cycle.
into a virion that buds from the host cell. The processgeneratesthe complete 5' LTR that functions as
After a retrovirus infects a cell, reversetranscription of a promoter for initiation of transcription preciselyat the 5,
its RNA genome by the retrovirus-encodedreverie tran- nucleotide of the R sequence,while the complete 3, LTR
scriptaseyields a double-strandedDNA containing complete functions as a poly(A) site leading to polyadenylation pre-
LTRs (Figure 6-14). This DNA synthesistakes place in the cisely at the 3' nucleotide of the R sequence.Consequentlg
cytosol. The double-strandedDNA with an LTR at each end no nucleotidesare lost from a LTR retrotransposonas it un-
is then transported into the nucleus in a complex with inte- dergoessuccessiverounds of insertion, transcription, reverse
grase, another enzyme encoded by retroviruses. Retroviral transcriprionand re-insertionat a new site.
integrasesare closelyrelatedto the transposases encodedby As noted above, LTR retrotransposons encode reverse
DNA transposonsand use a similar mechanismto insert the transcriptase and integrase. By analogy with retroviruses,
double-strandedretroviral DNA into the host-cell genome. these mobile elementsmove by a "copy-and-paste" mecha-
In this process,short direct repeats of the rarget-sltese- nism whereby reversetranscriptaseconverts an RNA copy
quenceare generatedat either end of rhe insertedviral DNA of a donor elementinto DNA, which is inserted into a target
sequence.Although the mechanism of reversetranscription site by integrase.The experiments depicted in Figure 6-15
provided strong evidencefor the role of an RNA intermedi-
ate in transposition of Ty elements.
LTR Coding region LTR Host-cellDNA The most common LIR rerrotransposons in humans are
Integrated calledERVs,for endogenousretroziruses.Most of the 443,000
retroviral ERV-relatedDNA sequencesin the human genome consist
DNA only of isolated LIRs. Theseare derived from fullJength provi-
Start site PolV(A)site ral DNA by homologous recombination between the two
,
I LIRs, resultingin deletionofthe internal retroviral sequences.
R N AO o l y m e r a lsl e
I IsolatedLTRs suchas thesecannot be transposedto a new po-
Primary
+ sition in the genome, but recombination between homologous
transcript c .n.n.n.n.n.n 3' LTRs at different positions in the genome have likely con-
I tributed to the chromosomalDNA rearrangementsleadingto
RNA-processing
enzymes
I geneand exon duplications, the evolution of proteins with new
I PolY(A)
PolYmerase
Retroviral + combinationsof exons, and, as we will seein Chapter 7, the
evolution of complex control of geneexpression.
RNA - .n.n.n,n.n (A)"
genome R-Us U3-R
A FIGURE 6-13 Generationof retroviralgenomicRNAfrom Non-LTRRetrotransposons Transpose
integratedretroviral DNA.Theleft LTRdirectscellularRNA b y a D i s t i n c tM e c h a n i s m
polymerase to initiate transcription at thefirstnucleotide of the
leftR regionTheresulting primary The most abundant mobile elementsin mammals are retro-
transcript extends beyondthe
rightLTR. TherightLTR, now present in the RNAprimary transposons that lack LTRs, sometimes called nonuiral
transcript,
directs cellular enzymes to cleave the primary transcript at the last retrotransposozs. These moderately repeated DNA se-
nucleotide of the rightR regionandto adda poly(A) tail,yielding a quences form two classesin mammalian genomes: LINEs
retroviral RNAgenomewiththe structure shownat thetop of (long interspersedelements)and SINEs (short interspersed
F i g u r6e- 1 4 A
. s i m i l amr e c h a n i si m
st h o u g htto g e n e r a t eh eR N A elements).In humans, full-length LINEs are-6 kb long, and
intermedrate duringtransposition of retrotransposons Theshort SINEsare:300 bp long (seeTable 6-1). Repeatedsequences
drrect-repeat sequences (black) of target-site DNAaregenerated with the characteristicsof LINEs have been observedin pro-
duringintegration of the retroviral DNAintothe host-cell qenome tozoans, insects,and plants, but for unknown reasonsthey

230 . c H A p r EG
R I G E N E cs E
, N o M t c sA,N Dc H R o M o s o M E s
llll| pe6usAnimation:RetroviralReverse
Transcription

(A)n3',

DNA

I tnrun extended
to form DNA copy (A)"3',
5',
of 5' endof
g e n o m i cR N A

!l nruaof DNA-RNA
hybrid digested

p Firstjump: DNA hybridized

w i t h r e m a i n i n gR N A R s e q u e n c e

E DNA strand extended

from 3' end

[l Most hybrid RNA digested

!l 3' enOof secondDNAstrand

synthesized

PBS R 5',
I hybrid
tnruain DNA-RNA
digested
s,ffis'

p S e c o n dj u m p

5',

p eotn strands 3'

completedby
synthesisfrom c
3' ends
Retroviral DNA

. 231
T R A N S P O S A B L( E
M O B I L E )D N A E L E M E N T S
 are particularly abundant in the genomes of mammals.
SINEs also are found primarily in mammalian DNA. Large
Tyetement
Ef---E numbers of LINEs and SINEs in higher eukaryoteshave
Ty mRNA accumulated over evolutionary time by repeatedcopying of
tv v^er\\,r\^>
sequencesat a few positions in the genome and insertion of
Galactose-sensitive Intron from
f--_-1
| | Promoter n the copiesinto new positions.
| | anotheroene

LINEs Human DNA contains three major families of LINE

Engn
i eer sequencesthat are similar in their mechanismof transposition,
recombinantTy but differ in their sequences:L1.,L2, and L3. Only membersof
in plasmidvectors the L1 family transposein the contemporary human genome.
Apparently there are no remaining functional copies of L2 or
L3. LINE sequences arepresentat :900,000 sitesin the human
genome,accounting for a staggering21 percent of total human
DNA. The generalstructure of a completeLINE is diagrammed
in Figure 6-15. LINEs usually are flanked by short direct re-
Gal-responsive
Ty Gal-responsive Ty peats, the hallmark of mobile elements,and contain two long
with unrelated open readingframes (ORFs).ORF1, -1 kb long, encodesan
Transformyeast cells; added intron RNA-binding protein. ORF2, -4 kb long, encodesa protein
grow in galactose-and I
n o n g a l a c t o s e - c o n t a i n im
negd i a that has a long region of homology with the reversetranscrip-
tases of retroviruses and LTR retrotransposons.but also
I
exhibitsDNA endonuclease activiry.
Results in galactose. Results in galactose-
containingmedium containingmedium
1 .T y m R N As y n t h e s i s 1 . T y m R N A sl a c k Evidence for the mobility of L1 elements first came
i ncreased i ntron from analysisof DNA cloned from humans with cer-
2. Transpositionof Ty 2. TransposedTy
e l e m e n t si n c r e a s e d elementslack intron
tain genetic diseasessuch as hemophilia and myotonic
dystrophy. DNA from these patients was found to carry
t*^->
mutations resulting from insertion of an L1 elementinto a
Ty mRNA
Primarytranscript gene,whereasno such elementoccurredwirhin this genein
I RryI. either parent. About 1 in 600 mutarions rhat causesignifi-
+spilcrng cant diseasein humans are due to L1 transpositions or
l%->

Ty mRNA SINE transpositionsthat are catalyzedby L1-encodedpro-

I Reverse teins. Later experiments similar to those just described
+transcription with yeastTy elements(seeFigure 6-15) confirmed that L1
r----I: elementstransposethrough an RNA intermediate.In these
TransposedTy experiments,an intron was introducedinto a cloned mouse
L1 element, and the recombinant L1 element was stably
EXPERIMENTAL FTGURE G-l5 The yeastTy element transformed into cultured hamster cells. After severalcell
transposes throughan RNAintermediate. Whenyeastcellsare doublings, a PCR-amplified fragment corresponding to the
transformed with a Ty-containing plasmid, theTyelement can L1 elementbut lacking the insertedintron was detectedin
transpose to newsites,although normally thisoccursat a low rate
Usingthe elements diagrammed at thetop,researchers engineered
two differentrecombinant plasmid vectors containing recomornanr Long interspersedelement (LINE)(-6 161
Tyelements adjacent to a galactose-sensitive promoterThese
plasmids weretransformed intoyeastcells,whichweregrownin a
g a l a c t o s e - c o n t a ranni dnagn o n g a l a c t oms e d i u mI ne x p e r i m e 1
n ,t
growthof cellsin galactose-containing mediumresulted in many
moretranspositions thanin nongalactose medium,indicating that
transcription tntoan mRNAintermediate is required for Ty FIGURE 6-16 Generalstructureof a UNE,a non-LTR
transposition In experiment 2, an intronfroman unrelated yeast retrotransposon. Mammalian DNAcarries two classesof non_LTR
genewasinserted intothe putative protein-coding regionof the retrotransposons,LINES (notshown)Thelengthof the
andSINES
recombinant galactose-responsive Tyelement. Theobserveo aosence target-site
directrepeats varies
amongcopiesof a LINEat differentsites
of the intronin transposed Tyelements isstrongevidence that in thegenomeAlthough thefull-length
L1sequence is=6 kb long,
transposition involves an mRNAintermediate fromwhichthe intron variableamountsof the leftendareabsentat over90 percentof the
wasremoved by RNAsplicing, asdepicted in the boxin the lower siteswherethismobileelement isfound Theshorter openreading
right In contrast, eukaryotic DNAtransposons, liketheAc element of frame(ORF1), -1 kb in length,
encodes an RNA-binding proteinThe
maize,containintronswithinthetransposase gene,indicating that longerORF2, -4 kb in length,encodes a bifunctional
proteinwith
theydo nottranspose viaan RNAintermediate. [See J.Boeke etal.. reversetranscriptase
andDNAendonuclease activity.
Notethat LINEs
1985. Cell 4O:491 I
lackthe longterminalrepeats foundin LTRretrotransposons.

232 o c H A p r E R6 | c E N E sG
, E N o M t c sA, N D c H R o M o s o M E s
 ORF2 < FIGURE 6-17 Proposedmechanism of LINEreverse
protein transcriptionand integration.Aftersynthesis of ORFlandORF2
C h r o m o s o m aD
l NA in thecytosol, a complex of LINE RNA (red), multiple copies of ORFl
5', AAATACT to the Poly(A) tail moves intothe
andone copy of ORF2 bound
TTTATGA 5',
nucleusOnlyORF2protein, a reverse transcrlptase, is represented
L I N ER N A Newlysynthesized LINEDNAisshownin blackStep[: ORF2makes
staggered nicksin chromosomal DNAon eithersrdeof anyaccessible
A/f-richsequence in the genome. Stepfl: Reverse transcription of
LINE RNA by ORF2 is primed by the single-stranded T-richsequence
E *,.n'"n generated bythe nickin the bottomstrand,whichhybridizes to the
f Nicksite Nicksite LINE poly(A) tail StepsB-E: ORF2
andthencontinues thisnewDNA
reverse-transcribes
strand, switching to
the LINE
the
RNA
single-
5', stranded regionof the upperchromosomal strandasa template
3', thenhydrolyze the RNAandextendthe3' end
Step6: Cellular enzymes
of thechromosomal DNAtopstrand, replacing theLINE RNAstrand
withDNA StepZ: Finally, the5' and 3' ends of the DNA strandsare
completing
ligated, the insertion The lasttwo steps probably are
catalyzedbythesamecellular enzymes that removeRNAprimers and
a P r i m i n g o f r e v e r s et r a n s c r i p t i o n
b v c h r o m o s o m aD l NA
ligateOkazaki fragments
{romD D Luan
duringDNAreplication
etal, 1993, Cell72:5951
(seeFigure 4-30)
lAdapted

AAA the cells. This finding strongly suggeststhat over time the
recombinantL1 elementcontaining the insertedintron had
transposedto new sitesin the hamster genomethrough an
I RNA intermediate that underwent RNA splicing to remove
S
- I R e v e r s et r a n s c r i p t i o n
o t L I N ER N A b y O R F 2 the intron. I
I

J
Since LINEs do not contain LTRs, their mechanism of
TGA 5', transpositionthrough an RNA intermediatediffers from that
of LTR retrotransposons.ORF1 and ORF2 proteins are
translated from a LINE RNA. In vitro studies indicate that

L I N ER N A

TTTATGA.,..

orotein then bind to the LINE RNA, and ORF2 protein

tindr ,o the poly(A) tail. The LINE RNA is then transported
orchromosomal back into the nucleus as a complex with ORF1 and ORF2
E SR?';f oroteins. and is reverse-transcribedinto LINE DNA in the
I .rr.r.letr,by ORF2. The mechanism involves staggeredcleav-
5' AAA age of cellular DNA at the insertion site followed by priming
3' TTTATGA 5' o] ,.u.r.. transcription by the resulting cleaved cellular
DNA as detailedin Figure 6-L7. The completeprocessre-
sults in insertion of a copy of the original LINE retrotrans-
.,. I Insertion completed by ooson into a new site in chromosomal DNA' A short direct
lI c e l l u l aer n z y m e s ,.p.", is generatedat the insertion site becauseof the initial
I 3. staggeredcleavageof the two chromosomal DNA strands'
AAATACT!,\/^/>TNAAAA
5',
TTTATGA\AAAAATTTATGA As noted abeady,the DNA form of an ITR retrotrans-

I
El
*
'rNE DNA
5',
al

L I N ED N A a reversetranscriptase,is primed by the 3'-end of cleaved

Directrepeats chromosomalDNA' which basepairs with the poly(A) tail

T R A N S P O S A B(LMEO B I L E D
) NA ELEMENTS 233
 of the non-LTR retroviral RNA (seeFigure 6-17, step Z).
Sinceits synthesisis primed by the cut end of a cleavedchro-
mosome, and synthesisof the other strand of the non-LTR
retrotransposonDNA is primed by the 3,-end of chromoso-
mal DNA on the other side of the initial cut in chromosomal
DNA (step E), rhe mechanismof synthesisresultsin inte-
gration of the non-LTR retrotransposon DNA. There is no
need for an integraseto insert the non-LTR rerrorransposon
DNA.
The vast majority of LINEs in the human genome are
truncated at their 5' end, suggestingthat reverserranscrip-
tion terminated before completion, and the resulting frag-
ments extending variable distancesfrom the poly(A) tail
intermediatein transposition.In addition to the fact that
most L1 insertions are truncared, nearly all the full-length
elementscontain stop codons and frameshift mutations in
ORFl and ORF2; thesemutations probably have accumu-
lated in most LINE sequences over eutl.rtionary time. As a re-
sult, only :0.01 percentof the LINE sequencesin the human
genome are full-length with intact open reading frames for
ORF1 and ORF2, representing-60-100 in total number.
SINEs The secondmost abundanrclassof mobile elements
in the human genome,SINEs constitute :13 percent of total
human DNA. Varying in length from about 100 to 400 base
pairs, theseretrotransposonsdo not encodeprotein, but most
contain a 3' A/T:rich sequencesimilar to thar in LINEs. SINEs
are transcribed by the same nuclear RNA polymerase that
transcribes genes encoding tRNAs, 55 rRNAs, and other
ing and reverse transcription/integration by LINE encoded
ORF1 and ORF2.
SINEs occur at about 1.6 million sites in the human
g e n o m e . O f t h e s e , - 1 . 1 m i l l i o n a r e A l u e l e m e n t s ,s o
named becausemost of them contain a single recognition
site for the restriction enzyme AluI. Alw elementsexhibit
considerable sequencehomology with and probably
evolved from 7SL RNA, a cytosolic RNA in a ribonucleo-
protein complex called the signal recognition particle.
This abundant cytosolic ribonucleoproteinparticle aids in
targetlng cerrain polypeptides to the membranes of the
endoplasmic reticulum (Chapter 13). Alu elemenrs are
scattered throughout the human genome at sites where
their insertion has not disrupted geneexpression:between
genes,within introns, and in the 3, untranslatedregionsof
some mRNAs. For instance,nine Alu elementsare located
y1t\in the human B-globin gene clusrer (seeFigure 6-4a).
Of the one new germ-line non-LTR retrotranspositionthat
is estimated to occur in about every eight individuals,
234 . c H A p r E6R | G E N EG
s ,E N o M t cAsN
, Dc H R o M o s o M E s
-40 percent involve L1 and 50 percent involve SINEs, of
which -90 percent areAlw elements.(Note that nearly all
new insertionsin human DNA involve retrotransposons.)
Similar to other mobile elements,most SINEs have ac-
cumulated mutations from the time of their insertion in
the germ line of an ancient ancestor of modern humans.
Like LINEs, many SINEs also are truncated at their 5,
end.
Other Retrotransposed RNAsAre Found
i n G e n o m i cD N A
In addition to the mobile elementslisted in Table 6-1, DNA
copies of a wide variety of mRNAs appear to have inte-
grated into chromosomalDNA. Sincethesesequences lack
introns and do not have flanking sequencessimilar to those
of the functional genecopies,they clearly are nor simply du-
plicated genesthat have drifted into nonfunctionality and
becomepseudogenes, as discussedearlier (seeFigure 6-4a).
Instead, theseDNA segmentsappear to be retrotransposed
copies of spliced and polyadenylatedmRNA. Compared
with normal genes encoding mRNAs, these inserted seg-
ments generally contain multiple mutations, which are
thought to have accumulated since their mRNAs were first
reverse-transcribedand randomly integrated into the
genome of a germ cell in an ancient ancestor.These non-
functional genomic copies of mRNAs are referred to as
processedpsewdogenes. Most processedpseudogenesare
flanked by short direct repeats, supporting the hypothesis
that they were generated by rare retrotransposition events
involving cellular mRNAs.
Other interspersedrepeatsrepresentingpartial or mutant
copiesof genesencoding small nuclear RNAs (snRNAs) and
tRNAs are found in mammalian genomes. Like processed
pseudogenesderived from mRNAs, these nonfunctional
copies of small RNA genes are flanked by short direct re-
peats and most likely result from rare retrotransposition
events that have accumulated through the course of evolu-
tion. Enzymes expressedfrom a LINE are thought to have
carried out all these retrotransposition events involving
mRNAs, snRNAs. and tRNAs.
M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l y
I n f l u e n c e dE v o l u t i o n
Although mobile DNA elements appear to have no direct
function other than to maintain their own existence,their
presencehas had a profound impact on the evolution of
modern-day organisms.As mentioned earlier,about half the
spontaneousmutations in Drosophila result from insertion
of a mobile DNA elemenrinto or near a transcription unit.
In mammals, mobile elementscause a much smaller pro-
portion of spontaneousmutations: -10 percent in mice,
and only 0.1-0.2 percentin humans. Still, mobile elements
have been found in mutant allelesassociatedwith several
human genetic diseases.For example, insertions into the
clotting factor IX genecausehemophilia, and insertionsinto
the gene encoding the muscle protein dystrophin lead to
myotonic dystrophy,commonly known as Duchennemuscu-
lar dystrophy. The genesencoding factor IX and dystrophin
are both on the X chromosome. Becausethe male genome
has only one copy of the X chromosome, transposition in-
sertionsinto thesegenespredominantly affect males.
In lineagesleading to higher eukaryotes'homologous re-
combination between mobile DNA elements dispersed
throughout ancestralgenomesmay have generatedgene du-
plications and other DNA rearrangementsduring evolution
(seeFigure 6-2b).For instance,cloning and sequencingof the
B-globin gene cluster from various primate specieshas pro-
vided strong evidencethat the human G" and A" genesarose
from an unequal homologous crossoverbetweentwo L1 se-
quencesflanking an ancestralglobin gene.Subsequentdiver-
genceof suchduplicatedgenescould leadto acquisitionof dis-
tinct, beneficialfunctions associatedwith each member of a
gene family. Unequal crossing over between mobile elements
located within introns of a particular gene could lead to the
duplication of exons within that gene (seeFigure 6-2a). This
processmost likely influencedthe evolution of genesthat con-
tain multiple copiesof similar exons encodingsimilar protein
domains,such as the fibronectin gene(seeFigure 4-16).
Some evidence suggeststhat during the evolution of
higher eukaryotes,recombination betweenmobile DNA ele-
ments (e.g., Alu elements)in introns of two sepdrdte genes
also occurred, generatingnew genesmade from novel com-
binationsof preexistingexons (Figure6-18).This evolution-
ary process,termed exon shuffling, maY have occurred dur-
ing evolution of the genes encoding tissue plasminogen
activator, the Neu receptor, and epidermal growth factor'
which all contain an EGF domain (seeFigure 3-11). In this
case, exon shuffling presumably resulted in insertion of an
EGF domain-encoding exon into an intron of the ancestral
form of each of thesegenes.
Both DNA transposonsand LINE retrotransposonshave
been shown to occasionallycarry unrelated flanking se-
quenceswhen they transposeto new sitesby the mechanisms
diagrammedin Figure 6-19. These mechanismslikely also
contributed to exon shuffling during the evolution of con-
temporarygenes.
In addition to causingchangesin coding sequencesin the
genome,recombination between mobile elementsand trans-
position of DNA adiacent to DNA transposonsand retro-
i."nrporon, likely played a significantrole in the evolution of
regulatory sequencesthat control gene expression'As noted
earlier, eukaryotic genes have transcription-control regions
> FIGURE6-18 Exon shuffling via
recombination between homologous
interspersedrepeats. Recombination
betweeninterspersed repeatsin the intronsof
separategenesproducestranscriptionunits
with a new combinationof exons(greenand
blue) ln the exampleshown here,a double
crossoverbetween two setsof A/u elements,
t I N E isn h u m a n s r) e s u l t s
( t h em o s ta b u n d a n S
in an exchangeof exonsbetweenthe two
9enes
called enhancersthat can operate over distancesof tens of
thousandsof basepairs. Transcription of many genesis con-
trolled through the combined effectsof severalenhancerele-
ments.Insertionof mobile elementsnear such transcription-
control regionsprobably contributed to the evolution of new
combinatr,onsof enhancersequences.These in turn control
which specificgenesare expressedin particular cell types and
the amount oi the encoded protein produced in modern
organisms,as we discussin the next chapter'
Theseconsiderationssuggestthat the early view of mobile
DNA elements as completely selfish molecular parasites
missesthe mark. Rather,they have contributed profoundly to
the evolution of higher organismsby promoting (1) the gen-
eration of genefamilies via geneduplication, (2) the creation
of new genesvia shuffling of preexistingexons' and (3) for-
mation of -o.. complex regulatory regionsthat provide mul-
tifaceted control of gene expression.Today, researchersare
attempting to harnesstranspositionmechanismsfor inserting
therapeuticgenesinto patients as a form of genetherapy'
Transposable(Mobile) DNA Elements
r Transposable DNA elements are moderately repeated
sequencestnterspersedat multiple sites throughout the
g.r,o-., of highir eukaryotes. They are present less fre-
quently in prokarYotic genomes.
r DNA transposonsmove to new sites directly as DNA;
first transcribed into an RNA copy of
retrotransporo.t.
which
".. then is reverse-transcribedinto DNA
the element,
( s e eF i g u r e6 - 8 ) '
r A common feature of all mobile elementsis the presence
of short direct repeatsflanking the sequence'
r Enzymesencodedby transposonsthemselvescatalyzein-
sertion of thesesequencesat new sitesin genomic DNA'
r Although DNA transposons'similar in structure to bac-
terial IS elements,occur in eukaryotes(e'g', the Drosophila
P element), retrotransposonsgenerally are much more
abundant, especiallyin vertebrates.
r LTR retrotransposonsare flanked by long terminal re-
peats (LTRs), similar to those in retroviral DNA; like
retroviruses,they encode reversetranscriptaseand inte-
grase. They move in the genome by being transcribed
into RNA, which then undergoesreversetranscription in
G e n e1 l l ]
G e n e2 I I I
I Doublecrossover
betweenA/uelements
{
+
T R A N S P O S A B (LM ) NA ELEMENTS
EO B I L E D 235
 (a)
< FIGURE 5-19 Exonshufflingvia
G e n e1 l l l
transpositionof a DNAtransposonor LINE
retrotransposon. (a)Transposition of an
Trun.ooru."excisionfrom gene exon(blue)flankedby homologous DNA
I transposons intoan intronof a second gene
As we sawin Figure 6-10,step[, transposase
canrecognize andcleave the DNAat the ends
tIsarfllo I S te of thetransposon inverted repeatsln gene1,
if thetransposase cleaves at the leftendof the
G e n e2 = : = ::: transposon on the leftandat the rightendof
I
I Trencnn, s a s ei n s e r t i o ni n t o g e n e2 thetransposon on the right,it cantranspose
y a l lt h ei n t e r v e n i n DgN A i,n c l u d i nt g
h ee x o n
fromgene1,to a newsitein an intronof
gene2 Thenetresultisan insertion of the
exonfromgene1 intogene2 (b)lntegration
(b)
W e a kp o l y ( A ) G e n e ' sp o l y ( A ) of an exonintoanothergeneviatransposition
srgnat srgnat
of a LINE c o n t a i n i nagw e a kp o l y ( As)i g n a l .f
s u c ha L I N E i s i n t h e3 ' - m o sitn t r o no f g e n e
G e n e1 l l ]
1 , t r a n s c r i p t i o fnt h eL I N Ei n t oa n R N A
3'exon
i n t e r m e d i am t ea yc o n t i n ubee y o n di t so w n
T r a n s c r i p t i oann d p o l y a d e n y l a t i o n poly(A) siteandextendintothe 3, exon,
at end of downstreamexon
t r a n s c r i b i tnhgec l e a v a gaen dp o l y a d e n y l a t i o n
%AAAA s i t eo f g e n e1 i t s e l fT h i sR N Ac a nt h e nb e
reverse-transcribed andintegrated by the
l l t s c n t o r ts t e
L I N EO R F 2 p r o t e i n( s e eF i g u r 6
e- ' 7
l ) i n t oa n
i n t r o no n g e n e2 , i n t r o d u c i naqn e w3 , e x o n
G e n e2 : : : ::: ( f r o mg e n e1 )i n t og e n e2
I
I ORF2reversetranscription
and insertion
I
:: = :::

the cytosol, nuclear import of the resulting DNA with

LTRs, and integration into a host-cell chromosome (see @ OrganelleDNAs
F i g u r e6 - 1 4 ) . Although the vast majority of DNA in most eukaryotesis
found in the nucleus,some DNA is presentwithin the mi-
tochondria of animals, planrs, and fungi and within the
chloroplastsof plants. Theseorganellesare the main cellu-
lar sitesfor ATP formation, during oxidative phosphoryla-
tion in mitochondria and photosynthesisin chloroplasts
(Chapter 12). Many lines of evidenceindicate that mito-
( s e eF i g u r e6 - 1 7 ) . chondria and chloroplastsevolvedfrom bacteriathat were
endocytosedinto ancestral cells containing a eukaryotic
sequencesexhibit extensivehomology with small
nucleus,forming endosymbionts(Figure 6-20). Over evo_
RNAs and are rranscribedby the sameRNA poly_
lutionary time, most of the bacterialgeneswere lost from
A/z elements,the most common SINEsin humans,
0-bp sequences found scatteredthroughout the hu_
man genome.
r Some interspersedrepeats are derived from cellular
RNAs that were reverse-transcribedand inserted rnto ge-
nomic DNA at some time in evolutionarv history. nucleus. However, mitochondria and chloroplasts in to_
Processed pseudogenes derivedfrom mRNAs lack tntrons, day's eukaryotesretain DNAs encoding some prorernses-
a featurethat distinguishesthem from pseudogenes,which sentialfor organellarfunction, as well as the ribosomal and
aroseby sequencedrift of duplicatedgenes. transfer RNAs required for synthesis of these proteins.
bile DNA elementsmost likely influencedevolution Thus eukaryotic cells have multiple geneticsysrems:a pre-
cantly by servingas recombinationsitesand by mo_ dominant nuclear systemand secondarysystemswith their
g adjacentDNA sequences. own DNA, ribosomes, and tRNAs in mitochondria and
chloroplasts.

236 CHAPTER
6 | G E N E SG
, E N O M t C SA, N D C H R O M O S O M E S
 Endocytosisof bacterium Endocytosisof bacterium
, Ancestral capableof photosYnthesis
caoableof oxidative cell
phosphorylation
ATPsynthase BacteriaI
t, ./ genome
Bacterial \/
genome
E u k a r y o t i cp l a s m am e m b r a n e

Mitochondrial Mitochondrial Stroma

matrix genome
FIGURE 6-20 Modelfor endosymbiotic origin of membrane suchthatthe portionof the
retaintheirorientation,
mitochondriaand chloroplasts. Endocytosisof a bacteriumby an spacenowfacesthe
proteinoncefacingthe extracellular
cellwouldgenerate an organellewith two intermembranespace. Budding fromthe innerchloroplast
of vesicles
eukaryotic
ancestral
membrane, suchasoccursduringdevelopment in
of chloroplasts
membranes, theoutermembrane derivedfromthe eukaryotic
(gray) plasma contemporaryplants,would generatethe thylakoidmembranes of
plasmamembrane andthe inneronefromthe bacterial
(red)Proteins to the ancestral
localized bacterial The
chloroplasts DNAs
organellar areindicated
membrane

M i t o c h o n d r i aC o n t a i nM u l t i p l e colonies. Genetic crossesbetween different (haploid) yeast

strains showed that the petite mutation does not segregate
m t D N AM o l e c u l e s
with any known nuclear geneor chromosome' In later stud-
Individual mitochondria arclarge enough to be seenunder ies, most petite mutants were found to contain deletions of
the light microscope, and even the mitochondrial DNA MtDNA.
(mtDNA) can be detectedby fluorescencemicroscopy.The
mtDNA is located in the interior of the mitochondrion, the
region known as the matrix (seeFigure 12-6). As judged by
the number of yellow fluorescent "dots" of mtDNA' a
Euglena gracilis cell contains at least 30 mtDNA molecules
( F i g u r e6 - 2 1 ) .
Replication of mtDNA and division of the mitochondrial
network can be followed in living cells using time-lapsemi-
croscopy.Such studiesshow that in most organismsmtDNA
replicates throughout interphase. At mitosis each daughter
cell receivesapproximately the same number of mitochon-
dria, but since there is no mechanism for apportioning ex-
actly equal numbers of mitochondria to the daughter cells,
some cells contain more mtDNA than others. By isolating
mitochondria from cells and analyztngthe DNA extracted
from them, it can be seenthat each mitochondrion contains
multiple mtDNA molecules. Thus the total amount of
mtDNA in a cell dependson the number of mitochondria,
the size of the mtDNA, and the number of mtDNA mole-
cules per mitochondrion. Each of these parametersvaries
greatly between different cell types. 10tr-
, t

A EXPERIMENTAL FIGURE 6-21 Dualstainingrevealsthe

m t D N A l s l n h e r i t e dC y t o p l a s m i c a l l y
muftipfemitochondrialDNAmoleculesin a growing Euglena
Studies of mutants in yeasts and other single-celledorgan- graciliscell.Cellsweretreatedwith a mixtureof two dyes:ethidium
isms first indicated that mitochondria exhibit cytoplasmic bromide, whichbindsto DNA andemits a red and
fluorescence,
inheritance and thus must contain their own genetic system D|OC6, which is into
specifically
incorporated mitochondriaand emits
(Figure 6-22). For instance,petite yeast mutants exhibit a greenfluorescence. Thusthe nucleusemitsa redfluorescence,and
structurally abnormal mitochondria and are incapable of areasrichin mitochondrialDNAfluoresce yellow-a combination of
redDNAandgreenmitochondrial fluorescence [FromY Hayashiand
oxidative phosphorylation. As a result, petite cells grow
more slowly than wild-type yeasts and form smaller K Ueda,1989, J CellSci93:565l

o R G A N E L L ED N A s t 237
 (a) Haploidparentswith < FIGURE 6-22 Cytoplasmic inheritanceof the pefite mutation
wild-type nucleargenes
in yeast.Petite-strain
mitochondria aredefective in oxidative
Normal "Petite" phosphorylation owingto a deletion in mtDNA(a)Haploid cellsfuse
mitochondrion mitochondrion to produce a diploidcellthatundergoes meiosis,duringwhichrandom
segregation of parental
chromosomes andmitochondria containing
mtDNAoccursNotethatalleles for genesin nuclear DNA(represented
by largeandsmallnuclear chromosomes colored redandblue)
segregate 2:2duringmeiosis (seeFigure 5-5) Incontrast, sinceyeast
normallycontain -50 mtDNAmolecules percell,allproducts of meiosis
usually
containbothnormalandpetitemtDNAs andarecapable of
respiration(b)Asthesehaploidcellsgrowanddividemitotically, the
cytoplasm (including
themitochondria) israndomly distributedto the
daughter cellsOccasionally,a cellisgenerated thatcontains only
petitemtDNAandyieldsa petitecolony.
defective Thusformation of
Diploid suchpetitecellsisindependent of anynuclear geneticmarker.
zygore
In the mating by fusion of haploid yeastcells,both parents
contribute equally to the cytoplasm of the resulting diploid;
thus inheritance of mitochondria is biparental. In mammals
M e i o s i s :r a n d o md i s t r i b u t i o n and most other multicellular organisms,however,the sperm
of mitochondriato contributeslittle (if any) cytoplasmto the zygote,and virtually
d a u g h t e rc e l l s all the mitochondria in the embryo are derived from those
in the egg, not the sperm. Studiesin mice have shown that
99.99 percentof mtDNA is maternally inherited, but a small
part (0.01 percent)is inheritedfrom the male parent. In higher
plants, mtDNA is inherited exclusivelyin a uniparental fash-
ion through the femaleparent (egg),not the male (pollen).

The Size,Structure,and CodingCapacity

of mtDNA Vary ConsiderablyBetweenOrganisms
Surprisingly,the size of the mtDNA, the number and nature
of the proteins it encodes,and eventhe mitochondrial genetic
code itself vary gre^iy between different organisms. The
(b) mtDNAs of most multicellular animals are :16-kb circular
moleculesthat encodeintron-lessgenescompactly arranged
on both DNA strands. VertebratemtDNAs encode the two
rRNAs found in mitochondrial ribosomes, the 22 tRNAs
used to translatemitochondrial mRNAs, and 13 proteins in-
volved in electron transport and AIP synthesis(Chapter 12).
The smallestmitochondrial genomesknown arein plasmod-
ium, single-celledobligate intracellular parasitesrhat cause
malaria in humans. Plasmodium mtDNAs are only :6 kb,
encoding five proteins and the mitochondrial rRNAs.
The entire mitochondrial genomesfrom a number of
different metazoan organisms (i.e., multicellular animals)
have now been cloned and sequenced,and mtDNAs from
all these sourcesencode essentialmitochondrial proteins
(Figure 6-23). All proteins encodedby mtDNA are synthe-
\ sized on mitochondrial ribosomes. Most mitochondria-
\Mitosis synthesizedpolypeptidesidentified thus far are subunits of
\ multimeric complexesused in electrontransport. ATp svn-
t h e s i s ,o r i n s e r r i o no f p r o r e i n si n t o t h e i n n e r m i t o c h o n d i i a l
membrane or intermembranespace.However, most of the
proteins localizedin mitochondria, such as those involved
in the processeslisted at the top of Figure 6-23, are en-
coded by nuclear genes, synthesizedon cytosolic ribo-
Respiratory-proficient Petite Respiratory- somes, and imported into the organelle by processesdis-
proficient c u s s e di n C h a p t e r1 3 .

238 . C H A P T E6R I G E N E SG
, E N O M I C SA, N D C H R O M O S O M E S
 L i p i dm e t a b o l i s m C a r b o h y d r a tm
e etabolism U b i q u i n o n es y n t h e s i s Chaperones
N u c l e o t i d em e t a b o l i s m H e m es y n t h e s i s Co-factorsynthesis S i g n a l i n gp a t h w a y s
A m i n o a c i dm e t a b o l i s m Fe-Ssynthesis Proteases DNA repair,replication,etc.

Heme
ryase

RNA
polymerase

TIM

c
Cvtochrome

A FIGURE 6-23 Proteinsencodedin mitochondrialDNA translocases areinvolved in proteinimportandexport,andinsertion

and their involvementin mitochondrialprocesses. Onlythe o f p r o t e i nisn t ot h e i n n e rm e m b r a n(eC h a p t e1r3 ) R N a sP e i sa
m i t o c h o n d rm i aal t r i xa n di n n e r
m e m b r a naer ed e p i c t e dM o s tm i t o - ribozyme that processes the 5'-endof tRNAs(Chapter 8) lt should
areencoded bythenucleus (blue); mitochondrial be notedthat the majorityof eukaryotes havea multi-subunit
chondrial components
components complex I as depicted here, with three subunits invariantly encoded
processes carriedout by exclusively nucleus-encoded
by mtDNA However, in a few organisms (e g , Saccharomyces'
a r el i s t e da t t h et o p M i t o c h o n d r ci aolm p o n e nst sh o w ni n p i n ka r e
by mtDNAin someeukaryotes but bythe nuclear genomein Schizosaccharomyces, andPlasmodium), thiscomplexis replacedby
encoded
few components invariably encoded in a nucleus-encoded, single-polypeptide enzyme. Formoredetailson
othereukaryotes. The relatively
l-V areinvolved in electron mitochondrial m e t a b o l i sam n dt r a n s p o rst e
, eC h a p t e r1s2 a n d1 3
mtDNAareshownin orangeComplexes
phosphorylation TlM,Sec,Tat,andOxal lAdapted from G Burger et al , 2003, Trends Genet 19:709 ]
transport andoxidative

In contrast to metazoan mtDNAs, plant mtDNAs are
many times larger,and most of the DNA doesnot encodepro-
tein. For instance,the mtDNA in the important model plant
Arabidopsis thaliana is 366,924 base pairs, and the largest
known mtDNA is =2 Mb, found in cucurbit plants (e.g.,
melon and cucumber).Most plant mtDNA consistsof long in-
trons, pseudogenes, mobile DNA elementsrestrictedto the mi-
tochondrial compartment, and piecesof foreign (chloroplast,
nuclearand viral) DNA that were probably insertedinto plant
mitochondrial genomesduring their evolution' Duplicated se-
quencesalso contributeto the greaterlength of plant mtDNAs.
Differences in the number of genes encoded by the
mtDNA from various organisms most likely reflect the
movement of DNA between mitochondria and the nucleus
during evolution. Direct evidencefor this movement comes
from the observation that several proteins encoded by
mtDNA in some speciesare encoded by nuclear DNA in
other, closely related species.The most striking example of
this phenomenon involves the cor 11 gene, which encodes
subunit 2 of cytochrome c oxidase, which constitutes com-
plex IV in the mitochondrial electron transport chain (see
Figure 12-1.6).This gene is found in mtDNA in all multicel-
lular plants studied except for certain related speciesof
legumes,including the mung bean and soybeans,in which
the cox 11geneis nuclear.The cox 1I geneis completely miss-
ing from mung beanmtDNA, but a defectivecox 1/ pseudo-
gene that has accumulatedmany mutations can still be rec-
ognized in soybeanmtDNA.
Many RNA transcripts of plant mitochondrial genesare
edited, mainly by the enzyme-catalyzedconversion of se-
lected C residuesto U, and occasionallyU to C' (RNA edit-
ing is discussedin Chapter 8.) The nuclear cox lI gene of
mrng bean corresponds more closely to the edited cox II
RNA transcripts than to the mitochondrial cox 11 genes
found in other legumes. These observations are strong
evidencethat the cox ll genemoved from the mitochondrion
to the nucleusduring mung bean evolution by a processthat
involved an RNA intermediate. Presumablythis movement
involved a reverse-transcriptionmechanism similar to that
by which processedpseudogenesare generatedin the nuclear
genome from nucleus-encodedmRNAs.
In addition to the large differences in the sizes of
mtDNAs in different eukaryotes' the structure of the
mtDNA also varies greatly.As mentioned above, mtDNA in
most animals is a circular molecule -16 kb' However' the
mtDNA of many organismssuch as the protist Tetrahymena
exists as linear head-to-tail concatomersof repeating se-
quence. In the most extreme examples, the mtDNA of the
protist Amoebidium parasiticum is composed of several
'h,rndred
distinct short linear molecules'And the mtDNA of
Trypanosoma is comprised of multiple maxicircles concate-
,r"t.d (ittt.tlocked) to thousands of minicircles encoding

o R G A N E L L ED N A s o 239
 guide RNAs involved in editing the sequenceof the mito-
chondrial mRNAs encodedin the maxicircles.
P r o d u c t so f M i t o c h o n d r i aG
l enes
Are Not Exported
As far as is known, all RNA transcripts of mtDNA and their
translationproducts remain in the mitochondrion in which
they are produced, and all mtDNA-encoded proteins are
synthesizedon mitochondrial ribosomes. Mitochondrial
DNA encodesthe rRNAs that form mitochondrial ribo-
somes, although most of the ribosomal proteins are im-
ported from the cytosol.In animalsand fungi, all the tRNAs
used for protein synthesisin mitochondria also are encoded
by mtDNAs. However,in plantsand many prorozoans,most
mitochondrialtRNAs are encodedby the nuclearDNA and
imported into the mitochondrion.
Reflectingthe bacterialancestryof mitochondria, mito-
chondrial ribosomesresemblebacterial ribosomesand
differ from eukaryotic cytosolic ribosomesin their RNA and
protein compositions,their size,and their sensitivityto certain
antibiotics (seeFigure 4-22). For insrance,chloramphenicol
blocks protein synthesisby bacterialand mitochondrial ribo-
somesfrom most organisms,but cycloheximidedoesnot. This
sensitivity of mitochondrial ribosomes ro the imoortant
aminoglycosideclass of antibiotics that includ., .hlo."--
phenicol is the main causeof the toxicity that theseantibiotics
can cause. Conversely,cytosolic ribosomes are sensitiveto
cycloheximideand resisrantto chloramohenicol.I
M i t o c h o n d r i aE v o l v e df r o m a S i n g l e
E n d o s y m b i o t iEc v e n tI n v o l v i n ga
-l
Rickettsi a i ke Bacteriu m
Analysisof the mtDNA sequences from various eukaryotes,
including single-celledprotists that diverged from other eu,
karyotes early in evolurion, provides rtrong support for the
idea that the mitochondrion had a singleorigin. Mitochon-
dria most likely arosefrom a bacterialsymbiontwhoseclos-
est contemporary relatives are in the Rickettsiacedegroup.
Bacteria in this group are obligate intracellular parasitei.
Thus, the ancestor of the mitochondrion probably also had
an intracellular life style, putting it in a good location for
evolving into an intracellularuylbion,. ihe mtDNA with
the largest number of encoded genesso far found is in the
protist speciesReclinomonasamericana. All other mtDNAs
have a subset of the R. amertcana genes)strongly implying
that they evolved from a common ancestor with R. i*rrl-
cqna, losing different groups of mitochondrial genesby dele-
tion and/or transfer to the nucleusover time.
In organismswhose mtDNA includesonly a limited num_
ber of genes, rhe same set of mitochondrial genes are re-
tained, independent of the phyla that includes these organ_
rsms (seeFigure 6-23, orangeproteins). One hypothesisfor
why thesegeneswere never successfullytransferredto the nu-
clear genome is that their encoded polypeptides are too hy-
drophobic to cross the outer mitochondrial membrane. and
240 . c H A p r EbR I G E N EcsE, N o M t c A
s ,N Dc H R o M o s o M E s
thereforewould not be imported back into the mitochondria
if they were synthesizedin the cytosol. Similarly,the large size
of rRNAs may interferewith their transport from the nucleus
through the cytosol into mitochondria. Alternatively, these
genesmay not have been transferred to the nucleus during
evolution becauseregulation of their expressionrn response
to conditions within individual mitochondria may be advan-
tageous.If thesegeneswere locatedin the nucleus,conditions
within each mitochondria could not influencethe exoression
of proteins found in that particular mitochondrion.
M i t o c h o n d r i aG
l e n e t i cC o d e sD i f f e r f r o m t h e
S t a n d a r dN u c l e a rC o d e
The geneticcode used in animal and fungal mitochondria is
different from the standard code used in l[ prokaryotic and
eukaryotic nuclear genes;remarkably, the code even differs
in mitochondria from different species(Table 6-3). '$7hyand
how these differencesarose during evolution is mysterious.
UGA, for example, is normally a stop codon, but is read as
tryptophan by human and fungal mitochondrial translation
systems;however,in plant mitochondria, UGA is still recog-
nized as a stop codon. AGA and AGG, the standardnuclear
codons for arginine, also code for arginine in fungal and
plant mtDNA, but they are stop codons in mammalian
mtDNA and serinecodons in Droso,bila mtDNA.
As shown in Table 6-3, plant mitochondria appear ro
E utilize the standard genetic code. However, compar-
isons of the amino acid sequencesof plant mitochondrial
proteins with the nucleotidesequences of plant mtDNAs sug-
gestedthat CGG could code for either arginine (the ,,stan-
d a r d " a m i n o a c i d ) o r t r y p r o p h a n .T h i s a f p a r e n r n o n s p e c i -
ficity of the plant mitochondrial code is explained by editing
of mitochondrial RNA rranscripts,which can converr cyro-
sine residuesto uracil residues.If a CGG sequenceis edited to
UGG, the codon specifiestryptophan, the standard amino
acid for UGG, whereas unedited CGG codons encode the
standard arginine. Thus the translation systemin plant mito-
chondria doesutilize the standard qeneticcode. I
M u t a t i o n si n M i t o c h o n d r i aD
l N A C a u s eS e v e r a l
G e n e t i cD i s e a s e isn H u m a n s
The severity of diseasecausedby a mutation in mtDNA de-
pends on the nature of the mutation and on the proportion
of mutant and wild-type mtDNAs presentin a particular cell
type. Generally when mutations in mtDNA are found, cells
contain mixtures of wild-type and mutant mtDNAs-a con-
dition known as heteroplasmy.Each time a mammalian so-
matic or germ-line cell divides, the mutant and wild-type
mtDNAs segregaterandomly into the daughter cells, as oc-
curs in yeast cells (seeFigure 6-2Zb). Thus, the mtDNA
genotype, which fluctuates from one generation and from
one cell division to the next, can drift toward predominantlv
w i l d - t y p eo r p r e d o m i n a n r l ym u r a n r m r D N A ; . S i n c ea l l e n -
zymes required for the replication and growth of mam-
malian mitochondria, such as the mitochondrial DNA and
 MITOCHOilDBIA

SIANOARO-
C[)DE MAMMATS DBOSI]PHITA NEUR()SPORAYEASTS PTANTS
CODON

UGA Stop Trn Trn

^_r Trp Trp Stop

AGA,AGG A.g Stop Ser Arg Arg Arg

AUA Ile Met Met Ile Met IIe

AUU Ile Met Met Met Met IIe

CUU,CUC,CUA,CUG Leu Leu Leu Leu Thr Leu

"For nuclear-encodedproteins.
S. Andersonet al., 1981,Nature 290:457;P.Borst,in InternationalCell Biology 1980-1981,H. G' Schweiger,ed', Springer-Verlag.p' 239;
souRCEs:
and U. L. Raj Bhandary,
1985, Trends Biochem. Sci.10:478483; V. K. and
Eckenrode C' S. 1'986,
Levings, In Vitro Cell Deu' Biol'
C. Breitenberger
22:169-176;J.M. Gualberetal., 1989,Nature34'1.:660-662;andP. S. Covelloand M. W. Gtay,1,989,Nature34l:662-666.

RNA polymerases,are encodedin the nucleusand imported

from the cytosol, a mutant mtDNA should not be at a "repli- (a) Wild-typemouse Homozygousmutant
cation disadvantage"lmutants that involve large deletionsof
mtDNA might even be at a selectiveadvantagein replication
becausethey can replicate faster.
Recent researchsuggeststhat the accumulation of muta-
tions in mtDNA is an important component of aging in
mammals. Mutations in mtDNA have been observedto ac-
cumulate with aging, perhaps due to a decreasein the proof-
reading ability of DNA polymerase.To study this hypothe-
sis, researchersused gene "knock-in" techniquesto replace
the nuclear gene encoding mitochondrial DNA polymerase (b) 100
with normal proofreading activity (seeFigure 4-34) with a 90
mutant gene encoding a polymerasedefectivein proofread- 80
ing. Mutations in mtDNA accumulatedmuch more rapidly 970
in homozygous mutant mice than in wild-type mice, and the t60
mutant mice aged at a highly acceleratedrate (Frgure6-24). 250
E oano
With few exceptions,all human cells have mitochon- d
;i

dria, yet mutations in mtDNA affect only some tis- zv

sues. Those most commonly affectedare tissuesthat have a 10

high requirementfor ATP produced by oxidative phospho- 0
o 100 2oo 3oo 400 500 600 700 800 900 1000
rylation and tissuesthat require most of or all the mtDNA
Age (days)
in the cell to synthesizesufficient amounts of functional
mitochondrial proteins. For instance, Leber's hereditary a EXPERIMENTAL FIGURE 6-24 Micewith a mitochondrial
optic neuropathy (degeneration of the optic nerve) is DNA polymerasedefectivefor proofreadingexhibit
p r e m a t u r ea g i n g .A l i n eo f " k n o c k - i nm " i c ew e r ep r e p a r e d
causedby a missensemutation in the mtDNA geneencod- mutation inthe
b y m e t h o dds i s c u s s ei ndC h a p t e5r w i t h a
ing subunit 4 of the NADH-CoQ reductase(complex I)' a D N Ap o l y m e r a t
s h
e a t i nactivates
g e n ee n c o d i n gm i t o c h o n d r i a l
protein required for ATP production by mitochondria. Any e r' so o f r e a d i nf ugn c t i o n(.a ) W i l d - t y paen d
t h e p o l y m e r a sp
of severallarge deletionsin mtDNA causesanother set of h o m o z y g o umsu t a n m t i c ea t 3 9 0d a y so l d( 1 3m o n t h s T ) he
diseasesincluding chronic progressiueexternal ophthalmo' mutanm t o u s ed i s p l a yms a n yo f t h ef e a t u r eosf a n a g e dm o u s e
plegia, characterizedby eye defects, and Kearns-Sayresyn- (>720 days,or 24 monthsof age) (b) Plotof survival versusage
drome, characterized by eye defects, abnormal heartbeat of wild-type mice and heterozygous and homozygous muranls'
and central nervous system degeneration.A third condi- T h eh o m o z y g o umsu t a n t cs l e a r lhy a v ea m u c hs h o r t elri f es p a n
tion, causing "ragged" muscle fibers (with improperly as- thanwild-typemice [From G C Kujoth et al, 2005,Sclence 309:481
sembled mitochondria) and associateduncontrolled jerky Part(a)courtesy of JeffMiller/Universrty of Wisconsin-Madison and Gregory
movements,is due to a single mutation in the TIICG loop Kuioth PhD l

DNAS
ORGANELLE 241
 of the mitochondrial lysine IRNA. As a result of this muta-
tion, the translation of several mitochondrial proteins
apparentlyis inhibited. I
ChloroplastsContain LargeDNAsOften
E n c o d i n gM o r e T h a n a H u n d r e dp r o t e i n s
Like mitochondria, chloroplasts are thought to have
evolved from an ancestral endosymbiotic photosvn-
thetic bacterium(seeFigure6-20). However,the endosymbi-
otic event giving rise to chloroplasts occurred more recently
(L2-1,.5 billion yearsago) than the eventleadingto the evo-
lution of mitochondria (1.5-2.2 billion years ago). Conse-
quentlg contemporary chloroplast DNAs show less struc-
tural diversity than do mtDNAs. Also similar to
mitochondria, chloroplasts contain multiple copies of the
organellar DNA and ribosomes, which synthesizesome
chloroplast-encodedproteins using the standard genetic
code. Like plant mtDNA, chloroplastDNA is inherited ex-
clusively in a uniparental fashion through the female parent
(egg). Other chloroplast proteins are encoded by nuclear
genes,synthesizedon cytosolic ribosomes,and then incorpo-
rated into the organelle(Chapter 13). I
In higher plants, chloroplast DNAs are 120-160 kb long,
depending on the species.They initially were thought to be
circular DNA molecules becausein genetically tractable or-
ganisms like the model plant protozoan Chlamydomonas
reinhardtii, the geneticmap is circular. However, recentstud-
ies have revealed that plant chloroplast DNAs are actually
long head-to-tail linear concatomersplus recombination in-
termediates between these long linear molecules. In these
studies,researchershave used techniquesthat minimize me-
chanical breakageof long DNA molecules during isolation
and gel electrophoresis,permitting analysisof megabase-size
DNA.
The complere sequencesof several chloroplast DNAs
from higher plants have been determined in the past several
years. They contain 120-135 genes, 130 in the important
model plant Arabidopsis thaliana. A. thaliana chloroplast
DNA encodes 76 protein-coding genes and 54 geneswith
RNA products such as rRNAs and tRNAs. Chloroplast
DNAs encode the subunits of a bacterial-like RNA
foly-
meraseand expressmany of their genesfrom polycistronic
operons as in bacteria (seeFigure 4-l3a). Some chloroplast
genescontain introns, but theseare similar to the soecialized
introns found in some bacterial genesand in mirochondrial
genesfrom fungi and protozoans, rather than the introns of
nuclear genes. As in the evolution of mitochondrial
genomes,many genesin the ancestralchloroplast endosym_
biont that were redundant with nuclear genes
-g.n., have beenlost
from chloroplast DNA. Also, many .rr..rtial for
chloroplast function have been transferred to rhe nuclear
genome of plants over evolutionary time. Recent estimates
from sequenceanalysisof the A. thaliana and cyanobacterial
genomesindicate that -4,500 geneshave been transferred
from the original endosymbiont to the nuclear genome.
242 CHAPTER
5 | G E N E sG
, E N O M t C SA, N D C H R O M O S O M E S
Methods similar to those used for the transformation
of yeast cells (Chapter 5) have been developedfor sta-
bly introducing foreign DNA into the chloroplasts of higher
plants. The large number of chloroplast DNA moleculesper
cell permits the introduction of thousandsof copiesof an en-
gineeredgeneinto eachcell, resulting in extraordinarily high
levels of foreign protein production. Chloroplast transfor-
mation has recently led to the engineeringof plants that are
resistant to bacterial and fungal infections, drought, and
herbicides. The level of production of foreign proteins is
comparable with that achievedwith engineeredbacteria,
making it likely that chloroplast transformation will be used
for the production of human pharmaceuticalsand possibly
for the engineeringof food crops containing high levelsof all
the amino acids essentialto humans. I
Organelle DNAs
r Mitochondria and chloroplastsmost likely evolved from
bacteria that formed a symbiotic relationship with ances-
tral cells containing a eukaryotic nucleus (seeFigure 6-20).
r Most of the genes originally within mitochondria and
chloroplastswere either lost becausetheir funcdons were re-
dundant with nucleargenesor moved to the nucleargenome
over evolutionary time, leaving different gene sets in the or-
ganellarDNAs of different organisms(seeFigure 6-23).
r Animal mtDNAs are circular molecules,reflecting their
probable bacterial origin. Plant mtDNAs and chloroplast
DNAs generally are longer than mtDNAs from other
eukaryotes, Iargely becausethey contain more noncoding
regions and repetitive sequences.
r All mtDNAs and chloroplast DNAs encoderRNAs and
some of the proteins involved in mitochondrial or photo-
synthetic electron transport and ATP synthesis.Most ani-
mal mtDNAs and chloroplast DNAs also encode the
tRNAs necessaryto translate the organellar mRNAs.
r Becausemost mtDNA is inherited from egg cells rather
than sperm, mutations in mtDNA exhibit a maternal cyto-
plasmic pattern of inheritance. Similarlg chloroplast DNA
is exclusivelyinherited from the maternal parent.
r Mitochondrial ribosomes resemble bacterial ribosomes
in their structure, sensitivity to chloramphenicol, and re-
sistanceto cycloheximide.
r The genetic code of animal and fungal mtDNAs differs
slightly from that of bacteria and the nuclear genome and
varies between different animals and fungi (seeTable 6-3).
In contrast, plant mtDNAs and chloroplast DNAs appear
to conform to the standard geneticcode.
r Severalhuman neuromusculardisordersresult from mu-
tations in mtDNA. Patients generally have a mixture of
wild-type and mutant mtDNA in their cells (heteroplasmy):
the higher the fraction of mutant mtDNA, the more severe
the mutant phenotype.
 The most widely usedcomputer program for this purpose
ff,| Genomics:Genome-wide Analysis is known as BLAST (basic/ocal alignment searchrool). The
of GeneStructureand Expression BLAST algorithm dividesthe "new" protein sequence(known
as the query sequence) into shorter segments and then
Using automated DNA sequencingtechniques,methods for
searchesthe databasefor significant matches to any of the
cloning DNA fragments on the order of 100 kb in length,
stored sequences. The matching program assignsa high score
and computer algorithms to piece together the stored se-
to identically matched amino acids and a lower score to
quence data, researchershave determined vast amounts of
matchesbetweenamino acidsthat are related (e.g.,hydropho-
DNA sequenceincluding nearly the entire genomic sequence
bic, polar, positively charged, negatively charged) but not
of humans and many key experimental organisms. This
identical.When a significantmatch is found for a segment'the
enormous volume of data,which is growing at a rapid pace,
BLAST algorithm will searchlocally to extend the region of
has been stored and organized in two primary data banks:
similarity.After searchingis completed,the program ranks the
the GenBank at the National Institutes of Health, Bethesda,
matches betweenthe query protein and various known pro-
Maryland, and the EMBL SequenceData Base at the
teins accordingto their p-ualwes.This parameteris a measure
European Molecular Biology Laboratory in Heidelberg,
of the probability of finding such a degreeof similarity be-
Germany. These databasescontinuously exchange newly
tween two protein sequencesby chance. The lower the p-
reported sequencesand make them available to scientists
value, the greater the sequencesimilarity between two se-
throughout the world on the Internet. By now, the genome
quences.A p-value lessthan about 10-' usually is considered
sequenceshave been completeln or nearly completely,deter-
as significantevidencethat two proteins sharea common an-
mined for hundreds of viruses and bacteria, scores of
cestor.Many alternativecomputer programs have beendevel-
archaea,yeasts (eukaryotes),and model multicellular eu-
oped in addition to BLAST that can detect relationshipsbe-
karyotes such as the roundworm C. elegans, the fruit fly
tween proteins that are more distantly related to each other
Drosophila melanogaster,mice, and humans.
than can be detected by BLAST. The development of such
The cost of sequencinga megabaseof DNA has fallen so
methodsis currently an active areaof bioinformaticsresearch.
low that projects are underway to sequencethe entire
genome in cancer cells and compare it to the genomein nor-
To illustrate the power of this approach, we consider
mal cells from the samepatient in order to determine all the
the human geneNF1 . Mutations in NF1 are associated
mutations that have accumulatedin that patient's tumor
with the inherited diseaseneurofibromatosis f in which
cells. This approach may reveal genes that are commonly
multiple tumors develop in the peripheral nervous system'
mutated in all cancers,as well as genesthat are commonly
causinglarge protuberancesin the skin. After a cDNA clone
mutated in tumor cells from different patients with the same
of NFl was isolated and sequenced,the deducedsequenceof
type of cancer (e.g., breast versus colon cancer). Such
the NF1 protein was checked against all other protein se-
detailed information also may eventually lead to highly indi-
quencesin GenBank. A region of NF1 protein was discov-
vidualized cancer treatments tailored to the specific muta-
ered to have considerablehomology to a portion of the yeast
tions in the tumor cells of a particular patient.
protein calledIra (Figure6-25). Previousstudieshad shown
In this section, we examine some of the ways re-
that Ira is a GTPase-activatingprotein (GAP) that modulates
searchersare mining this treasuretrove of data to provide
the GTPaseactivity of the monomeric G protein called Ras
insights about gene function and evolutionary relation-
(seeFigure 3-32). As we examine in detail in Chapter 16,
ships, to identify new geneswhose encodedproteins have
GAP and Ras proteins normally function to control cell
never been isolated, and to determine when and where
genes are expressed.This use of computers to analyze
sequencedata has led to the emergenceof a new field of
biology: b ioinformatics.

StoredSequencesSuggestFunctionsof Newly
ldentified Genesand Proteins
As discussedin Chapter 3, proteins with similar functions
often contain similar amino acid sequencesthat correspond
to important functional domains in the three-dimensional
structure of the proteins. By comparing the amino acid se-
quence of the protein encoded by a newly cloned gene with
the sequencesof proteins of known function, an investigator
can look for sequencesimilarities that provide clues to the
function of the encoded protein. Becauseof the degeneracy
in the geneticcode, related proteins invariably exhibit more
sequencesimilarity than the genesencoding them. For this
reason, protein sequencesrather than the corresponding
DNA seouences are usuallvcompared.
characteristicof the disease.I
Even when a protein shows no significant similarity to
other proteins with the BLAST algorithm, it may neverthe-
less share a short sequencethat is functionally important'
Such short segmentsrecurring in many different proteins,
referred to as structural motifs, generally have similar func-
tions. Several such motifs are described in Chapter 3 and
illustratedin Figure3-9.To searchfor theseand other motifs
in a new protein, researcherscompare the query protein
sequencewith a databaseof known motif sequences.

GENoM|CS:GENoME-W|DEANALYS|SoFGENESTRUCTUREANDEXPRESSIoN
243
 NF1 841 TRATFMEVLTK I LOOGTE FDTLAETVLADRFERLVE LVTMMGDOGE LP IA 890
aaaaaoaaaaaa
lra 1500 lR IAFLRVF ID lV. . TNYPVNPEKHEMDKMLA iDDFir<vi tr<wp TLAFF 1546
891 MA LANVV P C SOWD E L AR V L V T L F DS R H L L YO L LWNM F S K E V E LADSMOT L 940
1 5 4 7c s L A . . c s P A D V D L Y A G c i ir.rAFDrnriasH I Lire i ir<oe i KRAARSDDi rsg+
941 FRGNS LASKIMT FC FKVYGATYLOKL LDP L L R IV ITSSDWOHVS FEVDPT 990
.aaaa..a

1595 LRRNSCATRALS LYTRSRGNKYL IKTLRPVLoG IVDNKE . . sFE iD. 1638

991 RLEPSESLEENORNLLOMTEKF. FHAI ISSSSEFPPOLRSVCHCLYO 1036
aaaaaaaaaa
1639KMKPG. SENSEKMLDLFEKYMTRL I DAITSSIDDFP I ELVDICKTIYN 1685
1 0 3 7V V S O R F P O N S I GAVGSAMF LR F I NPA I VSPYEAG I LDKKPPPR I ERGLKL 1086
aaaaoaa
1 6 8 6A A S V N F P E Y A Y TAVGSFVFLRF tGpALVSpDSEtrt ti. tvrHAHDRrpF tr fi34
1 0 8 7M S K I L O S I A N . HVLFTKEEHMRPFND. FVKSNFDAARRFF 1124
aaaaaaaa
1735 LAKV I OSLANGREN I FKKD I LVSKEE F LKTCSDK I FNF LSE LCK I PTNN F 1]84
1125L D IASDCPTSDAVNHS L S F ISDGNVLALHR L LWNN. 1159
.aaaaa
1785TVNVREDPTP I SFDySFLHKFFyLNEFT I RKE I t rueSrIpGEFSFLKNTV 1834
1160 . OE K I GOYLSSNRDHKAVGR RPF DKMAT L LAYLGP PE HKPVA 12OO
aaaaaa
1 8 3 5M L N D K I L G V L G O P S I V I E I K N E I PPFVVENREKYPSLYE FMSRYAFKKVD 1882
F I G U R6E- 2 5 C o m p a r i s oonf t h e r e g i o n so f h u m a nN F 1 b y a b l u ed o t A m i n oa c i dn u m b e risn t h e p r o t e i ns e q u e n c e
a sr e
protein and 5. cereylsiaelra protein that show significant shownat the leftandrightendsof eachrow Blackdotsindicate
sequence s i m i l a r i t yT. h eN F 1a n dt h e l r as e q u e n c e
a sr es h o w n gapsin the proteinsequence inserted in orderto maximize the
o n t h e t o p a n d b o t t o m l i n e so f e a c hr o w , r e s p e c t i v e liyn,t h e o n e - a l i g n m e not f h o m o l o g o uas m i n oa c i d sT h e B L A S T p - v a l u ef o r t h e s e
l e t t e ra m i n o a c i dc o d e ( s e eF i q u r e2 - 1 4 ) A m i n o a c i d st h a t a r e t w o s e q u e n c ei s 1 O - 2 8i,n d i c a t i n ag h i g h d e g r e eo f s i m i l a r i t y[ F r o m
i d e n t i c ailn t h e t w o p r o t e i n sa r e h i g h l i g h t e di n y e l l o w A m i n o a c i d s c X u e t a l , 1 9 9 0C, eil62:5991
w i t h c h e m i c a l isyi m i l a rb u t n o n i d e n t i c asli d ec h a i n sa r e c o n n e c t e d

C o m p a r i s o no f R e l a t e dS e q u e n c efsr o m lins present in different organisms today evolutionary rela-

D i f f e r e n tS p e c i e sC a nG i v e C l u e st o tionshipscan be deduced,as illustratedin Figure 6-26b. Of
E v o l u t i o n a r yR e l a t i o n s h i pAs m o n g p r o t e i n s the three types of sequencerelationships,orthologous se-
quencesare the most likely to share the same function.
BLAST searchesfor related protein sequencesmay reveal
that proteins belong to a protein family. Earlier,we consid-
ered gene families in a single organism, using the B-globin G e n e sC a nB e l d e n t i f i e dW i t h i n G e n o m i c
genesin humans as an example (seeFigure 6-4a). But in a DNASequences
databasethat includes the genome sequencesof multiple The complete genomic sequenceof an organism contains
organisms,protein families also can be recognizedas being within it the information neededto deducethe sequenceof
shared among related organisms. Consider, for example, every protein made by the cells of that organis-. Fo. or-
the tubulin proteins; rheseare the basic subunits of micro- ganisms such as bacteria and yeast, whose genomeshave
tubules,which are important componentsof the cytoskele- few introns and short intergenic regions, mosr proreln-
ton (Chapter 18). According to the simplified schemein coding sequencescan be found simply by scanningthe ge-
Figure 6-26a, the earliest eukaryotic cells are thought to nomic sequencefor open reading frames (ORFs) of signif-
have contained a single tubulin gene that was duplicated icant length. An ORF usually is defined as a srretch of
early in evolution; subsequentdivergenceof the different DNA containing at least 100 codons that begins with a
copies of the original tubulin gene formed the ancestral start codon and ends with a stop codon. Becausethe prob-
versionsof the ct- and p-tubulin genes.As different species ability that a random DNA sequencewill contain no stop
diverged from these early eukaryotic cells, each of these codons for 100 codons in a row is very small, most ORFs
gene sequencesfurther diverged,giving rise ro the slightly encodeprotein.
different forms of ct-tubulin and B-tubulin now found in ORF analysiscorrectly identifiesmore than 90 percent of
each species. the genesin yeast and bacteria.Some of the very shortest
All the different membersof the tubulin family of genes genes,however,are missedby this method, and occasionally
(or proteins)are sufficienrlysimilar in sequencero suggesta long open reading frames that are not actually genesarise by
common ancestralsequence.Thus all these sequencesare chance. Both types of mis-assignmentscan be corrected by
consideredto be homologous.More specificallSsequences more sophisticatedanalysis of the sequenceand by genetic
that presumably diverged as a result of gene duplication tests for gene function. Of the Saccharomycesgenesidenti-
(e.g.,the ct- and B-tubulin sequences) are describedas paral- fied in this manner,about half were alreadyknown by some
ogous. Sequences that arosebecauseof speciation(e.g.,the functional criterion such as mutant phenotype. The func,
ct-tubulin genesin different species)are describedas irthol- tions of some of the proteins encodedby the remainingpu-
ogous.From the degreeof sequencerelatedness of the tubu- tative (suspected)genes identified by ORF analysis have

244 . c H A p r EG
R I G E N EG
SE, N o M t cAsN
, Dc H R o M o s o M E S
 (al (b) Orthologous

I
Ancestra cr-Tubulin
{human)
cell
a-Tubulin(fly)
I G e n ed u p l i c a t i o n (worm)
cr-Tubulin
I a n dd i v e r g e n c e !
*
s-Tubulin( yeast)
Orthologous

-..
p -l u b u l r n( n u m a n )

(fly)
F-Tubulin
p-Tubulin(worm)

p-Tubulin( yeast)

Species 1 S p e c i e s2

FIGURE 6-26 Generationof diversetubulin sequences during sequences divergedForexample, node1 representstheduplication
the evolutionof eukaryotes.(a)Probablemechanism givingriseto eventthatgaveriseto theo-tubulin andB-tubulin andnode2
families,
thetubulingenesfoundin existing
specieslt ispossibleto deduce that representsthe divergenceof yeastfrom species
multicellular Bracesand
a geneduplicationeventoccurredbeforespeciation because thecr- arrowsindicate, theorthologous
respectively, which
tubulin9enes,
tubulinsequencesfromdifferent
species(eg , humans andyeast) are andthe paralogous
drfferasa resultof speciatron, genes,whichdiffer
morealikethanarethect-tubulinandP-tubulin sequences withina asa resultof geneduplication Thisdiagram somewhat
issimplified
species(b)A phylogenetic therelationship
treerepresenting between because eachof thespecies actually
represented multiple
contains
thetubulinsequences Thebranch points(nodes), indicatedbysmall andB-tubulin
ct-tubulin genes thatarosefromlatergeneduplication
numbers, representcommon genes
ancestral at thetimethattwo events

been assignedbased on their sequencesimilarity to known exons, transcription-control regions, or sequenceswith

proteinsin other organlsms.
Identification of genesin organismswith a more com-
plex genome structure requires more sophisticatedalgo-
rithms than searching for open reading frames. Because
most genesin higher eukaryotesare composedof multiple,
relatively short exons separatedby often quite long non-
coding introns, scanning for ORFs is a poor method for
finding genes.The best gene-finding algorithms combine
all the available data that might suggestthe presenceof a
gene at a particular genomic site. Relevant data include
alignment or hybridization of the query sequenceto a full-
length cDNA; alignment to a partial cDNA sequence,gen-
erally 200-400 bp in length, known as afl expressedse'
quence tag (EST); fitting to models for exon, intron, and
splice site sequences;and sequencesimilarity to other or-
ganisms.Using thesecomputer-basedbioinformatic meth-
ods, computational biologistshave identified approxi-
mately 25,000 genesin the human genome. However, for
some 10,000 of these putative genesthere is not yet con-
c l u s i v e e v i d e n c et h a t t h e y a c t u a l l y e n c o d e p r o t e i n s o r
RNAs.
A particularly powerful method for identifying human
genesis to compare the human genomic sequencewith that
of the mouse. Humans and mice are sufficiently related to
have most genesin common, although largely nonfunctional
DNA sequences, such as intergenicregionsand introns,will
tend to be very differentbecausethesesequences are not un-
der strong selectivepressure.Thus correspondingsegments
of the human and mousegenomethat exhibit high sequence
similarity are likely to be functional coding regions,that is,
other functions that are not yet understood.
T h e N u m b e ro f P r o t e i n - C o d i nG
genes
in an Organism'G s e n o m el s N o t D i r e c t l y
Relatedto lts Biological Complexity
The combination of genomic sequencingand gene-finding
computer algorithms has yielded the complete inventory of
protein-coding genesfor a variety of organisms.Figure 6-27
iho*t the total number of protein-coding genesin several
eukaryotic genomes that have been completely sequenced.
The functions of about half the proteins encoded in these
genomesare known or have been predicted on the basis of
sequencecompansons.One of the surprising featuresof this
comparison is that the number of protein-coding genes
within different organisms does not seem proportional to
our intuitive senseof their biological complexity. For exam-
ple, the roundworm C. elegansapparently has more genes
than the fruit fly Drosophila, which has a much more com-
plex body plan and more complex behavior. And humans
have fewer than one and one-half the number of genesas C'
elegans.\fhen it first became apparent that humans have
fewer than twice the number of protein-coding genesas the
simple roundworm. it was difficult to understand how such
a small increasein the number of proteins could generate
such a staggeringdifferencein complexity.
Clearly, simple quantitative differencesin the number
of genes in the genomes of different organisms are inade-
quate for explaining differences in biological complexity'
Howeveq severalphenomenacan generatemore complexity

GENoM|CS:GENoME-W|DEANALYS|SoFGENESTRUCTUREANDEXPRESS|oN
245
 Organism Human
Genes -25,000
Organism Drosophila (flVl
Genes 13,338
A FIGURE 6-27 Comparison of the numberand typesof
proteinsencodedin the genomesof different eukaryotes.For
eachorganism, the areaof theentirepiechartrepresents thetotal
numberof protein-coding genes, allshownat roughly the same
scaleIn mostcases, thefunctions of the proteins
encoded by about
halfthe genesarestillunknown(lightblue)Thefunctions of the
remainder, whichareknownor havebeenpredicted by sequence
similarity
to genesof knownfunction, areclassified
assnownIn
the colorkey lAdapted fromInternational
Human Genome Seouencino
Consortium,2001,Nature 409:860
l
in the expressedproteins of higher eukaryotes than is pre-
dicted from their genomes.First, alternativesplicing of a
kinds of proteins. Larger numbers of cells can interact in
more complex combinations, as in comparing the cerebral
cortex from mouse to man. Similar cells are presentin both
the mouse and human cerebralcortex, but in humans more
of them make more complex connections.Evolution of the
increasing biological complexity of multicellular organisms
likely required increasinglycomplex regulation of cell repli-
cation and geneexpression,leading to increasingcomplexity
of embryological development.
The specificfunctions of many genesand proteins iden-
tified by analysisof genomic sequencesstill have not been
determined.As researchersunravel the functions of indi-
vidual proteins in different organisms and further detail
their interactions with other proteins, the resulting ad-
vanceswill becomeimmediately applicableto all homolo-
gous proteins in other organisms. When the function of
every protein is known, no doubt, a more sophisticatedun-
246 CHAPTER
5 | c E N E S ,G E N O M | C SA, N D C H R O M O S O M E S
Arabidopsis (plantl C. elegans (roundworm)
25,706 18,266
Metabolism
DNA replication/modification
Transcription/translation
I n t r a c e l l u l asri g n a l i n g
Saccharomyces(yeast) C e l l - c e lcl o m m u n i c a t i o n
-6000
P r o t e i nf o l d i n g a n d d e g r a d a t i o n
Transport
I M u l t i f u n c t i o n apl r o t e i n s
Cytoskeleton/structu
re
D e f e n s ea n d i m m u n i t y
M i s c e l l a n e o ufsu n c t i o n
Unknown
derstandingof the molecular basis of complex biological
systemswill emerge.
S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n e
Copy-NumberVariationAre lmportant
Determinantsof DifferencesBetween
I n d i v i d u a l so f a S p e c i e s
The DNA sequencebetweenindividual humans who are not
closelyrelateddiffers at about L-2 percentof the 3 x 10ebase
pairs in the human genome.Most of thesedifferences,called
single nucleotide polymorpbisms (SNPs), are probably not
functionally significant becausethey occur in long introns or
betweengenes,or result in synonymouscodon changesin cod-
ing regions. Nonetheless,such SNPs are important markers
for measuringthe frequencyof recombination betweengenes
and can be used to link a specificgenewith a trait or pheno-
type as discussedin Chapter 5 (seeFigure 5-36). On the other
hand, someSNPsmay be functionally significantbecausethey
result in amino acid changes in protein-coding regions or
base-pairchangesin control regionsthat affect the binding of
transcription factors. Thesesingle nucleotidepolymorphisms
clearly contribute to differencesbetweenindividuals.
 A secondhighly significant type of geneticvariation, differ-
encesin gene-copynumber, was discoveredvery recently. Re-
cent analysesof the number of copiesof DNA sequences per
cell in different individualsrevealedwidespreaddeletions,tan-
dem duplications,and complex combinationsof deletionsand
duplicationsthat vary benveenindividuals over a remarkably
high:12 percentof the genome.The deletionsaverage:40 kb
in length, and the tandem duplicationsavenge:120 kb, but
some deletionsand duplicationsare much longer.Thesevary-
ing deletionsand duplications probably arose from unequal
crossingover befweenchromosomesduring meiotic recombi-
nation in a direct ancestor(seeFigure 6-2). This resultsin dif-
ferencesin gene-copynumbers betweenindividuals.
In some individuals. for instance.a deletion of DNA se-
quenceoccurson one chromosomebut the normal sequenceis
presenton the homologouschromosome;as a result they have
only a single copy of genesin the deletedregion. Likewise,
some individuals contain a duplication of some geneson one
chromosomethat is not presenton the homologous chromo-
some, resulting in three copies of genesin the duplicated re-
gion. Another possibility found in someindividuals is a dupli-
cation on both homologous chromosomes,generating four
gene copies;additional duplications on one or both chromo-
somescan lead to genecopy numbersgreaterthan four. These
copy-numberuariationsare inherited in a Mendelian manner,
as for other alleles,and are occasionallygeneratedas a new
variation not observedin the DNA of either parent.
Copy-number variations are evenmore common between
individuals than differencesin DNA sequence(SNPs).Since
variations in genecopy number can affect the amount of pro-
tein expressedfrom a gene,copy-number variations may be
among the most important determinantsof individual differ-
encesbetweenhumans, including differencesin susceptibility
to various diseases.Studiesare currently underway to deter-
mine the influence of gene copy number variations on indi-
vidual traits including diseasesusceptibility.
Genomics:Genome-wide Analysis of Gene Structure
and Expression
r The function of a protein that has not been isolated (a
query protein) often can be predicted on the basis of simi-
larity of its amino acid sequenceto the sequencesof pro-
teins of known function.
r A computer algorithm known as BLAST rapidly searches
databasesof known protein sequencesto find those with
significantsimilarity to a query protein.
r Proteinswith common functional motifs, which often can
be quite short, may not be identified in a typical BLAST
search.Such short sequencesmay be located by searchesof
motif databases.
r A protein family comprisesmultiple proteins all derived
from the same ancestralprotein. The genesencoding these
proteins, which constitute the corresponding gene family,
arose by an initial gene duplication event and subsequent
divergenceduring speciation (seeFigure 6-26).
S T R U C T U R AOL R G A N I Z A T I O N
r Relatedgenesand their encodedproteins that derive from
a gene duplication event are paralogous' such as the o and
B-globinsthat combine in hemoglobin (ct2B2);those that
derive from mutations that accumulated during speciation
are orthologous. Proteinsthat are orthologous usually have
a similar function in different organisms,such as the mouse
and human adult B-globins.
r Open reading frames (ORFs) are regions of genomic
DNA containing at least 100 codons located between a
start codon and stop codon.
r Computer search of the entire bacterial and yeast ge-
nomic sequencesfor open reading frames (ORFs) correctly
identifies most protein-coding genes.Severaltypes of addi-
tional data must be used to identify probable (putative)
genesin the genomic sequencesof humans and other higher
eukaryotesbecauseof their more complex genestructure in
which relatively short coding exons are separatedby rela-
tively long, noncoding rntrons.
r Analysis of the complete genome sequencesfor several
different organisms indicates that biological complexity is
not directly related to the number of protein-coding genes
(seeFigure6'27).
f[ StructuralOrganization
of EukaryoticChromosomes
Now that we have examined the various types of DNA
sequences found in eukaryotic genomesand how they are or-
ganizedwithin it, we turn to the question of how DNA mole-
cules as a whole are organizedwithin eukaryotic cells. Because
the total length of cellular DNA is up to a hundred thousand
times a cell'sdiameter,the packing of DNA is crucial to cell ar-
chitecture.It is also essentialto prevent the long DNA mole-
cules from getting knotted or tangled with each other during
cell division when they must be preciselysegregatedto daugh-
ter cells.The task of compacting and organizingchromosomal
DNA is performed by abundant nuclear proteins called his-
tones. As noted previously'the complex of histones,nonhis-
tone proteins, and DNA constituteschromatin' which exists
in various degreesof folding or compaction (seeFigure 6-1)'
Chromatin, which is about half DNA and half protein by
mass, is dispersedthroughout much of the nucleus in inter-
phase cells (those that are not undergoing mitosis). Further
folding and compaction of chromatin during mitosis pro-
ducesthe visible metaphasechromosomes,whose morphol-
ogy and staining characteristicswere detailed by early cyto-
geneticists.Although every eukaryotic chromosome includes
millions of individual protein molecules,each chromosome
contains iust one, extremely long, linear DNA molecule.The
lonqest DNA molecules in human chromosomes,for in-
st"n.., are2.8 x 108 basepairs, or almost 10 cm, in length!
The structuraI organization of chromatin allows this vast
length of DNA to be compacted into the microscopic con-
straints of a cell nucleus.Yet chromatin is organized in such
a way that specificDNA sequenceswithin the chromatin are
CHROMOSOMES
OF EUKARYOTIC
247
 < EXPERIMENTAL FIGURE 6-28 Theextended
and condensedforms of extractedchromatin
have very different appearancesin electron
micrographs. (a)Chromatin isolated in low-ionic-
strengthbufferhasan extended "beads-on-a-
string"appearance The"beads"arenucleosomes
(10-nmdiameter) andthe "string"isconnecting
(linker)DNA (b)Chromatin isolated in bufferwith
a physiological ionicstrength (0 15M KCI)appears
asa condensed fiber30 nm in diameter. lpart(a)
courtesy of 5 McKnight andO Miller, Jr.Part(b)courtesy
o f B H a m k a l o a nBdRJa t t n e r l

readily availablefor cellularprocesses like the transcription,
replication, repair, and recombination of DNA molecules.In
this section,we consider the properties of chromatin and its
organizationinto chromosomes.Important featuresof chro-
mosomesin their entirety are covered in the next section.
ChromatinExistsin Extended
a n d C o n d e n s e dF o r m s
lfhen the DNA from eukaryotic nuclei is isolated using a
method that preservesnative protein-DNA interactions,it is
associatedwith an equal mass of protein in the nucleopro-
tein complex known as chromatin.Histones,the most abun-
dant proteins in chromarin, constitute a family of small,
basic proteins. The five major types of histone proteins-
termed H1, H2A, H2B, H3, and H4-are rich in positively
charged basic amino acids, which interacr with the nega-
tively charged phosphategroups in DNA.
'When
chromatin is extracted from nuclei and examined
in the electronmicroscope,its appearancedependson the salt
concentration to which it is exposed.At low salt concentra-
tion in the absenceof divalent cations such as Mg*', isolated
chromatin resembles"beadson a string" (Figure 6-28a).In
this extendedform, the string is composedof free DNA called
"linker" DNA connectingbeadlike structurestermed nucleo-
somes. Composed of DNA and histones, nucleosomesare

FIGURE 5-29 Structureof the nucleosome basedon x-ray sideof the nucleosome. Thismodelshowsmoreclearly thatDNA
crystallography. (a)Nucleosome with space-filling
modelof the "covers"muchof the proteinon the nucleosomes lateral
surface.
histones. Thesugar-phosphate backbonesof the DNAstrands are H2Asubunits aregold;H2Bsarered;H3sareblue;H4saregreen
represented asgraytubesto allowbettervjsualizatton of the TheN-terminal tailsof the eighthistones andthetwo H2AandH2B
histonesNucleosome shownfromthetop (/eft)andfromthe side C-terminaltailsinvolved in condensation of chromatin arenotvisible
(right',the sideviewis rotatedclockwise90. f romthe rop vrew.,r because theyaredisordered in the crystal.
[AfterK Lugeretal, 1997,
(b)SpaceJilling modelof histonesandDNA(white)viewedfromthe Nature
389:251 l

248 CHAPTER
5 | G E N E SG
, E N O M | C SA, N D C H R O M O S O M E S
about 10 nm in diameter and are the primary structural units from the histone amino acid sequenceswere selectedagainst
of chromatin. If chromatin is isolated at physiological salt strongly during evolution. The amino acid sequence'ofH1,
concentration, it assumesa more condensedfiberlike form however, varies more from organism to organism than do the
that is 30 nm in diameter(Figure6-28b). sequencesof the other major histones.The similarity in se-
quence among histones from all eukaryotes suggeststhat they
Structure of Nucleosomes The DNA componentof nucle- fold into very similar three-dimensionalconformations, which
osomesis much lesssusceptible to nucleasedigestionthan is the were optimized for histone function early in evolution in a com-
linker DNA between them. If nucleasetreatment is carefully mon ancestorof all modern eukaryotes'
controlled,all the linker DNA can be digested,releasingindi-
vidual nucleosomeswith their DNA component. A nucleosome
consistsof a protein core with DNA wound around its surface
like thread around a spool. The core is an octamercontaining
two copieseach of histonesHZA, H2B, H3, and H4. X-ray
crystallography has shown that the octameric histone core is a
roughly disk-shapedstructure made of interlocking histonesub-
units (Figure6-29). Nucleosomesfrom all eukaryotescontain C h a i no f
nucleosomes
147 base pairs of DNA wrapped one and nvo-thirds turns
around the protein core. The length of the linker DNA is more
variable among species,and even betweendifferent cells of one
organism,ranging from about 10 to 90 basepairs. During cell
replication,DNA is assembledinto nucleosomesshortly after
the replicationfork passes(seeFigure 4-33). This processde-
pendson specificchaperonesthat bind to histonesand assemble
them together with newly replicated DNA into nucleosomes.
II
Structure of the 30-nm Fiber When extracted from cells Zig-zag
in isotonic buffers (i.e.. buffers with the samesalt concentra- ribbon
tion found in cells, :0.15 M KCl, 0.004 M MgCl2), most
chromatin appearsas fibers :30 nm in diameter(seeFigure
II
6-28b). Current research,includingX-ray crystallographyof
nucleosomesassembledfrom recombinant histones,indicate
that the 30-nm fiber has a "zig-zagribbon" structurethat is
wound into a "two-start" helix made from two "strands" of
nucleosomesstacked on top of each other like coins (Figure
6-30). The two "strands" of stackednucleosomesare then
wound into a double helix similarly to the two strands in a
DNA double helix, except that the helix is left handed,
rather than right handed as it is in DNA. The 30-nm fibers Two-start
helix
also includeH1, the fifth major histone.Hl is bound to the
DNA as it entersand exits the nucleosomecore, but its struc-
ture in the 30-nm fiber is not known at atomic resolution.
The chromatin in chromosomalregionsthat are not being
transcribed or replicated exists predominantly in the con-
densed,30-nm fiber form and in higher-order folded struc-
tures whose detailed conformation is not currently under-
stood. The regions of chromatin actively being transcribed (a) (b)
are thought to assumethe extendedbeads-on-a-stringform.
FIGURE 6-30 Structureof the 30-nmchromatinfiber' (a)Model
for thefoldingof a nucleosomalchainat top intoa "zig-zag ribbon"
Conservation of Chromatin Structure The generalstruc- " In each"strand"the
of nucleosomes two "strands
containing
ture of chromatin is remarkably similar in the cells of all eu-
nucleosomes arealignedwith eachotherlikea stackof coins.These
karyotes, including fungi, plants, and animals, indicating that "strands" nucleosomesarethenwoundintoa left-handed
two of
the structure of chromatin was optimized early in the evolution doublehelixcalleda "two-start"helixForsimplicity, DNAisnot
of eukaryotic cells.The amino acid sequencesfor four histones represented helix(b)Modelof the 30-nmfiber
in thetwo-start
(H2A, IJ'ZB, H3, and H4) are highly conservedbetween dis- basedon x-raycrystallographyof a tetranucleosome (a shortstretch
tantly related species.For example,the sequencesof histone H3 of four nucleosomes) DNA on nucleosomes
alternating iscolored
from sea urchin tissue and calf thymus differ by only a single distinguishing
to simplify
lightanddarkblue,respectively, (a)
them [Part
amino acid, and H3 from the garden pea and calf thymus differ adapted from C L F.Woodcocket al , 1984, J CellBiol 99:42 Part(b) from
only in four amino acids. Apparently significant deviations T S c h a l c he t a l , 2 0 0 5 , N a t u r e4 3 5 : 13 8 l

CHROMOSOMES
OF EUKARYOTIC
S T R U C T U R AOL R G A N I Z A T I O N 249
 Minor histone variants encodedby genesthat differ from
the highly conservedmajor types also exist, particularly in
vertebrates.For example, a specialform of H2A, designated
H2AX, is incorporated into nucleosomesin place of H2A in
a small fraction of nucleosomesin all regionsof chromatin.
At sites of DNA double-strandedbreaks in chromosomal
DNA, H2AX becomesphosphorylatedand participaresin
the chromosome-repairprocess,probably by functioning as
a binding site for repair proteins. In the nucleosomesat cen-
tromeres,H3 is replacedby another variant histone called
CENP-A, which participatesin the binding of spindle micro-
tubules during mitosis. Most minor histone variants differ
only slightly in sequencefrom the major histones.These
slight changesin histonesequence may influencethe stability
of the nucleosome as well as its rendency to fold into the
30-nm fiber and other hieher-orderstructures.
Modificationsof HistoneTailsControl
C h r o m a t i nC o n d e n s a t i o a
nn d F u n c t i o n
Each of the histone proteins making up the nucleosome
core contains a flexible N-terminus of 19-39 residuesex-
tending from the globular structureof the nucleosome;the
H2A and H2B proteins also contain a flexible C-terminus
extending from the globular histone octamericcore. These
termini, called histone tails, are represented in the model
shown in Figure 6-31.a.The histone tails are required for
chromatin to condensefrom the beads-on-a-stringconfor-
mation into the 30-nm fiber. For example, recent experi-
ments indicate that the N-terminal tails of histone H4,
particularly lysine 16, are critical for forming the 30-nm
fiber. This positively charged lysine interacts with a nega-
tive patch at the H2A-H2B intefiace of the nexr nucleo-
some in the stacked nucleosomesof the 30-nm fiber (see
F i g u r e6 - 3 0 ) .
Histone tails are subject to multiple post-translational
modifications such as acetylation, methylation, phosphory-
lation, and ubiquitination. Figure 6-31b summarizesthe
types of post-translationalmodifications observedin human
histones.A particular histone protein never has all of these
modifications simultaneously, but the histones in a single
nucleosomemay collectivelycontain a severaldifferent types
of modifications. The particular combinations of post-
transcriptional modifications found in different regions of
chromatin have been suggestedto constitute a bistone code
that influenceschromatin function by creating or removing
binding sitesfor chromatin-associatedproteins. Here we de-
scribethe most abundant kinds of modifications found in hi-
stone tails and how these modifications control chromatin
condensation and function. We end with a discussionof a

(a) (b)
(F)AcAcAc
I
uol
-.::. .H24.,
Irrr
SGRG KOGCKARAKAKTRSS .:-VLLPKKT'ESHHKAKGK
1 5 9 13
119

i,^)-\
Me
H4]

t
I

I
IJ'Q I

t
I
H2B'I @ tr,,te nc Ac Ac Ac @;Me
I T{rrrYl
H2A' S' f G R G K G G K G L G K G G A K R H R K V L R D N I O G I T
3 5 8 12 16 18 20

Ac G) Phosphorylationmark
I
vel
rl
t-vrrrenKSAGAAK M e M e t h y l a t i o nm a r k
I
'7u AC Acetylationmark
I

Ub U b i q u i t i n a t i o m
n ark

FIGURE6-31 Histone tails and their post-translational (b)Summary of post-translational

modificationsobserved in human
modifications. (a) Model of a nucleosome viewedfrom top with histonesHistone-tail
sequences areshownin theone-letter aminoacid
histonesdepictedas ribbondiagramsThismodeldepictsthe lengths code(seeFigure2-14)Themainportionof eachhistoneisdepicted as
of the histonetails(dottedlines),which are not visiblein the crystal an ovalThesemodifications
do notalloccursimultaneously on a single
structure(seeFigure6-29).The H2A N-terminaltailsare at the histonemoleculeRather,
specificcombinationsof a few modifications
bottom, and C-terminaltails,at the top The H2B N-terminaltatls of onehistone
areobserved in anyparticular
nucleosome [part(a)from
are on the right and left, and C-terminaltailsat the bottom center K Luger
andT.J Richmond,
1998, CurrOpinGenet.& Deret8:140part(b)
HistonesH3 and H4 haveshortC-terminaltailsthat are not modified adapted
fromR Margueronetal,2005,Curr.
OpinGenet& Devel. 15:1631

250 CHAPTER
6 | G E N E SG
, E N O M t C SA,N D C H R O M O S O M E S
special case of chromatin condensation,the inactivation of (a) Decondensedchromatin Condensedchromatin
X chromosomesin femalemammals.

Histone Acetylation Histone-tail lysines undergo re-

versible acetylation and deacetylation by enzymesthat act
on specific lysines in the N-termini. In the acetylatedform, 4.6 kb 4.6 kb
the positive charge of the lysine e-amino group is neutral-
ized. As mentioned above, lysine 16 in histone H4 is partic-
ularly important for the folding of the 30-nm fiber becauseit
interacts with a negatively charged patch on the surface of
the neighboring nucleosome in the fiber. Consequently,
14-dayerythroblast MSB
when H4 lysine 16 is acetylated,the chromatin tends to form
the less condensed "beads-on-a-string" conformation con-
ducive for transcription and replication. (b) DNA from
14-day DNA
Histone acetylation at other sitesin H4 and in other his- from
erythroblasts
tones (seeFigure6-31b) is correlatedwith increasedsensitiv- MSB
ity of chromatin DNA to digestion by nucleases.This phe-
DNase(pg/ml) 0 .01 .05 .1 .5 .1 1.5 1.5
nomenon can be demonstrated by digesting isolated nuclei
with DNase L Following digestion, the DNA is completely
separated from chromatin protein, digested to completion
with a restriction enzyme, and analyzed by Southern blot-
ting. An intact gene treated with a restriction enzyme yields
', 4'6 P5
fragments of characteristicsizes.If a gene is exposedfirst to {P+
DNase, it is cleavedat random siteswithin the boundariesof
the restriction enzymecut sites.Consequently,any Southern
blot bands normally seen with that gene will be lost. This
method has been used to show that the transcriptionally in-
active B-globingenein non-erythroidcells,where it is asso- r** f}**t
ciated with relatively unacetylated histones, is much more
resistant to DNase I than is the active, transcribed B-globin
gene in erythroid precursor cells, where it is associatedwith
acetylatedhistones(Figure6-32). Theseresultsindicatethat EXPERIMENTAL FIGURE 6-32 Nontranscribed genesare less
the chromatin structure of nontranscribedDNA is more susceptibleto DNaseI digestionthan activegenes.Chickembryo
erythroblastsat 14 daysactively synthesizeglobin,whereas cultured
condensed,and therefore more protected from DNase diges-
undifferentiatedMSBcellsdo not (a)Nuclei fromeachtypeof cell
tion, than that of transcribed DNA. In condensedchro-
wereisolated andexposed to increasing concentrationsof DNase l.
matin, the DNA is largely inaccessibleto DNase I becauseof
ThenuclearDNAwasthenextracted andtreatedwith the restriction
its close associationwith histones and other chromatin- the DNAaroundtheglobinsequence
enzyme 8amHl, whichcleaves
associatedproteins that bind to unacetylatedhistone tails. In andnormally releasesa 4 6-kbglobinfragment(b)TheDNase l- and
contrast, actively transcribed DNA is much more accessible BamHl-digested DNAwassubjected to Southern blotanalysis witha
to DNase I digestion becauseit is present in the extended, probeof labeled clonedadultglobinDNA,whichhybridizes to the
beads-on-a-stringform of chromatin. 4 6-kbEarnHl fragment. lf theglobingeneissusceptible to the initial
Genetic studies in yeast indicate that histone acetylases DNase digestion, it wouldbe cleaved repeatedly andwouldnot be
(HATs), which acetylatespecific lysine residuesin histones, expected to showthisfragmentAs seenin theSouthern blot,the
are required for the full activation of transcription of a num- active
transcriptionally DNA from the 14-day globin-synthesizing cells
ber of genes.Consequently,the control of acetylation of his- wassensitive to DNase I digestion, indicatedbytheabsence of the
tone N-termini in specific chromosomal regions is thought 4 6-kbbandat highernuclease concentrations. the
In contrast,
DNAfromMSBcellswasresistant
inactive to digestion.These results
to contribute to the transcriptional control of gene expres-
suggestthatthe inactive DNAisin a morecondensed formof
sion by mechanismsdescribedbelow and in the next chapter.
chromatin in whichthe globingeneisshielded fromDNase digestion.
Just as genesin condensed,folded regions of chromatin are Cell 19:973; photographcourtesyofH Weintraub]
[SeeJStalderetal ,1980,
less accessibleto exogenouslyadded DNase I than genesin
decondensed, extended regions of chromatin, RNA
polymeraseand other proteins required for transcription are groups can be methylated, a processthat prevents acetyla-
also inhibited from interactins with DNA in condensed tion, thus maintaining their positive charge. Moreover, ly-
chromatin. sine e-amino groups can be methylated once, twice, or three
times. Arginine side chains can also be methylated. Serine
Other Histone Modifications As shown in Figure6-31.b, and threonine side chains can be reversibly phosphorylated,
histone tails in chromatin can undergo a variety of other co- introducing a negative charge. Finally, a single 76-amino-
valent modificationsat specificamino acids.Lysinee-amino acid ubiquitin moleculecan be reversiblyadded to a lysine in

OF EUKARYOTIC HROMOSOMES
S T R U C T U R AOL R G A N I Z A T I O N 251
the C-terminal tails of H2A and H2B. Recall that addition of
multiple linked ubiquitin moleculesto a protein can mark it
for degradationby the proteasome(seeFigure3-Z9b).In this
case, however, the addition of a single ubiquitin molecule
does not affect the stability of a hisrone, although it does in-
fluence chromatin strucrure.
As mentioned previouslS it is the precisecombination of
modified amino acids in histone tails that helps control the
condensation,or compaction,of chromatin and its ability to
be transcribed,replicated,and repaired.This can be illus-
trated by comparing the specific modifications observed in
highly condensedchromarin, known as heterochromatin,
with those in less condensedchromatin, known as euchro-
matin (Figure6-33a).Heterochromatindoesnot fully decon-
densefollowing mitosis, remaining in a compactedstate dur-
ing interphase.It is typically found at the cenrromeresand
telomeresof chromosomes,as well as some other discretelo-
cations.'Whencellsare subjectedto dyesthat bind DNA, re-
gions of heterochromatinstain very darkly. In contrast, areas llpm I
of euchromatin, which are in a less compacted state during
(b) Heterochromatin{inactive/condensedl
interphase,stain lightly with DNA dyes.Typically,most tran-
t,"'
scribedregionsof DNA are found in euchromatin,while het-
erochromatin remains transcriptionally inactive. H3 AR TKOTARK STGGKAPRKOLATKAARKSAPAT
9

t,"'
Reading the Histone Code The histonecodeis "read" by
proteinsthat bind to the modified histonetails and in turn pro- H3 ARTKOTARKSTGG
KAPRKOLATKAARKSAPAT
27
mote condensationor decondensationof chromatin, forming
"closed" or "open" chromatin structures.Higher eukaryotes
Euchromatin (active/openl
expressa number of proteins containing a so-calledchromo-
A:Q Ac
domain that binds to histone rails when they are methylated at
specific lysines. One example is heterochromatin protein 1 H3 ARTKOTARKSTGGKAPRKOLATKAARKSAPAT
(HP1).In addition to histones,HP1 is one of rhemajor proteins
Me. Ac
associatedwith heterochromatin. The HP1 chromodomain tl
binds the H3 N-terminal tail only when it is tri-methylated at ly- H3 ARTKOTARKSTGGKAPRKOLATKAARKSAPAT
4 14

sine9 (Figure6-33b).HP1 alsoconrainsa seconddomain called

FIGURE6-33 Heterochromatin versuseuchromatin.(a)Inthis
a chromosbadow domain becauseit is frequently found in pro- electronmicrographof a bonemarrowstemcell,thedark-staining areas
teins that contain a chromodomain. The chromoshadowdo- in thenucleus(N)outside thenucleolus(n)areheterochromatin The
main binds to other chromoshadow domains. Consequentlg light-staining,
whitishareas areeuchromatin (b)Themodificationsof
chromatincontainingH3 tri-methylatedat lysine9 (H3K9Me3) histoneN-terminaltailsin heterochromatin
andeuchromatin differ,
as
is assembledinto a condensedchromatin structure by HP1, illustrated
herefor histone H3 Notein oarticularthathistone
tarlsare
although the srructure of this chromatin is not well understood generallymuchmoreextensively acetylatedin euchromatincompared
(Figure6-34a). with heterochromatin Heterochromatin ismuchmorecondensed (thus
In addition to binding to itself, the chromoshadow do- lessaccessible
to proteins) andismuchlesstranscriptionally
activethan
main also binds the enzyme that methylatesH3 lysine 9, iseuchromatin PC CrossandK
[Part(a) L Mercer, 1993,Cell
andTissue
H3K9 histone methyl transferase (HMT). As a conse- Ultrastructure,
W H Freeman andCompany,p 165 Part(b)adapted
fromT.
quence,nucleosomesadjacentto a region of HP1-contain- JenuweinandC D Allis,
2001, Science293:10741
ing heterochromatin also become methylated at lysine 9
(Figure 6-34b). This createsa binding site for another Hp1
that can bind the H3K9 histone merhyl transferase, matic regions of a chromosome are re-establishedfollowing
resulting in "spreading" of the heterochromatinstrucrure DNA replication during the S phase of the cell cycle. \fhen
along the chromosome until a boundary element is en- DNA in heterochromatin is replicated,the histone octamers
countered that blocks further spreading. Boundary ele- that are tri-methylated at H3 lysine 9 becomedistributed to
ments so far characterizedare generally regions in chro- both daughter chromosomesalong with an equal number of
matin where several nonhistone proteins bind to DNA. newly assembled histone octamers. The H3K9 histone
p o s s i b l yb l o c k i n gh i s t o n em e t h y l a i i o no n r h e o r h e r s i d e o f methyl transferaseassociatedwith the H3K9 tri,methylated
the boundary. nucleosomesmethylate lysine 9 of the newly assembled
Significantly,the model of heterochromatin formation in nucleosomes,regenerating the heterochromatin in both
Figure 6-34b provides an explanation for how heterochro- daughterchromosomes.

252 CHAPTER
6 | c E N E S ,G E N O M t C SA, N D C H R O M O S O M E S
 < FIGURE5-34 Model for the formation of heterochromatin
by binding of HP1 to histone H3 tri-methylated at lysine 9. (a)
HP1contributesto the condensation of heterochromatin by binding
to histoneH3 N-terminaltailstri-methylated at lysine9 (H3K9Mer),
I ui.,on"H 3 K 9 followedby association of histone-boundHP'lmolecules with each
methvl
transferase other (b) Heterochromatin condensation can spreadalonga
I chromosomebecauseHP1bindsa histonemethyltransferase (HMT)
that methylateslysine9 of histoneH3 Thiscreatesa bindingsitefor
HPl on the neighboringnucleosomeThe spreadingprocess
continuesuntil a "boundaryelement" is encounteredlPart(a)adapted
from G Thielet al . 2004,Eur.J Biochem271:2855Part(b)adaptedfrom
et al . 2001,Nature
A J Bannister 410i120
I

I a i n u i nosf H e t
chromodomain to H3K9Me3
J In summarS multiple types of covalent modifications of
Herorisorn"rization
f X-Chromosome lnactivation in Mammalian Females
Heterochromatin
Ac Ac Ac Ac
Active chromatin
!l S p r e a d i n go f s i l e n c e da n d
HP1-coatedheterochromati
Other protein domainsassociatewith histone-tailmodi-
fications typical of euchromatin. For example, the bromod-
omain binds to acetylatedhistonetails and thereforeis asso-
ciated with transcriptionally active chromatin. TFIID, a
protein involved in transcription, contains two closely
spacedbromodomains,which probably help TFIID to asso-
ciate with transcriptionallyactive chromatin (i.e., euchro-
matin). This protein also has histone acetylaseactivity,
which may maintain the chromatin in a hyperacetylated
s t a t ec o n d u c i v et o t r a n s c r i o t i o n .
histone tails can influence chromatin structure by altering
nucleosome-nucleosomeinteractions and interactions with
additional proteins that participatein or regulateprocesses
suchas transcriptionand DNA replication.The mechanisms
and molecular processesgoverning chromatin modifications
that regulatetranscriptionare discussedin greaterdetail in
the next chapter.
One important case of heterochromatin formation that
correlateswith geneinactivation in mammals is the random in-
activation and condensationof one of the two female sex chro-
mosomes(the X chromosomes)in virtually all the diploid cells
clf adult females.Inactivationof one X chromosomein females
results in dosagecompensation,a processthat generatesequal
expressionof genes on the sex chromosome in males and
females.The inactive X appears as heterochromatin in inter-
phasecells.It is visible as a dark-staining,peripheralnuclear
structure called the Barr body, named after its discoverer'
Each female mammal has two X chromosomes?one con-
tributed by the egg from which they developed(X-) and one
contributedby the sperm(Xo). Early during embryologicde-
velopment, random inactivation of either the X* or the Xp
chromosome occurs in each cell. In the female embryo,
about half the cells have an inactive X*, and the other half
have an inactiveXe. All subsequentdaughtercellsmaintain
the same inactive X chromosomesas their parent cells. As a
result,the adult femaleis a mosaic of clones,some express-
ing the genesfrom the X- and the rest expressingthe genes
from the Xo. Histones associatedwith the inactive X chro-
mosome have post-translationalmodifications character-
n istic of other regions of heterochromatin:hypoacetylation
of lysines,di- and tri-methylation of histone H3 lysine 9,
tri-methylation of H3 lysine27, and a lack of methylation
a t h i s t o n eH 3 l y s i n e4 ( s e eF i g u r e 6 - 3 3 b ) . X - c h r o m o s o m e
inactivation at an early stagein embryonic developmentis
controlled by the X-inactivation center, a complex locus
on the X chromosomethat determineswhich of the two X
chromosomeswill be inactivated and in which cells. The
X-inactivation center also contains the Xlsr gene, which
encodesa remarkableRNA that coats only the X chromo-
some it was transcribedfrom, thereby triggering silencing
of the chromosome.

OF EUKARYOTIC HROMOSOMES
S T R U C T U R AOL R G A N I Z A T I O N 253
 Although the mechanismof X-chromosome inactivation
is not fully understood, it involves severalprocessesinclud-
ing the action of Polycomb protein complexesthat are dis-
cussedfurther in Chapter 7. One subunit of the Polycomb
complex contains a chromodomain that binds to histone H3
tails when they are tri-methylated at lysine 27. The Poly-
comb complex also contains a histone methyl transferase
specificfor H3 lysrne27. This finding helps to explain how
the X-inactivationprocessspreadsalong largeregionsof the
X chromosome and how it is maintained through DNA Loops
replication,similar to heterochromatizationby the binding of
DNA
of HPl to histone H3 tails methylaredat lysine 9 (seeFig-
ure 6-34b).
X-chromosomeinactivationis an epigeneticprocess:that
is, a processthat affects the expressionof specificgenesand
is inheritedby daughtercells,but is not the resultof a change
in DNA sequence.Instead,the activity of geneson the X
chromosomein femalemammalsis controlled by chromatin
structurerather than the nucleotidesequenceof the underly, protein
i n g D N A . A n d t h e i n a c t i v a t e dX c h r o m o s o m el e i r h e rX - o r scaffold
Xu) is maintainedas the inactivechromosomein the progeny
of all future cell divisions becausethe histones are modified
in a specific, repressingmanner that is faithfully inherited
through eachcell divisron.

N o n h i s t o n eP r o t e i n sP r o v i d ea S t r u c t u r a l A EXPERIMENTAL FIGURE 6-35 An electronmicrograph of a

Scaffoldfor Long ChromatinLoops
Although histonesare the predominantproteinsin chromatin,
lessabundant, nonhistonechromatin-associated proteins,and
eventhe DNA moleculeitself, are also crucial to chromosome
structure.Electronmicrographsof histone-depleted metaphase
chromosomesfrom HeLa cellsreveallong loops of DNA an-
chored to what appearsto be a protein chromosomescaffold
composedof nonhistoneproteins (Figure6-35). Although this
chromosomescaffoldhas the shapeof the metaphasechromo-
some, recent results indicate that it is not protein alone that
givesa metaphasechromosomelts structure.
Micromechanical studies of large metaphasechromo-
somesfrom newts in the presenceof proteasesor nucleases
indicate that DNA, nor protein, is responsiblefor the me-
chanical integrity of a metaphasechromosome when it is
pulled from its ends. These results are inconsistent with a
continuous protein scaffold at the chromosome axis. Rather,
the integrity of chromosome strucrure requires the complete
chromatin complex of DNA, histoneoctamers,and nonhis-
tone chromatin-associated proteins.
In situ hybridization experimenrswith several different
fluorescent-labeled probesto the DNA of one chromosomein
human interphasecellssupport a model in which chromatin is
arrangedin large loops. In theseexperiments,some probe se-
quencesseparatedby millions of basepairs in linear DNA ap-
peared reproducibly very close to one another in interphase
nuclei from different cells of rhe same type (Figure 6-36).
Thesecloselyspacedprobe sitesare postulatedto lie closeto
regions of chromatin, called scaffoLd-associatedregions
/SARs/ or matrix-attacbment regions (MARs), that are lo-
cated at the basesof the DNA loops observed in histone-
histone-depleted metaphasechromosomerevealsan apparent
scaffoldaroundwhich the DNAappearsto be organized.Long
loopsof DNAarevisible extendingfromthe nonhistoneprotein
"scaffold"(thedarkstructure)
thatreflects
theshapeof the metaphase
chromosome However,recentstudies
indicate
thatthesenonntsrone
proteinsdo notforma continuous structure responsible
solely for
determining theshapeof a metaphase chromosome asonemight
expectfor a truescaffold
structureThechromosome wasprepared
f romHeLacellsbytreatment witha milddetergent I R paulson
[From
andU K Laemml1
i ,9 7 7 , C e 1
l l 2 : 8 1 7C o p y r i g h t l 9 7 7 t V [ ]
depleted metaphase chromosomes (see Figure 6-35).
SARs/MARs have beenmapped by digestinghistone-depleted
chromosomeswith restriction enzymesand then recovenng
the fragments that remain associated with the histone-
depletedpreparation.The measureddistancesbetweenprobes
are consistentwith chromatin loops ranging in size from 1
million to 4 million basepairs in mammalian interphasecells.
In general,SARs/MARs are found betweentranscription
units, and genesare located primarily within the chromatin
loops. As discussedbeloq the loops are tetheredat their bases
by a mechanismthat does not break the duplex DNA mole-
cule, which extendsthe entire length of the chromosome.Evi-
denceindicatesthat SARs/MARs may affect transcription of
neighboringgenes.Experimentswith transgenicmice indicate
that in somecasesSARs/MARs are requiredfor high-levelex-
pression of genes in the vicinity of SARs/MARs. And in
Drosophila, some SARs/MARs function as insulators,that is,
DNA sequences of tens to hundredsof basepairs that insulate
transcription units from each other. Proteinsregulating tran-
scription of one gene cannot influence the transcription of a
neighboringgenethat is separatedfrom it by an insulator.

254 . cHAprER
6 | c E N E SG, E N o M t c sA,N D c H R o M o s o M E s
 An SMC monomer contains two globular domains, a
OF
head domain and hinge domain, that are separatedby a very
long coiled-coil domain. The head domain is formed from
EO ', z L o o p o t
OG 30-nm the N- and C-termini of the polypeptide, which are folded
chromatin together in the native protein structure. The hinge domain
DO fiber
forms where the polypeptide folds back on itself. The hinge
,H
domain of one monomer binds to the hinge domain of a sec-
// 'B co ond monomer, forming a roughly U-shapeddimeric complex
// / (Figure 6-38a). The head domains of the monomers have
ATPaseactivity and are linked by membersof another small
Chromosome
scaffold protein family calledkleisins.

Interphase Chromosome Structure Studieshave indi-

cated that SMC proteins can link two circular DNA molecules
probes by a mechanism that does not require direct protein-DNA
EXPERIMENTAL FIGURE 5-36 Fluorescent-labeled
hybridizedto interphasechromosomesdemonstratechromatin binding. Rather, the two DNA molecules are topologically
loopsand permittheir measurement. In situhybridizationof linked and can be separatedby either cleavageof the SMC com-
interphasecellswascarried out with several
different orobessoecific plex with a protease,or cleavageof one of the circular DNA
for sequencesseparated by knowndistances in linear,clonedDNA moleculesby a restriction enzyme.Theseresults,combined with
Letteredredcircles
represent probes. Measurement of thedistances the U-shaped structure of an SMC complex, suggestthat an
between differenthybridizedprobes, whichcouldbe distinguished SMC complex can link two 30-nm chromatin fibers by encir-
bytheircolor,showedthatsomesequences (e.g.,A, B,andC), cling both of them as depicted in Figure 6-38b. Using the tech-
separatedfromoneanotherby millions of basepairs,appearlocated nique of chromatin immunoprecipitation, discussedin the next
nearoneanotherwithinnucleiForsomesetsof sequences, the chapter, researchershave demonstrated that SMC proteins in
measured distancesin nucleibetweenoneprobe(e g , C)and yeast interphasecells associatewith chromatin primarily at
sequences successively
fartherawayinitiallyappearto increase (eg ,
regions between genes.It is possible that ringlike SMC protein
D, E,andF)andthenappearto decrease (e g , G andH).[Adapted
complexes are "pushed" into these regions by RNA poly-
fromH Yokotaetal. 1995.J CellBiol130:1239I merasestranscribing the regionsof chromatin betweenthem.

Individual interphasechromosomes,which are less con-

densedthan metaphasechromosomes,cannot be resolvedby
standardmicroscopyor electronmicroscopy.Nonetheless,the
chromatin of one chromosomein interphasecellsis not spread
throughout the nucleus.Rather,interphasechromatin is organ-
ized into chromosometerritories.As illustratedin Figure 6-37,
in situ hybridization of interphasenuclei with chromosome-
specificfluorescent-labeled probes shows that the probes are
visualizedwithin restrictedregionsof the nucleusrather than
appearingthroughout the nucleus.Use of probes specificfor
different chromosomesshows that there is little overlap be-
tween chromosomesin interphasenuclei.However,the precise
positionsof chromosomesare not reproduciblebetweencells.

Ringlike Structure of SMC Protein Complexes Charac-

terizationof proteinsassociatedwith metaphasechromosomes
identified a small family of proteins called structwral mainte-
nance of chromosome proteins, or SMC proteins. These non-
histoneproteinsare critical for maintainingthe morphological
structureof chromosomes.In yeastwith mutations in certain
SMC proteinschromosomecondensationduring the prophase FIGURE 6-37 Duringinterphase, human
a EXPERIMENTAL
period of mitosisdoesnot occur.Mutants with defectsin other remainin specific territoriesin the nucleus. Fixed
chromosomes
SMC proteins fail to properly associatedaughter chromatids interphasehumanfibroblasts werehybridized in situto f luorescently
following DNA replicationin the S phase.As a result,chromo- labeledprobesspecificfor sequencesalongthef ull lengthof human
somesdo not properly segregate to daughtercellsduring mito- chromosomes 7 (cyan)and8 (purple)DNAwasstained bluewith
sis. Related SMC proteins are required for proper segregation DAPIInthisdiploidcell,eachof thetwo chromosome 7sandtwo
of chromosomesin bacteriaand archaea,indicatingthat this is chromosome 8scanbe seento be restricted to a territoryor domain
an ancientclassof proteinsvital to chromosomestructureand withinthe nucleus,ratherthanstretchingthroughout the entire
segregationin all kingdoms of life. nucleus.[Courtesy andT.Cremerl
of DrsI Solovei

CHROMOSOMES .
OF EUKARYOTIC
S T R U C T U R AOL R G A N I Z A T I O N 255
(a) Hinge many more loops of chromatin, so that the length of each
domain
Ioop is greatly reduced compared with that in interphase
cells. However, the folding of chromatin in metaphasechro-
mosomes is not well understood. Microscopic analysis of
mammalian chromosomesas they condenseduring prophase
indicatesthat the 30-nm fiber folds into a 100- to 130-nm
Head fiber called a chromonema fiber. As depicted in Figure 6-39,
domain a chromonema fiber then folds into a structure with a diam-
eter of 200-250-nm called a middle prophase chromatid,
Kleisin which then folds into the 500- to 750,nm diameter chro-
matids observedduring metaphase.

C h r o m a t i nl o o p c o n t a i n i n g A d d i t i o n a lN o n h i s t o n eP r o t e i n sR e g u l a t e
a transcriotionunit
Transcriptionand Replication
The total mass of the histones associated with DNA in
chromatin is about equal to that of the DNA. Interphase
chromatin and metaphasechromosomesalso contain small
amounts of a complex set of other proteins. For instance,
hundreds to thousands of different transcription factors are
associated with interphase chromatin. The strucrure and
function of these critical nonhistone proteins, which help
regulate transcription, are examined in Chapter 7. Other
FIGURE 6-38 Modelsof SMCcomplexesand their association low-abundance nonhistone proteins associatedwith chro-
with 30-nmchromatinfibersin interphasecells.(a)An SMCprotein matin regulate DNA replication during the eukaryotic cell
complex consists
of two monomers, SMC2(blue)andSMC4(red), cycle (Chapter 20).
whosehingedomains associateTheheaddomains, whichhaveATpase A few other nonhistone DNA-binding proteins are pres-
activity,
areIinkedbya kleisin
protein,forminga ringlikestructure ent in much larger amounts than the transcription or repli-
(b)TheringlikeSMCcomplex topologicallylinkstwo chromatinfibers cation factors. Some of these exhibit high mobility during
(graycylinders).
Thecylinderdiameter representsthediameterof a electrophoretic separation and thus have been designated
nucleosome andisto scalerelative
to thedimensions of theSMC
complex(c)Loopsof transcriptionallyactivechromatin maybetethered
at theirbasebyseveralSMCcomplexes, forminga topological
knot
30 nm
[Adapted from K Nasmythand C H Haering, ZOO5,Ann Rev_Biochem 74:595]

Basedon these various lines of evidence,a recent model 100-130nm

( c h r o m o n e mf iab e r )
proposesthat the long loops of chromatin detectedin inter-
phase chromosomes(seeFigure 5-36) are tethered at the
baseof eachloop by severalSMC complexes(Figure6-38c).
These topological knots of SMC proteins and chromatin at 200-250nm
( m i d d l ep r o p h a sceh r o m a t i d )
the base of each loop are probably linked together in some
way to produce the apparent protein scaffold shape visual-
ized in histone-depletedmetaphasechromosomes (seeFig-
ure 6-35). The linkage may require additional types of
proteins, or may result from linkage of SMC complexes
alone. In either case,the model in Figure 5-38c can explain
why cleavageof the DNA at a relatively small number of
sites leads to rapid dissolution of chromosome srructure, 500-750nm
whereas proteasecleavagehas only a minor effect on chro- (metaphase
chromatid)
mosome structure until most of the protein is digested:
When the DNA is cut anywhere in a chromatin loop, the
broken ends can slip through the SMC protein rings, ,.unty-
ing" the topological knots that constrain the loops of chro-
matin. In contrast, most of the individual rings of SMC pro-
teins must be broken before the topological constraints FIGURE 6-39 Modelfor the folding of the 30-nmchromatin
holding the baseof the loops together is released. fiber in a metaphasechromosome. Thisdrawingdepicts the
sequential foldingof a single30-nmfiberintoa singlechromatidof
Metaphase Chromosome Structure Condensation of a metaphase chromosome fromN Kireeva
[Adapted et-al
,2004,J Cell
chromosomesduring prophasemay involve the formation of Biol.166:775l

255 CHAPTER
6 I G E N E 5G
, E N O M I C SA, N D C H R O M O S O M E S
HMG (bigh-mobility group) proteins. !7hen genesencoding
the most abundant HMG proteins are deleted from yeast
cells, normal transcription is disturbed in most genesexam-
ined. SomeHMG proteins have been found to bind to DNA
cooperativelywith transcription factors that bind to specific
DNA sequences,stabilizing multiprotein complexesthat reg-
ulate transcription of a neighboring gene.
Structural Organization of Eukaryotic Chromosomes
r In eukaryoticcells,DNA is associatedwith about an equal
massof histoneproteinsin a highly condensednucleoprotein
complex called chromatin. The building block of chromatin
is the nucleosome,consistingof a histone octamer around
which is wrapped 1.47bp of DNA (seeFigure 6-29).
r The chromatin in transcriptionally inactive regions of
DNA within cells is thought to exist in a condensed,30-nm
fiber form and higher-order structures built from it (see
Figures6-30b and 6-39).
r The chromatin in transcriptionally active regionsof DNA
within cells is thought to exist in an open, extended form
(seeFigure6-28a).
r The histone H4 tails, particularly H4 lysine 16, arc rc-
quired for beads-on-a-stringchromatin (the 10-nm chro-
matin fiber) to fold into a 30-nm fiber.
r Histone tails can be modified by acetylation, methyla-
tion, phosphorylation, and monoubiquitination (see Fig-
ure 6-31). Thesemodificationsinfluencechromatin struc-
ture by regulating the binding of histone tails to other less
abundant chromatin-associatedproteins.
r The reversible acetylation and deacetylation of Iysine
residues in the N-termini of the core histones regulates
chromatin condensation.Proteins involved in transcrip-
tion, replication, and repair, and enzymeslike DNaseI can
more easily accesschromatin with hyperacetylatedhistone
tails (euchromatin)than chromatin with hypoacetylatedhi-
stone tails (heterochromatin).
r'When metaphasechromosomesdecondenseduring inter-
phase, areas of heterochromatin remain much more con-
densedthan regions of euchromatin.
r Heterochromatin protein 1 (HP1) usesa chromodomain
to bind to histone H3 tri-methylated on lysine 9. The chro-
moshadow domain of HP1 also associateswith itself and
with the histone methyl transferasethat methylatesH3 ly-
sine 9. Theseinteractions causecondensationof the 30-nm
chromatin fiber and spreading of the heterochromatic
structure along the chromosome until a boundary element
is encountered(seeFigure 6-34).
r One X chromosome in nearly every cell of mammalian
females is highly condensedheterochromatin, resulting in
repressionof expressionof nearly all geneson the inactive
chromosome. This inactivation results in dosagecompen-
sation so that geneson the X chromosome are expressedat
the same level in both males and females.
AND FUNCTIONAL
MORPHOLOGY
r Each eukaryotic chromosome contains a single DNA
molecule packaged into nucleosomesand folded into a
3O-nm chromatin fiber, which is associatedwith a protein
scaffold made up in part of structural maintenanceof chro-
mosome (SMC) proteins at sites between transcription
units (seeFigure 6-38c). Additional folding of the scaffold
further compacts the structure into the highly condensed
form of metaphasechromosomes(seeFigure 6-39).
Wl andFunctional
Morphology
Elementsof EukaryoticChromosomes
Having examined the detailed structural organization of
chromosomes in the previous section' we now view them
from a more global perspective.Early microscopic observa-
tions on the number and sizeof chromosomesand their stain-
ing patterns led to the discovery of many important general
characteristicsof chromosome structure. Researcherssubse-
quently identified specific chromosomal regions critical to
their replication and segregationto daughter cells during cell
division. In this sectionwe discussthesefunctional elements
of chromosomes and consider how chromosomes evolved
through rare rcarrangementsof ancestralchromosomes.
ChromosomeNumber,Size,and ShaPe
at MetaphaseAre Species-Specific
As noted previously in nondividing cells individual chromo-
somesare not visible,evenwith the aid of histologic stainsfor
DNA (e.g.,Feulgenor Giemsastains)or electronmicroscopy.
During mitosis and meiosis,however' the chromosomescon-
denseand becomevisible in the light microscope.Therefore,
almost all cytogenetic work (i.e., studies of chromosome
morphology) has beendone with condensedmetaphasechro-
mosomes obtained from dividing cells-either somatic cells
in mitosis or dividing gametesduring meiosis'
The condensationof metaphasechromosomesprobably
results from several orders of folding of 30-nm chromatin
fibers (seeFigure 6-39\. At the time of mitosis, cells have al-
ready progressedthrough the S phase of the cell cycle and
have replicated their DNA. Consequently,the chromosomes
that becomevisible during metaphase are duplicated struc-
tures. Each metaphase chromosome consists of two sister
chromatids,which are linked at a constrictedregion, the cen-
tromere (Figure 5-40). The number, sizes,and shapesof the
metaphasechromosomesconstitute the karyotype, which is
distinctive for each species.In most organisms' all cells have
the same karyotype. However, species that appear quite
similar can have very different karyotypes, indicating that
similar genetic potential can be organized on chromosomes
in very different ways. For example, two speciesof small
deer-the Indian muntjac and Reeves muntjac-contain
about the same total amount of genomic DNA. In one
species,this DNA is organized into 22 pairs of homologous
autosomesand two physically separatesex chromosomes.In
contrast, the other speciescontains the smallest number of
chromosomesin any mammal, only three pairs of autosomes;
OF EUKARYOTIC HROMOSOMES
ELEMENTS
257
 reagent produces R bands in a pattern that is approximately
the reverseof the G-band pattern. The distinctive banding pat-
terns of each chromosome permit cytologists to identify spe-
cific parts of a chromosome and to locate the sitesof chromo-
somal breaks and translocations(Figure 6-42a).In addition,
cloned DNA probes that have hybridized to specificsequences
in the chromosomescan be located in particular bands.
The method of spectral karyotyping or chromosome
painting greatly simplifies differentiating chromosomes of
similar size and shape. This technique, a variation of fluores-
cence in situ hybridization (FISH), makes use of probes spe-
cific for sites scatteredalong the length of each chromosome.
The probes are labeled with several different fluorescent dyes
with distinct excitation and emissionwavelengths.Probesspe-
cific for each chromosome are labeled with a predetermined
fraction of each of the dyes.After the probes are hybridized to
chromosomesand the excessremoved,the sampleis observed
with a fluorescent microscope in which a detector determines
the fraction of each dye present at each fluorescing position in
the microscopic field. This information is conveyed to a com-
puter, and a special program assigns a false color image to
each type of chromosome. A related technique called mubi-
color FISH can detectchromosomaltranslocations(Figure5-
42b). The much more detailed analysispossible with these
techniques permits detection of chromosomal translocations
that banding analysis does not reveal. The photograph at the
beginning of the chapter illustrates the use of multicolor FISH
in preparing the karyotype of a human female.

FIGURE 6-40 Typicalmetaphasechromosome.As seenin this

scanningelectron
micrograph, eachchromosome hasreplicatedand
comprisestwo chromatids, eachcontainingoneof two identical DNA
molecules.Thecentromere, wherechromatids areattachedat a
constriction,
isrequiredfor theirseparation
latein mitosis.
Special
telomeresequencesat theendsfunctionin preventing chromosome
shortening[AndrewSyred/photoResearchers,
Inc]

one sex chromosome is physically separate,but the other is

joined to the end of one autosome.

During Metaphase,ChromosomesCan Be
b y B a n d i n gp a t t e r n s
Distinguished
and Chromosome Painting
Certain dyes selectivelystain some regions of metaphasechro-
mosomesmore intenselythan other regions,producing charac-
teristic banding patterns that are specific for individual chro-
mosomes.The regularity of chromosomal bands serveas useful
visible landmarks along the length of each chromosome and
can help to distinguishchromosomesof similar sizeand shaoe.
EXPERIMENTAL FIGURE 6-41 G bandsproducedwith Giemsa
G bands are produced when metaphasechromoso..,
"ra stainsare useful markersfor identifying specificchromosomes.
subjected briefly to mild heat or proteolysis and then stained
Shownherearethechromosomes froma humanmalethatwere
with Giemsareagenr,. p.r-un.ni DNA dye (Figure6-41). G
subjectedto briefproteolytic
treatmentandthenstaining with
bands correspond to large regions of the human genome that Giemsa reagent. Theresulting
darkbandsat characteristic
places
are
have an unusually low G + C content. Treatment of chromo- distinctive
for eachchromosome. Mal,phD , Manrtoba
of Sabine
[Courtesy
someswith a hot alkaline solution before staining with Giemsa I n s t i t u t eo f C e l lB i o l o g yC
, anadal

258 CHAPTER
6 | G E N E SG
, E N O M t C SA, N D C H R O M O S O M E S
 to the Iong arm of chromosome 10 (Figure 6-43b). Further'
when multiple probes from the long arm of human chromo-
P h i l a d e l p h i a some 10 with different fluorescentdye labelswere hybridized
chromosome
to human chromosome 10 and tree shrew metaphasechro-
mosomes,tree shrew sequenceshomologous to each of these
probes were found along tree shrew chromosome 16 in the
sameorder that they occur on human chromosome 10.
der (221 These results indicate that during the evolution of hu-
mans and tree shrews from a common ancestor that lived
-85 million yearsago, a long, continuous DNA sequenceon
one of the ancestralchromosomesbecamechromosome 16
in tree shrews,but evolvedinto the long arm of chromosome
10 in humans. The phenomenon of genesoccurring in the
same order on a chromosome in rwo different speciesis re-
ferred to as conservedsynteny (derived from Latin for "on
the same ribbon"). The presenceof two or more genesin a
der (9) common chromosomal region in two or more speciesindi-
catesa conservedsyntenic segment.
The relationships between the chromosomes of many
primates have been determined by cross-specieshybridiza-
tions of chromosome paint probes as shown for human and
tree shrew in Figure 6-43a and b. From these relationships
and higher resolution analysesof regionsof synteny by DNA
sequencingand other methods, it has been possible to pro-
poie the karyotype of the common ancestor of all primates
basedon the minimum number of chromosomal rearrange-
ments necessaryto generatethe regions of synteny in chro-
mosomesof contemporaryprimates.
Human chromosomes are thought to have derived from a
common primate ancestor with23 autosomesplus the X and
Y sex chromosomes by several different mechanisms (Fig-
ure 6-43c). Somehuman chromosomeswere derivedwithout
large scale rearrangementsof chromosome structure. Others
arethought to have evolvedby breakageof an ancestralchro-
mosome into two chromosomes or' conversely, by fusion of
tvvo ancestral chromosomes' Still other human chromosomes
EXPERIMENTAL FIGURE 5-42 Chromosomal translocations
appear to have been generated by exchangesof parts of the
can be analyzedusingbandingpatternsand multicolorFl5H.
aims of distinct chromosomes,that is, by reciprocal translo-
chromosomal
Characteristic translocationsareassociated with certain
two ancestralchromosomes.Analysis of re-
geneticdisorders andspecifictypesof cancers. Forexample, in nearly cation involving
the leukemic cells gions of conserved synteny between the chromosomes of
allpatientswith chronic myelogenous leukemia,
containthe Philadelphia chromosome, a shortened chromosome 22 many mammals indicatesthat chromosomal rearrangements
[der(22)],andan abnormally longchromosome 9 [der(9)]("der" such as breakage,fusion and translocationsoccurredrarely in
standsfor derivative).Theseresultfrom a translocation between mammalian evolution, about once every five million years'
normalchromosomes 9 and22 Thistranslocation canbe detected \fhen such chromosomal rearrangementsdid occur, they very
bandinganalysis
by classical (a)andby multicolor (b).
FISH IPart (a) likely contributed to the evolution of new speciesthat cannot
fromJ Kuby, 1997, lmmunology,3d ed,W H Freeman andCompany, p interbreed with the speciesfrom which they evolved'
(b)
578 Part courtesy of J RowleyandR Espinosa
l Chromosomal rearrangementssimilar to those inferred for

Chromosome P a i n t i n ga n d D N A S e q u e n c i n g
Revealthe Evolutionof Chromosomes
Analysis of chromosomesfrom different specieshas provided
considerableinsight about how chromosomesevolved. For
example, hybridization of chromosome paint probes for
chromosome 16 of the tree shrew (Tupia belangeri) to tree
shrew metaphasechromosomesrevealed the two copies of
chromosome 16, as expected(Figure 6-43a). However, when
the same chromosome paint probes were hybridized to hu-
man metaphasechromosomes,most of the probeshybridized
ture (i.e., among mammals, among insectswith similar body
organization, among similar plants, etc.) and the evolutionary
reLtionships basedon the fossil record and on the extent of di-
vergenceof DNA sequencesfor homologous genesis a strong
argument for the validity of evolution as the processthat gen-
erated the diversity of contemporary organisms.

OF EUKARYOTIC HROMOSOMES O
ELEMENTS
AND FUNCTIONAL
MORPHOLOGY
259
 (c)
Primate ancestor

1
I
23456 X

7891011121314

15 16 17 18 19 20 21 22 23

Homo sapiens

123456X

7891011121314
tt_ tt19- -
ll' l,l-lrol-lrz
ltrll"a, 7)tr
15 16 17 18 19 20 21 22
EXPERIMENTAL FIGURE 543 Evolutionof primatechromo-
somes.(a)Chromosome paintprobes for chromosome 16of thetree
shrew(7fbelanger|a primate-like animaldistantly relatedto humans)
werehybridized (yellow)to treeshrewmetaphase chromosomes (red).
These probes"painted"theentirety of bothcopies of chromosome 16
(b)Thesametreeshrewchromosome 16 paintprobeswerehybridized
to humanmetaphase chromosomes Theseprobesweretargety
localized
to the longarmsof thetwo chromosome 10s (c)proposed
evolutronof humanchromosomes (bottom)fromthe chromosomes of
the commonancestor of all primates(top)Theproposed common
primateancestor chromosomes arenumbered according to theirsizes,
with eachchromosome represented by a differentcolor.Thehuman
chromosomes arealsonumbered according to theirrelativesizes
with colorstakenfromthe colorsof the proposed commonprimate
ancestor chromosomes fromwhichtheywerederivedSmallnumbers
to therightof thecolored regionsof thehumanchromosomes indicate
thenumberof theancestral chromosome fromwhichtheregron
wasderived. Humanchromosomes werederived fromthe proposed
InterphasePolyteneChromosomes Arise chromosomes of the commonpnmateancestor in severarways:
b y D N AA m p l i f i c a t i o n withoutsignificant rearrangements (e.g, humanchromosome 1);by
fusion(e.g, humanchromosome 2 byfusionof ancestral
The larval salivary glands of Drosopbila species and other chromosomes 9 and11);breakage (eg , humanchromosomes 14and
dipteran insects contain enlarged interphase chromosomes 15by breakage of ancestralchromosome 5),andchromosomar
that are visible in the light microscope. When fixed and translocations(e9.,humanchromosomes 12 and22bya reciprocal
stained, these polytene chromosomes are characterized by a translocationbetweenancestral chromosomes 14 and2j) lparts (a)and
large number of reproducible, well-demarcated bands that (b)courtesy
of Professor
Dr.Johannes Weinberg, Institute
forHuman Genetics
and Anthropology,Universityof Munich part(c) courtesyof LutzFroenicke
Ph D , Schoolof VeterinaryMedicine,Universityof California,Davisl

glands (Figure 6-44b). Chromosomal translocarionsand in-

versionsalso are readily detectablein polytenechromosomes,
and specificchromosomalproteins can be localizedon inter-
phase polytene chromosomesby immunostaining with spe-
cific antibodiesraised against them (seeFigure 7-11). Insect
polytene chromosomes offer one of the only experimental

260 . cHAprER
G I c E N E sG
, E N o M t c sA,N D c H R o M o s o M E s
> EXPERIMENTAT FIGURE 644 Bandingon Drosophilapolytene (a)

salivaryglandchromosomes and in situ hybridizationare used Chromocenter

togetherto localizegenesequences. (a)Inthislightmicrograph of

Drosophila melanogaster larvalsalivaryglandchromosomes, four
chromosomes canbe observed (X,2, 3, and4),with a totalof approxi-
mately5000distinguishable bands. Thebandingpatternresults from
reproducible packing of DNAandproteinwithineachamplified site
alongthechromosome. Darkbandsareregions of morehighlycom-
oactedchromatinThecentromeres of allfourchromosomes often
appearfusedat thechromocenter Thetipsof chromosomes 2 and3 are
labeled (L : leftarm;R : rightarm),asisthetip of theX chromosome.
(b)A particular DNAsequence canbe mappedon Drosophila salivary
glandchromosomes by in situhybridization. Thisphotomicrograph
showsa portionof a chromosome thatwashybridized with a cloned
DNAsequence labeled with biotin-derivatized nucleotides. Hybridization
isdetected with the biotin-binding proteinavidinthatiscovalently
boundto theenzyme alkaline phosphatase Onadditionof a soluble
substrate, theenzyme catalyzes a reaction thatresultsin formation of
an insoluble coloredprecipitate at thesiteof hybridization (asterisk).
Sincetheveryreproducible bandingpatterns arecharacteristic of each
Drosophila polytene chromosome, the hybridizedsequence canbe
locatedon a particular chromosome Thenumbers indicatemajorbands
Bandsbetween thoseindicated aredesrgnated with numbers andletters
(notshown). [Part(a)courtesyofJ GallPart(b)courtesy of F.Pignoni]

systemsin all of nature where such immuno-localizationstud-

ies on decondensedinterphasechromosomesare possible.
A generalizedamplification of DNA gives rise to the poly-
tene chromosomesfound in the salivary glands of Drosophila.
This process,termed polytenizatioin,occurs when the DNA re-
peatedly replicateseverywhereexcept at the telomeresand cen-
tromere, but the daughter chromosomesdo not separate.The
result is an enlarged chromosome composed of many parallel
copiesof itself (Figure6-45). The amplification of chromosomal somes.The yeastgenomecontainsmany -100-bp sequences'
DNA greatly increasesgenecopy number,presumablyto supply caIIedautonomously replicating sequences(ARSs/, that act as
sufficient mRNA for protein synthesisin the massivesalivary replication origins. The observationthat insertion of an ARS
gland cells. Although the bands seen in G-banded human into a circular plasmid allows the plasmid to replicatein yeast
metaphasechromosomesprobably representvery long folded cells provided the first functional identification of origin se-
or compacted stretchesof DNA containing about 10' base quencesin eukaryotic DNA (seeFigure 6-46a).
pairs, the bands in Drosophila polytene chromosomesrepresent Even though circular ARS-containingplasmidscan repli-
much shorterstretchesof only 50,000-100,000basepairs. cate in yeast cells, only about 5-20 percent of progeny cells
contain the plasmid becausemitotic segregationof the plas-
Three FunctionalElementsAre Required mids is faulty. However, plasmids that also carry a CEN
f o r R e p l i c a t i o na n d S t a b l eI n h e r i t a n c e sequence,derived from the centromeresof yeastchromosomes'
of Chromosomes
Although chromosomes differ in length and number between
species,cytogeneticstudieshave shown that they all behave
similarly at the time of cell division. Moreover, any eukaryotic
chromosomemust contain three functional elementsin order
to replicate and segregatecorrectly: (1) replication origins at
which DNA polymerasesand other proteins initiate synthesis
of DNA (seeFigures 4-31 and a.33); (2) the centromere' the
constricted region required for proper segregationof daughter A FIGURE 645 The Patternof generalizedDNAamplificationof
chromosomes;and (3) the two ends, or telomeres.The yeast one polytenechromosomeduring five replications'Double-stranded
transformation studiesdepictedin Figure 6-45 demonstrated DNAisrepresented telomere
bya singleline'Duringpolytenization, and
the functions of thesethree chromosomalelementsand estab- centromereDNAarenotamplified andthedaughter chromosomes do
lishedtheir importance for chromosomefunction. Insalivary
notseparate. glandpolytene chromosomes, eachparental
As discussedin Chapter 4, replication of DNA begins chromosome undergoes -10 replications(210: 1024strands)'
[Adapted
fromC D et
Laird al, 1973, Spring
Cold HarborSymp Quant' 1l
38:31
Biol'
from sitesthat are scatteredthroughout eukaryotic chromo-

MoRPHoLoGYANDFUNcT|oNALELEMENTSoFEUKARYoT|CcHRoMo5oMES
261
 Plasmid with Transfected Progenyof transfected cell Conclusion
sequencefrom leu- cell
normal yeast Growth Mitotic
(a) without segregation
leucine

ARS required
No for plasmid
replication

No
Poor In presence
vL \ $-20% ofARS.
Lo ') of cells olasmid
have replicationoccurs,
plasmid) but mitotic
Yes segregationis
fa u ltv

(b)

Yes
Good Genomic
{\ (>90%
5J fragment
of cells CEN required
have for good
plasmid) segregation

(c)

'?dL E U -

l -'ARS
l
No L i n e a rp l a s m i d
lackingTEL
Restriction is unstable
enzyme
p r o d u c e sl i n e a r
plasmid

Yes L i n e a rp l a s m i d s
c o n t a i n i n gA R S
TEL TEL and CEN behave
** ARs- rEU- cEN *-* uooo l i k en o r m a l
,/ ..L' E chromosomesif
c
Yes genomic fragment
TEL is added
to both ends
A EXPERIMENTAL FIGURE 6-46 yeast transfection pieces of genomic yeastDNAareinserted intoplasmros
experimentsidentify the functionalchromosomalelements containing ARSandLEU,someof thesubsequently transfected
necessaryfor normal chromosomereplicationand cellsproduce largecolonies, indicating thata highrateof mitotic
segregation. Intheseexperiments, plasmids containing theIEU segregation amongtheirplasmids isfacilitating the continuous
genefromnormalyeastcellsareconstructed andintroduced into growthof daughter cells. TheDNArecovered fromplasmids in
/eu cellsbytransfection. lf the plasmid is maintained in the/eu- theselargecolonies contains yeastcentromere (CEN)
cells,theyaretransformed to LEU*by the LEUgeneon the sequences. (c)When/eu- yeastcellsaretransfected with
plasmid andcanformcolonies on mediumlackinoleucrne. l i n e a r i z epdl a s m i dcso n t a i n i ntgE U ,A R Sa, n dC E Nn, o c o l o n i e s
(a)Sequences thatallowautonomous replicationinns)of . grow.Additionof telomere (TEL) sequences to the endsof the
plasmid wereidentified because theirinsertion intoa plasmid linearDNAgivesthe linearized plasmids the abilityto replicate as
vectorcontatning a clonedLEUgeneresulted in a highfrequency new chromosomes that behaveverymuchlikea normal
of transformationto IEU* However, evenplasmids with ARS chromosome in both mitosisand meiosis. [See A W Murray and
exhibitpoorsegregation duringmitosis, andtherefore do not J W,Szostak, 1983,Nature 305:89, andL Clarke andJ.Carbon. 1985.
appearin eachof the daughter cells.(b)Whenrandomlv broken Ann Rev.Genet19i29l

262 C H A P T E R6 | G E N E SG
, E N O M | C SA, N D C H R O M O S O M E S
 AAT
YeastCEN GTCACGTG 78-86bp TGTTTCTGNTTTCCGAAA

6-47 Yeastcentromere(CEN)sequence.The fairlyconstant Yeast

in lengthandisrichin A andT residues.
A FIGURE
yeastCENsequence
consensus shownhere,basedon analysis of chromosomes arequiteshortandtheirCENsequences areslmpler
S.cerevisiae
from 10 different chromosomes, includes thanthosein othereukaryotes [See andJ Carbon,
L Clarke Ann
1985,
centromeres
regionsRegion
threeconserved variable
ll,although is
in sequence, Genet19:291
Rev.

segregateequally or nearly so to both mother and daughter bound by nucleosomescontaining the CENP-A histone H3
variant, as well as other repeatedsimple-sequence DNA.
cellsduring mitosis (seeFigure 6-46b1.
If circular plasmidscontaining an ARS and CEN sequence In higher eukaryotes,a complex protein structure called
are cut once with a restriction enzyme) the resulting linear the kinetochoreassemblesat centromeresand associateswith
plasmids do not produce LEU, coloniesunlessthey contain multiple mitotic spindle fibers during mitosis. Homologs of
special telomeric (TEL) sequencesligated to their ends (see most of the centromericproteins found in the yeastsoccur in
Figure 6-46c). The first successfulexperiments involving humans and other higher eukaryotesand are thought to be
transfectionof yeast cellswith linear plasmidswere achieved componentsof kinetochores.The role of the centromereand
by usingthe endsof a DNA moleculethat was known to repli- prot;ins that bind to it in the segregationof sisterchromatids
cate as a linear molecule in the ciliated protozoan Tetrahy- during mitosis is describedin Chapters 18 and 20.
mena. During part of the life cycle of Tetrahymena, much of
the nuclear DNA is repeatedlycopied in short piecesto form
Addition of TelomericSequencesby Telomerase
a so-calledmacronucleus.One of these repeatedfragments PreventsShorteningof Chromosomes
was identified as a dimer of ribosomal DNA, the ends of Sequencingof telomeresfrom multiple organisms,including hu-
which containeda repeatedsequence(G+Tzl".\fhen a section mans, has shown that most are repetitive oligomerswith a high
of this repeatedTEL sequencewas ligatedto the endsof linear G content in the strand with its 3' end at the end of the chro-
yeast plasmids containing ARS and CEN, replication and mosome. The telomere repeat sequencein humans and other
good segregationof the linear plasmidsoccurred. vertebratesis TTAGGG. Thesesimple sequencesare repeatedat
the very termini of chromosomesfor a total of a few hundred
CentromereSequencesVary Greatlyin Length base pairs in yeastsand protozoans, and a few thousand base
Once the yeast centromereregions that confer mitotic segrega- pairs in vertebrates.The 3' end of the G-rich strand extends
tion were cloned, their sequencescould be determinedand com- 12-16 nucleotidesbeyond the 5' end of the complementary C-
pared, revealingthree regions(I, II, and III) that are conserved rich strand.This region is bound by specificproteins that protect
among different chromosomes(Figure 6-47). Short, fairly well the endsof linear chromosomesfrom attack by exonucleases'
conservednucleotide sequencesare presentin regionsI and III. The needfor a specializedregion at the endsof eukaryotic
Although region II seemsto have a fairly constant length, it con-
tains no definite consensussequence;however,it is rich in A and
T residues.RegionsI and III are bound by proteinsthat interact
with a set of more than 30 other proteins, which in turn bind to
microtubules.As a result of theseinteractions,each of the S.
cereuisiaechromosomesbecomesattached to one microtubule
of the spindleapparatusduring mitosis.RegionII is bound to a
nucleosomethat hasa variant form of histoneH3 replacingthe
usualH3. Centromeresfrom all eukaryotessimilarlyare bound
by nucleosomeswith this specialized,centromere-specificform
of histoneH3, called CENP-A in humans,that is essentialfor
centromere function. S. cereuisiaehas by far the simplest cen-
tromeresequence known in nature. would be shortenedat eachcell division.
In the fission yeastS. pombe, centromeresare :40 kb in The problem of telomereshorteningis solved by an enzyme
length and are composedof repeatedcopiesof sequences sim- that addi telomeric (TEL) sequencesto the ends of each chro-
ilar to those in S. cereuisiaecentromeres.Multiple copies of mosome. The enzyme is a protein-RNA complex called telo-
proteins homologous to those that interact with S. cereuisiae mere terminal transferase,ot telomerase.Becausethe sequence
centromeres bind to these complex S. pombe centromeres of the telomerase-associatedRNA, as we will see,servesas the
and in turn bind the much longer S. pombe chromosomesto
severalmicrotubules of the mitotic spindle apparatus.In
plants and animals, centromeresare megabasesin length and
are composed of multiple repeatsof simple-sequence DNA.
In humans,centromerescontain 2t to 4-megabasearrays of a
771-bp simple-sequenceDNA called alphoid DNA that is

MoRPHoLoGYANDFUNcT|oNALELEMENTSoFEUKARYoT|ccHRoMosoMEs
263

Drosopbila speciesmaintain telomere lengths by the regu-
lated insertion of non-LTR retrotransposonsinto telomeres.
This is one of the few instancesin which a mobile element
L a g g i n gs t r a n d
has a specificfunction in its host organism. I
5',
'11
J
L a g g i n gs t r a n d
l-Shortenedl
J
mutated RNA sequenceto the ends of telomeric primers. Thus
telomeraseis a specializedform of a reversetranscriptasethat
carriesits own internal RNA template to direcr DNA synthesis.
Figure 6-49 depicts how telomerase,by reversetran-
scription of its associatedRNA, elongatesthe 3, end of the
single-strandedDNA at the end of the G-rich srrand men-
tioned above. Cells from knockout mice that cannot produce
the telomerase-associated RNA exhibit no telomeraseactiv-
ity, and their telomeres shorten successivelywith each cell
generation.Such mice can breed and reproduce normally for
three generations before the long telomere repeats become
substantiallyeroded.Then, the absenceof telomereDNA re-
sults in adverseeffects,including fusion of chromosome ter-
mini and chromosomal loss. By the fourth generation, the
reproductive potential of theseknockout mice declines,and
they cannot produce offspring after the sixth generation.
The human genes expressing the telomerase protein
and the telomerase-associated
RNA are active in germ
cells and stem cells, but are turned off in most cells of adult
tissuesthat replicate only a limited number of times, or will
never replicate again (such cells are called postmitotic).
However, these genes are activated in most human cancer
cells, where telomeraseis required for the multiple cell
divisions necessaryto form a tumor. This phenomenon has
stimulated a search for inhibitors of human telomeraseas
potential therapeutic agentsfor treatrng cancer.
While telomerase prevents telomere shortening in
most eukaryotes, some organisms use alternative strategies.
264 CHAPTER
6 | G E N E SG, E N O M t C SA, N D C H R O M O 5 O M E S
FocusAnimation:TelomereReplication
flltt
< FIGURE 6-48 StandardDNA replicationleadsto lossof DNA
at the 5' end of eachstrandof a linearDNAmolecule.
Replication of the rightendof a linearDNAisshown;thesame
process occurs at the leftend(shownby inverting thefigure)As the
replicationforkapproaches theendof the parentalDNAmolecule,
the leading strandcanbe synthesized allthewayto theendof the
parentaltemplate strandwithoutthe lossof deoxyribonucleotides.
However, sincesynthesis of the laggingstrandrequiresRNAprimers,
the rightendof the laggingdaughter DNAstrandwouldremainas
ribonucleotides whichcannotserveasthetemplate for a replicative
DNApolymerase Alternative mechanisms mustbe utilizedby cells
(andviruses with linearDNAgenomes) to prevent
successive
shortening of the laggingstrandwith eachroundof replication.
Morphology and Functional Elements
of Eukaryotic Chromosomes
r During metaphase,eukaryotic chromosomes become
sufficiently condensedthat they can be visualizedindividu-
ally in the light microscope.
r The chromosomal karyotype is characteristic of each
species.Closely related speciescan have dramatically dif-
ferent karyotypes, indicating that similar genetic informa-
tion can be organized on chromosomesin different ways.
r Banding analysis and chromosome painting are used to
identify the different human metaphasechromosomesand
to detect translocationsand deletions (seeFigure 6-42).
r Analysis of chromosomal rearrangementsand regions of
conservedsynteny betweenrelated speciesallows scientists
to make predictions about the evolution of chromosomes
(seeFigure 6-43c). The evolutionary relationshipsbetween
organisms indicated by these studies are consistent with
proposed evolutionary relationships based on the fossil
record and DNA sequenceanalysis.
The highly reproducible banding patterns of polytene
romosomes make it possibleto localize cloned Drosophila
DNA on a Drosophila chromosome by in situ hybridization
(seeFigure 6-44) andto visualizechromosomal deletionsand
rearrangementsas changesin the normal pattern of bands.
r Three types of DNA sequencesare required for a long
linear DNA moleculeto function as a chromosome:a reoli-
cation origin, called ARS in yeast; a centromere (CEN) se-
quence; and two telomere (TEL) sequencesat the ends of
the DNA (seeFigure5-46).
r Telomerase,a protein-RNA complex, has a special re-
verse transcriptase activity that completes replication of
telomeresduring DNA synthesis(seeFigure 6-49).In the
absenceof telomerase,the daughter DNA strand resulting
from lagging-strandsynthesiswould be shortened at each
cell division in mosr eukaryotes(seeFigure 6-48).
llll) pe6usAnimation:TelomereReplication
Catalyticsite for dNTP addition < FIGURE 649 Mechanismof actionof telomerase.Thesingle-
stranded 3' terminus of a telomere isextended bytelomerase,
Telomerase counteracting the inability of the DNAreplication mechanism to
protein terminus of linear DNA. Telomerase elongates
synthesize the extreme
thissingle-stranded endby a reiterative reverse-transcription
Telomerase- mechanism. Theactionof thetelomerase fromthe protozoan
associated Oxytricha, whichaddsa TaGarepeatunit,isdepicted; other
RNA template sequences. The telomerase contains
telomerases addslightlydifferent
an RNA template (red)that base-pairs to the 3' end of the lagging-
strandtemplate. Thetelomerase catalytic site(green) thenadds
deoxyribonucleotides (blue)usingtheRNAmolecule asa template; this
reverse transcription proceeds to position 35 of the RNA template
(steptr). Thestrands of the resulting DNA-RNA duplexarethen
thought to sliprelativeto each other, leading to displacement of a single-
G Q Q GT DNAstrandandto uncovering of part
Cccc44 stranded regionof the telomeric
(step
of the RNAtemplatesequence E) Thelagging-strand telomeric
sequence isagainextended to position35 by telomerase, andthe
DNA-RNA duplex undergoes translocation and hybridization asbefore
(stepsB and 4). Telomerases canaddmultiplerepeats by repetition
of stepsB and El. DNApolymerase a-primase canprimesynthesis of
newOkazaki fragments on thisextended template strand The net
Dissociationof 3' end of DNA resultprevents shortening of the laggingstrandat eachcycleof DNA
replication. [Adapted from D Shippen-Lentz andE H Blackburn, 1990, Nature
2471550 |

The human genome sequenceis a goldmine for new discover-

ies in molecular cell biology in identifying new proteins that

5',
nl
I
Il I Translocationand hybridization
I
Y
terization of cDNA copies of mRNAs isolated from the hun-
dreds of human cell types, will likely lead to the discovery of
GGGTTT new proteins, to a better understanding of biological processes,
u
T and may lead to applications in medicine and agriculture'
T We have seen that although most transposons do not
T
T function directly in cellular processes'they have helped to
N
N -3',
N
5'
of reverse-transcription
I Repetition
p I andtranslocation-hybridization
I steps
Y

history. Large numbers of these interspersed repeats are

P E R S P E C T I VF
EOS RT H E F U T U R E . 265
 polymorphic within populations, occurring at a particular 4. Mobile DNA elementsthat can move or transposeto a
site in some individuals and not others. Individuals sharing new site directly as DNA are called DNA transposons.De-
an insertion at a particular site descendedfrom a common scribethe mechanismby which a bacterial DNA transposon,
ancestorthat developedfrom an egg or sperm in which that called an insertion sequence,can transpose.
insertion occurred. The time elapsedfrom the initial inser-
5. Retrotransposons are a class of mobile elements that
tion can be estimated by the differencesin sequencesof the
transposevia a RNA intermediate. Contrast the mechanism
element that arose from the accumulation of random muta-
of transposition betweenretrotransposonsthat contain long
tions. Analysis of retrotransposon polymorphisms will un-
terminal repeats(LTRs) and those that lack LTRs.
doubtedly add immensely to our understanding both of hu-
6. Discussthe role that transposonsmay have played in the
man migrations sinceHomo sapiensfirst evolved, as well as
evolution of modern organisms.!7hat is exon shuffling? What
the history of contemporarypopulations.
role do transposonsplay in the processof exon shuffling?
7. Mitochondria contain their own DNA molecules.Describe
KeyTerms
the typesof genesencodedin the mitochondrial genome.How do
the mitochondrial genomesof plants, fungi, and animalsdiffer?
Barr body 253 matrix-associatedregions
(MARs)254 8. Mitochondria and chloroplasts are thought to have
bioinformatics 243
evolvedfrom symbiotic bacteriapresentin nucleatedcells.Re-
centromere 262 monocistronic245
view the experimental evidencethat supports this hypothesis.
cnromatld li / nucleosome248
9. Why is screeningof sequencedatabasesfor genesbasedon
chromatin 216 open reading frame the presenceof ORFs (open reading frames)more useful for
cytoplasmic inheritance 2 3 7 (oRF) 244 bacterialgenomesthan for eukaryoticgenomes?\Jfhatare par-
DNA transposons227 polytene chromosome 25-1 alogousand orthologousgenes?What are some of the expla-
eprgenetic 254 protein famlly 220 nations for the finding that humans are a much more complex
pseudogene 220 organism than the roundworm C. elegans,yet have only fewer
eachromatin 252
than one and a half as many genes(25,000 versus18,000)?
exon shuffling 235 retrotransposons 227
10. The DNA in a cell associateswith proteins to form chro-
fluorescencein situ repetitiousDNA 215
matin. What is a nucleosome?What role do histonesplay in
hybridization (FISH) 2'l8 scaffold-associatedregrons
nucleosomes?How are nucleosomesarranged in condensed
gene famrly 220 (SARs)254
30-nm fibers?
genomics216 simple-sequence(satellite)
11. Vhat post-translation modifications of histones are
heterochromatin 252 DNA 224
associatedwith transcribed genes (euchromatin) and with re-
histones 247 SINEs230 pressedgenes(heterochromatin)?What protein is associated
histone code250 SMC proteins255 with heterochromatin in most eukaryotes?How doesthis affect
synteny 259 heterochromatin formation over a region of a chromosome?
insulator 254
| ^ -- telomere 252 12. Describethe general organization of a eukaryotic chro,
KaryotypezJ / 'Sfhat
transcription unit 217 mosome. structural role do scaffold-associated
regions
LINEs 230 (SARs) or matrix artachment regions (MARs) play? rX/here
long terminal repeats transposableDNA
are genesprimarily locatedrelative to chromosomestructure?
(I:fP.s) 229 element216
13. FISH is a powerful diagnostic tool readily used by cyto,
geneticists.\fhat is FISH? Briefly describehow it is used to
Review the Concepts charaaerize chromosomal translocations associated with
certain geneticdisordersand specifictypes of cancers.
1. Cenescan be transcribedinto mRNA for protein-coding 14. Metaphasechromosomescan be identified by character-
genes or RNA for genes such as ribosomal or transfei istic banding paterns. What are G bands and R bands?
RNAs. Define a gene. Describehow a complex transcrip- \fhat is chromosome painting, and how is this technique
tion unit can be alternativelyprocessedto geneiatea variety tf useful?How can chromosome paint probes be used to
m R N A s a n d u l r i m a t e l yp r o t e i n s . lyze the evolution of mammalian chromosomes? "rru-
2. Sequencingof the human genome has revealedmuch 15. Certain organismscontain cells that possesspolytene
about the organization of genes.Describe the differences chromosomes.ril/hat are polytene chromosomes,where are
between solitary genes, gene families, pseudogenes,and they found and what function do they serve?
tandemlyrepeatedgenes.
16. Replication and segregationof eukaryotic chromosomes
3. Much of the human genomeconsistsof repetitiousDNA. require three functional elements:replication origins, a cen-
Describethe differenceberweenmicrosatelliteand minisatel_ tromere, and telomeres.Describe how these three elements
lite DNA. How is this repetitiousDNA useful for identifying function. What is the role of telomerasein maintainins chro-
individuals by the techniqueof DNA fingerprinting? mosome structure?

266 c H A p r E R6 | c E N E S ,c E N O M | C SA, N D C H R O M O S O M E s
 10 M 10
Analyzethe Data M

To determineif genetransferfrom an organellegenometo the t-

-
nucleus can be observed in the laboratory' a chloroplast
transformation vector was constructed that contained two
selectableantibiotic-resistancemarkerseachwith its own pro-
moter: the spectinomycin-resistance geneand the kanamycin-
resistancegene (seeS. Stegemannet al., 2003, Proc. Ndt'|.
Acad. Sci. USA 100:8828-8833). The spectinomycin-
resistancegene was controlled by a chloroplast promoter,
yielding a chloroplast-specificselectablemarker. Plants
Kan resistance Kan resistance
grown on spectinomycin are white unless they expressthe I P C Rp r o d u c t su s i n g I P C Rp r o d u c t su s i n g
spectinomycin-resistancegene in the chloroplast. The k an a m y c n
i -resistance spectinomycin-resistance
geneprimersl geneprimersl
kanamycin-resistancegene, inserted into the plasmid adla-
cent to the spectinomycin-resistance gene' was under the
control of a strong nuclearpromoter.Tiansgenic,spectinomycin- c. V4ren the original transgenic plants, which were se-
resistanttobacco plants were selectedfollowing transforma- lected on spectinomycin but not on kanamycin, were used to
tion with this plasmid by identifying green plants grown on pollinate wild-type plants,none of the offspring were kanamycin
medium with spectinomycin. These plants contain the two iesistant. !7hat can be deducedfrom theseobservations?
antibiotic-resistancegenes inserted into the chloroplast
genome by a recombination eventl however, kanamycin
resistanceis not expressedbecauseit is under the control of
References
a nuclear promoter. These spectinomycin-resistant plants
grown for multiple generationsand used in the follow- EukaryoticGeneStructure
were
ing studies. Blencowe,B. J.2006. Alternative splicing:new insightsfrom

a. Leavesfrom the spectinomycin-resistanttransgenrc

plants were placed in a plant regenerationmedium contain-
ing kanamycin. Some of the leaf cells were resistant to
kanamycin, and grew into kanamycin-resistantplants.
Pollen (paternal) from kanamycin-resistantplants was used
to pollinate wild-type (nontransgenic)plants. In tobacco, no
chloroplasts are inherited from pollen. The resulting seeds
were germinated on media with and without kanamycin.
Half of the resulting seedlingswere kanamycin resistant.
lfhen thesekanamycin-resistantplants were allowed to self-
pollinate, the offspring exhibited a 3:1 ratio of kanamycin-
resistantto sensitivephenotypes.\[hat can be deducedfrom
these data about the location of the kanamycin-resistance
gene?
b. To determineif transfer of the kanamycin-resistance
gene to the nucleus was mediated via DNA or an RNA
intermediate,DNA was extractedfrom 10 seedlingplants
germinated from seedsproduced by a wild-type plant polli-
nated with a kanamycin-resistantplant. The 10 seedling
plants, numbered1-10 in the correspondinggel lanesin the
figure below, consist of 5 kanamycin-resistant(+) and 5
kanamycin-sensitive(-) plants. Each DNA sample was
subjected to PCR analysis using primers to amplify the
kanamycin-resistancegene (gel at left) or the spectinomycin-
resistancegene (gel at right). The lane marked M shows
molecular weight markers. -What does the correspondence
between the presenceor absenceof PCR products gener-
ated in the same plant with both sets of primers suggest
about the mode of transfer of the kanamycin gene to the
nucleus?
Trends Genet. 19:640-648'
Suzuki,Y., and S. Sugano.2006. Transcriptomeanalysesof
human genesand applicationsfor proteomeanalyses'Curr' Protein
PeptideSci.7:147-163.
Chromosomal Organization of Genes and Nontoding DNA
InternationalHuman GenomeSequencingConsortium' 2004'
Finishingthe euchromaticsequenceof the human genome'Nature
431:931- 945.
Paterson,A.H. 2006. Leafing through the.genomesof our maior
crop plants: strategiesfor capturing unique informatron' Nature
Reu.Genet.7z174-184.
Pearson.C. E, K. Nichol Edamura, and J' D' Cleary' 2005'
Repeatinstability: mechanismsof dynamic mutations' Natwre Reu'
Genet.6:729-742.
Ranum, L. P.,and T. A. Cooper.2006' RNA-mediatedneuro-
musculardisorders'Annu. Reu.Neurosci.29:259-277'
Transposable (Mobile) DNA Elements
Feschotte,C., N. Jiang, and S. R'
lilessler'2002'Plant transpos-
able elements,where geneiicsmeets genomics' Natwre Reu'Genet'
3:329-341'.
Gray, Y. H. 2000' It takestwo transposonsto tango: transpos-
able-ele#ent-mediated chromosomalrearrangement s' TrendsG enet'
16:461-468.
elements:the full
Jones,R. N. 2005. McClintock's controlling
ttotl. Cytogrnet. GenomeRes.109:90-103'
Kazazian,H. H.' Jr. 1999. An estimatedfrequencyof endoge-
nous insertionalmutations in humans.Nature Genet' 22:130'
Kazazian,H.H., Jr. 2004. Mobile elements:drivers of genome
evolution. Science 303z\ 626-1632'

REFERENCES 267
 Kleckner,N., et al. 1996.TnI} and IS10 transpositionand
.
chromosomerearrangements:mechanismand requlation in vivo and
in vitro. Curr. TopicsMicrohiol. Immunol. 204?49-92.
Mahillon, J., and M. Chandler.1998. Insertion sequences.
Ml_
crobiol. Mol. Biol. Reu.62:725-774.
. Morgante,M.2006. Plantgenomeorganisationand diversity:
the year of the;unk! Cur. Opin. Biotechiol. 17:169-173.
E. M., and H.H.Kazazian, Jr. 2001. Biologyof mam_
..Ost_errag,
malian L1 rerrotransposons. Ann. Reu.Ginet.35:501-5jg.
Steiniger-\?hiteM, L Rayment,and W. S. Reznikoff. 2004.
Structu_relfunctioninsightsinto TnS transposition.Curr. Opin.
Struc.Biol. 14:50-57.
Organelle DNAs
Bendich,A. J.2004. Circularchloroplastchromosomes:
g r a n di l l u s i o nP. l a n tC e l l l 6 t l 6 6 l - 1 6 6 6 .
Chan,D. C.2006. Mitochondria:dynamicorganelles
aging, and development.Cell 125:1241-1252.
. Clayton, D. A. 2000. Transcriptionand replication of mitochon_
drial DNA. Hum. Reprod.2(Suppi.):11-17.
Daniell,H., M. S. Khan, and L. Allison.2002. Milestonesin
chloroplastgenericengin_eering: an environmentallyfriendly era in
brotechnology. Trcnd s Plant Sci. 7 zB4-91.
W., G. Burger,and B. F. Lang.2001. The origin and early
,Glry,Y.
evolutionof mitochondria.GenomeBft.,/. g.1-101g.5.'
2(Reviews):101
Shutt, T. E., and M. V. Gray. 2006. Baceriophageorigins of
m^itochondrialreplication and transcriprionproteins.-Tren1s G enet.
t t.gn-g ( Schalch,T., et al. 2005. X-ray strucure of a tetranucleosome
Sugiura,M., T. Hirose,and M. Sugita.199g. Evolutionand mech_
anismof translarionin chloroplasts.Ain. Reu.Genet.32:437459.
Genomics: Genome-wide Analysis of Gene Structure
and Expression
BLAST Information can be found ar: hftp://www.ncbi.nlm.nih.gov/
Education/BLASTinfo/information3.htm
Binnewies,T. T., et aL.2006.Ten yearsof bacterialgenomese_
quencing:c_omp_arative-genomics-based
discoveries.Fuict. Integr.
Genomics6:165-185.
Celniker,S. E., and G. M. Rubin. 2003. The Drosoohila
melanogastergenome.Annu. Reu.GenomicsHum. Geiet. 4:g9_177.
ChimpanzeeSequencingand AnalysisConsorrium.2005. Initial
sequenceof the chimpanzeegenomeand comparisonwith the
human genome.Nature 437:69-87.
Gebhardt,C., R. Schmidt,and K. Schneider. 2005. plant
genomeanalysis:the stateof the art. Int'\. Reu.Cttot.
247:223-284.
Gele
Q119logyConsortium. 2006. The Gene Ontology (GO)
projectin 2006. NucleicAcids Res.34:D322-D326.
Guigo,R., et a|.2006.EGASp:the human ENCODE genome
annotationassessmenr project. G enome B iot. 7(Suppl 1):S2:.
1_S2.3 1.
Harris, T. \7., and L. D. Stein.2006. WormBase:methodsfor data
mining and comparativegenomics.Methods Mol. Biol.351:31_50.
InternationalHapMap Consortium. 2003. The international
HapMap project. N ature 426:789-79 6.
InternationalHuman GenomeSequencingConsortium.2004.
Finishingthe euchromaticsequenceof the huiran genome.Nature
431:931.-945.
. Lander,E. S.,et al. 2001. Initial sequencing and analysisof rhe
human genome.Nature 409:860-927.
Ness,S. A. 2006. Basicmicroarrayanalysis:strategies for suc_
cessfulexperiments.Methods Mol. Biol. 316:13_33.
__ Shianna,K. V., and H. F. Willard. 2006.|n searchof normality.
Nature 444:428-429.
_ Sioblom,T.2006. The consensus codingsequences of human
breastand colorectalcancers.Science3l4,i6g-274.
. Wat€rston,R. H., et al.2002.Initial sequencingano compara_
tive analysisof the mousegenome.Nature AZO:SZi_SAZ.
\7indsor,A. J., and T. Mitchell-Olds.2006.Comparatrvegenomrcs
as a tool for genediscovery.Curr. Opin. Biotechnol.iZrrcttZZ.
268 CHAPTER
5 | G E N E SG
, E N O M t C SA, N D C H R O M O S o M E S
Structural Organization of Eukaryotic Chromosomes
Carroll, C. I7., and A. F. Straight.2006. Centromereformation:
from epigeneticsto self-assemb\y. TrendsCell Biol. 16:70-78.
Dehghani,H., G. Dellaire,and D. P.Bazen-Jones. 2005. Organiza-
tion of chromatinin the inrerphasemammalianiell. Miuon 36:t5-10g.
Dillon, N. 2004. Heterochromatinstructureand function. Blol.
Cell.2004. 96:631-637.
Henikoff, S., and Y. Dalal. 2005. Centromericchromatin: what
makesit unique?Curr. Opin. Genet.Deu. 15:177-1,84.
Horn, P.J., and C. L. Peterson.2002. Molecular biology. Chro-
matin higher order folding-wrapping up rranscription. Sience
29721.824-'1.827.
Luger,K. 2003. Structureand dynamic behaviorof nucleo-
tne somes.Curr. Opin. Genet.Deu. 13:127-135.
Luger,K., and T. J. Richmond. 1998. The histonetails of the
in disease, nucleosome.Curr. Opin. Genet.Deu. 8:1.40-146.
. Margueron, R., P. Trojer, and D. Reinberg.2005. The key to
development:interpretingthe histone code?Curr. Opin. Genet.Deu.
15:163-176.
McBryant,S.J., V. H. Adams,and J. C. Hansen.2006. Chro-
matin architecturalproteins. ChromosomeRes.l4:39-51..
Nasmyth,K., and C. H. Haering.2005. The strucrureand func-
tion of SMC and kleisincomplexes.Annu. Reu.Biochem.T4:595-64g.
Sarma,K., and D. Reinberg.2005. Histone variants meertheir
match.Nature Reu.Mol. Cell Biol. 6:139-149.
and its implicarionsfor the chromatin fibre.Nature 436:13g-14I.
'Woodcock,
C. L. 2006. Chromatin architecture.Curr. Onin.
Struc. Biol. 16:2 | 3-220.
'Woodcock,
C. L., A. I. Skoultchi,and y. Fan. 2006. Role of
linker histonein chromatin structureand function: H1 stoichiome-
try and nucleosomerepeatlength. ChromosomeRes.14:17-25.
Morphology and Functional Elements of Eukaryotic
Chromosomes
Armanios, M., and C. $7. Greider.2005. Telomeraseano cancer
stem celis.Cold Spring Harb. Symp. euant. Biol.70:205-20g.
Belmont,A. S. 2002. Mitotic chromosomescaffold structure:
New approachesto an old conrroversy.Proc. Nat'|. Acad. Sci.USA
9 9 : 1 5 8 5 5 -5
18 5 7 .
Belmont, A. S., et al. 1999. Large-scalechromatin structureand
function. Curr. Opin. Cell Biol. 11307-31I.
Blackburn, E. H. 2005. Telomeresand telomerase:their mecha-
nisms of acrion and the effectsof altering their functions. FEBSLett.
5792859-862.
Carroll, C. W., and A. F. Straight.2006. Cenrromereformarion:
from epigeneticsto self-assembly. TrendsCetl. Biol. 16:70-7g.
C., and C. \X/alter.2005. Eukaryotic origins of DNA
.Cvetic, J.
replicafion:could you pleasebe more specific?Semin.Cell Deu.
Biol. 16:343-353.
de Lange,T.2006. Lasker laurelsfor telomerase.Cel/
126:1.017-1.020.
Froenicke,L. 2005. Origins of primate chromosomesas delin_
eatedby Zoo-FISH and alignmentsof human and mousedraft
genomesequences. CytogenetGenome Res.l}8z122-I3g.
Gassmann,R., et al. 2004. Mitotic chromosomeformation and
the condensinparadox. Exp. Cell Res.296:35-42.
MacAlpine, D. M, and S. P. Bell. 2005. A genomicview of eu_
karyotic DNA replication. ChromosomeRes.7l:SOg-SZe.
Malik, H. S. 2006. A hitchhiker'sguide ro survival finally makes
CENs. /. Cell Biol. 1742747-749.
Marko, J. F., and M. G. Poirier.2003. Micromechanicsof chro_
matin and chromosomes.Biochem. Cell Biol. St:209-220.
Robinson,N.. P.,and S._D.Bell. 2005. Origins of DNA replica-
tion in the three domains of life. FEBS/ . 272t3757-3766.
\Tarburron, P. F,.2004. Chromosomaldynamicsof human neo_
centromereformation. ChromosomeRes.12617 -626.
 CHAPTER

TRANSCRIPTIONAL
OF GENE
CONTROL
EXPRESSION

polytene
Drosophila chromosomes stained against
withantibodies
a remodeling
chromatin ATPase calledKismet(blue),
RNA
ll wrthlowCTDphosphorylation
polymerase (red),
andRNA
ll withhighCTDphosphorylation
polymerase (green) of
[Courtesy
JohnTamkun; et al, 2005,Development
seeS Srinivasan 132:1623
I

coded into a particular protein, are reviewed in Chapter 4'

I n previous chapters we have seenthat the properties and
I functions of eachcell type are determined by the proteins it
I contains. In this and the next chapter,we consider how the
kinds and amounts of the various proteins produced by a
particular cell type in a multicellular organism are regulated.
This regulation of gene expression is the fundamental
processthat controls the developmentof a multicellular or- newly formed RNA transcripts, which function as mRNA
ganism such as ourselvesfrom a singlefertilized egg cell into
'W'hen
the thousands of cell types from which we are made.
gene expressiongoes awr5 cellular properties are altered, a OUTLINE
processthat all too often leadsto the developmentof cancer.
As discussedfurther in Chapter 25, genesencoding proteins 7.1 Control of Gene Expressionin Bacteria 271
that restrain cell growth are abnormally repressedin cancer
cells, whereas genesencoding proteins that promote cell 7.2 Overviewof EukaryoticGene Control and
growth and replication are inappropriately activated in can- RNA Polymerases 276
cer cells. Abnormalities in gene expressionalso result in de-
7.3 RegulatorySequences Genes 282
in Protein-Coding
velopmental defectssuch as cleft pallet' tetrology of Fallot (a
common, serious developmentaldefect of the heart that can 7.4 of Transcription
Activatorsand Repressors 286
be treated surgically), and many others. Regulation of gene
expressionalso plays a vital role in bacteria and other single- 7.5 TranscriptionInitiation by RNA Polymerasell 295
celled microorganisms, where it allows cells to adjust their
enzymaticmachinery and structural componentsin response 7.6 MolecularMechanismsof Transcription
and Activation
Reoression 299
to their changing nutritional and physical environment.
Consequently,to understand how microorganisms respond 7.7 Activity
Regulationof Transcription-Factor 311
to their environment and how multicellular organisms nor-
mally develop, as well as how pathological abnormalities of 7.8 RegulatedElongationand Terminationof
gene expressionoccur, it is essentialto understand the mo- Transcription 314
lecular interactions that control protein production.
7.9 Other EukaryoticTranscriptionSystems 316
The basic stepsin geneexpression,i.e.' the entire process
whereby the information encodedin a particular gene is de-

269
 without further modification. In eukaryotes.however.the ini-
tial RNA transcript is subyected,o pio..rring that yields a
functional mRNA (see Figure 4-15). The mRNA then is
transported from its site of synthesisin the nucleusto the cy-
toplasm, where it is translatedinto protein with the aid of ri-
bosomes,tRNAs, and translationfactors(seeFigure4-25).
TheoreticallS regulation at any one of the various steps
in gene expressionoutlined above could lead to differential
production of proteins in different cell types or developmen-
tal stagesor in responseto external conditions. Although
examples of regulation at each step in gene expressionhave
been found, conrrol of transcription initiation-the first
step-is the most important mechanism for determining
whether most genesare expressedand how much of the en-
coded mRNAs and, consequently,proteins are produced.
The molecular mechanismsthat regulate transcription initi-
ation are critical to numerous biological phenomena,includ-
ing the development of a multicellular organism from a sin-
gle fertilized egg cell as mentioned above, the immune
responsesthat protect us from pathogenic microorganisms,
and neurological processessuch as learning and memory.
Vhen theseregulatory mechanismscontrolliig transcription
function improperly, pathological processesmay occur. For
example, reduced activity of the pax6 gene causesaniridia,
failure to develop an iris. Pax6 is a transcription factor that
normally regulatestranscription of genesinvolved in eye de-
velopment(seeFigure 1-26e).In other organisms,mutarions
of transcription factors causean extra pair of wings to de-
v e l o pi n D r o s o p h i l a( s e eF i g u r e2 Z - 3 1 ) , t h " n g . t h e i t r u c t u r e
of flowers in plants (seeFigure 22-36), and are responsible
for multiple other developmentalabnormalities.
Transcriptionis a complex processinvolving many lay_
ers of regulation. In this chapter, we focus on the molecular
eventsthat determinewhen transcription of a gene occurs.
First, we consider the relatively basic mechanismsof gene
expressionin bacteria,where repressorand activator pro_
> FIGURE7-1 Overview of eukaryotic
transcription control. Activatorproteinsbind to
specificDNA controlelementsin chromatinand
interactwith multiproteinco-activator machines,
suchas mediator,to decondense chromatinand
assembleRNApolymerase and general
transcription factorson promotersInactivegenes
are assembled into regionsof condensed
chromatlnthat lnhibitRNApolymerases and their
assocrated generaltranscription factors(GTFs)
from interactingwith promotersAlternatively,
repressor proteinsbind to other controlelements
to inhibitrnitiationby RNAporymerase ano
interactwith multiproteinco-repressor complexes
to condensechromatin
270 c H A P T E R7 | TRANSCRtpTtoNC
ALONTROL
O F G E N EE X p R E S S T O N
teins recognize and bind to specific regions of DNA to con-
trol the transcription of a nearby gene.The remainder of the
chapter focuseson eukaryotic transcription regulation and
how the basic tenets of bacterial regulation are applied in
more complex ways in higher organisms. Figure 7-1. pro-
vides an overview of eukaryotic gene regulation and the
processesoutlined in this chapter. \Ve discusshow specific
DNA sequencesfunction as transcription-control regions
by serving as the binding sites for transcription factors (re-
pressorsand activators)and how the RNA polvmerasesre-
sponsiblefor transcription bind ,o pro-ot.. sequences ini-
tiate the synthesisof an RNA molecule complementary to
template DNA. Next, we consider how activators and re-
pressors influence transcription through interactions with
large, multiprotein complexes. Some of these multiprotein
complexes modify chromatin condensation, altering access
of chromosomal DNA to transcription factors and RNA
polymerases.Other complexes influence the rate at which
RNA polymerase binds to DNA at the site of transcription
initiation, as well as the frequency of initiation. Then we
discusshow transcription of specific genescan be specified
by particular combinationsof the -2,000 transcriptionfac-
tors encoded in the human genome, giving rise to cell-type-
specific gene expression.rWealso further consider the vari-
ous ways in which the activities of transcription factors
themselvesare controlled to ensuregenesare expressedonly
at the right time and in the right place. Finally, we'll exam-
ine control of transcription elongation and termination and
the transcription of nonprotein-coding RNAs. RNA pro-
cessingand various post-transcriptional mechanismsfor
controlling eukaryotic gene expression are covered in the
GENE
"OFF"
,f
*"Or"arora o",,u,.o,.,
f
Decondensed
chromatin
GENE
"ON" Activators
RNA
General polymerase
transcription
factors

Jn Controlof GeneExpression
in Bacteria
Since the structure and function of a cell are determined by
the proteins it contains, the control of gene expressionis a
fundamental aspectof molecular cell biology. Most commonly,
the "decision" to initiate transcription of the gene encoding
a particular protein is the major mechanism for controlling
production of the encoded protein in a cell. By controlling
transcription initiation, a cell can regulate which proteins it
'When
produces and how rapidly. transcription of a gene is
repressed, the corresponding mRNA and encodedprotein or
proteins are synthesizedat low rates. Conversely,when tran-
scription of a geneis actiuated,both the mRNA and encoded
protein or proteins are produced at much higher rates.
In most bacteria and other single-celledorganisms,gene
expressionis highly regulatedin order to adjust the cell'sen-
zymatic machinery and structural componentsto changesin
the nutritional and physical environment. Thus, at any given
time, a bacterial cell normally synthesizesonly those pro-
teins of its entire proteome required for survival under the
particular conditions. Here we describethe basic featuresof
transcription control in bacteria, using the lac operon and
the glutamine synthetasegene in E. coli as our primary ex-
amples. Many of the same processes,as well as others, are
involved in eukaryotic transcription control, which is the
subject of the remainder of this chapter.
TranscriptionInitiation by BacterialRNA
PolymeraseRequiresAssociationwith a
SigmaFactor
ln E. coli, about half the genesare clustered into operons,
each of which encodesenzymesinvolved in a particular
metabolic pathway or proteins that interact to form one
multisubunit protein. For instance,the trp operon discussed
in Chapter 4 encodesfive enzymesneededin the biosynthe-
sis of tryptophan (seeFigure 4-13). Similarly the lac operon
encodesthree enzymesrequired for the metabolism of lac-
tose, a sugar presentin milk. Sincea bacterial operon is tran-
scribed from one start site into a singlemRNA, all the genes
within an operon are coordinately regulated;that is, they are
all activated or repressedto the sameextent.
Transcription of operons, as well as of isolated genes,is
controlled by an interplay between RNA polymerase and
specific repressorand activator proteins. In order to initiate
transcription, however,E. coli RNA polymerasemust be as-
sociated with one of a small number of o (sigma) factors.
The most common one in eubacterialcells is oto. oto bi.tds
to RNA polymeraseand to promoter DNA sequences,bring-
ing the RNA polymerase enzyme to a promot r. o70 recog-
nizes and binds to both a six-basepair sequencecenteredat
-1,0 and a seven-base pair sequencecenteredat -35 from
the +1 transcriptionstart. Consequently,the -10 plus the
-35 sequenceconstitute a promoter for E. coli RNA poly-
meraseassociatedwith o70 (seeFigure4-10b). Although the
promoter sequencescontactedby o'o are located at -35 and
- 10, E. coll RNA polymerasebinds to the promoter region
DNA from:-50 to:*20 through interactionswith DNA
that do not dependon the sequencr. o'0 also assiststhe RNA
polymerasein separatingthe DNA strands at the transcrip-
tion start site and inserting the coding strand into the active
site of the polymeraseso that transcription starts at +1 (see
Figure4-11, step2).The optimal o7O-RNApolymerasepro-
moter sequence,determined as the consensussequenceof
multiple strongpromoters,is:
-35 region -10 region
a16trQ,\1- 15 -17 bp-1a-t66t
The sizeof the font indicatesthe importance of that baseat this
position. The sequenceshows the strand of DNA that has the
same5'-+3' orientation as the transcribedRNA (i.e.,the non-
template strand). However, the o7O-RNA polymerase initially
binds to double-strandedDNA. After the polymerasetran-
scribesa few tensof basepairs,o70is released.Thus o7oactsas
an initiation factor required for transcription initiation but not
for RNA-strand elongation once initiation has taken place.
Initiation of lacOperonTranscriptionCan Be
Repressedand Activated
When E. coli is in an environment that lacks lactose' synthe-
sis of lac mRNA is repressedso that cellular energy is not
wasted synthesizingenzymesthe cellscannot use.In an envi-
ronment containing both lactose and glucose, E- coli cells
preferentially metabolize glucose, the central molecule of
carbohydrate metabolism. Lactose is metabolized at a high
rate only when lactose is present and glucose is largely de-
pleted from the medium. This metabolic adjustment is
achievedby repressingtranscription of the lac operon until
lactoseis presentand allowing synthesisof only low levelsof
/ac mRNA until the cytosolic concentration of glucosefalls
to low levels.Transcription ofthe lac operon under different
conditions is controlled by lac tepressor and catabolite acti-
vator protein (CAP) (also called CRP for catabolite /eceptor
protein), each of which binds to a specificDNA sequencein
the lac transcription-control region (Figure 7-2, top). -^
For transcription of the lacipeton to begin, the o70sub-
unit of the RNA polymerasemust bind to the lac promoter
'When no Iactoseis
at the -35 and - 10 promoter sequences.
present, the lac repressor binds to a sequence called the lac
operator, which overlaps the transcription start site' There-
fore, lac repressorbound to the operator site blocks binding
and hencetranscription initiation by RNA polymerase(Fig-
ure 7-2a\. -Whenlactoseis present, it binds to specific bind-
ing sitesin each subunit of the tetrametic lac repressor,caus-
ing a conformational change in the protein that makes it
diisociate from the lac operatot.As a result, the polymerase
. 27'I
C O N T R O LO F G E N EE X P R E S S I OIN
N BACTERIA
 +1 (transcriptionstart site) In fact, the lac operon is more complex than depicted in
PromoterV
-
-_l
F F -- the sirnplified model of Figure 7-2. The tetrameric lac re-
CAPsite
pressoractually binds to two sitessimultaneously,one at the
Ooerator
E. coli lac transcription-control
primary operator (lacO1) that overlapsrhe region of DNA
regions
bound by RNA polymerase at the promoter and at one of
two secondaryoperatorscenreredat +412 (lacO2) and -82
(lacO3) (Figure7-3). The /ac repressortetrameris a dimer of
(4, dimers. Each dimer binds to one operaror. Simultaneous
- lactose binding of the tetram eric lac repressorto the prima ry lac op-
+ glucose erator O1 and one of the two secondaryoperators is possi-
(lowcAMP) N o m R N At r a n s c r i p t i o n ble becauseDNA is quite flexible, as we saw in the wrapping
of DNA around the surfaceof a histone octomer in the nu-
cleosomesof eukaryotes(Figure6-29l.These secondaryop-
(b) erators function to increasethe local concentration of lac re-
pressor in the micro-vicinity of the primary operator where
+ lactose repressorbinding blocks RNA polymerasebinding. Sincethe
+ glucose equilibrium of binding reactions dependson the concentra-
( l o wc A M P ) Low transcription
tions of the binding partners, the resulting increasedlocal
concentrationof ldc repressorin the vicinity of Ol increases
repressorbinding to OL. There are approximatelyI0 lac re-
(c) cAMP pressor tetramers per E. coli cell. Becauseof binding to 02
and 03, there is nearly always a /ac repressortetramer much
+ lactose closerto 01 than would otherwisebe the caseif the 10 re-
- grucose pressorswere diffusing randomly through the cell. If both
( h i s hc A M P )
02 and 03 are mutated so that the lac repressorno longer
binds to them with high affinity, repressionat the lac pro-
moter is reduced by a factor of 70. Mutation of only 02 or
A FIGURE 7-2 Regulationof transcription from the /acoperon only 03 reducesrepressiontwofold, indicating that either
ol E. coli.(Top)The transcription-control region, composed of -100 one of thesesecondaryoperators provides most of the stim-
basepairs,rncludes threeprotein-binding regions: theCApsite,which ulation of repression.
bindscatabolite activator protein;
the/acpromoter, whichbindsthe Although the promoters for different E. coli genesexhibit
o70-RNA polymerase complex; andthe/acoperator, whichbinds/ac considerablehomology, their exact sequencesdiffer. The pro-
repressor The/acZgene, thefirstof threegenesin theoperon, is moter sequencedeterminesthe intrinsic rate at which an
shownto the right (a)In theabsence of lactose, verylittle/acmRNA RNA polymerase-o complex initiates transcription of a gene
isproduced because the/acrepressor bindsto theoperator, inhibiting in the absenceof a repressoror activator protein. Promoters
transcription initiation by,I7o-RNA polymerase. (b)Inthe presence of
glucose that support a high rate of transcription initiation have -10
andlactose, /acrepressor bindslactose anddissociates from
theoperator, and -35 sequencessimilar to the ideal promoter shown pre-
allowing o7o-RNA polymerase to initiaterranscnptton at
a low rate (c)Maxlmal viously and are calledstrong promoters. Those that support
transcriptionof the/acoperon occurs in the
presence of lactose andabsence of glucoseInthissrtuation, a low rate of transcription initiation differ from this ideal se-
cAMp
increasesin response to the lowglucose concentration andformsthe quenceand are called weak promoters. The lac operon, for
CAP-cAMP complex, whichbindsto theCApsite,wherert interacts instance,has a weak promoter. Its sequencediffers from the
with RNApolymerase to stimulatethe rateof transcription initiation consensusstrong promoter at severalpositions. This low in-
trinsic rate of initiation is further reducedby the lac repressor
and substantiallyincreasedby the cAMP-CAP acuvaror.
the -35 and - 10 sequences in the lac promorer differ from
the ideal o"'-binding sequences shown previously.
Once glucoseis depletedfrom the media and the intra- 02l+412]r os (-821
cellular glucoseconcentrationfalls, E. coli cellsrespond by
+
synthesizingcyclic AMP, or cAMp. As the concentration of
cAMP increases,it binds to a sire in each subunit of the
dimeric CAP protein, causinga conformarionalchangethat
allows the protein to bind to the CAp site in the lac tran-
scription-control region. The bound CAp-cAMp complex
t
01(+111
interacts with the polymerase bound to the promoter,
greatly stimulating the rate of transcriptioninitiation. This FIGURE 7-3 Lacrepressor-operator interactions.The
tetrameric
/acrepressorbindsto the primary (O/) and
/acoperator
activationleadsto synthesisof hieh levelsof /ac mRNA and
oneof two secondaryoperators (O2or C.3) simultaneously.
Thetwo
subsequentlyof the enzymesencoded by the lac operon
structures
arein equilibrrum
IAdapted fromB Muller-Hill,
1998,Curr.
Op
(Frgure7-2c1.
Microbiol
1:1451

272 . c H A p r E R7 T R A N S c R t p I o N A Lc o N T R o L o F G E N EE X e R E S s t o N
|
 nf M a n y
S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i o o cellular needsby binding specificsmall moleculeligands (e.g.,
l e n e sv i a D N A - B I n d i n g
B a c t e r i aG Repressors cAMP) or by post-translationalmodifications, such as phos-
phorylation, that alter the conformation of the activator.
and Activators
Transcription of most E. coli genesis regulated by processes
TranscriptionInitiation from SomePromoters
similar to those described for the lac operon, although the
detailed interactions differ at each promoter. The general
RequiresAlternative SigmaFactors
mechanisminvolves a specificrepressorthat binds to the op- Most E. coli promoters interact with o7O-RNA polymerase,
erator region of a gene or operon, thereby blocking tran- the major initiating form of the bacterial enzyme. Tran-
scription initiation. A small molecule ligand (or ligands) scription of certain groups of genes,however, is initiated by
binds to the repressor,controlling its DNA-binding activity E. coli RNA polymerasescontaining one of severalalter-
and consequentlythe rate of transcription as appropriate for native sigmafactorsthat recognizedifferent consensuspro-
the needsof the cell. As for the lac operon,many eubacterial moter sequences than o70does(Table7-1). Thesealternative
transcription-control regionscontain one or more secondary o-factors are required for the transcription of sets of genes
operators that contribute to the level of represslon. with related functions such as those involved in the re-
Specificactivator proteins, such as CAP in the lac operon, sponse to heat shock or nutrient deprivation, motility, or
also control transcription of a subsetof bacterial genesthat sporulation in gram-positive eubacteria.In E. coli there are
have binding sitesfor the activator.Like CAP,other activators six alternativeo-factors in addition to the major "house-
bind to DNA together with RNA polymerase, stimulating keeping" o-factor, o70. The genome of the gram-positive,
transcription from a specific promoter. The DNA-binding sporulating bacterium Streptomycescoelicolor encodes 63
activity of an activator can be modulated in response to o-factors,the current record, basedon sequenceanalysisof

IONSENSUS
PROMOTER

- 35REGION - I OREGION
FACTOR
SIGMA RECt)GNIZED
PROMOTERS
70
Housekeeping genes,most genesin TTGACA TATAAT
exponentially replicating cells

('s Stationary-phasegenesand general TTGACA TAIAAT

stressresponse

o't2 Induced by unfolded proteins in the TCTCNCCCTTGAA CCCCATNTA

cytoplasm; genesencoding
chaperonesthat refold unfolded
proteins and protease systems
leading to the degradation of
unfolded proteins in the cytoplasm

C'-
F
Activated by unfolded proteins in the GAACTT TCTGA
periplasmic spaceand cell
membrane;genesencodingproteins
that restore integrity to the cellular
envelope

o
F
Genes involved in flagellum assembly CTAAA CCGATAT

FecI Genesrequired for iron uptake TTGGAAA GTAATG

Genes for nitrogen metabolism and

other functions
eds', Cold Spring
Souncr-s: C. A. Gross, M Lonetto, and R. Losick, 1992, rn Transcriptional Regulation, S L. McKnight and K' R, Yamamoto,
1 . 9 8 9 , P r o c . N a t ' l . A c a d . S c l . U S A 8 6 : 8 3 0 ; K . T a n a k a e t a l . , 1 9 93,Proc'Nat'l'
HarborLaboratoryPress;D N.ArnostiandM. I.Chamberlin,
169:483'
A c a d . S c i . ,{ / S A 9 0 : 3 5 1 1 ; C . D a r t i g a l o n g u e e t a l . , 2 0 0 1 , J . B i o l . C h e m . 2 7 6 2 2 0 8 6 6 ; A . A n g e r e r a n d V B r a u n , 1 9 9 8 , A r c h ' M i c r o b i o l '

N BACTERIA
C O N T R O LO F G E N EE X P R E S S I OIN . 273
 tal
P a i ro f p h o s p h o r y l a t e d
NtrC dimers
\ o54- RNA
polymerase
( - 1 4 0a n d
- 10 8 )
EXPERIMENTAL FTGURE 7-4 DNAloopingpermitsinteraction
of bound NtrCand oso-RNApolymerase.(a)Drawing (/eft)and
electron (right)oI DNArestrrction
mrcrograph fragmentwith
phosphorylated
NtrCdimersbindingto theenhancerregionnearone
endandoso-RNA polymerase boundto theqtnApromoternearthe
nearly 100 eubacterialgenomes.Most are structurally and
functionally relatedto o70.But one classis unrelated,repre-
sentedin E. coli by oto. Transcription initiation by RNA
polymerasescontaining o70-likefactors is regulatedby re-
pressorsand activatorsthat bind to DNA near the region
where the polymerasebinds, similar to initiation by ot0-
R N A p o l y m e r a s ei t s e l f .
Transcriptionby oto-RNApolymerasels
Controlledby ActivatorsThat Bind Farfrom
the Promoter
The sequenceof one E. coli sigma factor, o54, is distinctly
different from that of all the o7o-like facrors. Transcription
of genesby RNA polymerasescontaining o5a is regulated
solely by activators whose binding sitesin DNA, referred to
as enhancers,generallyare located 80-160 basepairs up-
stream from the start site. Even when enhancers -ou.d
"r.
more than a kilobase away from a start site, o-5a-activators
can activate transcription.
The best-characterizedosa-activator-the NtrC protein
(nitrogen regulatory protein C)-stimulates transcription of
the glnA gene.glnA encodesthe enzymeglutamine synthetase,
274 . c H A p r E R7 | T R A N s c R t p l o N AcLo N r R o L o F G E N EE X p R E s s t o N
N t r Cd i m e r s
promoter
NtrC dimers osa- RNA polymerase
otherend (b)Drawing(/eff)andelectronmicrograph (flght)of the
samefragmentpreparation showingNtrCdimersandosa-RNA
polymerasebindingto eachotherwith the intervening
DNAforming
a loopbetweenthem [Micrographs fromW Suetal, 1990,procNat':.
Acad.SciU5A87:5505;courtesv
of S Kustu
I
which synthesizesthe amino acid glutamine from glutamic
acid and ammonia. The osa-RNA polymerase binds to the
glnA promoter but does not melt the DNA strands and initi-
ate transcription until it is activated by NtrC, a dimeric
protein. NtrC, in rurn, is regulatedby a protein kinase called
NtrB. In responseto low levels of glutamine, NtrB phos-
phorylaresdimeric NtrC, which then binds to an enhancer
upstream of the glnA promoter. Enhancer-bound phospho-
rylated NtrC then stimulatesthe osa-polymerasebound at
the promoter to separate the DNA strands and initiate
transcrlptron.
Electron microscopy studies have shown that phospho-
rylated NtrC bound ar enhancersand osa-polymerasebound
at the promoter directly interact, forming a loop in the DNA
between the binding sites (Figure 7-4). As discussedlater
in this chapter, this activation mechanism resemblesthe
predominant mechanism of transcriptional activation in
eukaryotes.
NtrC has MPase activirl, and ATP hydrolysis is required
for activation of bound osa-polymeraseby phosphorylated
NtrC. Evidence for this is that mutants with an NtrC
defectivein ATP hydrolysis are invariably defectivein stimu-
lating the osa-polymeraseto melt the DNA strands at the
transcription start site. It is postulated that AIP hydrolysis
supplies the energy required for melting the DNA strands.
In contrast, the o70-polymerasedoes not require ATP
hydrolysis to separatethe strands at a start site.
Are Controlledby
Many BacterialResponses
Two-ComponentRegulatorySystems
As we have just seen,control of the E. coli glnA gene de-
pends on two proteins, NtrC and NtrB. Such two-
component regulatory systemscontrol many responsesof
bacteria to changesin their environment. Another example
involves the E. coli proteins PhoR and PhoB, which regulate
transcription in responseto the concentration of free phos-
phate. PhoR is a transmembraneprotein, located in the inner
(plasma)membrane, whose periplasmic domain binds phos-
phate with moderate affinity and whose cytosolic domain
has protein kinase activity; PhoB is a cytosolic protein.
Large protein pores in the E. coli outer membrane allow
ions to diffuse freely between the external environment and
the periplasmic space. Consequently,when the phosphate
concentration in the environment falls, it also falls in the
periplasmic space,causing phosphateto dissociatefrom the
PhoR periplasmicdomain, as depictedin Figure 7-5. This
causesa conformational changein the PhoR cytoplasmic do-
main that activatesits protein kinase activity. The activated
PhoR initially transfers a 1-phosphate from ATP to a histi-
dine (H) side chain in the PhoR kinase domain itself. The
samephosphateis then transferredto a specificaspartic acid
(D) side chain in PhoB, converting PhoB from an inactive to
an active transcriptional activator. Phosphorylated' active
PhoB then inducestranscription from severalgenesthat help
the cell cope with low phosphateconditions.
Many other bacterial responsesare regulated by two
proteins with homology to PhoR and PhoB. In each of these
regulatory systems,one protein, called a sensor,contains a
transmitter domain homologous to the PhoR protein kinase
domain. The transmitter domain of the sensorprotein is reg-
ulatedby a secondunique protein domain (e.g.,the periplas-
mic domain of PhoR) that sensesenvironmental changes.
The second protein, called a responseregulator, contains a
receiver domain homologous to the region of PhoB that is
phosphorylated by activated PhoR. The receiver domain of
the responseregulator is associatedwith a second domain
that determinesthe protein's function. The activity of this
second functional domain is regulated by phosphorylation
of the receiverdomain. Although all transmitter domains are
homologous (as are receiverdomains),the transmitter do-
main of a specificsensorprotein will phosphorylate only the
receiver domains of specific responseregulators, allowing
specific responsesto different environmental changes.Note
that NtrB and NtrC, discussedabove, function as sensor
and response regulator proteins, respectively,in the two-
component regulatory systemthat controls transcription of
glnA. Simrlar two-component histidyl-aspartyl phospho-
relay regulatory systemsare also found in plants.

< FIGURE 7-5 The PhoR/PhoB two-

componentregulatorysystemin E. coli'
In responseto low phosphate concentrations In
the environment andperiplasmic space, a
Cytoplasm 'i phosphate iondissociatesf romthe periplasmic
'll
'il
'*. domainof the inactive sensor proteinPhoR.
Thiscauses a conformational change that
:rl,
a proteinkinase
activates transmitter domainin
ii
i;
thecytosolic regionof PhoRTheactivated
,; transmitterdomaintransfers an ATPry
::; ohosohate to a conserved histidine(H)in the
:l
il
transmitterdomainThisphosphate isthen
// , \ transferred acid(D)in the receiver
to an aspartic
1i
domainof the response regulator PhoB. Several
l:
1! PhoBproteins canbe phosphorylated by one
activatedPhoR. Phosphorylated PhoBproteins
thenactivate fromgenesencoding
transcription
that helpthecellto respond
proteins to low
phosphate, phoA,phoS,phoE,and
including
/rnoE response
ugpB
regulator(active)

H
, PhoB resoonse
lit regulator(inactive)
;!

lnner (cytoplasmic)membrane ,,.t:t:' .t1'

,!i'

I
O u t e rm e m b r a n e

NBACTERIA
O F G E N EE X P R E S S I OI N
CONTROL 275

Control of Gene Expressionin Bacteria
r Gene expressionin both prokaryotes and eukaryotes is
regulated primarily by mechanismsthat control the initia-
tion of transcription.
r The first step in the initiation of transcription in E. coli
is binding of the o subunit complexed with an RNA poly-
meraseto a promoter.
r The nucleotide sequenceof a promoter determines its
strength,that is, how frequently different RNA polymerase
moleculescan bind and initiate transcription per minute.
r Repressorsare proteins that bind to operator sequences,
which overlap or lie adjacentro promorers.Binding of a
repressorto an operator inhibits transcription initiation.
r The DNA-binding activity of most bacterial repressorsis
modulated by small molecule ligands.This allows bacterial
cells to regulate transcription of specificgenesrn response
to changesin the concentration of various nutrients in the
environment and metabolitesin the cytoplasm.
r The lac operon and some other bacterial genesalso are
regulated by activator proteins that bind next ro promorers
and increase the rate of transcription initiation by RNA
polymerase.
r The major sigma factor in E. coli is o70, but severalother
less abundant sigma factors are also found, each recogniz-
ing different consensuspromoter sequences.
r Transcription initiation by all E. coli RNA polymerases,
except those containing osa, can be regulated by repressors
and activators that bind near the transcriotion start site
( s e eF i g u r e7 - 2 ) .
Genes transcribed by osa-RNA polymerase are regu-
ted by activators that bind to enhancerslocated :100
basepairs upstream from the start site. When the activator
and osa-RNA polymeraseinteracr, the DNA betweentheir
binding sitesforms a loop (seeFigure 7-4).
r In two-component regulatory systems,one prorern acts
as a senso! monitoring the level of nutrients or other com-
ponents in the environment. Under appropriate conditions,
the "y-phosphateof an ATP is transferredfirst to a histidine
in the sensorprotein and then to an aspartic acid in a sec-
ond protein, the responseregulator. The phosphorylated
response regulator then binds to DNA regulatory se-
quences,thereby stimulating or repressingtranscription of
specificgenes(seeFigure 7-5).
JA Overviewof EukaryoticGene
Controland RNAPolymerases
In bacteria,geneconrrol servesmainly to allow a singlecell to
adjust to changesin its environmentso that its growth and di-
vision can be optimized. In multicellular organisms,environ-
mental changesalso induce changesin geneexpression.An ex-
276 CHAPTER
7 | T R A N S C R | P T | O NCAOL N T R O L
O F G E N EE X P R E S S | O N
ample is the responseto low oxygen (hypoxia) in which a spe-
cific set of genesis rapidly induced that help the cell survive un-
der the hypoxic conditions. These include secretedangiogenic
proteins that stimulate the growth and penetration of new cap-
illaries into the surrounding tissue.However, the most charac-
teristic and biologically far-reachingpurpose ofgene control in
multicellular organisms is execution of the genetic program
that underlies embryological development. Generation of the
many different cell types that collectively form a multicellular
organism depends on the right genes being activated in the
right cells at the right time during the developmentalperiod.
In most cases,once a developmentalstep has been taken
by a cell, it is not reversed. Thus these decisions are
fundamentally different from the reversible activation and
repression of bacterial genesin responseto environmental
conditions. In executingtheir geneticprograms, many differ-
entiated cells (e.g., skin cells, red blood cells, and antibody-
producing cells) march down a pathway to final cell death,
leaving no progeny behind. The fixed patterns of gene con-
trol leading to differentiation serve the needs of the whole
organism and not the survival of an individual cell. Despite
the differencesin the purposes of gene control in bacteria
and eukaryotes, two key features of transcription control
first discoveredin bacteriaand describedin the previous sec-
tion also apply to eukaryotic cells.First, protein-binding reg-
ulatory DNA sequences,or control elements,are associated
with genes.Second, specific proteins that bind to a gene's
regulatory sequencesdetermine where transcription will
start and either activate or repressits transcription. As rep-
resentedin Figure 7-1, in multicellular eukaryotes, inactive
genes are assembledinto condensedchromatin, which in-
hibits the binding of RNA polymerasesand general tran-
scription factors required for transcription initiation. Activa-
tor proteins bind to control elementsnear the transcription
start site of a gene as well as kilobases away and promote
chromatin decondensationand binding of RNA polymerase
to the promoter. Repressorproteins bind to alternative con-
trol elements,causing condensation of chromatin and inhi-
bition of polymerasebinding. In this section,we discussgen-
eral principles of eukaryotic genecontrol and point out some
similarities and differencesbetweenprokaryotic and eukary-
otic systems.Subsequentsectionsof this chapter will address
specificaspectsof eukaryotic transcription in greater detail.
RegulatoryElementsin EukaryoticDNA Are
Found Both Closeto and Many KilobasesAway
from TranscriptionStart Sites
Direct measurementsof the transcription rates of multiple
genesin different cell types have shown that regulation of
transcription initiation is the most widespread form of gene
control in eukaryotes,as it is in bacteria.In eukaryotes,as in
bacteria, a DNA sequencethat specifieswhere RNA poly-
merasebinds and initiates transcription of a gene is called a
promoter. Transcription from a particular promoter is con-
trolled by DNA-binding proteins that are functionally equiv-
alent to bacterial repressorsand activators. Sincethesetran-
scriptional regulatory proteins can often function either to
 G 56 7 8 I 1 01 1
fl r n H T TI
P a n c r e a s L e n s ea n d Telencephalon Retina Retina D i -a n d r h o m b o -
cornea e n c e p h aol n

T r a n s c r i pat

T r a n s c r i pbt

T r a n s c r i pct

25 30 kb

(b) < FIGURE 7-6 Analysisof transcription-control regionsof the

mousePax6genein transgenicmice.(a)Threealternative Pax6
promoters areutilizedat distinct timesduringembryogenesis indifferent
tissues
specific of thedeveloping embryoTranscription-control regtons
. / -t 'p regulatingexpression oI Pax6 in different tissues are indicated bycolored
{ Thetelencephalon-specific controlregionin intron1 between
rectangles
t exonsOandt hasnotbeenmapped to highresolution Theother
controlregions shownare:200-500basepairs in length(b)B-
l\ galactosidaseexpressed intissues of a mouseembryo with a
\- r B-galactosidasereporter transgene 1 0 5 days afterfertilization The
genome of themouse embryo contained a transgene withB kbof DNA
upstream fromexonOfusedto theB-galactosidase codingregionLense
pit(LP)
isthetissue thatwill develop into the lense of theeye Expression
wasalsoobserved intissue thatwilldevelop intothepancreas (p) (c)B-
galactosidaseexpression ina 13,5-day embryo witha B-galactosidase
activateor to represstranscriptiondependingon their asso- reportergeneundercontrolof thesequence in part(a)between exons4
ciation with other proteins, they are more generallycalled and5 marked Retina,Arrowpoints to nasal andtemporal regions of the
transcriptionfactors.The DNA control elementsin eukary- developing relina Pax6 transcription-control regions have alsobeen
otic genomesthat bind transcription factors often are lo- found:17 kbdownstream fromthe3' exonin an intronof the
neighboring geneIPart (a)adapted fromB Kammendal etal, 1999, DeuBiol
cated much farther from the promoter they regulatethan is
the casein prokaryotic genomes.In some cases,transcrlp- patstbranotcr:Couflesv
205:79; ofPete" Grussl
tion factors;that regulateexpressionof protein-codinggenes
in higher eukaryotesbind at regulatory sitestens of thou-
sandsof basepairs either upstream (oppositeto the direc- -5-43)containing a B-galactosiddserepotter gene fused to 8
tion of transcription)or downstream(in the samedirection kb of DNA upstreamhom Pax6 exon 0, B-galactosidase is
a s t r a n s c r i p t i o n )f r o m t h e p r o m o t e r . A s a r e s u l t o f t h i s observed in the developinglense,cornea' and pancreas of the
arrallgemernt,transcription of a single gene may be regu- embryo halfway through gestation(Figure7-6b).Analysisof
lated by binding of multiple transcriptionfactorsto alterna- transgenicmice with smallerfragmentsof DNA from this re-
tive control elements,directing expressionof the samegene gion allowed the mapping of separatetranscription-control
in different types of cells and at different times during regions regulatingtranscriptionin the pancreasand in the
develclpment. lenseand cornea.Transgenicmice with other reporter gene
F o r e x a m p l e , s e v e r a l s e p a r a t et r a n s c r i p t i o n - c o n t r o l constructsrevealedadditional transcription-controlregions
DNA sequ,:nces regulateexpressionof the mammaliangene (Figure7-6a).Thesecontrolledtranscriptionin the develop-
encoding the transcription factor Pax6. Pax6 protein is re- ing retina and different regions of the brain (encephalon).
quired for developmentof the eye, certain regions of the Sorneof thesetranscription-controlregionsare in introns be-
brain and spinal cord, and the cellsin the pancreasthat se- tween exons4 and 5 and betweenexons 7 and 8. For exam-
crete horrnonessuch as insulin. Heterozygoushumanswith ple, a reporter geneunder control of the region labeledretina
only one ftrnctional Pax6 geneare born with aniridia, a lack in Figure 7-6a betweenexons 4 and 5 led to reporter gene
of irisesin the eyes(Figure1-26).The Pax6 geneis expressed expressionspecificallyin the retina (Figure7-6c).
from at leastthreealternativepromotersthat function in dif- Control regionsfor many genesare found severalhun-
ferentcell rrypesand at different timesduring embryogenesis dreds of kilobasesaway from the coding exons of the gene.
c i c ea r ep r e p a r e d( s e eF i g u r e
( F i g u r e7 - 6 a ) .W h e n t r a n s g e n im Or.remethodfor identifyingsuchdistantcontrol regionsis to

L ND RNAPOLYMERASES
CE N EC O N T R O A
O F E U K A R Y O T IG
OVERVIEW 277
 (a) Comparativeanalysis < EXPERIMENTAL FIGURE 7-7 The
human SALLIgeneenhanceractivates
expressionof a reportergene in limb
budsof the developingmouseembryo.
(a)Graphicrepresentation of the conservation
E vtouse . ^., ^
f
of DNAsequence rna regionof the human
c genome (from50214-50220 5 kb of the
o
chromosome 16sequence) =500 kb
downstream fromtheSAlt 7 geneencoding a
o Chicken zincfingertranscription repressor.A regionof
E :500 bp of non-coding
6 sequence isconserved
o fromfishto human.900bp including
o this
o
f
conserved regionwereinserted intoa plasmid
0) nextto thecodingregionfor E.coli
@
B-galactosidase (b)Theplasmid was
microinjected intoa pronucleus of a fertilized
mouseeggandimplanted in the uterus of a
pseudo-pregnant mouseto generate a
5 0 2 15 50217 transgenic mouseembryowith the "reporter
C h r o m o s o m e16 ( k i l o b a s e s ) gene"on the injected plasmidincorporated
intoitsgenome(seeFigure5-43).(c)After
(b) (c) 11.5daysof development whenlimbbuds
develop, thefixedandpermeabilized embryo
wasincubated in X-galwhichisconverted into
an insoluble intensely bluecompound by B-
galactosidase The= 900 bp regionof human
DNAcontained an enhancer thatstimulated
strongtranscription of the B-galactosidase
Forelimb reporter genein limbbudsspecifically. fFrom
bud
theVISTA Enhancer Browser http://enhancer.
lblgov;
(b)and(c):Courtesy
Parts of LenA Pennacchio,
Joint
Hindlimb Genome Institute,
LawrenceBerkeley
National
bud
Laboratoryl

rvouseegg
Mouse egg microin.jection
mtcroinjection E11.5 reporterstaining

compare the sequencesof distantly related organisms.Tran- Three EukaryoticPolymerasesCatalyze

scription control regions for a conservedgene are also often
conservedand can be recognizedin the background of non-
functional sequencethat divergesduring evolution. For ex-
ample, there is a human DNA sequence:500 kilobases
downstream of the SALLI gene thai is highly conservedin
mice, frogs, and fish (Figure 7-7a).This geneencodesa tran-
scription repressorrequired for normal development of the
lower intestine, kidneys, limbs, and ears. Vhen transgenic
mice were generated containing this conserved DNA se-
quence linked to a B-galactosidasereporter gene (Figure 7-
7b), the transgenic embryos expresseda very high level of
the B-galactosidasereporrer gene specificallyin the develop-
ing limb buds (Figure 7-7c).Human patients with deletions
in this region of the genome develop with limb abnormali-
ties. Theseresults indicate that this conservedregion directs
transcription of the SALLl genein the developinglimb. pre-
sumably, other enhancerscontrol expressionof this gene in
other types of cells where it functions in the normal diveloo-
ment of the lower intestine.kidneys and ears.
Formationof Different RNAs
The nuclei of all eukaryotic cells examined so far (e.g.,ver-
tebrate, Drosophila, yeast, and plant cells) contain three
different RNA polymerases, designated I, II, and III. These
enzymesare eluted at different salt concentrations during ion-
exchangechromatography, reflecting the polymerases'various
net charges.The three polymerasesalso differ in their sensi-
tivity to o'-amanitin, a poisonous cyclic octapeptide pro-
duced by somemushrooms (Figure7-8). RNA polymeraseI is
very insensitiveto o-amanitin, but RNA polymeraseII is very
sensitive-the drug binds near the active site of the enzyme
and inhibits translocarion of the enzyme along the DNA
template.RNA polymeraseIII has intermediatesensitivity.
Each eukaryotic RNA polymerase catalyzestranscription
of genesencodingdifferent classesof RNA (Table 7-2). RNA
polymerase1, located in the nucleolus,transcribesgenesen-
coding precursor rRNA (pre-rRNA), which is processedinto
28S, 5.8S, and 18S rRNAs. RNA polymerase III transcribes
genes encoding tRNAs, 55 rRNA, and an array of small,

278 . c H A p r E R7 T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X e R E s s t o N
|
 lNaCll--> subunits and 1.0-14smallersubunits,some of which are com-
mon betweentwo or all three of the polymerases.The best-
characterizedeukaryotic RNA polymerasesare from the yeast
,A
II Saccbaromycescereuisiae.Each of the yeast genes encoding
I
the polymerasesubunits has been cloned and sequencedand
O

c
the effects of gene-knockout mutations have been character-
O
ized. In addition, the three-dimensional structure of yeast
o:
A RNA polymeraseII has beendetermined(Figure7-9b, c).The
I
aE
I co
three nuclear RNA polymerasesfrom all eukaryotes so far ex-
o
ACt o. amined are very similar to those of yeast.
t; T-
tc EE The two large subunits (RPB1 and RPB2) of all three eu-
->
karyotic RNA polymerasesare related to each other and are
9< z* similar to the E. coli B' and p subunits, respectively(Figure
YZ
7-10). Each of the eukaryotic polymerasesalso contains an
co-likeand two nonidentical a-like subunits. The extensive
10 20 30 40 50
similarity in the structures of these core subunits in RNA
Fractionnumber
polymerasesfrom various sourcesindicatesthat this enzyme
A EXPERIMENTAL FIGURE 7-8 Columnchromatography arose early in evolution and was largely conserved. This
separatesand identifiesthe three eukaryoticRNApolymerases, seemslogical for an enzyme catalyzing a process so basic as
eachwith its own sensitivityto a-amanitin.A proteinextract
copying RNA from DNA.
fromthe nucleiof cultured eukaryotic cellsispassed througha DEAE
In addition to their core subunits related to the E. coli
Sephadex columnandadsorbed proteineluted(black curve)witha
Threefractions RNA polymerasesubunits, all three yeast RNA polymerases
solution of constantly increasingNaClconcentration
fromthe eluatesubsequently showedRNApolymerase activity(red contain four additional small subunits, common to them but
curve)At a concentration of 1 pglml,o-amanitin polymerase not to the bacterial RNA polymerase.FinallS each eukary-
inhibits
ll activitybut hasno effecton polymerases I andlll(greenshading) otic nuclear RNA polymerase has several enzyme-specific
Polymerase by 10 pglmlof o-amanitin,
lll isinhibited whereas subunits that are not present in the other two nuclear RNA
polymerase I is unaffectedevenat thishigherconcentration [See R polymerases. Gene-knockout experiments in yeast indicate
G Roeder. 1974. J BiolChen249:241 I that most of these subunits are essentialfor cell viability.
Disruption of the few polymerasesubunit genesthat are not
absolutely essentialfor viability (subunits4 andT) neverthe-
stable RNAs, including one involved in RNA splicing (U6) lessresults in very poorly growing cells.Thus it seemslikely
and the RNA component of the signal-recognitionparticle that all the subunits are necessaryfor eukaryotic RNA poly-
(SRP)involved in directing nascentproteins to the endoplas- merasesto function normally.
mic reticulum (Chapter 13). RNA polymerase11 transcribes
all protein-codinggenes;that is, it functions in production of
The LargestSubunit in RNA Polymerasell Has
mRNAs. RNA polymeraseII also produces four of the five
an EssentialCarboxyl-Terminal Repeat
small nuclearRNAs that take part in RNA splicing.
Each of the three eukaryotic RNA polymerasesis more The carboxyl end of the largest subunit of RNA polymerase II
complex than E. coli RNA polymerase,although their struc- (RPB1)contains a stretch of sevenamino acids that is nearly
tures are similar (Figure 7-9a,b). All three contain two large precisely repeated multiple times' Neither RNA polymerase I

POLYMERASE BIIATRANSCBIBED RilAFUNCTII)N

RNA polymeraseI Prer-RNA (28S,185,5.8SrRNAs) Ribosomecomponents'proteinsynthesis

RNA polymeraseII mRNA Encodesprotein

snRNAs RNA Splicing
miRNAs genecontrol
Post-transcriptional

III
RNA oolvmerase tRNAs Proteinsynthesis
55 rRNA Ribosomecomponent,proteinsynthesis
snRNA U6 RNA Splicing
75 RNA Signal-recognitionparticlefor insertionof
polypeptidesinto the endoplasmicreticulum
Other stableshort RNAs Variousfunctions,unknown for many

G E N EC O N T R O LA N D R N A P O L Y M E R A S E S
O V E R V I E WO F E U K A R Y O T I C 279
 ( a ) B a c t e r i aRl N A p o l y m e r a s e (b)YeastRNA polymerasell (c)YeastRNA polymerasell

DNA
llllltl

RNA Polymerasell

A FIGURE 7-9 €omparisonof three-dimensional structures of RNApolymerase ll ) (c)Space-filling modelof yeastRNApolymerase

bacterialand eukaryoticRNApolymerases. (a,b) TheseC. trace including subunits 4 andT These subunits extendfromthecore
models arebasedon x-raycrystallographic analysis of RNApolymerase portionof theenzyme shownin (b)neartheregionof the C-terminal
fromthe bacterium T.aquaticus andcoreRNApolymerase ll from domaio n f t h el a r g es u b u n iC
t l a m pi sa d o m a i o
n f R P Btlh a ts w i n g s
(a)Thefivesubunits
5 cereyr''siae of the bacterial enzymeare on a hingewhenRNArsin theexitchannel to closeoverupstream
distinguished bycolorOnlythe N-terminal domains of thecrsubunits DNAsothatthe polymerase cannotrelease fromthetemplate until
areincluded in thismodel(b)Tenof the 12subunits constituting transcription hasterminated Wallisthedomainof RpB2 thatforces
yeastRNApolymerase ll areshownin thismodelSubunits thatare DNAenteringthejawsof the polymerase fromthe leftto bendbefore
similar
in conformation to thosein the bacterral enzyme areshownin it exitsthe polymerase TheRNAexitchannel isindicated Ipart (a)
thesamecolorsTheC-terminal domainof the largesubunitRpBl based oncrystal structures fromG Zhang etal, 1999,Ce// 98:81 1 part(b)
wasnotobserved in thecrystalstructure, but it isknownto extend fromP Crameret al , 200'1,Science2g2:1863Part(c)fromK J Armacheet
fromtheposition marked witha redarrow(RpBistheabbreviation al , 2003,PNAS100:6964, andD A Bushnell andR D Kornberg, 2003,?NAS
for "RNApolymerase 8," whichisan alternative wayof referring to 100:6969; coloredmodelcourtesvof Steven
HahnI

nor III contains these repeating units. This heptapeptide
repeat,with a consensussequenceof Tyr-Ser-Pro-Thr-Ser-pro-
Ser,is known asthe carboxyl-terminaldomain (CTD).yeast
RNA polymeraseII contains 26 or more repears,vertebrate
enzymes have 52 repeats, and an intermediate number of
repeats occur in RNA polymerase II from nearly all other
eukaryotes. The CTD is critical for viability, and at least
10 copiesof the repeatmust be presentfor yeastto survlve.
In vitro experimentswith model promoters first showed
that RNA polymerasell moleculesthat initiaretranscription
have an unphosphorylated CTD. Once the polymerase
initiatestranscription and beginsto move away from the pro-
moter, many of the serineand some tyrosine residuesin the
CTD are phosphorylated.Analysis of polytene chromosomes
from Drosophila salivaryglandspreparedjust beforemolting
of the larva, a time of active transcription, indicate that the
CTD also is phosphorylatedduring in vivo transcription.The
large chromosomal "puffs" induced at this time in develop-
ment are regions where the genome is very actively tran-
scribed.Staining with antibodiesspecificfor the phosphory-
lated or unphosphorylated CTD demonstrared thai RNR
polymeraseII associatedwith the highly transcribedpuffed re-
gions contains a phosphorylatedCTD (Figure 7-11).
RNA Polymerasell InitiatesTranscriptionat
D N A S e q u e n c eC
s o r r e s p o n d i ntgo t h e 5 ' C a p
of mRNAs
In vitro transcription experiments involving RNA poly-
meraseII, a protein extract prepared from the nuclei of cul-
tured cells, and DNA templatescontaining sequencesencod-
ing the 5' ends of mRNAs for a number of abundantly
expressedgenes revealed that the transcripts produced al-
ways contained a cap structure at their 5' endsidentical with
that present at the 5' end of nearly all eukaryotic mRNAs
(seeFigure4-14).lnthese experimenrs,the 5'cap is addedto
the 5' end of the nascentRNA by enzymesin the nuclear ex-
tract, which can only add a cap to an RNA that has a 5, tri-
or diphosphate.Becausea 5' end generatedby cleavageof a
longer RNA would have a 5' monophosphate,it would not
be capped. Consequently,researchersconcluded that the
cappednucleotidesgeneratedin the in vitro transcription re-
actions must have been the nucleotides with which tran-
scription was initiated. Sequenceanalysisrevealedthat for a
given gene,the sequenceat the 5' end of the RNA transcriprs
produced in vitro is the same as that at the 5' end of the
mRNAs isolated from cells, confirming that the capped

280 o c H A p r E R7 T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E s s t o N
|
 E. coli core RNA polymerase(a2pp'rrl)

EukaryoticRNApolymerases

r il ill

0'-
ano
B-likesubunits EEEEEE \ cto

a-likesubunits tr >\
>,.
o-likesubunit

.\
FIGURE 7-11 Antibodystaining
Common
r--) O .-) A EXPERIMENTAL
demonstratesthat the carboxyl-terminaldomain (CTD)of RNA
s ub u n i t s during in vivo transcription'
polymerase ll is phosphorylated
H
lt .1l 1 li-:;l
l ::l
Salivaryglandpolytenechromosomes wereprepared tromDrosophila
larvaejustbefore molting.The preparation was treatedwith a rabbit
antibody specific for phosphorylated CTDandwith a goatantibody
Additional for unphosphorylated
specific CTDThepreparation thenwasstained
enzyme-specif ic +5 +3 +7 withfluorescein-labeled anti-goatantibody (green) andrhodamine-
s ub u n i t s (red).Thuspolymerase with an
labeledanti-rabbit antibody molecules
FIGURE 7-10 Schematic representation of the subunit unphosphorylated CTDstaingreen,andthosewith a phosphorylated
structureof the E coli RNAcore polymeraseand yeast nuclear CTDstainred Themoltinghormone ecdysone induces veryhigh
RNApolymerases. All threeyeastpolymerases havefivecore ratesof transcription in the puffedregions labeled 74EFand758;
subunits homologous to the B, B', two o, ando subunits of E coli notethatonlyphosphorylated CTDis present in theseregions
RNApolymerase Thelargest subunit(RPB1) of RNApolymerase ll Smaller puffed regions transcribed at highrates alsoarevisible.
alsocontains an essential C-terminal domain(CTD)RNApolymerases Nonpuffed sitesthatstainred(uparrow)or green(horizontal arrow)
I andlllcontainthe sametwo nonidentical ct-like
subunits, whereas alsoareindicated, asisa sitestaining bothredandgreen,producing
RNApolymerase ll contains two othernonidentical cr-likesubunits a yellowcolor(downarrow)lFrom J R Weeks etal, 1993,Genes Dev
All threepolymerases sharethesameo-likesubunitandfourother 7:2329;courtesy ofJ R Weeks and A L Greenleafl
commonsubunitsIn addition, eachyeastpolymerase contains three
to seven unique s m a l l esru b u n i t s

nucleotideof eukaryoticmRNAs coincideswith the transcrip-
tion start site. Today, the transcription start site for a newly
characterizedmRNA generallyis determinedsimply by iden-
tifying the DNA sequenceencodingthe 5' end of the mRNA.
Overview of Eukaryotic Gene Control and RNA
Polymerases
r The primary purpose of genecontrol in multicellular or-
ganismsis the execution of precisedevelopmentaldecisions
so that the proper genesare expressedin the proper cells
during developmentand cellular differentiation.
r Transcriptional control is the primary means of regulat-
ing gene expressionin eukaryotes,as it is in bacteria.
r In eukaryotic genomes,DNA transcription-control ele-
ments may be located many kilobasesaway from the pro-
moter they regulate. Different control regions can control
transcription of the samegene in different cell types.
r Eukaryotes contain three types of nuclear RNA poly-
merases.All three contain two large and three smaller core
subunits with homology to the F" F, o, and to subunits of
E. coli RNA polymerase, as well several additional small
subunits(seeFigure 7-10).
r RNA polymeraseI synthesizesonly pre-rRNA. RNA
polymerase II synthesizesmRNAs and some of the small
nuclear RNAs that participate in mRNA splicing' RNA
polymerase III synthesizestRNAs' 55 rRNA, and several
other relatively short, stable RNAs (seeTable 7-2).
r The carboxyl-terminal domain (CTD) in the largest sub-
unit of RNA polymeraseII becomesphosphorylatedduring
transcription initiation and remains phosphorylated as the
enzymetranscribesthe template.
r RNA polymerase II initiates transcription of genes
at the nucleotide in the DNA template that corres-
p o n d s t o t h e 5 ' n u c l e o t i d e t h a t i s c a p p e di n t h e e n c o d e d
mRNA.

G E N EC O N T R O LA N D R N A P O L Y M E R A S E S
O V E R V I E WO F E U K A R Y O T I C 281

A21 to 491
Base c23 100
frequency
t%l G 2 8 35 3 0
T 2810 83 9100 5 33 0 36 10 11 9
ArAfn+
5',
_3; Consensus
to -25
FIGURE7-12 Determination of consensusTATAbox
sequence.The nucleotidesequences upstreamof the startsite in
900 differenteukaryoticprotein-coding
geneswere alrgneoro
maximizehomologyin the regionfrom -35 to -25 Thetabulated
numbersare the percentage frequencyof eachbaseat eachposition
lE RegulatorySequences
in Protein-
CodingGenes
As noted in the previous section,expressionof eukaryotic
protein-codinggenesis regulatedby multiple protein-bind-
ing DNA sequences, genericallyreferredto as rranscription-
control regions.Theseinclude promotersand other typesof
control elementslocated near transcription start sites, as
well as sequences locatedfar from the genesthey regulate.In
this section,we take a closerlook at the propertiesof various
control elementsfound in eukaryotic protein-codinggenes
and sometechniquesusedto identify them.
The TATABox, Initiators,and CpGlslands
F u n c t i o na s P r o m o t e r si n E u k a r y o t i cD N A
The first genesto be sequencedand studiedthrough in vitro
transcriptionsystemswere viral genesand cellular protein,
coding genesthat are very activelytranscribedeither at par-
ticular times of the cell cycle or in specificdifferentiated cell
types.In all thesehighly transcribedgenes,a conservedse-
quencecalledthe TAIA box was found :25-35 basepairs
upstreamof the start site (Figure7-12). Mutagenesisstudies
have shown that a single-base changein this nucleotidese-
quence drastically decreasesin vitro transcription by RNA
polymerase II of genes adjacent to a TAIA box. In mosr
cases,sequencechangesbetweenthe TAIA box and start site
do not significantly affect the transcription rate. If the base
pairs between the TAIA box and the normal start site are
deleted,transcriptionof the altered,shortenedtemplate be-
gins at a new site -25 base pairs downstream from the
TAIA box. Consequently,the TATA box acts similarly to an
E. coli promoter to position RNA polymerase II for tran-
scriptioninitiation (seeFigure4-12).
Instead of a TATA box, some eukaryotic genesconraln
an alternative promoter element called an initiator. Most
naturally occurringinitiator elementshave a cytosine(C) at
the - 1 position and an adenine(A) residueat the transcrip-
tion start site (+1). Directed muragenesisof mammalian
geneswith an initiator-containingpromoter revealedthat
the nucleotide sequenceimmediately surrounding the start
282 . c H A p r E R7 | T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E s s t o N
TATAbox mRNAstarts
09567975241 16 24 A=50%
G=25%
00 0 0 0 9 35 37 C , U= 2 5 %
0 0 0 31240 38 30
v
I
Transcription
rAAA-/9
A
G
sequence
Maximum homology occursoveran eight-base
region,referred
to as
the TATAbox,whoseconsensus sequenceisshownat the bottom,
Theinitialbasein mRNAs encoded by genescontaininga TATAbox
mostfrequently isan A [SeeP Bucher,
1990,J Mol.Biol212:5631
site determinesthe strength of such promoters. Unlike the
conservedTAIA box sequence,however, only an extremely
degenerateinitiator consensussequencehas been defined:
(5') Y-Y-A+ r-N-T/A-Y-Y-Y (3')
where A*' is the baseat which transcriptionstarts,Y is a
pyrimidine (C or T), N is any of the four bases,and T/A is T
or A at position *3.
Transcription of genes with promoters containing a
TAIA box or initiator element beginsat a well-defined initi-
ation site. However, transcription of many protein-coding
geneshas been shown to begin at any one of multiple possi-
ble sitesover an extendedregion,often20-200 basepairs in
length. As a result, such genesgive rise to mRNAs with mul-
tiple alternative 5' ends. These genes,which generally are
transcribedat low rates(e.g.,genesencodingthe enzymesre-
quired for basic metabolic processesrequired in all cells, of-
ten called "housekeeping genes"), do not contain a TATA
box or an initiator. Most genesof this type contain a CG-
rich stretchof 20-50 nucleotideswithin :100 basepairs up-
stream of the start-siteregion. The dinucleotide CG is statis-
tically underrepresentedin vertebrate DNAs, and the
presenceof a CG-rich region, or CpG island, just upstream
from a start site is a distinctly nonrandom distribution. For
this reason, the presenceof a CpG island in genomic DNA
suggeststhat it may contain a transcription-initiation region.
Promoter-Proximal ElementsHelp Regulate
E u k a r y o t i cG e n e s
Recombinant DNA techniqueshave been used to systemati-
cally mutate the nucleotide sequencesof various eukaryotic
genes in order to identify transcription-control regions.
For example, alternative transcription-control elementsreg-
ulate expression of the mammalian gene that encodes
transthyretin (TTR), which transports thyroid hormone in
blood and the cerebrospinalfluid that surrounds the brain
and spinal cord. Transthyretin is expressedin hepatocytes,
which synthesizeand secretemost of the blood serum pro-
teins, and in choroid plexus cells in the brain, which secrete
cerebrospinalfluid and its constituent proteins. The control
 Upstreanl region Start DNA techniques.Reporter genesexpressenzymesthat are
of r7F DNA r-->
s'ffis' easily assayedin cell extracts. Commonly used reporter
genesinclude the E. coli lacZ geneencoding B-galactosidase;
[ | neco-uinanD t NA
Reporter the firefly gene encoding luciferase,which converts energy
techniaues
from ATP hydrolysis into light; and the jellyfish geneencod-
I ing green fluorescentprotein (GFP).
1 T--------------Tl
2 l------ .-a By constructing and analyzing a S'-deletion series up-
3 T-----l stream of the T?R gene,researchersidentified two control el-
4 l-----;-1 ementsthat stimulate reporter-geneexpressionin cultured he-
5 f-;-l patocytes but not in other cell types. One region mapped
5'-deletionseries betweenthe transcription start site and -200 basepairs up-
z Ligate into vector
carrying reporter gene
streamof the start site; the other mapped between:1.85 and
2.01 kb upstream of the ?TR gene transcription start site.
E Transform E. coli
and isolateolasmid DNAs
Further studiesdemonstratedthat alternativeDNA sequences
control TTR transcription in choroid plexus cells, demon-
strating again that alternative control elementsoften regulate
transcription of eukaryotic genesin different cell types.
By now, hundreds of eukaryotic genes have been ana-
lyzed, and scoresof transcription-control regions have been
identified. These control elements,together with the TATA
5'-deletion mutants box or initiator, often are referred to as the promoter of the
gene they regulate. However, we prefer to reservethe term
- | Transfecteach type of
E I nlasmio (1-5) separatelyinto promoter for the TATA box or initiator sequencesthat
'We
c u l t u r e dc e l l s determine the initiation site on the template. use the
I regions lying
term promoter-proximal elements for control
Reporter In
Reporter within 100-200 base pairs upstream of the start site.
plasmid
enzyme some cases, promoter-proximal elements are cell-type spe-
cific; that is, they function only in specific differentiated cell
Reporter types. In eukaryotes the term enhancer is used to refer to
mRNA transcription-control regions greater than -200 bp from
I Pr"o.r" cellextractand the transcriptionstart site.
E | ...iy activityof reporter Once a transcription-control region has been identified,
enzyme
J analysis of linker scanning mutations can pinpoint the se-
Plasmidno. Reporter-geneexpression quenceswithin the regulatory region that function to con-
1 +++ trol transcription. In this approach, a set of constructs with
2 +++ contiguous overlapping mutations are assayedfor their ef-
3+ fect on expressionof a reporter gene or production of a spe-
4+
5- cific mRNA (Figure 7 -1.4a). One of the first usesof this type
of analysis identified promoter-proximal elements of the
A EXPERIIVIENTAL FIGURE 7-13 S'-Deletion analysiscan
thymidine kinase (lk) genefrom herpessimplex virus (HSV).
identify transcription-controlsequencesin DNA upstreamof a
The results demonstratedthat the DNA region upstream
eukaryoti<gene.Step[: Recombinant DNAtechniques areused
of the HSV rft gene contains three separate transcription-
to prepare a series of DNAfragments thatextendfromthe 5'- -32 to
untranslated regionof a genevarious distances upstream. StepE: control sequences:a TAIA box in the interval from
-'1,6 and two other control elementsfarther upstream (Fig-
TheDNAfragments areligatedintoa reporter plasmid upstream of
an easilyassayed reporter gene StepB: TheDNAistransformed into ure7-14b\.
E.colito isolateplasmids with deletions of variouslengths5' to the To test the spacingconstraintson control elementsin the
transcription startsite Step4: Eachplasmid isthentransfected into HSV lA promoter region identified by analysisof linker scan-
culturedcells(or usedto prepare transgenic organisms) andexpression ning mutations, researchersprepared and assayedconstructs
of the reporter (step
geneisassayed E) Theresults of thishypo- containing small deletions and insertions between the ele-
theticalexample (bottom)indicate thatthetestfragmentcontains two ments. Changes in spacing between the promoter and
controlelements. The5' endof oneliesbetween deletions2 and3; promoter-proximal control elements of 20 nucleotides or
the 5' endof the otherliesbetweendeletions 4 and5 fewer had little effect. However, insertions of 30 to 50 base
pairs between a promoter-proximal element and the TATA
elements required for transcription of the TTR gene were box was equivalent to deleting the element. Similar analyses
identified by the procedure outlined in Figure 7-13. In this of other eukaryotic promoters have also indicated that con-
experimental approach, DNA fragments with varying ex- siderableflexibility in the spacingbetween promoter-proximal
tents of sequenceupstream of a start site are cloned in front elements is generally tolerated, but separations of several
of a reporter gene in a bacterial plasmid using recombinant tens of basepairs may decreasetranscription.

N P R O T E I N - C O D I NGGE N E S
Y E Q U E N C EIS
R E G U L A T O RS 283
 (a)
fiePortergene
VectorDNA fk mRNA
- | controlresion | // i +++
..
Mutant
no.
1 +++

2 +

4 +++

+++

+++

9 +++

Controlelements

Control region of fk gene

A E X P E R I M E N TFA
IGL U R7E- 1 4 L i n k e r s c a n n i nmgu t a t i o n s Inthe hypothetical example shownhere,LSmutations 1,4, 6,7, and
identify transcription-control elements.(a)A regionof eukaryotic t havelittleor no effecton expression of the reportergene,indicating
DNA(tan)thatsupports high-level expression of a reporter gene(light thatthe regions alteredin thesemutants containno controlelements
purple)isclonedin a plasmid vectorasdiagrammed at thetop. Reporter-gene expressionissignificantly
reducedin mutants2, 3, 5,
Overlapping linkerscanning (15)mutations (crosshatch) areintroduced and8, indicatingthatcontrolelements (brown)liein the intervals
shown
fromoneendof the regionbeinganalyzed to the other.These at the bottom(b)Analysis of LSmutations in thetranscription-control
mutations resultfromscrambling the nucleotide sequence in a short regionof the thymidinekinase (tk)genefromherpes simplexvirus(HSV)
stretchof theDNA Afterthe mutantplasmids aretransfected separately identifieda TATAboxandtwo promoter-proximal elements (PE-1 and
intoculturedcells,the activityof the reporter-gene productisassayed PE-2)[Part (b)seeS L McKnight andR Kingsbury 1982,Science217:316]

Distant EnhancersOften Stim u late Transcription
by RNA Polymerasell
As noted earlier, transcription from many eukaryotic pro-
moters can be stimulated by control elementslocated thou-
sands of base pairs away from the start site. Such long-
distance transcription-control elements, referred to as
enhancers, are common in eukaryotic genomes but fairly
rare in bacterial genomes.The first enhancer to be discov-
ered that stimulatestranscription of eukaryotic geneswas in
a 366-bp fragment of the simian virus 40 (SV40) genome
(Figure 7-15). Further analysisof this region of SV40 DNA
revealedthat an :100-bp sequencelying -1gg basepairs
upstream of the SV40 early transcription start site was re-
sponsible for its ability to enhance transcription. In SV40,
this enhancer sequencefunctions ro stimulate transcription
from viral promoters. The SV40 enhancer,however, ,ii-u-
lates transcription from all mammalian promoters that have
beentestedwhen it is insertedin either oiientation anywhere
on a plasmid carrying the test promoter, even when it is
thousands of base pairs from the start site. An extensive
linker scanningmutational analysisof rhe SV40 enhancerin-
dicated that it is composed of multiple individual elements,
each of which contributes to the total activity of the en-
hancer.As discussedlater, each of theseregulatory elements
is a protein-binding site.
Soon after discovery of the SV40 enhancer, enhancers
were identified in other viral genomesand in eukaryotic cel-
lular DNA. Some of these control elementsare located 50
or more kilobasesfrom the promoter they control. Analyses
of many different eukaryotic cellular enhancershave shown
that they can occur upstream from a promoter, downstream
from a promoter within an intron, or even downstream
from the final exon of a gene,as in the caseof the Pax6 gene
(see Figure 7-6). Llke promoter-proximal elements, many
enhancersare cell-type specific. For example, an enhancer
controlling Pax6 expressionin the retina has been charac-
terized in the intron between exons 4 and 5 (seeFigureT-6).
Analysesof the effects of deletions and linker scanningmu-
tations in cellular enhancershave shown that. like the SV40

284 C H A P T E R7 | T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S T O N
 enhancer,they generally are composed of multiple elements
that contribute to the overall activity of the enhancer.

p - gl o b i n Most EukaryoticGenesAre Regulatedby

Elements
Multiple Transcription-Control
Initially, enhancers and promoter-proximal elements were
thought to be distinct types of transcription-controlelements.
Howeve! as more enhancers and promoter-proximal ele-
ments were analyzed, the distinctions between them became
less clear. For example, both types of element generallycan
A.3' stimulate transcription even when inverted, and both types
p - g l o b i nm R N A often are cell-typespecific.The generalconsensusnow is that
a spectrum of control elements regulates transcription by
Treat with S1 nuclease RNA polymeraseII. At one extremeare enhancers,which can
and then denature stimulate transcription from a promoter tens of thousandsof
base pairs away (e.g., the SV40 enhancer).At the other ex-
treme are promoter-proximal elements,such as the upstream
elementscontrolling the HSV tk gene,which lose their influ-
Perform gel electrophoresis ence when moved an additional 30-50 base pairs farther
and autoradiography
from the promoter. Researchershave identified a large num-
ber of transcription-control elementsthat can stimulate tran-
scription from distancesbetweenthesetwo extremes.
c12 Figure 7-16a summarizesthe locations of transcription-
control sequencesfor a hypothetical mammalian gene. The
527 start site at which transcription initiates encodesthe first (5')
nucleotide of the first exon of an mRNA, the nucleotidethat
404 is capped. For many genes,especiallythose encoding abun-
**t dantly expressedproteins, a TATA box located approxi-
mately 25-35 basepairs upstream from the start site directs
309*
RNA polymeraseII to begin transcription at the proper nu-
cleotide. Promoter-proximal elements,which are relatively
2'r?
238 short (:lQ base pairs), are located within the first -200
basepairs upstream of the start site. Enhancers,in contrast,
usually are about 50-200 basepairs long and are composed
of multiple elementsof :10 base pairs. Enhancersmay be
A E X P E R I M E N TFA IGL U R7E- 1 5 P l a s m i dcso n t a i n i n a
g located up to 50 kilobasesor more upstream or downstream
particular5V40DNAfragment showed markedincreasein from the start site or within an intron. Many mammalian
mRNAproductioncomparedwith plasmidslackingthis genesare controlled by more than one enhancerregion.
enhancer. Plasmids containing the B-globin genewith or withouta The S. cereuisiaegenome contains regulatory elements
366-bpfragment of SV40DNAwereconstructed. Theseplasmids
called upstream activating sequences(UASs),which function
weretransfected intoculturedcells,andanyresulting RNAwas
similarly to enhancersand promoter-proximal elementsin
h y b r i d i z et od a B - g l o b iD
n N A p r o b (
e s t e p s
B a n d Z ) T h ea m o u n t
higher eukaryotes.Most yeast genescontain only one UAS,
of B-globinmRNAsynthesized by cellstransfected with oneor the
otherplasmid wasassayed by the S1nuclease-protection method which generally lies within a few hundred base pairs of the
(stepB) Therestriction fragmentprobe,generated froma B- start site. In addition, S. cereuisiaegenescontain a TATA box
g l o b i nc D N Ac l o n ew :90 base pairs upstream from the transcription start site
, a sc o m p l e m e n t at or yt h e 5 ' e n do f B - g l o b i n
mRNAThe5' endof the probewaslabeled with 32P(reddot) (Figure7-1.6b).
Hybridization of B-globinmRNAto the probeprotected an -340-
nucleotide fragmentof the probe fromdigestion by S1 nuclease,
w h i c hd i g e s tssi n g l e - s t r a n dDeNdAb u t n o t D N Ai n a n R N A - D N A
hybridAutoradiography of electrophoresed 51-protected Regulatory Sequencesin Protein-CodingGenes
fragments (step4) revealed that cellstransfected with plasmid1 r Expressionof eukaryotic protein-coding genesgenerally
( l a n e1 ) p r o d u c em d u c hm o r eB - g l o b im n R N At h a nt h o s e is regulated through multiple protein-binding control re-
transfected with plasmid2 (lane2) LaneC is a controlassay of B- gions that are located close to or distant from the start site
globinmRNAisolated from reticulocytes, whichactively synthesize ( F i g u r e7 - 1 6 ) .
p-globinTheseresults showthatthe SV40DNAfragmentin
plasmi1 d c o n t a i nasn e l e m e nt h, ee n h a n c et rh,a tg r e a t l y r Promotersdirect binding of RNA polymeraseII to DNA,
stimulates synthesis of B-globinmRNA[Adapted fromJ Banerji et al, determine the site of transcription initiation, and influence
1981, Cell 27:299 l transcrlptlon rate.

N P R O T E I N - C O D I NGGE N E S
Y E Q U E N C EIS
R E G U L A T O RS 285
 (a) Mammalian gene
up to
-50 kb or more
(b) S. cerevrblaegene
=-90
A FIGURE 7-16 Generalorganizationof control elementsthat
regulategene expressionin multicellulareukaryotesand
yeast.(a)Genes of multicellular
organisms containbothpromoter-
proximal elements andenhancers, aswellasa TATAboxor other
promoter elementThepromoter elements positionRNApolymerase
ll to initiatetranscription
at thestartsiteandinfluencethe rateof
transcription Enhancersmaybe eitherupstream or downstream and
r Three principal types of promorer sequenceshave been
identified in eukaryotic DNA. The TAIA box, the most
common, is prevalent in highly transcribed genes.Initiator
promoters are found in some genes,and CpG islands are
characteristicof genestranscribed at a low rate.
r Promoter-proximal elements occur within -200 base
pairs upstream of a start site. Severalsuch elements.con-
taining -10 basepairs, may help regulatea particular gene.
r Enhancers, which contain multiple short control ele-
ments, may be located from 200 basepairs to tens of kilo-
basesupstream or downstream from a promoter, within an
intron, or downstream from the final exon of a gene.
r Promoter-proximal elements and enhancers often are
cell-type specific,functioning only in specificdifferentiated
cell types.
lA Activatorsand Repressors
of
Transcription
The various transcription-control elementsfound in eukary-
otic DNA are binding sitesfor regulatory proteins. The sim-
plest eukaryotic cells encode hundreds of transcription fac-
tors, and the human genome encodes over 2000. The
transcription of each gene in the genome is independently
regulated by combinations of specific transcription factors
that bind to its transcription-control regions.The number of
possible combinations of this many transcription factors is
astronomical, sufficient to generate unique controls for
every geneencodedin the genome.In this section,we discuss
the identification, purification, and structures of these tran-
scription factors, which function to activate or repressex-
pressionof eukaryotic protein-coding genes.
Footprintingand Gel-5hiftAssaysDetect
Protein-DNAInteractions
In yeast,Drosophila, and other geneticallytractable eukary-
otes, numerous genes encoding transcriptional activators
and repressors have been identified by classical genetic
286 C H A P T E R7 | T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S | O N
+ 1 0t o
+50 kb or more
Exon fl Intron l-l rnrn uo*
Promoter-proximal
l-l Enhancer;
- yeast
element UAS
asfar awayas 50 kb or morefromthe transcription startsite In
somecases, enhancersliewithinintronsForsomegenes,promoter-
proximalelements occurdownstream fromthe startsiteaswellas
upstream(b) MostS.cerevisiae genescontainonlyone regulatory
region,calledan upstreamactivatingsequence(UAS)and a TATA
box,whichis :90 basepairsupstream fromthe startsite.
analyses like those described in Chapter 5. However, in
mammals and other vertebrates,which are lessamenableto
such genetic analysis,most transcription factors have been
detected initially and subsequentlypurified by biochemical
techniques.In this approach, a DNA regulatory elementthat
has been identified by the kinds of mutational analysesde-
scribed in the previous section is used to identify cognate
proteins that bind specificallyto it. Two common techniques
for detectingsuch cognateproteins are DNase I footprinting
and the electrophoreticmobility shift assay.
DNase I footprinting takes advantage of the fact that
when a protein is bound to a region of DNA, it protects that
DNA sequencefrom digestion by nucleases.As illustrated in
Figure 7-17, when samples of a DNA fragmenr that is la-
beled at one end are digestedunder carefully controlled con-
ditions in the presenceand absenceof a DNA-binding pro-
tein and then denatured, electrophoresed,and the resulting
gel subjectedto autoradiographg the region protected by the
bound protein appearsas a gap, or "footprint," in the array
of bands resulting from digestion in the absenceof protein.
When footprinting is performed with a DNA fragment con-
taining a known DNA control element,the appearanceof a
footprint indicatesthe presenceof a transcription factor that
bincis that control element in the protein sample being as-
sayed. Footprinting also identifies the specific DNA se-
quenceto which the transcription factor binds.
The electrophoretic mobility shift assay (EMSA), also
called the gel-shift or band-shift assay,is more useful than
the footprinting assay for quantitative analysis of DNA-
binding proteins. In general,the electrophoreticmobility of
a DNA fragment is reducedwhen it is complexed to protein,
causing a shift in the location of the fragment band. This as-
say can be used to detect a transcription factor in protein
fractions incubated with a radiolabeled DNA fragment con-
taining a known control element (Figure 7-18).
In the biochemical isolation of a transcription factor, an
extract of cell nuclei commonly is subjectedsequentiallyto
severaltypes of column chromatography (Chapter 3). Frac-
tions eluted from the columns are assayedby DNase I foot-
printing or EMSA using DNA fragmentscontaining an iden-
tified regulatory element (see Figures 7-"1.7 and 7-18).
 SampleA Sample B < EXPERIMENTAL FIGURE 7-'17DNaselfootprinting reveals
(DNA-binding protein absentl (DNA-binding protein present) control-elementsequencesand can be usedas an assayin
transcriptionfactor purification.(a)DNaseI footprintingcan
Sequ e nce-specific control-element sequences A DNAfragmentknownto
Protein-binding identify
b i n d i n gp r o t e i n
sequence containthecontrolelementislabeled at oneendwith 12P(reddot)
Portions of the labeled DNAsample thenaredigested with DNase I in
the presence andabsence of proteinsamples thoughtto containa
J cognate protein. DNase I randomly hydrolyzes the phosphodiester
bondsof DNAbetweenthe 3' oxygenon the deoxyribose of one
nucleotide andthe 5' phosphate of the nextnucleotide A low
concentration of DNase I isusedsothaton average, eachDNA
molecule iscleaved justonce(vertical arrows). lf the proteinsample
doesnot containa cognateDNA-binding protein, the DNAfragment
iscleaved at multiple positions betweenthe labeled andunlabeled
endsof theoriginal fragment, asin sample A on the left lf the
proteinsample contains a cognate protein, asin sample B on the
right,the protein binds to the DNA, thereby protecting a portionof
thefragment fromdigestionFollowing DNase treatment, the DNAis
separated fromprotein, denatured to separate thestrands, and
electrophoresed Autoradiography of the resulting geldetects only
labeled strands andreveals fragments extending fromthe labeled
endto thesiteof cleavage by DNase L Cleavage fragments
containing thecontrolsequence showup on thegelfor sample A
but aremissing in sample B because the boundcognateprotein
blocked cleavages withinthatsequence andthusproduction of the
corresponding fragments The missing bands on the gelconstitute
thefootprint(b)A proteinfractioncontaining a sequence-specific
DNA-binding proteincanbe purifiedby columnchromatography.
DNase I footprinting canthenidentify whichof the elutedfractions
(b) Fraction
contarn thecognateproteinInthe absence of addedprotein(NE,no
MNEOFTl 8 9 1 0 1 11 2 1 3 1 4 1 51 6 1 8 2 02 2
extract), DNase I cleaves the DNA fragment at multiple sites,
producing multiple bandson the gelshownhereA cognateprotein
present in the nuclear extractapplied to the column(O,onput)
generated a footprintThisproteinwasboundto the column,since
footprinting activity wasnot detected in theflow-through protein
fraction(FT)Aftera saltgradient wasapplied to the column,mostof
the cognateproteinelutedin fractions 9-12,asevidenced by the
missing bands(footprints). Thesequence of the protein-binding
regioncanbe determined bycomparison with markerDNA
fragments of known length analyzed on the samegel(M).Ipart (b)
fromS Yoshinaga et al, 1989, J BiolChem264:105291

Fraction o N 1 2 3 4 5 6 7 8 9 1011121416182022 < EXPERIMENTAL FIGURE 7-18 Electrophoretic mobilityshift

assaycan be usedto detecttranscriptionfactorsduring
purification.Inthisexample, proteinfractions separated bycolumn
Bound chromatography wereassayed to bindto a radiolabeled
for theirability
p r o b e*
;l' DNA-fragment probecontaining
aliquotof theproteinsample
a knownregulatory
wasloaded
elementAfteran
ontothecolumn(ON)and
successive columnfractions (numbers) wereincubated withthelabeled
probe,the samples wereelectrophoresed under conditions thatdo not
disruptprotein-DNA interactionsThe freeprobe not bound to protein
migrated to the bottomof the gel A proteinin the preparation applied
to thecolumnandin fractions 7 and8 boundto theprobe,forminga
Free DNA-protein complex that migratedmoreslowlythanthefreeprobe
PrObe+ Thesefractions thereforelikelycontainthe regulatory proteinbeing
souqht[From 5 Yoshinagaetal, 1989,,/Biol.Chem264:10529]

OS
SND REPRESSOR
ACTIVATORA F TRANSCRIPTION 287
 Adenovirus SV40
DNA DNA

*t
rt

SP1:-+-+
A EXPERIMENTAL FIGURE 7-19 Transcription factorscan be Reporter-gene
identified by in vitro assayfor transcriptionactivity.Sp1was tra nscri pts
identified
basedon itsabilityto bindto a regionof the SV40genome
thatcontains sixcopies of a GC-rich promoter-proximal elementand
waspurified by columnchromatography Totestthetranscription-
activating
abilityof purified5P1,it wasincubated in vitrowithtemplate
DNA,a proteinfractron containing RNApolymerase ll andassociated
generaltranscriptionfactors, A EXPERIMENTAL FIGURE 7-20 ln vivo transfectionassay
andlabeled ribonucleoside triphosphates
Thelabeled RNAproducts weresubjected measurestranscriptionactivity to evaluateproteinsbelieved
to electrophoresis and
autoradiography Shownhereareautoradiograms to be transcriptionfactors.Theassaysystemrequires two
fromassays with
adenovirus andSV40DNAin the absence (-) andpresence plasmids Oneplasmid contains the geneencoding the putative
(+) of
SP1SP1hadno significant effecton transcription
transcriptionfactor(protein X) Thesecondplasmid containsa
fromtheadenovirus
promoter,whichcontains reportergene(eg,lac\ andoneor morebindingsitesfor proteinX.
no SP1-binding sitesln contrast, Sp1
stimulatedtranscription fromthe SV40promoter Bothplasmids aresimultaneously introduced intocellsthat lackthe
abouttenfold
geneencoding proteinX. Theproduction of reporter-gene RNA
[Adaptedfrom M R Briggset al , 1986, Science234:4jI
transcripts
ismeasured; alternatively,
theactivity of theencoded
proteincanbe assayedlf reporter-gene transcription isgreaterin the
Fractionscontaining protein that binds to the regulatory ele- presence of theX-encoding plasmid thanin itsabsence, thenthe
proteinisan activator;if transcriptionisless,thenit isa repressor.By
ment in these assaysprobably contain a putative transcrip-
useof plasmids encoding a mutatedor rearranged transcription
tion factor. A powerful technique commonly used for the
factor,importantdomains of the proteincanbe identified
final stepin purifying transcriptionfacrorsis sequence-specific
DNA affinity cbromatography, a particular type of affinity
chromatography in which long DNA strands containing tose, was identified by complementation analysis of gal4
multiple copies of the transcription factor-binding site are mutants (Chapter 5). Directed mutagenesisstudieslike those
coupled to a column matrix. As a final test that an isolated describedpreviously identified UASs for the genesactivated
protein is in fact a transcription factor, its ability to modu-
by GAL4. Each of these UASs was found to contain one or
late transcription of a template containing the corresponding
more copies of a related 17-bp sequencecalled UAS6AL.
protein-binding sites is assayedin an in vitro transcription
DNase I footprinting assayswith recombinant GAL4 pro-
reaction. Figure 7-19 shows the results of such an assayfor
tein produced in E. coli from the yeast GAL4 gene showed
SP1, a transcription factor that binds to GC-rich sequences,
that GAL4 protein binds to UAS6al sequences.rWhen a
thereby activating transcription from nearby promoters. copy of UAS541 w4s cloned upstream of a TAIA box followed
Once a transcription factor is isolated and purified, its by a lacZ reporter gene, expression of lacZ was activated in
partial amino acid sequencecan be determined and used to
galactosemedia in wild-type cells but not in gal4 mutants.
clone the geneor cDNA encodingit, as outlined in Chapter 5.
These results showed that UAS6a1 is a transcription-control
The isolated gene can then be used to test the ability of the
element activated by the GAL4 protein in galactosemedia.
encodedprotein to activate or represstranscription in an in
A remarkable set of experiments with gal4 deletion
vivo transfection assay(Figure 7-20).
mutants demonstrated that the GAL4 transcription factor
is composedof separablefunctional domains: an N-terminal
ActivatorsAre Modular ProteinsComposedof DNA-binding domain, which binds to specific DNA se-
l o m a i n sa n d p r o m o t e
D i s t i n c tF u n c t i o n aD quences,and a C-terminal actiuation domain, which inter-
acts with other proteins to stimulate transcription from a
Transcription
nearby promoter (Figure 7-21). Sfhen the N-terminal
Studieswith a yeast transcription activator called GAL4 pro- DNA-binding domain of GAL4 was fused directly ro vari-
vided early insight into the domain structure of transcripiion ous portions of its own C-terminal region, the resulting
factors. The gene encoding the GAL4 protein, whichpro- truncated proteins retained the ability to stimulate expres-
motes expression of enzymes needed to metabolize galac- sion of a reporter gene in an in vivo assay like that

288 C H A P T E R7 | T R A N S C R T P T T O NCAOLN T R O LO F G E N E E X P R E S S t O N
(a) Reporter-gene construct < EXPERIMENTAL FIGURE 7-21 Deletion
mutantsol the GAL4gene in yeastwith a UASGAT
reporter-gene constructdemonstratethe seParate
UASGAT TATA (a)Diagram of
functionaldomainsin an activator.
oox
DNAconstruct containing a laczreporler geneand
TATAboxligatedto UASCAL a regulatory elementthat
(b) Wild-type and mutant GAL4 proteins Binding B - g a l a c t o s i d a s e containsseveral GAL4-binding sitesThereporter-gene
to UASor, activity
constructandDNAencoding wild-type or mutant
1 74 738 823 into
(deleted)GAL4weresimultaneously introduced
Wild-type N +++
mutant(9al4)yeastcells,andthe activityof B-
DNA-binding Activation Activity
galactosidase expressed fromlacZwasassayed
domain domain
willbe high if the introduced GAL4 DNA encodes a
50 881 protein(b)Schematic diagrams of wild-type
f unctional
GAL4andvarious mutantformsSmallnumbers refer
+++
Fe+e to positionsin thewild-type sequence Deletion of 50
+++ aminoacidsfromthe N-terminal enddestroyed the
N-and of GAL4to bindto UAS6a1 andto stimulate
ability
C-terminal
deletion expressionof B-galactosidase fromthe reporter gene
m u t an t s Proteinswithextensive deletions fromtheC-terminal
endstillboundto UASGAT. These resultslocalize the
DNA-binding domain to the N-termrnal end of GAL4
Theabilityto activate F-galactosidase expression was
notentirely eliminated unless somewhere between 126
f-l,o '1
and 89 or moreaminoacidsweredeleted fromthe
C-terminal end Thustheactivation domainliesin the
74 oeol-----l eer +++
C-terminal regionof GAL4Proteins with internal
I n t e r na I deletions(bottom)alsowereableto stimulate
deletion f_l zq zeel--_-] esr +++
expression of B-galactosidase, indicating thatthe
murants
zoaf-l ear ++ centralregionof GAL4isnotcrucial for itsfunctionin
f-],0 thisassay. J MaandM Ptashne, 1987, Cell48:847 'I
[See
A H o o ea n d K S t r u h l .1 9 8 6 ,C e / / 4 6 : 8 8 5a; n d R B r e n ta n d M
Ptashne,1985. Cell43:729I

depicted in Figure 7-20. Thus the internal portion of the

protein is not required for functioning of GAL4 as a tran-
Examples
scription factor. Similar experiments with another yeast
transcription factor, GCN4, which regulatesgenesrequired N C GAL4
for synthesisof many amino acids,indicated that it contains
an -60-aa DNA-binding domain at its C-terminusand an
N C GCN4
:20-aa activation domain near the middle of its sequence.
Further evidencefor the existenceof distinct activation
N CGR
domains in GAL4 and GCN4 came from experimentsin
which their activation domains were fused to a DNA-
binding domain from an entirely unrelated E. cctli DNA- NCSP1
binding protein. \7hen thesefusion proteins were assayedin
vivo, they activated transcription of a reporter genecontain-
DNA-binding
ing the cognate site for the E. coli protein. Thus functional domain
transcriptionfactorscan be constructedfrom entirely novel
Activation
combinationsof prokaryotic and eukaryoticelements.
domain
Studiessuchas thesehavenow beencarried out with many
eukaryotic activators.The structural model of eukaryotic F l e x i b l ep r o t e i n
domain
activatorsthat has emergedfrom thesestudiesis a modular
one in which one or more activationdomainsare connected FfGURE7-22 Schematicdiagrams illustrating the modular
to a sequence-specific DNA-binding domain through flexi- structure of eukaryotic transcription activators. These
ble protein domains (Figure 7-22). In some cases,amlno transcription factorsmay containmore than one activationdomain
acids included in the DNA-binding domain also contribute b u t r a r e l yc o n t a i nm o r et h a n o n e D N A - b i n d i ndgo m a i n G A L 4a n d
to transcriptionalactivation.As discussedin a later section, GCN4are yeasttranscription activators. The glucocorticord receptor
activationdomainsare thought to function by binding other (GR)promotestranscription of target geneswhen certainhormones
proteins involved in transcription.The presenceof flexible a r eb o u n dt o t h e C - t e r m i n aalc t i v a t i o dn o m a i n S P 1b i n d st o G C - r i c h
domainsconnectingthe DNA-binding domainsto activation p r o m o t ee r l e m e n t si n a l a r g en u m b e ro f m a m m a l r agne n e s

OS
AND REPRESSOR
ACTIVATORS F TRANSCRIPTION 289
domains may explain why alterationsin the spacingbetween
control elementsare so well tolerated in eukaryotic control
regions. Thus even when the positions of transcription fac-
tors bound to DNA are shifted relative to each other, their
activation domains may still be able to interact becausethey
are attachedto their DNA,binding domains through flexible
protein regions.
RepressorsInhibit Transcriptionand Are the
FunctionalConverseof Activators
Eukaryotic transcription is regulated by repressorsas well as
activators. For example, geneticistshave identified muta-
tions in yeast that result in continuously high expressionof
certain genes.This type of unregulated,abnormally high ex-
pression is called constitutive expression and results from
the inactivation of a repressorthat normally inhibits the
transcription of these genes. Similarly, mutants of
Drosophila and Caenorhabditis eleganshave been isolated
that are defective in embryonic development becausethey
expressgenesin embryonic cells where those genesare nor-
mally repressed.The mutations in these mutanrs inactivate
repressors,leading to abnormal development.
Repressor-bindingsites in DNA have been identified by
systematiclinker scanning mutation analysis similar to that
depictedin Figure 7-14.In this type of analysis,mutation of
an activator-binding site leadsto decreasedexpressionof the
linked reporter gene, whereas mutation of a repressor-
binding site leads to increasedexpressionof a reporter gene.
Repressorproteins that bind such sites can be purified and
assayedusing the same biochemicaltechniquesdescribed
earlier for activator proteins.
Eukaryotic transcriptionrepressorsare the functional con-
verseof activators.They can inhibit transcription from a gene
they do not normally regulatewhen their cognatebinding sites
are placedwithin a few hundred basepairs of the gene'sstart
site. Like activators,most eukaryotic repressorsare modular
proteins that have two functional domains: a DNA-binding
domain and a repressiondomain. Similar to activation do-
mains,repressiondomainscontinue to function when fusedto
another type of DNA-binding domain. If binding sitesfor this
secondDNA-binding domain are insertedwithin a few hun-
dred basepairs of a promoter,expressionof the fusion protein
inhibits transcription from the promoter. Also like activation
domains, repression domains function by interacting with
other proteins, as discussedlater in this chapter.
The absence of appropriate repressor activity can
have devastatingconsequences. For instance,the pro-
-Wilms'
tein encodedby the tumor (WT1)gene rs a repres-
sor that is expressedpreferentially in the developingkid-
ney. Children who inherit mutations in both the maternal
and paternal WT1 genes,so that they produce no func-
tional WT1 prorein, invariably develop kidney tumors
early in life. The WT1 protein binds to the control region
of the gene encoding EGR1, which itself is a transcripiion
factor as well (Figure7-23).This gene,like many other eu-
karyotic genes,is subjectto both repressionand activation.
290 c H A P T E R7 | T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X p R E S S t O N
W T 1 b i n d i n gs i t e
! snrncr binding
site
FGR-7control
regron ner binding
site
I
A FIGURE 7-23 Diagramof the controlregionof the gene
encodingEGR-1. a transcriptionactivator.Thebindingsitesfor WT1,
a eukaryotic
repressor protein,
do notoverlap the bindingsitesfor
theactivator
AP1or thecomposite bindingsitefor the activators
SRF
andTCF. Thusrepression byWT1doesnot involve directinterference
with bindingof otherproteins
asin thecaseof bacterial repressors.
Experiments have shown that binding by \7T1 represses
transcription of the EGRi genewithout inhibiting binding
of the activatorsthat normally stimulateexpressionof this
gene. These experiments demonstrate that WT1 can func-
tion as a transcriptional repressor.It is likely that WT1
functions to represstranscription of multiple other genesin
addition to EGRl. Consequently,tumor formation in pa-
tients with homozygous mutations of the'WT1 gene may
result in part from abnormal activation of multiple genes
such as the EGRI gene.I
DNA-B|ndingD o m a i n sC a n B e C l a s s i f i e d
into
NumerousStructuralTypes
The DNA-binding domains of eukaryotic activarors and re-
pressorscontain a variety of structural motifs that bind spe-
cific DNA sequences.The ability of DNA-binding proteins
to bind to specific DNA sequencescommonly results from
noncovalent interactions betweenatoms in an ct helix in the
DNA-binding domain and atoms on the edgesof the bases
within a major groove in the DNA. Interactions with sugar-
phosphate backbone atoms and, in some cases,with atoms
in a DNA minor groove also contribute to binding.
The principles of specificprotein-DNA interactionswere
first discoveredduring the study of bacterialrepressors.Many
bacterial repressorsare dimeric proteins in which an cr helix
from each monomer insertsinto a major groove in the DNA
helix (Figure 7-24).This ct helix is referredto as the recogni-
tion helix or sequence-readinghelix becausemost of the
amino acid sidechainsthat contact DNA extend from this he-
Iix. The recognition helix that protrudes from the surfaceof
bacterialrepressorsto enter the DNA major groove and make
multiple, specificinteractionswith atoms in the DNA is usu-
ally supported in the protein structure in part by hydropho-
bic interactions with a secondct helix just N-terminal to it.
This structural element, which is present in many bacterial
repressors,is called a helix-turn-helix motif.
Many additional motifs that can presentan cr helix to the
major groove of DNA are found in eukaryotic transcription
factors, which often are classifiedaccording to the type of
DNA-binding domain they contain. Becausemost of these
motifs have characteristicconsensusamino acid sequences,
newly characterizedtranscription factors frequently can be
classifiedonce the correspondinggenesor cDNAs are cloned
and sequenced.The genomes of higher eukaryotes encode
(a)
FIGURE 7-24 lnteractionof bacteriophage434 repressor
with DNA.(a)Ribbon diagram of 434 repressorboundto itsspecific
operatorDNA,Repressor monomers arein yellowandgreenThe
helices
recognition areindicatedby asterisks. modelof
A space-filling
complex (b)shows how the proteininteracts
the repressor-operator
with onesideof the DNAmolecule
intimately overa lengthof 1 5
fromA K Aggarwal
turns [Adapted etal, 1988,Science242:8991
dozensof classesof DNA-binding domains and hundredsto
thousands of transcription factors. The human genome' for
instance,encodes-2000 transcription factors.
Here we introduce several common classesof DNA-
binding proteins whose three-dimensional structures have
been determined.In all theseexamplesand many other tran-
scription factors, at least one ct helix is insertedinto a major
groove of DNA. However. some transcription factors con-
tain alternative structural motifs (e.g., F strands and loops)
that interact with DNA.
Homeodomain Proteins Many eukaryotic transcription
factors that function during developmentcontain a con-
served 60-residue DNA-binding motif, called a home-
odomain that is similar to the helix-turn-helixmotif of bac-
terial repressors.These transcription factors were first
identified in Drosophila mutants in which one body part
was transformed into another during development(Chap-
ter 22). The conserved homeodomain sequence has also
been found in vertebrate transcription factors' including
those that have similar master-controlfunctions in human
development.
Zinc-Finger Proteins A number of different eukaryotic
proteins have regions that fold around a central Zn"- ion,
producing a compact domain from a relatively short length
of the polypeptide chain (Figure 7-25a). Termed a zir.c fin-
ger, this structural motif was first recognizedin DNA-bind-
ing domains but now is known to occur also in proteins that
do not bind to DNA. Here we describe two of the several
classesof zinc-finger motifs that have been identified in eu-
karvotic transcriptionfactors.
The C2H2 zinc finger is the most common DNA-binding
motif encoded in the human genome and the genomes of
most other multicellular animals. It is also common in mul-
ticellular plants but is not the dominant type of DNA-
binding domain in plants as it is in animals. This motif has a
23- to 26-residue consensussequencecontarnrng two con-
servedcysteine(C) and two conservedhistidine (H) residues,
whose side chains bind one Znz* ion (Figure 3-9c). The
name "zinc finger" was coined becausea two-dimensional
diagram of the structure resemblesa finger. Sfhen the three-
dirnensional structure was solved, it became clear that the
binding of the Znz* ion by the two cysteineand two histi-
dine residuesfolds the relatively short polypeptide sequence
into a compact domain, which can insert its ct helix into the
zinc finger (becauseit has four conservedcysteinesin contact
with the Zn2*\. is found in -50 human transcription fac-
tors. The first members of this class were identified as spe-
cific intracellular high-affinity binding proteins, or "recep-
tors," for steroid hormones, leading to the name steroid
receptor swperfamily.Becausesimilar intracellular receptors
for nonsteroid hormones subsequently were found, these
transcription factors are now commonly called nuclear re-
."ptott. The characteristicfeature of Ca zinc fingers is the
p..r..t.. of two groups of four critical cysteines'one toward
each end of the 55- or 56-residuedomain' Although the Ca
three or more repeating finger units and bind as monomers'
whereas Ca zinc-finger proteins generally contain only two
finger units and generally bind to DNA as homodimers or
heterodimers.Homodimers of Ca zinc-finger DNA-binding
domains have twofold rotational symmetry (Figure 7-25c)'
ConsequentlS homodimeric nuclear receptors bind to con-
sensusDNA sequencesthat are inverted repeats'
Leucine-Zipper Proteins Another structuralmotif present
in the DNA-binding domains of alarge classof transcrip-
tion factors contains the hydrophobic amino acid leucine
at every seventh position in the sequence'These proteins
bind to DNA as dimers, and mutagenesis of the leucines
showed that they were required for dimerization' Conse-
quently, the name leucine zipper was coined to denote this
structural motif.
The DNA-binding domain of the yeast GCN4 transcrip-
tion factor mentioned earlier is a leucine-zipperdomain' X-
ray crystallographicanalysisof complexesbetweenDNA and
the GCN4 DNA-binding domain has shown that the dimeric
protein contains two extendeda helicesthat "grip" the DNA
holecule, much like a pair of scissors,at two adjacentmajor
grooves separatedby about half a turn of the double helix
OS
AND REPRESSOR
ACTIVATORS F TRANSCRIPTION O 291
 > FfGURE 7-25 Zinc-Fingerproteins.(a)This (a)
two-dimensional depiction oI C2H2 zincfingers
illustratesthe shapethatgavethismotifits ,*d #;o op
+p@ e0 f,,l
n a m el n d i v i d uaam
l i n oa c i d a
s r ei n p u r p l et ;h e €\.} o{} (D {}
aminoacidsthatcontacttheZn*2ionarein €'*aCiD{3@
blue (b)GL1isa monomeric proteinthat
contains fiveC2H2 zincf ingersa-Helices are
shownascylinders, Zn*2ionsasspheresFinger
'1
doesnot interact with DNA,whereas the
otherfourfingersdo (c)Theglucocorticoid
receptor isa homodimeric Cozinc-finger
proteino-Helices areshownaspurpleribbons,
B-strands asgreenarrows, Zn*2ionsasspheres
Twoo helices (darker shade). onerneach
monomer, interact with the DNA LikeallCa
zinc-finger homodimers, thistranscription factor
hastwofoldrotational symmetry; the centerof
symmetry isshownby theyellowellipse, In
contrast, heterodimeric nuclear receptors do not (b)
exhibitrotational symmetry [See N p pavletich
a n d C O P a b o , 1 9 9 3 ,S c i e n c e 2 6 l : 1 7 0 1a, n d B F
Lursiet al , 1991, Nature352:49j l

F i n g e r5
F i n g e r4

F i n g e3
r

. Finoer 2
. . /1 N
C
\dt

F r n g e 1r

3',
JJJ 5', 3'
5'

7;26a). The portions of the o helicescontacting the
!{qure
DNA include positively charged (basic)residuesthat interact
with phosphates in the DNA backbone and additional
residuesthat interact with specificbasesin the major groove.
GCN4 forms dimers via hydrophobic interactions be-
subsequentlywere identified. Like leucine-zipper proreins,
they form dimers containing a C-terminal coiLi-coil dimer-
izationregion and an N-terminal DNA-binding domain. The
term basi zipper (bZIP)now is frequently ,ri.d to refer to
all proteins with these common structural features. Many
basic-zipper transcription factors are heterodimers of two
different polypeptide chains, each containing one basic_
zipper domain.

Basic Helix-Loop-Helix (bHLH) proteins The DNA_binding

domain of another classof dimeric transcription factors con-

coiled-coildimer (seeFigure 3-9a).

Although the first leucine-zippertranscription factors to be
analyzedcontainedleucineresidues seventhposition
"t.u..y
in the dimerization region, additional DNA-binding proteins
containing other hydrophobic amino acids in thesepositions contain an N-terminal a helix with basicresiduesthat interact

292 o c H A p r E R7 | T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E S s t o N
(a) (b) tion domains marked by an unusually high percentageof par-
ticular amino acids. GAL4, GCN4, and most other yeast tran-
scription factors, for instance'have activation domains that are
rich in acidic amino acids (aspartic and glutamic acids).These
so-called acidic actiuation domains generally are capable of
stimulating transcription in nearly all types of eukaryotic
cells-fungal, animal, and plant cells.Activation domains from
some Drosophila and mammalian transcription factors are
glutamine-rich, and some are proline-rich; still others are rich
in the closely related amino acids serineand threonine, both of
which have hydroxyl groups. However, some strong activation
-. I domains are not particularly rich in any specificamino acid'
, Biophysical studies indicate that acidic activation do-
mains have an unstructured, random-coil conformation'
I
Thesedomains stimulate transcription when they are bound
to a protein co-actiuator.The interaction with a co-activator
A FIGURE 7-26 Interactionof homodimericleucine-zipperand ."nr.t the activation domain to assumea more structured ct-
basichelix-loop-helix(bHLH)proteinswith DNA.(a)In leucine-
helical conformation in the activation domain-co-activator
zipperproteins, basicresidues in the extended regions
cr-helical of
complex. A well-studied example of a transcription factor
the monomers interactwith the DNAbackbone at adjacentmajor
domainisstabilized by with an acidic activation domain is the mammalian CREB
groovesThecoiled-coil dimerization
hydrophobic interactions between the monomers. (b)In bHLH protein, which is phosphorylated in responseto increased
proteins,the DNA-binding helices at the bottom(N-termini of the ievels of cAMP. This regulated phosphorylation is required
(CREB binding
monomers) areseparated by nonhelical loopsfroma leucine-zipper- for CREB to bind to its co-activator CBP
a coiled-coil dimerizationdomainlPart (a)see protein), resulting in the transcription of genes whose con-
likeregioncontaining
T.E Ellenbergerelal,1992,Cell71:1223; part(b)seeA R Ferre-D'Amare trol regionscontain a CREB-binding site (seeFigure 1'6-31')'
etal , 1993,Nature353:38l \Thenihe phosphorylatedrandom coil activation domain of
CREB interacts with CBR it undergoes a conformational
change to form two ct helices linked by a short loop that
with DNA, a middle loop region, and a C-terminal region
wrap around the interacting domain of CBP.
with hydrophobic amino acids spacedat intervals characteris-
Someactivation domains arelarget and more highly struc-
tic of an amphipathic a helix. As with basic-zipper proteins'
tured than acidic activation domains. For example' the ligand-
different bHLH proteins can form heterodimers.
binding domains of nuclear receptors function as activation
domains when they bind their specific ligand (Figure 7-27)'
StructurallyDiverseActivation and Repression Binding of ligand induces alarge conformational change that
DomainsRegulateTranscription allows the ligand-binding domain with bound hormone to in-
Experiments with fusionproteinscomposed of the GAL4 teract with a short ct helix in nuclear-receptor co-activators;
DNA-binding domain and random segmentsof E. coli pro- the resulting complex then can activate transcription of genes
teins demonstrated that a diverse group of amino acid se- whose control regions bind the nuclear receptor.
quencescan function as activation domains, -1 percent of Thus the acidic activation domain in CREB and the lig-
all E. coli sequences,even though they evolved to perform and-binding activation domains in nuclear receptors repre-
other functions. Many transcription factors contain activa- sent two structural extremes. The CREB acidic activation

> FIGURE 7-27 Eftectof ligand binding on conformation (a)

of the estrogenreceptor.Onlythe ligand-binding domarnof
the receptorisshown.(a)Whenestrogenis boundto the
domain, the greeno helixinteractswith the ligand,generating
a hydrophobic aroovein theligand-bindingdomain(darkbrown
helices) thatbindsan amphipathic cthelixin a co-activator
subunit(blue).(b)Theconformation of the estrogenreceptorin
theabsence of hormoneisthoughtto bestabilized by binding
of theestrogen antagonisttamoxifen.Inthisconformation, the
greenhelixof the receptorfoldsinto a conformation that o-helixfrom
interacts bindinggrooveof the active
with the co-activator interacting
receptor, blockingbindingof co-activators.
sterically [From AK co-activator
Shrau et al, 1998,Cell95:927) Estrogen
(agonist)

A C T I V A T O R SA N D R E P R E S S O ROSF T R A N S C R I P T I O N 293
 > FIGURE 7-28 Combinatorial possibilities due to formationof (a)
heterodimerictranscriptionfactors.(a)In someheterodimeric Factor Factor Factor
transcrrption factors, eachmonomer recognizes thesameDNA A C z Activation
sequence In the hypothetical example shown,transcription factors O (D/ domain
\\
A, B,andC canallinteract with oneanother; creating sixdifferent E RDNA-binding
alternativecombinations of activationdomains thatcanallbindat domain
thesamesite Eachcomposite bindingsiteisdivided intotwo half-
sites,andeachheterodimeric factorcontains theactivation domains .)r)
of itstwo constituent monomers(b)Whentranscription factor YY
-r-- --fl
??
monomers recognize differentDNAsequences, alternative
combinations of the threefactorsbindto sixdifferentDNAsequences
(sites1-6),eachwith a uniquecombination of activation domarns(c) ( b )
Expression of an inhibitory factor(red)thatinteracts onlywith factor Factor Factor Factor
A inhibits
binding;hencetranscriptional activationat sites1,4, and5 A B C -, Activation
isinhibited, but activation at sites2, 3. and6 is unaffected a) domain Inhibitory
tf factor
R DNA-binding
domain
domain is a random coil that folds into two a-heliceswhen
it binds to the surfaceof a globular domain in a co-activaror. oo oo
In contrast, the nuclear-receptorligand-binding activation r-l--t r-f--.]
domain is a structured globular domain that interactswith a
Site 1 Site 4 Site 5
short cr helix in a co-activator, which probably is a random
coil before it is bound. In both cases, however, specific
(c)
protein-protein interactions between co-activators and the
activation domains permit the transcription factors to stim-
ulate gene expression.
CurrentlS lessis known about the structure of repression
-tFttF
domains. The globular ligand-binding domains of some nu-
Site 1 Site 4 Site 5
clear receptorsfunction as repressiondomains in the absence
of their specific hormone ligand. Like activation domains,
repressiondomains may be relatively short, comprising 15
or fewer amino acids. Biochemical and genetic st;dies indi-
cate that repression domains also mediate protein-protein particular cell at a particular time and how their activities
interactions and bind to co-repressorproteins, forming a are regulated.
complex that inhibits transcription initiation by mechanisms In some heterodimeric transcription factors, however,
that are discussedlater in the chaoter. each monomer has a different DNA-binding specificity.The
resulting combinatorial possibilities increasethe number of
TranscriptionFactorInteractionsIncreaseGene- potential DNA sequencesthat a family of transcription fac-
Control Options tors can bind. Three different factor monomers theoretically
could combine to form six homo- and heterodimericfactors,
Two typesof DNA-binding proteins discussedpreviously- as illustrated in Figure 7-28b. Four different facor
basic-zipper proteins and bHLH proteins-oiten exisi in monomers could form a total of 10 dimeric factors; five
alternative heterodimeric combinations of monomers. monomers, 15 dimeric factors; and so forth. In addition, in-
Other classesof transcription factors not discussedhere hibitory factors are known that bind to some basic-zipper
also form heterodimeric proteins. In some heterodimeric and bHLH monomers, thereby blocking their binding to
transcription factors, each monomer recognizes the same DNA. Ifhen these inhibitory factors are expressed,they re-
sequence.In these proteins, the formation of alternative press transcriptional activation by the factors with which
heterodimers does not increase the number of different they interact (Figure 7-28c). The rules governing the interac-
sites on which the monomers can act but rather allows the tions of members of a heterodimeric transcription factor
activation domains associatedwith each monomer to be class are complex. This combinatorial complexity expands
brought together in alternative combinations that bind to both the number of DNA sites from which these factors
can activate transcription and the ways in which they can be
regulated.
Similar combinatorial transcriptional regulation is
achieved through the interaction of structurally unrelated
transcription factors bound to closely spaced binding sites
transcriptional responsesdepending on which 6ZIp or in DNA. An example is the interaction of two rranscrip-
bHLH monomers that bind to that ,it. expressedin a tion factors, NFAT and AP1, which bind to neighboring
"..

294 C H A P T E R7 | T R A N S C R | p T t o N ACLO N T R O LO F G E N E E X P R E S S | O N

Weak NFAT Weak AP1 Cooperativebinding
b i n d i n gs i t e b i n d i n gs i t e of NFATand AP1
sitesin a compositepromoter-proximal elementregulating
the gene encoding interleukon-Z (lL-Z). Expressionof the
IL-2 geneis critical to the immune response,but abnormal
expressionof IL-2 can lead to autoimmune diseasessuch
as rheumatoid arthritis. Neither NFAT nor AP1 binds to
its site in the lL-2 control region in the absence of the
other. The affinities of the factors for these particular
DNA sequencesare too low for the individual factors to
form a stable complex with DNA. However, when both
NFAT and AP1 are present, protein-protein interactions
between them stabilize the DNA ternary complex com-
posed of NFAT, AP1, and DNA (Figure 7-29). Such coop-
eratiue DNA binding of various transcription factors
results in considerablecombinatorial complexity of tran-
scription control. As a result, the approximately 2000
transcription factors encoded in the human genome can
bind to DNA through a much larger number of coopera-
tive interactions, resulting in unique transcriptional con-
trol for each of the several tens of thousands of human
genes.In the caseof IL-2, transcription occurs only when
both NFAT is activated,resulting in its transport from the
cytoplasm to the nucleus,and the two subunits of API' are
synthesized.These eventsare controlled by distinct signal
transductionpathways (Chapters15 and 16)' allowing
stringent control of IL-2 expression.
Cooperative binding by NFAT and AP1 occurs only
when their weak binding sites are positioned quite close to
each other in DNA. The sites must be located at a precise
distancefrom each other for effectivebinding. Recentstudies
have shown that the requirementsfor cooperativebinding are
not so stringent in the case of some other transcription
factors and control regions.For example, the EGR-1 con-
trol region contains a composite binding site to which the
SRF and TCF transcription factors bind cooperatively(see
Figure 7-23). Becausea TCF has a long' flexible domain
that interactswith SRF.the two proteins can bind cooper-
< FIGURE 7-29 Cooperativebinding of
two unrelatedtranscriptionfactorsto
neighboringsitesin a compositecontrol
element.Bythemselves, both monomeric NFAT
andheterodimeric factors
AP1transcription
binding
havelow affinityfor theirrespective
sitesin the /L-2promoter-proximalregion.
Protein-protei betweenNFATand
n interactions
AP1addto the overallstabilityof the NFAT-
AP1-DNA complex, sothatthe two proteins
bindto the composite [SeeL
sitecooperatively.
Chen etal.1998,Nature392:421
atively when their individual sites in DNA are separated
by any distanceup to 10 basepairs or are inverted relative
to each other.
Multiprotein ComplexesForm on Enhancers
As noted previouslS enhancers generally range in length
from aboui 50 to 200 basepairs and include binding sitesfor
severaltranscription factors. The multiple transcription fac-
tors that bind to a single enhancer are thought to interact'
Analysis of the :70-bp enhancer that regulatesexpression
of B-lnterferon, an important protein in defenseagainstviral
infections in humans, provides a good example of such tran-
scription-factor interactions.The B-interferonenhancercon-
tains four control elements that bind four different tran-
scription factors simultaneously.In the presenceof a small,
abundant protein associatedwith chromatin called HMGI,
binding of the transcription factors is highly cooperative'
similai to the binding of NFAT and AP1 to the composite
promoter-proximal site inthe IL-2 control region (FigureT-29)'
Thi, .oop.."tive binding produces a multiprotein complex
on the B-interferon enhancerDNA (FigureT-30)' The term
enhancesomehas been coined to describesuch large nucleo-
protein complexes that assemblefrom transcription factors
a. th.y bind cooperatively to their multiple binding sites in
an enhancer.
HMGI binds to the minor groove of DNA regardlessof
the sequence and, as a result, bends the DNA molecule
sharply. This bending of the enhancerDNA permits bound
tr"nr.riptio.t factors to interact properly' The inherently
weak, noncovalent protein-protein interactions between
transcription factors are strengthened by their binding to
neighboring DNA sites, which keeps the proteins at very
high relative concentrations.
Becauseof the presenceof flexible regions connectlng
the DNA-binding domains and activation or represston
OS
AND REPRESSOR
ACTIVATORS F TRANSCRIPTION 295

IRF.7
F
^\-
FIGURE7-30 Model of the enhancesomethat forms on the
p-interferon enhancer.Two monomerictranscription factors,IRF_3
and IRF-7,and two heterodimeric factors,)un/AfF-2and p50/ p65
(NF-rB),bind to the four controlelementsin this enhancer.
Cooperative bindingof thesetranscription factorsis facilitatedby
HMGI,which bindsto the minor grooveof DNA and alsointeracts
directlywith the dimericfactors.Bendingof the enhancersequence
resultingfrom HMGI bindingis critrcalto formationof an
enhancesome. DifferentDNA-bendingproteinsact similarlyat other
enhancers. fromD.Thanos
[Adapted andT.Maniatis,1995,Ce//g3:1091.
andM A Wathelet al , 1998,Mot Cett1:5OlI
subjectedto this evolutionaryexperimentationthan would
be the caseif constraintson the spacingbetweenregulatory
elementswere strict, as for most genesin bacteria.
Activatorsand Repressors
of Transcription
r Transcription factors, which stimulate or repress tran-
scription, bind to promoter-proximal regulatory elements
and enhancersin eukaryotic DNA.
r Transcription activators and repressorsare generally
modular proteins containing a singleDNA-binding domain
and one or a few activation domains (for activators) or re_
pression domains (for repressors).The different domains
frequently are linked through flexible polypeptide regions
(seeFigure 7-22).
r Among the most common structural motifs found in
the DNA-binding domains of eukaryotic transcription
factors are the C2H2 zinc finger,homeodomain, basichelix_
Ioop-helix (bHLH), and basic zipper (leucinezipper). All
these and many other DNA-binding motifs conrain one or
296 C H A P T E R7 | T R A N S C R | P T | o N ACLO N T R O LO F G E N EE X P R
more a helicesthat inreract malor grooves in their
cognatesite in DNA.
r Activation and repressiondomains in transcription fac-
tors exhibit a variety of amino acid sequencesand three-
dimensional structures. In general, these functional
domains interact with co-activators or co-repressors,
which are critical to the ability of transcription factors to
m o du l a r eg e n ee x p r e s s i o n .
r The transcription,control regions of most genescontain
binding sites for multiple transcription factors. Transcrip-
tion of such genesvaries dependingon the particular ..p.r-
toire of transcription factors that are expressedand acti-
vated in a particular cell at a particular time.
r Combinatorial complexity in transcription control re-
sults from alternative combination, of -ono-ers that
form heterodimeric transcription facors (seeFigure 7-2g)
and from cooperative binding of transcription factors to
composite control sites (seeFigure 7-29).
r Cooperative binding of multiple activators to nearby
sitesin an enhancerforms a multiprotein complex called
an enhancesome(seeFigure 7-30). Assemblyof enhance-
somesoften requiressmall proteins that bind to the DNA
minor groove and bend the DNA sharply, allowing
bound proteins on either side of the bend to rnteract
more readily.
lE Transcription
Initiationby RNA
Polymerasell
In previous sections,many of the eukaryotic proteins and
DNA sequencesthat participate in transcription and its
control have beenintroduced. In this section,we focus on as-
sembly of transuiption preinitiation complexe.s.The preiniti-
ation complex is an associationof RNA polymerase II and
several protein initiation factors that assembletogether at
the start site and begin to unwind the DNA in preparation
for transcription of the gene. Recall that this eukaryotic
RNA polymeraseII catalyzessynthesisof mRNAs and a few
small nuclear RNAs (snRNAs). Specific activator and
repressorproteins regulate the mechanismsthat control the
assembly of Pol II transcription preinitiation complexes-
and hencethe rate of transcription of protein-coding genes-
and are consideredin the nexr section.
GeneralTranscriptionFactorspositionRNA
Polymerasell at Start Sitesand Assistin
Initiation
In vitro transcriprion
by purifiedRNA polymerase
II requires
the addition of several initiation factors that are seoaiated
from the polymerase during purification. These initiation fac-
tors, which position polymerase molecules at transcription
start sites and help to melt the DNA strands so rhar the tem_
plate strand can enter the active site of the enzyme, are called
general transuiption factors. In contrast to the transcription
ES5IO
N
 Complex
ffi eod."rt: Assemblyof the Pol ll Preinitiation
> FIGURE 7-31 In vitro assemblyof RNApolymerase ll
preinitiationcomplex.Theindicated general transcriptton factors
andpurifiedRNApolymerase ll (Polll)bindsequentially to TATA-box
DNAto forma preinitiation complexATPhydrolysis thenprovides
the energy for unwinding of DNA at the startsite by a TFIIH subunit
As Polll initiates
transcrtptionin the resultingopencomplex, the
polymerase movesawayfromthe promoterand itsCTDbecomes
phosphorylated Invitro,thegeneral transcription factors(except for TFIIB
GL
TBP)dissociate from the TBP-promoter complex, but it is not yet
knownwhichfactorsremainassociated with promoter regions
followingeachroundof transcription initiationin vivo

factors discussedin the previous section' which bind to specific

sitesin a limited number of genes,generaltranscription factors
are required for synthesisof RNA from most genes.
The generaltranscription factors that assistPol II in initia- CTD
tion of transcription from most TATA-box promoters in vitro
have been isolated and characterized.Theseproteins are desig-
nated TFIIA, TFIIB, etc., and most are multimeric proteins.
The largest is TFIID, which consistsof a single 38-kDa TATA-
box-binding protein (TBP) and 13 TBP-associatedfactors
(TAFs). General transcription factors with similar activities
rF,,E
have been isolated from cultured human cells' rat liver'
Drosophila embryos, and yeast.The genesencoding thesepro-
J"
teins in yeasthave beensequencedas part of the completeyeast
genome sequence,and many of the cDNAs encoding human
and DrosophilaPol II general transcription factors have been
cloned and sequenced.In all cases,equivalentgeneraltranscrip-
tion factors from different eukaryotesare highly conserved.
rF,,H
SequentialAssemblyof ProteinsFormsthe Pol ll
TranscriptionPreinitiationComplexin Vitro
1,
The complex of RNA polymeraseII and its general transcrip- Preinitiation
tion factors bound to a promoter and ready to initiate tran- complex
scription is called a preinitiation complex. Understanding how
a preinitiation complex is assembledon a promoter is impor-
tant because,in principle, each step in the processcan be con- ATP
trolled, allowing complex regulation of the overall processof ADP
transcription initiation. Detailed biochemical studies using
DNase I footprinting and electrophoreticmobility shift assays
were used to determine the order in which Pol II and general
Nascent
transcription factors bound to TAIA-box promoters. Because RNA Elongating
the complete, multisubunit TFIID is difficult to purifS re- Pol ll with
searchersused only the isolatedTBP component of this general Releaseof phosphorylated
transcription factor in these experiments.Pol II can initiate generalfactors, CTD
exceptTBP
transcription in vitro in the absenceof the other TFIID subunits.
Figure 7-31 summarizesour current understandingof
the stepwiseassemblyof the Pol II transcription preinitiation
complex in vitro. TBP is the first protein to bind to a TATA-
box promoter. All eukaryotic TBPs analyzed to date have
very similar C-terminal domains of 180 residues'The se-
quenceof this region is 80 percent identical in the yeast and quence and length among different eukaryotes, functions in
hu-"n proteins, and most differencesare conservativesub- the Pol ll-catalyzed transcription of genes encodlng
stitutions. This conserved C-terminal domain functions as snRNAs.) TBP is a monomer that folds into a saddle-shape
well as the full-length protein does for in vitro transcription' structure; the two halves of the molecule exhibit an overall
(The N-terminal domain of TBP, which varies greatly in se- dyad symmetry but are not identical' Like the HMGI and

. 297
T R A N S C R I P T I OI N I T I A T I O NB Y R N A P O L Y M E R A S IEI
 Video: 3D Model of an RNA template strand is bound at the polymerase active site. If the
Polymerasell PreinitiationComplex remaining ribonucleosidetriphosphatesare present,pol II be-
gins transcribingthe templatestrand. As the polymerasetran-
scribes away from the promoter region, another subunit of
t
-t TFIIH phosphorylates the Pol II CTD at multiple sites (see
TFIIF ,- i Figure 7-31).In the minimal in vitro transcription assaycon-
taining only these general transcription factors and purified
TFirfi
RNA polymerase II, TBP remains bound to the TATA box as
the polymerase transcribes away from the promoter region,
but the other general transcription factors dissociate.

The first subunitsof TFIIH to be cloned from hu-

El mans were identified becausemutations in the genes
encoding them cause defects in the repair of damaged
DNA. In normal individuals, when a transcribing RNA
polymerase becomes stalled at a region of damaged tem-
plate DNA, a subcomplex of TFIIH is thought to recognize
the stalled polymerase and then recruit other proteins that
function with TFIIH in repairing the damaged DNA re-
gion. In patients with mutant forms of TFIIH subunits, such
repair of damaged DNA in transcriptionally active genesis
A FIGURE 7-32 Model for the structureof an RNApolymerase
impaired. As a result, affected individuals have extreme
ll preinitiationcomplex.yeastRNApolymerase ll issnownasa skin sensitivity to sunlight (a common cause of DNA dam-
space-fillingmodelwith the direction of transcription to the left.The
templatestrandof DNAisshownin darkblueandthe nontemolate a.ge)and exhibit a high incidence of cancer.Depending on
strandin red.Thestartsiteof transcription the severity of the defect in TFIIH function, theie individu-
isshownasa space-filling
redanddarkbluebasepair.TBpandTFIIB als may suffer from diseasessuch as xerode a pigmento-
areshownasgreenand
yellowwormtraces of thepolypeptide backbone. Structures for TFllE, sum and Cockayne'ssyndrome (Chapter 25
F,
andH havenot beendetermined to highresolution Theirapproximate
positions lyingoverthe DNAin the preinitiationcomplex areshownby In Vivo TranscriptionInitiation by pol ll Requires
ellipses (lightblue),TFilF
for TFIIE (red),andTFI|H (orange). [Adapted
fromG MillerandS Hahn,2006,Nature Additional Proteins
Strua.Biol..13:6031
Although the general transcription factors discussedabove
allow Pol II to initiate transcription in vitro, another general
transcription factor, TFIIA, is required for initiation by pol II
in vivo. Purified TFIIA forms a complex with TBp and
TATA-box DNA. X-ray crystallography of this complex
shows that TFIIA inreracts with the side of TBp that is
upstream from the direction of transcription. Biochemical
experimentssuggestthat in cells of higher eukaryotes,TFIIA
and TFIID, with its multiple TAF subunits, bind first to
TATA-box DNA and then the orher general transcription
factors subsequentlybind as indicated in Figure 7-31.
The TAF subunits of TFIID appear to play a role in initi_
ating transcription from promoters that lack a TAIA box.
For instance, some TAF subunits contact the initiator ele-
ment in promoters where it occurs,probably explaining how
such sequencescan replace a TATA box. Additional TFIID
TAF subunits can bind to a consensussequenceA/G-G-A/
T-C-G-T-G centered =30 base pairs downstream from the
transcription start site in many genesthat lack a TATA_box
promoter. Becauseof its position, this regulatory sequenceis
called the downstream promoter element (DpE). tt. Opn
facilitatestranscription of TAIA-less genesthat contain it by
increasingTFIID binding.
In addition to general transcription factors, specific
activators and repressorsregulate transcription of genesby
Pol II. In the next sectionwe will examine how thesi regula-
tory proteins influence Pol II transcription initiation.

298 ' c H A p r E R7 | T R A N s c R r p t o N AcLo N T R o Lo F G E N EE X p R E s s r o N


TranscriptionInitiation by RNA Polymerasell
r Transcription of protein-coding genes by Pol II can be
initiated in vitro by sequentialbinding of the following in
the indicated order: TBP, which binds to TMA-box DNA;
TFIIB; a complex of Pol II and TFIIF; TFIIE; and finally
TFIIH (seeFigure 7-31).
r The helicase activity of a TFIIH subunit separatesthe
template strands at the start site in most promoters' a
process that requires hydrolysis of ATP. As Pol II begins
transcribing away from the start site, its CTD is phospho-
rylated by another TFIIH subunrt.
r In vivo transcription initiation by Pol II also requires
TFIIA and, in metazoans,a completeTFIID protein, includ-
ing its multiple TAF subunits as well as the TBP subunit.
MolecularMechanisms of
lB
Transcription and Activation
Repression
The repressorsand activators that bind to specificsitesin DNA
and regulate expressionof the associatedprotein-coding genes
do so by trno generalmechanisms.First, theseregulatorypro-
teins act in concert with other proteins to modulate chromatin
structure, inhibiting or stimulating the ability of general tran-
scription factors to bind to promoters.Recall from Chapter 6
thai the DNA in eukaryotic cells is not free but is associated
with a roughly equal massof protein in the form of chromatin.
The basic structural unit of chromatin is the nucleosome,
which is composed of :147 base pairs of DNA wrapped
tightly around a disk-shapedcore of histoneproteins.Residues
within the N-terminal region of each histone, and the C-
terminal regionsof histonesH2A and H2B, calledhistonetails,
extend from the surface of the nucleosomeand can be re-
versiblymodified (seeFigure 6-31b). Suchmodifications'espe-
cially the acetylation of histone H3 and H4 tails, influence the
relative condensation of chromatin and thus its accessibilityto
proteins required for transcription initiation. In addition to
their role in such chromatin-mediatedtranscriptionalcontrol,
activators and repressorsinteract with a large multiprotein
complex called the mediator of transcription complex, or sim-
ply mediator. This complex in turn binds to Pol II and directly
regulatesassemblyof transcriptionpreinitiation complexes.
In this section,we review current understandingof how re-
pressorsand activators control chromatin structure and preini-
iiation complex assembly.In the next section of the chapter,we
discusshow the concentrations and activities of activators and
repressorsthemselvesare controlled,so that geneexpressionis
preciselyaftunedto the needsof the cell and organism.
Formationof HeterochromatinSilencesGene
Expressionat Telomeres,Near Centromeres,and
in Other Regions
For manl' years it has been clear that inactive genes in
eukaryotic cells are often associatedwith heterochromatin,
M O L E C U L A RM E C H A N I S M S
regions of chromatin that are more highly condensed and
stain more darkly with DNA dyes than euchromatin, where
most transcribed genes are located (see Figure 6-33a)'
Regions of chromosomes near the centromeresand telom-
er.i a.td additional specificregionsthat vary in different cell
types are organized into heterochromatin. The DNA in het-
ertchromatin is less accessibleto externally added proteins
than DNA in euchromatin and consequentlyis often referred
to as "closed" chromatin. For instance, in an experiment
describedin Chapter 6, the DNA of inactive genes was
found to be far more resistantto digestion by DNase I than
the DNA of transcribed genes(seeFigure 6-32)'
Chromatin-Mediated Repression in Yeast Study of DNA
regions in S. cereuisiaeth^t behavelike the heterochromatin
oi high.t eukaryotes provided early insight into the
chroiatin-mediated repressionof transcription' This yeast
can grow either as haploid or diploid cells' Haploid cells ex-
hibit one of two possiblemating types, called a and ct' Cells
of different mating type can "mate," or fuse, to generatea
diploid cell (seeFigure 1-6). Vhen a haploid cell dividesby
buddlng, the larger "mother" cell switches its mating type
(seeFigure 21-27). Genetic and molecular analyseshave re-
vealedlhat three genetic loci on yeast chromosome III con-
trol the mating type of yeast cells (Figure 7-33)' Only the
central mating-type locus, termed MAT, is actively tran-
scribed. How the proteins encoded at the MAT locws deter-
mine whether a cell has the a or ct phenotype is explained in
Chapter 21. The two additional loci, termed HML and
HMR, near the left and right telomere, respectively,contain
"silent" (nontranscribed)copies of the a or ct genes'These
seouencesare transferred alternately from HMLa or HMRa
into the MAT locus by a type of nonreciprocal recombina-
tion between sister chromatids during cell division' \fhen
the MAT locus contains the DNA sequencefrom HMLa, the
cells behave as ct cells. \fhen the MAT locus contains the
DNA sequencefrom HMRa, the cells behavelike a cells'
Our interest here is how transcription of the silent mat-
ing-type loci at HML and HMR is repressed'If the genesat
theseloci are expressed'as they are in yeastmutants with de-
fects in the repressingmechanism,both a and ct proteins are
expressed,causing the cells to behave like diploid cells,
which cannot mate. The promoters and UASs controlling
transcription of the a and a geneslie near the center of the
DNA sequencethat is transferred and are identical whether
the sequencesare at the MAT locus or at one of the silent
loci. Tilis indicatesthat the function of the transcription fac-
tors that interact with these sequencesmust somehow be
blocked ^t HML and HMR but not at the MAT locus' This
repressionof the silent loci dependson silencersequenceslo-
caied next to the region of transferred DNA at HML and
HMR (Figure 7-33).If the silenceris deleted,the adjacent
silent locris is transcribed.Remarkably' any geneplaced near
the yeast mating-type silencer sequence-by recombinant
DNA techniques is repressed,or "silenced"' even a tRNA
gene transcrited by RNA polymeraseIII, which usesa dif-
ierent set of general transcription factors than RNA poly-
meraseII uses.
O F T R A N S C R I P T I ORNE P R E S S I OANN D A C T I V A T I O N
299
 Yeast chromosome lll

Teromere "S i l"ei n"c' e r

___ ___l Teromere
e
"7ffi

n s e q u e n c e sa t M A T l o c u s a sequences
at MATlocus
-..l-----------L
-1-l-
LF-

;J-T* a1
FIGURE 7-33 Arrangementof mating-typelocion encoded proteinsspecify the mating-type
phenotype of thecell.The
chromosomelll in the yeastS. cerevisiae.
Silent(unexpresseo,t silencer
sequences nearHML andHMRbindproteinsthat arecritical
mating-typegenes(eithera or ct,depending
on the strain)
are for repression
of thesesilentloci.Haploid
cellscanswitchmating
located
at theHMLlocus. Theopposite mating-typegenesare typesrn a processthat transfersthe DNAsequence fromHMLor
present
at the silentHMRlocusWhentheo or a sequences are HMRIo the transcriptionally activeMATlocus
present
at the MAI locus,
theycanbetranscribed intomRNAs whose

Severallines of evidenceindicate that repressionof the
HML and HMR loci results from a condensedchromatin
silencersequences. For instance,when a geneis placedwithin
a few kilobases of any yeast telomere, its expression is
repressed.In addition, this repressionis relievedby the same
mutations in the H3 and H4 histone tails that interfere with
repressionat the silent mating-typeloci.

Theseresultsindicate that the DNA of the silent loci is inac_
peated multiple times at each yeast chromosome telomere.
Further biochemical studiesshowed that the SIR2 protein is
a,histone deacetylase;it removes acetyl groups on lysinesof
the histone tails. Also, the RAP1, and SIR2, 3, and 4 proteins
bind to one another, and SIR3 and SIR4 bind to the N_
terminal tails of histonesH3 and H4 that are maintained in
a-largely unacetylated state by the deacetylaseactivity
of SIR2. Several experiments using fluorescenceconfocal

( a ) N u c l e ia n d t e l o m e r e s (b)Telomeres (c) SIRSprotein

periphery. (b, c) Confocalmicrographsof yeastcellsIabeledwith a

telomere-specific hybridization
probe(b) and a fruorescent-rabered
yeast nuclei. (a)Confocalmicrograph0.3 pm thick
throughthree antibodyspecificfor SlR3(c) Note that SlR3is locailzedin the
diploidyeastcells,eachcontaining6g telomeres.Telomeres were repressed telomericheterochromatin. similarexperiments with RAp1,
labeledby hybridization
to a fluorescenttelomere_specific
probe SlR2,and SlR4haveshown that theseproteinsalsocolocalize with
(yellow).DNA was stainedred to revealthe nuclei.
The 6g telomeres the repressed telomericheterochromatin[Fromlvt cottaet al , 1996,
coalesceinto a much smallernumberof regionsnearthe nuclear
J CellBiol 134:1349;courtesyof M Gotta.T.Laroche,andS M Gasserl

300 . c H A p r E R7 TRANSCRIpIoNA
| c oL N r R o L o F G E N EE X P E
R5 S
I ON
microscopy of yeast cells either stained with fluorescent-
labeled antibody to any one of the SIR proteins or RAP1 or
hybridized to a labeled telomere-specificDNA probe re-
vealed that these proteins form large, condensedtelomeric
nucleoprotein structures resembling the heterochromatin
found in higher eukaryotes (Figure 7-34). Figure 7-35 de-
picts a model for the chromatin-mediated silencing at yeast
telomeres based on these and other studies. Formation of
heterochromatin at telomeresis nucleatedby multiple RAPl
proteins bound to repeated sequencesin a nucleosome-
free region at the extreme end of a telomere. A network of
protein-protein interactions involving telomere-bound
RAP1, three SIR proteins (2, 3, and 4), and hypoacetylated
histones H3 and H4 createsa stable, higher-order nucleo-
protein complex that includes several telomeres and in
which the DNA is largely inaccessibleto external proteins.
One additional protein, SIR1, is also required for silencing
of the silent mating-type loci. It binds to the silencerregions
associatedwith HML and HMR together with RAP1 and
other proteins to initiate assemblyof a similar multiprotein
silencingcomplex that encompassesHML and HMR.
Chapter 6 that acetylation of lysines neutralizes their positive
chargeand eliminatestheir interaction with DNA phosphate
groups, reducing chromatin condensation' Repressionat
ielome.es and at the silent mating-type loci was defectivein
the mutants with glycine substitutions but not in mutants
with arginine substitutions. Further, acetylation of H3 and
H4 lysinesinterfereswith binding by SIR3 and SIR4 and con-
sequentlypreventsrepressionat the silent loci and telomeres'
Chromatin Condensation in Higher Eukaryotes In
higher eukaryotes, a similar processleads to the formation
oicond.ns.d heterochromatinat centromeresand telom-
eres, as well as repressionof genesat internal chromosome
positions that differ depending on the cell type' But in

< FIGURE 7-35 Schematic modelof

Hypoacetylatedhistone
Si12 N - t e r m i n atla i l s silencing mechanism at yeast telomeres.
Si14
Si13 (Iop)Multiplecopies of RAPlbindto a simple
,,\ repeated sequence at eachtelomere region
Rapl thatlacksnucleosomes SlR3andSlR4bindto
R A P 1a,n dS l R 2b i n d st o S l R 4S l R 2i sa h i s t o n e
deacetylase thatdeacetylates thetailson the
histones neighboring therepeated RAPlbinding
Telomeric S i 1 2S , i 1 4p r o t e i n s
, i13S histonetails
DNA
stte(Middle)Thehypoacetylated
Hypoacetylatedhistone arealsobindingsitesfor SlR3andSlR4, which
N - t e r m i n atla i l s in turn binds additional SlR2, deacetylating
neighboring histonesRepetition of this
process results in spreading of the regionof
hypoacetylated histones with associated SlR2,
SlR3,and SlR4 (Bottom) Interactions between
comolexes of SlR2, SlR3, andSlR4cause the
chromatin to condense andseveral telomeres
to associate, asshownin Figure 7-34.fhe
Nucleosomescondense
a n d m u l t i p l et e l o m e r e s higher-order chromatin structure generated
associate blocks
sterically other proteins from interacting
with the underlying DNA.[Adapted fromM
Grunstein, 1997, Curr. OpinCellBiol9:383|

MoLECULARMECHANIsMsoFTRANSCR|PT|oNREPRESS|oNANDACT|VAT|oN
301
 multicellular organisms,methylation of specificlysineresidues
in histone H3 contribute to chromatin condensationin addi-
tion to the deacetylation of histone lysines. We learned in
Chapter 5 how HP1 (heterochromatinprotein 1) promotes
chromatin condensationby binding to nucleosomesthat are
methylated at lysine 9 of histone H3. BecauseHp1 also
binds to a bistone methyl transferasethat methylatesH3 ly-
sine 9 on neighboring nucleosomes,additional Hpl is re-
cruited to the area. Further associationsbetweenthe individ-
ual HP1 moleculesthemselvesremodel chromatin into a
more compact structure(seeFigure6-34).
sential for maintaining the repression of genes in specific
types of cells and in all of the subsequentcells that develop
from them throughout the life of an organism. Importani
genes regulated by Polycomb proteins include the Hox
genes,encoding master regulatory transcription factors. As
discussed in Chapter 22, different combinations of Hox
transcription factors help to direct the development of spe_
cific tissuesand organs in a developingembryo. Early during
embryogenesis,expressionof Hox genesis controlled by
typical activator and repressor proteins. However, the ex_
quent descendentsof that cell.
Remarkably, virtually all cells in the developing embryo
and adult expressa similar set of Polycomb and Trithorax
proteins and all cells contain the same set of Hox genes.yet
only the Hox genes in cells where they were initially re-
pressed in early embryogenesis remain repressed, even
though the same Hox genesin other cells remain active in
the presenceof the same Polycomb proteins. Consequentl5
as in the caseof the yeast silent-mating-typeloci, the expres-
sion of Hox genes is regulated by a process that involves
more than simply specific DNA sequencesinteracting with
proteins that diffuse through the nucleoplasm.The reason is
the samecollection of Polycomb and Trithorax proteins and
the same Hox DNA sequencesin all cells result in the ex-
pression of specific Hox genesin cells comprising the ante,
rior of an embryo and their repressionin cells located in the
posterior of the embryo.
A current model for repressionby polycomb proteins is
depictedin Figure 7-36. Most Polycomb proteins are subunits
of one of two multiprotein complexes, pRCl and pRC2.
PRC2 is thought to act initially by associatingwith specificre-
pressorsbound to their cognateDNA sequencesearly in em-
bryogenesis.The PRC2 complex contains a subunit with a
SET domain, the enzymaticallyactive domain of severalhis-
tone methyl transferases.This SET domain methylates histone
H3 on lysine 27. The PRC1 complex then binds the methy-
lated nucleosomesthrough dimeric Pc subunits eachcontain-
ing a binding domain (called a chromodomain\ soecjhc for
methylated H3 lysine 27. Binding of the dimeric pi to neigh-
boring nucleosomesis proposed to condensethe chromatin
into a structure rhat inhibits transcription. PRC2 or another
methyltransferase is postulatedto associatewith pRC1, main-
taining methylation of H3 lysine 27 in nucleosomesin the re-
gion. This resultsin associationof the chromatin with pRCl
and PRC2 complexeseven after expressionof the initial re-
pressorproteins in Figure 7-36ahas ceased.
A key feature of Polycomb repressionis its maintenance
in daughter cells through successivecell divisions for the life

PRCl complex

H3. H3. H3. H3

K27tMeK27tMeK27+Me
K

A FIGURE 7-36 Model for repressionby polycombcomplexes. Intoa repressed

chromatin pRC2complexes
structure. or another
(a)Duringearlyembryogenesis repressors
associatewith the pRC2 histonemethyltransferase
associates with pRClcomptexes to
complex.(b)Thisresults (Me)of neiqhborinq
in methylation maintainH3lysine27 methylationof neighboringnucreosomes (not
nucleosomeson histone H3 lysine
27 \K27)Oytfreiff-Oomiin_ shown)As a consequence,PRC 1 associationwith the regionrs
containing
subunitE(z).(c)ThepRClcomplexes bindnucreosomes maintainedwhenexpressionof the repressorproteinsin (a)ceases
methylatedat H3 lysine
27 througha dimeric,
chromodomain_ [Modified from A H Lund and M van Lohuizen,2OO4,Curr Op CettBiol.
subunitPc ThepRClcomplex
containing condenses the chromatin 16:239 l

302 o c H A p r E R7 T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E s s t o N
|
 < EXPERIMENTAL FIGURE 7-37ThC
Cross-linked chromatinimmunoprecipitationmethod
chromatin can revealthe acetylationstate of histones
in chromatin.Histones arelightlycross-linked
to DNAin vivousinga cell-permeable,
I chemical
reversible, cross-linkingagent.
E l s o t a t ea n d s h e a rc h r o m a t i nm e c h a n i c a l l y with acetylated histone tailsare
I Nucleosomes
specific for acetvlated N-terminal shownin green.StepE: Cross-linked
E I ffl*i:tibodv to an
chromatin isthenisolated andsheared
+ Antibody against average lengthof two to threenucleosomes
acetylatedhistone
StepZ: An antibody against a particular
N-terminaltail
acetylatedhistone tailsequence isadded,and
N u c l e o s o m ew i t h (stepE) bound nucleosomes are
acetylatedhistone immunoprecipitated Step4: DNAin the
tails immunoprecipitated chromatin fragments is
released by reversingthecross-link andthenis
quantitated usinga sensitive PCRmethod.The
methodcanbe usedto analyze the in vivo
I associationof anyproteinwith a specific
E I trnrnunoprecipitate sequence of DNAby usingan antibody against
+ the proteinof interestin stepE. [See SE
et al. 1998,
Rundlett Nature 392:831 l

of an organism (-199 years for some vertebrates,2'000
years for a sugar cone pine!). This stability of Hox gene ex-
pression state is thought to result from the distribution of
nucleosomeswith methylated H3 lysine 27 to both daughter
DNA molecules immediately following DNA replication.
Association of PRC1 complexes with these H3 lysine 27-
methylated nucleosomes and methylation of new nucleo-
somes assembledon the replicated DNA on H3 lysine
27 would allow the complete PRC1 complex to be re-
establishedover the sameregion of chromatin in both repli-
cated daughter chromosomes. Although Polycomb repres-
sion may involve additional mechanismsas well, this model
can explain how repressionof specificgenescan be maintained
in all daughter cells derived from an initial cell where the gene
was repressedby a transientlyexpressedset of repressors.
Alternatively, a complex of Trithorax proteins includes a
histone methyl transferasethat methylateshistone H3 lysine
4, a histone methylation associatedwith the promoters of
actively transcribed genes. This histone modification is
thought to create a binding site for histone acetylaseand
chromatin remodeling complexesthat promote transcription
and prevent methylation of histone H3 at lysine 9, prevent-
ing the binding of HP1, and at Iysine 27, preventing the
binding of the PRC1 repressingcomplex. Nucleosomes
marked with histone H3 lysine 4 methylation also are
thought to be distributed to both daughter DNA molecules
during DNA replication. Binding of Trithorax complexesto
nocleosomeswith the histone H3 lysine 4 methylation mark
may causethe same methylation at unmodified histones in-
corporated into the daughter chromatin, leading to perpetu-
ation of the chromatin mark in this region' In this way, in-
heritance of the expression status of Hox genes and other
genes regulated by the Polycomb/Irithorax system is tem-
plat.d through chromatin replication by post-translational
modifications on histones rather than DNA sequence'This
type of inheritance through modifications of chromatin
structure rather than modification of DNA sequenceis re-
ferred to as epigeneticinheritance.
Can DirectHistoneDeacetylation
Repressors
and Methylation at SPecificGenes
The importance of bistone deacetylation and methylation in
chro-atitt-tttediated gene repression has been further sup-
ported by studiesof eukaryotic repressorsthat regulategenes
at internal chromosomal positions. These proteins are now
known to act in part by causingdeacetylationof histonetails
in nucleosomesthat bind to the TATA box and promoter-
proximal region of the genesthey repress.In vitro studies
have shown that when promoter DNA is assembledonto a

MoLECULARMECHAN|SMsoFTRANSCR|PT|oNREPRESS|oNANDACT|VAT|oN
303
 nucleosomewith unacetylatedhistones,the generaltranscrip-
tion factors cannor bind to the TATA box and initiation re-
gion. In unacetylatedhistones,the N-terminal lysinesare pos-
itively charged and inreract strongly with DNA phosphates.
The unacetylatedhistonetails also inreractwith neighboring
histone octamers,favoring the folding of chromatin into con-
densed,higher-order structureswhose preciseconformation
is not well understood. The net effect is that general tran-
scription factors cannot assembleinto a preinitiation com-
plex on a promoter associatedwith hypoacetylatedhistones.
In contrast, binding of general transcription factors is re-
pressedmuch less by histones with hyperacetylatedtails in
which the positively charged lysinesare neutralizedand elec-
trostatic interactionswith DNA phosphatesare eliminated.
The connection between histone deacetylationand re-
pressionof transcriptionat specificyeastpromotersbecame
clearerwhen the cDNA encodinga human histonedeacety-
lase was found to have high homology to the yeast RpD3
gene,known to be required for the normal repressionof a
number of yeast genes.Further work showed that RpD3
protein has histonedeacetylase activity.The ability of RpD3
to deacetylate histonesat a number of promotersdependson
two other proteins:UME6, a repressorthat binds to a spe-
cific upstreamregulatorysequence(URS1),and SIN3, which
is part of a large, multiprotein complex that also contains
RPD3. SIN3 also binds to the repressiondomain of UME6,
thus positioning the RPD3 histonedeacetylasein the complex
so it can interact with nearby promoter-associatednucleo-
somesand remove acetylgroups from histonetail lysines.Ad-
ditional experiments,using the chromatin immunoprecipita-
tion technique outlined in Figure 7-37, demonstrated that in
wild-type yeast, one or two nucleosomesin the immediate
vicinity of UME6-binding sites are hypoacetylated. These
DNA regions include the promoters of genesrepressedby
UME6. In sin3 and rpd3 deletion mutants, not only were
these promoters derepressed,but the nucleosomesnear the
UME6-binding siteswere hyperacetylated.
All these findings provide considerable support for
the model of repressor-directeddeacetylationshown in Fig-
ureT-38a.In this model, the SIN3-RPD3complex funcions
as a co-repressor,Co-repressor complexes containing his-
tone deacetylasesalso have been found associatedwith
many repressorsfrom mammalian cells. Some of thesecom-
plexes contain the mammalian homolog of SIN3 (mSin3),
which interacts with the repressiondomain of the repressor,
as in yeast.Other histone deacetylasecomplexesidentified in
mammalian cells appear to contain additional or different
repressor-bindingproteins. These various repressorand co-
repressor combinations are thought to mediate histone
deacetylationat specificpromoters by a mechanism similar
to the yeast mechanism (Figure 7-38a).
In higher eukaryotes,some co-repressorcomplexesalso
contain histone methyl transferasesubunits that methylate his-
tone H3 at lysine 9, generatinga binding site for Hp1 protein,

> FIGURE7-38 Proposedmechanismof (a) Repressor-directedhistonedeacetvlation

histone deacetylation and hyperacetylation
in yeast transcription control. (a) Repressor-
d i r e c t e dd e a c e t y l a t i oonf h i s t o n eN - t e r m i n a l Deacetvlationof histone
t a i l s .T h e D N A - b i n d i n gd o m a i n( D B D )o f t h e
repressorUfvlE6interactswith a specific
u p s t r e a mc o n t r o le l e m e n t( U R S 1o) f t h e g e n e s
i t r e g u l a t e sT h e U M E 6r e p r e s s i odno m a i n
( R D )b i n d sS l N 3 ,a s u b u n i to f a m u l t i p r o t e i n
c o m p l e xt h a t i n c l u d e sR p D 3 ,a h i s t o n e
deacetylaseDeacetylation of histone
N - t e r m i n atla i l so n n u c l e o s o m ei sn t h e r e g i o n
o f t h e U M E 6 - b i n d r nsgi t ei n h i b r t sb i n d i n go f
generaltranscriptionfactorsat the TATA
box, therebyrepressinggene expression
(b) Activator-directed hyperacetylation of
h i s t o n eN - t e r m i n atla i l s T h e D N A - b i n d i n g
d o m a i no f t h e a c t i v a t o G r C N 4i n t e r a c t sw i t h (b) Activator-di
rectedh istone hyperacetylation
specificupstreamactivatingsequences(UAS)
o f t h e g e n e si t r e g u l a t e sT h e G C N 4a c t i v a t i o n
Hyperacetylation of histone
d o m a i n( A D )t h e n i n t e r a c t sw i t h a m u l t i p r o t e r n GCNS tails
h i s t o n ea c e t y l a sceo m p l e xt h a t i n c l u d e st h e \ N - t e r m i n a l
\
G C N 5c a t a l y t i cs u b u n i t S u b s e q u e n t \
h y p e r a c e t y l a t i oonf h i s t o n eN - t e r m i n atla i l so n
n u c l e o s o m eisn t h e v i c i n i t yo f t h e G C N 4 -
b i n d i n gs i t ef a c i l i t a t e as c c e s so f t h e g e n e r a l
t r a n s c r i p t i ofna c t o r sr e q u i r e df o r i n i t i a t i o n
R e p r e s s i oann d a c t i v a t i o no f m a n yg e n e si n
h i g h e re u k a r y o t eosc c u r sb Vs i m i l a m r echanisms

304 CHAPTER
7 | TRANSCR|pTtoNA
C LO N T R O L
O F G E N EE X P R E S S T O N
 Active
Transgene Heterochromatin
FIGURE 7-39 Associationof a repressedtransgenewith
heterochromatin. Mousefibroblasts werestablytransformed with a
transgenewith bindingsitesfor an engineered repressor.
The
wasa fusionbetween
repressor a DNA-binding domain,a repression
domainthatinteractswith the KAP1co-repressor complex,andthe
domainof a nuclear
ligand-binding receptorthatallowsthe nuclear
importof thefusionproteinto be controlled (see
experimentally
Figure7-49)DNAwasstained blue with the dye
intercalating DAPI
regions
Brighter-staining areregions wherethe
of heterochromatin,
DNAconcentration ishigherthanin euchromatin Thetransgene was
as discussedearlier. For example, the KAP1 co-repressor
complex functions with a class of more than 200 zinc-finger
transcription factors encodedin the human genome.This co-
repressorcomplex includes an H3 lysine 9 methyl trans-
ferasethat methylatesnucleosomesover the promoter region
of repressedgenes,leading to HP1 binding and repressionof
transcription. An integrated transgenein cultured mouse fi-
broblaststhat was repressedthrough the action of the KAP1
co-repressorassociatedwith heterochromatin in most cells,
whereas the active form of the same transgene associated
with euchromatin (Figure 7-39). Chromatin immunoprecipi-
tation assays(seeFigure 7-37) showedthat the repressedgene
was associatedwith histone H3 methylated at lysine 9 and
HP1, whereasthe active genewas not.
Interestingly,in addition to methylation of histone pro-
teins, methylation of the DNA sequenceitself can also be a
trigger for chromatin condensation.The discoveryof mSin3-
containing histone deacetylasecomplexes provided an ex-
planation for earlier observations that in vertebratestran-
scriptionally inactive DNA regions often contain the
modified cytidine residue 5-metbylcytidine (mC) followed
immediately by a G, whereas transcriptionally active DNA
regions contain fewer mC residues.DNA containing 5-
methylcytidine has beenfound to bind a specificprotein that
M o L E C U L A R M E C H A N | s M s o F T R A N S C R | P T | o N R E P R E S S | o N A N D A C305
Repressed
Transgene
detectedby hybridizationof a fluorescently labeledcomplementary
probe(green)Whenthe recombinant repressor wasretained in the
cytoplasm,thetransgene wastranscribed (/eft)andwasassociated
with euchromatin in mostcells.Whenhormone wasaddedsothat
the recombinant repressor entered the nucleus, thetransgene was
repressed(right)and associatedwith heterochromatin-Chromatin
immunoprecipitation assays(seeFigure 7-37)showedthatthe
repressedgenewasassociated with histone H3 methylated at lysine
9 andHP1,whereas theactivegenewasnot.[Courtesy of Frank
fromAyyanathan
Rauscher et al, 2003Genes andDevl7:1855l
in turn interacts specifically with mSin3' This finding sug-
gests that association of mSin3-containing co-repressors
with methylated sites in DNA leads to deacetylationof his-
tones in neighboring nucleosomes,making these regions in-
accessibleto general transcription factors and Pol II and
hencetranscriptionallyinactive.
ActivatorsCan DirectHistoneAcetylationand
Methylation at SPecificGenes
bind
Just as repressorsfunction through co-repressorsthat
to their repressiondomains' the activation domains of DNA-
binding activators function by binding multisubunit co-
actiuator complexes.One of the first co-activator complexes
to be characterized was the yeast SAGA complex, which
functions with the GCN4 activator protein describedin Sec-
tion 7.4. Early genetic studies indicated that full activity of
the GCN4 activator required a protein called GCNS. The
clue to GCNS's function came from biochemical studiesof a
histone acetylasepurified from the protozoan Tetrahymena,
the first histone acetylaseto be purified. Sequenceanalysis
revealed homology between the Tetrahymena protein and
yeast GCN5, which was soon shown to have histone acety-
lase activity as well. Further geneticand biochemical studies
T|VAT|oN
 revealedthat GCN5 is one subunit of a multiprotein co-acti-
vator complex, named the SAGA complex after genes en-
coding someof the subunits.Another subunit of this histone
acetylasecomplex binds to acrivation domains in multiple
yeast activator proteins, including GCN4. The model shown
in Figure 7-38b is consistenrwith the observationthat nu-
cleosomesnear the promoter region of a gene regulated by
the GCN4 activator are specifically hyperacetylated com-
pared to most histonesin the cell. This activator-directedhy-
peracetylationof nucleosomesnear a promoter region opens
the chromatin strucure so as to facilitate the binding of
other proteins required for transcription initiation. The
chromatin structure is less condensedcompared to most
chromatin, as indicated by its sensitivity to digestion with
nucleasesin isolated nuclei. Also, the acetylation of specific
histone lysinesgeneratesbinding sitesfor proreins wiih bro-
modomains that bind them. For example.a subunit of the
general transcription factor TFIID contains two bromod-
omains that bind to acetylatednucleosomeswith high affin-
ity. Recall that TFIID binding ro a promoter initiates assem-
bly of an RNA polymeraseII preinitiation complex (see
Figure 7-31). Nucleosomesar promoter regionsof virtually
all active genesare hyperacetylated.
A similar activation mechanismoperatesin hieher eu-
k a r y o t e s .M a m m a l i a n c e l l sc o n r a i n m u l r i s u b u n i th i s r o n e
acetylaseco-activatorcomplexeshomologous to the yeast
SAGA complex. They also expresstwo related -400-kDa,
multidomain proteins called CBP and P300, which are
thought to function similarly. As noted earlier, one domain
of CBP bindsthe phosphorylatedacidicactivariondomain in
the CREB transcription factor. Other domains of CBp inter-
act with different activation domains in other activators. yet
another domain of CBP has histone acetylaseactivity, and
another CBP domain associateswith additional multisub-
unit histone acetylasecomplexes.CREB and many other
mammalian activators are thought to function in part by di-
recting CBP and the associatedhistone acetylasecomplex to
specific nucleosomes,where they acetylatehistone tails, fa-
cilitating the interaction of generaltranscription factors with
promoter DNA. In addition, the largest TFIID subunit also
has histone acetylaseactivity and may function to maintain
histone tail hyperacetylationin promoter regions.
Methylation of histone H3 lysines9 or 27 resultsin rran-
scriptional repression mediated by binding proteins of the
HP1 or Polycombclass,respectivelgas discussedabove.In
contrast, H3 lysine 4 methylation is observed in the pro-
moter regionsof active genesthrough the rargetingof methyl
transferasesspecificfor H3 lysine 4. One example of this in-
volves the Trithorax complex proteins. As discussedearlier,
Trithorax complex proteins maintain expressionof Hox
g e n e si n a p p r o p r i a t ec e l l s ,j u s t a s p o l y c o m bp r o t e i n sm a i n -
tain repressionof the same Hox genesin other cells. Tritho-
rax proteins, including one with a SET domain that merhy-
lates lysines, assembleinto a multiprotein complex that
tri-methylatesH3 lysine 4. H3 tails tri-methylated at lysine 4
then serveas a binding site for another subunit of the titho-
rax complex so that the methyl transferasesubunit of the
Trithorax complex can maintain H3 lysine 4 in the methy-
305 . c H A p r E R7 | T R A N s c R t p l o N AcLo N r R o L o F G E N EE X p R E s s t o N
lated state in chromatin associatedwith the complex. This is
a similar mechanismto the maintenanceof histone H3 lysine
27 methylation by Polycomb complexes (seeFigure 7-36).
The H3 amino-terminal tail tri-methylated on lysine 4 also
servesas a binding site for co-activator complexes.For ex-
ample, SAGA-like histone acetylasecomplexes also contain
a domain that binds specificallyto tri-methylated H3 lysine
4. This results in acetylation of histone tail lysines,thereby
generatinga chromatin structure conducive to transcription.
Many genesin multicellular organisms in addition to Hox
genesare expressedin lineage-specificexpressionprograms
regulated by Trithorax and Polycomb proteins. This is most
clearly revealedby staining Drosophila salivary gland poly-
tene chromosomeswith antibodies to Polycomb and Tritho-
rax proteins.This experimentrevealsbinding of theseproteins
to more than 100 siteson fly chromosomesin thesecells.
C h r o m a t i n - R e m o d e l i nFga c t o r sH e l pA c t i v a t eo r
RepressTranscription
In addition to histone acetylase complexes, multiprotein
chromatin-remodelingcomplexesalso are required for activa-
tion at many promoters. The first of thesecharacterizedwas
the yeastSWVSNFchromatin-remodelingcomplex.One of the
SWVSNF subunitshas homology to DNA helicases,enzymes
that use energy from AIP hydrolysis to disrupt interactions
between base-pairednucleic acids or between nucleic acids
and proteins. In vitro, the SVIiSNF complex is thought to
pump or push DNA into the nucleosomeso rhat DNA bound
to the surface of the histone octomer transiently dissociates
from the surfaceand translocates,causingthe nucleosomesto
"slide" along the DNA. The net result of such chromatin
remodelingis to facilitate the binding of transcription factors
to specific DNA sequencesin chromatin. Many activation
domains bind to chromatin-remodelingcomplexes,and this
binding stimulates in vitro transcription from chromatin
templates(DNA bound ro nucleosomes).Thus the SWSNF
complex representsanothertype of co-activatorcomplex.The
experiment shown in Figure 7-40 dramatically demonstrates
how an activation domain can cause decondensationof a
region of chromatin. This is thought to result from the inter-
action of the activation domain with chromatin-remodeling
and histoneacetylasecomplexes.
Chromatin-remodelingcomplexesare required for many
processesinvolving DNA in eukaryotic cells, including tran-
scription control, DNA replication, recombination, and re-
pair. Severaltypes of chromatin-remodeling complexes are
found in eukaryotic cells,all with homologous DNA helicase
domains. S\fVSNF complexes and related chromatin-
remodeling complexes in multicellular organisms contain
subunits with bromodomains that bind to acetylatedhistone
tails. Consequently,SI7VSNF complexes remain associated
with activated, acetylatedregions of chromatin, presumably
maintaining them in a decondensedconformation. Some
chromatin-remodelingcomplexescontain subunits that bind
to histone H3 methylated on lysine 4, contributing to rran-
scriptional activation by Trithorax proteins. Surprisingly,
chromatin-remodeling complexes can also participate in

EXPERIMENTAL FIGURE 7-40 Expression of fusionproteins
demonstrateschromatindecondensationin responseto an
activationdomain.A cultured hamster cellIinewasengineered to
containmultiple copies of a tandemarrayof E.colilacoperator
sequences integrated intoa chromosome in a regionof
heterochromatin (a)Whenan expression vectorfor the/acrepressor
wastransfected intothesecells,/acrepressors boundto the /ac
operatorsitescouldbevisualized in a regionof condensed chromatin
usingan antibody against the /acrepressor (red)DNAwasvisualized
by stainingwith DAPI(blue), revealing the nucleus. (b)Whenan
expressionvectorfor the /acrepressor fusedto an activation domain
wastransfected intothesecells,staining asin (a)revealed thatthe
activationdomaincauses thisregionof chromatin to decondense into
a thinnerchromatin fiberthatfillsa muchlarger volume of thenucleus
Bar: 1 pm [Courtesy of Andrew S Belmont, 1999, J CellBiol145:1341 ]
transcriptionalrepression.Thesechromatin-remodelingcom-
plexesbind to transcription repressiondomains of repressors
and contribute to repression,presumably, by folding chro-
matin into condensedstructures.Much remainsto be learned
about how this important classof proteins alters chromatin
structure to influencegeneexpressionand other processes.
HistoneModificationsVary Greatlyin
T h e i rS t a b i l i t i e s
Pulse-chase radiolabelingexperimentshave shown that acetyl
groups on histone lysinesturn over rapidly, whereasmethyl
groups are much more stable.The acetylationstate at a spe-
cific histone lysine on a particular nucleosomeresultsfrom a
dynamic equilibrium between acetylation and deacetylation
by histone acetylasesand histone deacetylases,respectively.
Acetylation of histonesin a localizedregion of chromatin pre-
dominateswhen local DNA-bound activatorstransientlybind
histone acetylasecomplexes. De-acetylation predominates
when repressorstransiently bind histone deacetylasecom-
plexes. In addition to these processes,localized to relatively
short lengths of chromatin, which include promoters and
other transcription-control regions, histone acetylasesand
deacetylases also function globally on all euchromatin, con-
stantly removing and replacinghistonelysine acetylgroups.
In contrast to acetyl groups, methyl groups on histone
lysinesare much more stableand turn over much lessrapidly
than acetyl groups. Histone lysine methyl groups can be
M O L E C U L A RM E C H A N T S MO
removed by recently discovered histone lysine demethylases.
But the resulting turnover of histone lysine methyl groups is
much slower than the turnover of histonelysine acetylgroups.
Multiple other post-translationalmodifications have been
characterizedon histones, many of them summarized in Fig-
ure 6-31b. Theseall have the potential to positively or nega-
tively regulate the binding of proteins that interact with the
chromatin fiber to regulatetranscription and other processes.
A picture of chromatin is emergingin which histone tails ex-
tending as random coils from the chromatin fiber are post-
translationally modified to generate one of many possible
combinations of modifications that regulate transcription
and other processesby regulating the binding of a large num-
ber of different protein complexes.Some of these modifica-
tions, like histone lysine acetylation, are rapidly reversible,
whereas others, like histone lysine methylation, can be tem-
plated through chromatin replication, generating epigenetic
inheritancein addition to inheritanceof DNA sequence.
T h e M e d i a t o rC o m p l e xF o r m sa M o l e c u l a r
Bridge BetweenActivation Domainsand Pol ll
Now let's shift our attention from how activators and re-
pressorscontrol chromatin structure to the other mechanism
of gene regulation outlined in the introduction to this
section-regulation of the assemblyof transcription preiniti-
ation complexes.
Interaction of activators with the multiprotein mediator
complex (Figure 7-41.1directly assistsin assemblyof Pol II
preinitiation complexes.Someof the -30 mediator subunits
bind to RNA polymerase II, and other mediator subunits
bind to activation domains in various activator proteins.
Thus mediator can form a molecular bridge between an ac-
tivator bound to its cognate site in DNA and Pol II at a pro-
moter. In addition, one of the mediator subunits has histone
acetylaseactivity and may function to maintain a promoter
region in a hyperacetylatedstate.
Experimentswith temperature-sensitive yeast mutants in-
dicate that some mediator subunits are required for transcrip-
tion of virtually all yeastgenes.Thesesubunits most likely help
maintain the overall structure of the mediator complex or bind
to Pol II and therefore are required for activation by all activa-
tors. In contrast, other mediator subunits are required for nor-
mal activation or repressionof specific subsetsof genes.DNA
microarray analysis of yeast gene expression in mutants with
defectsin these mediator subunits indicates that each such
subunit influencestranscription of -3-10 percent of all genes
to the extent that its deletion either increasesor decreases
mRNA expression by a factor of rwofold or more (seeFigure
5-29 for DNA microarray technique).Thesemediator subunits
are thought to interact with specific activation domains; thus
when one subunit is defective,transcription of genesregulated
by activators that bind to that subunit is severely depressed,
but transcription of other genesis unaffected. Consistent with
this explanation are binding studies showing that some activa-
tion domains do indeed interact with specific mediator sub-
units. However, recent studies suggestthat most activation
domains may interact with more than one mediator subunit.
' 307
SF T R A N S C R I P T I ORNE P R E S S I OANN D A C T I V A T I O N
 ( a ) Y e a s tm e d i a t o r - P ol ll c o m o l e x < FIGURE7-41 Structureof yeast and human mediator
complexes. (a) Reconstructed imageof mediatorfrom 5 cereyrsiae
boundto Poill N/ultipleelectronmicroscopy imageswerealignedand
computer-processed to producethis averageimagein whichthe three-
dimensional Polll structure(lightorange)is shownassociated with the
yeastmediatorcomplex(darkblue) (b) Diagrammatic representation of
mediatorsubunitsfrom 5 cereyrsr'ae Subunitsshownin the samecolor
arethoughtto form a module Mutationsin one subunitof a module
may inhibitassociatron of othersubunitsin the samemodulewith the
Exiting
restof the complex(c)Diagrammatic representation of human
DNA
mediatorsubunitsThe relativepositionof eachhumanmediator
subunitis arbitraryexceptfor the subunitsthat are homologousto 5
cerevisiae mediatorsubunits IPart(a)fromS Hahn,2004,Nat.Struct.Mol
E n t e r i n gD N A B i o l1 1 : 3 9 4b,a s e d o n D
J a v i s e t a , 2 O O 2 , MCoel l1l 0 : 4 0 9P a r t ( bf r)o m B
Gugliemi el al ,2004,Nuc AcidsRes32:5379Part(c)adapted fromS Malik
andR G Roeder, 2005,Trends BtochemScl 30:256l

or promoter-proximal elements can interact with mediator

associatedwith a promoter becausechromatin, like DNA, is
flexible and can form a loop bringing the regulatory regions
and the promoter close together, as observed for the E. coli
NtrC activator and oso-RNA polymerase (see Figure 7-4).
The multiprotein nucleoprotein complexes that form on eu-
karyotic promoters may comprrse as many as 100 polypep-
tides with a total mass of :3 megadaltons (MDa), as large as
a ribosome.

MED19 MED13 T r a n s c r i p t i o on f M a n y G e n e sR e q u i r e s
MED21
O r d e r e dB i n d i n ga n d F u n c t i o no f A c t i v a t o r s
and Co-activators
'We
can now extend the model of Pol II transcr:iotioninitia-
tion in Figure 7-31 to take into accountthe role of activarors
and co-activators.These accessoryproteins function not
MED18
oniy to make geneswithin nucleosomalDNA accessible to
generaltranscriptionfactors and Pol II but also directly re-
cruit Pol II to promoter regions.
Q C o Km o d u t e

DNA-binding
domain Mediator
Activation
Largemediatorcomplexes,isolatedfrom yeastand from domarn
culturedmammaliancells,are requiredfor mammalian acti-
vators to stimulate transcription by Pol II in vitro. Since
genesencodinghomologsof mediator subunitsare found in
the genomesol C. elegans,Drosophila, and plants, it ap-
pears that most multicellular organismshave homologous TAFs
mediator complexes.About half of the merazoan(multicel-
lular animals)mediator subunitsare clearly homologousto
yeastmediatorsubunits(Figure7-411r,c). But the remaining
TBP TFIIB Pol ll
subunits,which appear to be distinct from any yeasrpro-
t e l n s , m a y i n t e r a c t w i t h a c t i v a t i o nd o m a i n s t h a t a r e n o t
found in yeast.
The various experimentalresultsindicatingthat individ-
FIGURE7-42 Model of several DNA-bound activators
uai mediator subunits bind to specificactivation domains
interacting with a single mediator complex. The abilityof
suggestthat multiple acrivatorsinfluencetranscriptionfrom differentmediatorsubunitsto interactwith specific activation
a single promoter by interactingwith a mediator complex domainsmay contributeto the integrationof signalsfrom several
simultaneously(Figure7-42). Activatorsbound at enhancers activatorsat a singlepromoter Seethe text for drscussron

308 c H A P T E R7 | TRANSCRtpTtoNC
ALONTROL
O F G E N EE X P R E S S I O N

Condensed
chromatin
El
Preinitiation multiprotein co-activator complexes.Theseinclude chromatin-
complex
Recentstudieshave analyzedthe order in which activa-
tors bind to a transcription-control region and interact with
co-activatorsas a gene is induced. Such studiesshow that
assemblyof preinitiation complexesdependson multiple
protein-DNA and protein-protein interactions, as illus-
trated in Figure 7-43,which depictsactivation of the yeast
HO gene. This gene encodesa sequence-specific nuclease five activators are required and HNF1 and HNF3 are miss-
that initiates mating-type switching in haploid yeast cells
(seeFigure 7-33). Activation of the HO gene begins with
M O L E C U L A RM E C H A N I S M sO F T R A N S C R I P T I ORNE P R E S S I OANN D A C T I V A T I O N
< FIGURE 7-43 Orderedbinding and interactionof activators
and co-activatorsleadingto transcriptionof the yeast HO
gene.Stepn: Initially, the HOgeneis packaged intocondensed
chromatinActivation begins whenthe SWl5activator bindsto
enhancer sites1200-1400 basepairsupstream of the startsiteand
interactswith the SWI/SNF chromatin-remodeling complexStep[:
TheSWI/SNF complex actsto decondense the chromatin, thereby
exposing histone tails StepS: A GCN5-containing histone acetylase
complex associateswith boundSWl5andacetylates histone tailsin
the HOlocusasSWI/SNF continuesto decondense adlacent
chromatinStep4:SWl5 is released fromthe DNA,buttheSWI/SNF
andGCN5complexes
because of subunits
remainassociated
of bothcomplexes
withthe HOcontrolregion
thatbindacetylated histone
tailsthroughbromodomains Theiractionallowsthe SBFactivator to
bindseveral in
sites the promoter-proximal region Step E: SBFthen
bindsthe mediator complex. Step@: Subsequent bindingof Polll
andgeneral transcriptionfactorsresultsin assembly of a transcription
preinitiationcomplex whosecomponents aredetailed in Figure7-42-
[ A d a p t e df r o m C J F r ya n d C L P e t e r s o n2, 0 0 1 , C u r r . B i o l1 1 : R 1 8 5S e ea l s o
M P C o s m a e t a l, 1 9 9 9 . C e l l9 7 : 2 9 9 a, n d M P C o s m a e t a l, 2 O O l , M o l C e l l
7 : 1 2 1 3l
binding of the SS7I5 activator to an upstream enhancer.
Bound S\7I5 then interacts with the S\7VSNF chromatin-
remodeling complex and the GCNS-containing SAGA his-
tone acetylasecomplex. Once the chromatin in the HO con-
trol region is decondensedand hyperacetylated' a second
activator,SBE,can bind to severalsitesin the promoter-prox-
imal region. Subsequentbinding of the mediator complex by
SBF then leads to assemblyof the transcription preinitiation
complex containing Pol II and the generaltranscription fac-
tors shown in Figure 7-31.
N7e can now see that the assembly of a preinitiation
complex and stimulation of transcription at a promoter re-
sults from the interaction of severalactivators with various
remodeling complexes, histone acetylasecomplexes, and a
mediator complex. Although much remains to be learned
about these processes,it is clear that the net result of these
multiple molecular eventsis that activation of transcription
at a promoter depends on highly cooperative interactions
initiated by severalactivators. This allows genesto be regu-
lated in a cell-type-specificmanner by specificcombinations
of transcription factors.
The TTR gene,which encodestransthyretin in mammals,
is a good example of this. As noted earlier, transthyretin is
expressedin hepatocytesand in choroid plexus cells' Tran-
scription of the TTR gene in hepatocytesis controlled by at
least five different transcriptional activators (Figute 7-441.
Even though three of these activators-HNF4, C/EBP,and
AP1-are also expressedin cells of the intestine and kidney,
TTR transcription does not occur in these cells, becauseall
ing in kidney and intestine cells. Other hepatocyte-specific
enhancers and promoter-proximal regions that regulate
309
 Enhancer Promoter-proximalregion A two-step selectionprocessis used (Figure 7-45c).The
+TATA bait vector also expressesa wild-type TRP gene, and the
hybrid vector expressesa wild-type LEU gene.Transfected
cells are first grown in a medium that lacks tryptophan and
-1 86 200 bp -100 leucine but contains histidine. Only cells that have taken up
the bait vector and one of the fish plasmids will survive in
Activators
this medium. The cells that survive then are plated on a
H N F 1I medium that lacks histidine. Those cells expressinga fish
Expresseoonty
L
In hepatocytes hybrid that does not bind to the bait hybrid cannot tran-
HNF3
J scribe the HIS geneand consequentlywill not form a colony
on medium lacking histidine. The few cells that express a
A FIGURE 7-44 Transcription-control region of the mouse bait-binding fish hybrid will grow and form colonies in the
transthyretin(rlfl gene.Binding sitesfor thefiveactivatorsrequired absenceof histidine. Recoveryof the fish vectors from these
for transcription
of IIR in hepatocytes areindicated.Thecomplete set colonies yields cDNAs encoding protein domains that inter-
of activators
isexpressed at the required
concentrations to stimulate act with the bait domain.
transcription
onlyin hepatorytes A differentsetof activators
stimulates
transcription
in choroidplexus cells[SeeR Costa etal, 1989,
Mot.Cett
Biol
9:1415, and K Xanthopouluset al , 1989,Proc Nat'l.Acad. Sci USA86:4iil l

Molecular Mechanismsof TranscriptionRepression

additional genesexpressedonly in hepatocytescontain bind-
ing sitesfor other specificcombinations of transcription fac-
tors found only in thesecells, together with those expressed
ubiquitously.
The YeastTwo-HybridSystemExploitsActivator
Flexibilityto DetectcDNAsThat Encode
InteractingProteins
A powerful molecular genetic method called the yeast two-
hybrid systemexploits the flexibility in activator structuresto
identify geneswhose products bind to a specific protein of
interest. Becauseof the importance of protein-protein inter-
actions in virtually every biological process,the yeast two-
hybrid systemis usedwidely in biological research.
This method employs a yeast vector for expressing a
DNA-binding domain and flexible linker region without the
associated activation domain, such as the deleted GAL4
containing amino acids 1-592 (seeFigure 7-21). A cDNA
sequenceencoding a protein or protein domain of interest,
called the bait domain, is fused in frame to the flexible linker
region so that the vector will expressa hybrid protein com-
posed of the DNA-binding domain, linker region, and bait
domain (Figure 7-45a, Ieft). A cDNA library is cloned into
multiple copiesof a secondyeastvector that encodesa strong
activation domain and flexible linker to produce a vector
library expressingmultiple hybrid proteins, eachcontaining a
different fish domain (Figure 7-45a,rigbt).
The bait vector and library of fish vectors are then rrans-
fected into engineeredyeastcells in which the only copy of a
generequired for histidine synthesis(HIS) is under control of
a UAS with binding sitesfor the DNA-binding domain of the
hybrid bait protein. Transcription of the HlS gene requires
activation by proteins bound to the UAS. Transformed cells
that expressthe bait hybrid and an interacting fish hybrid
will be able to activate transcription of the H1S gene (Fig-
ure 7 -45b). This systemworks becauseof the flexibility in the
spacingbetweenthe DNA-binding and activation domains of
eukaryotic activators.
and Activation
r Eukaryotic transcription activators and repressorsexert
their effects largely by binding to multisubunit co-activa-
tors or co-repressorsthat influence assemblyof Pol II tran-
scription preinitiation complexes either by modulating
chromatin structure (indirect effect) or by interacting with
Pol II and generaltranscription factors (direct effect).
r The DNA in condensed regions of chromatin (hete-
rochromatin) is relatively inaccessibleto transcription fac-
tors and other proteins, so that geneexpressionis repressed.
r The interactions of severalproteins with each other and
with the hypoacetylated N-terminal tails of histones H3
and H4 are responsiblefor the chromatin-mediatedrepres-
sion of transcription that occurs in the telomeres and the
silent mating-type loci in S. cereuisiae(seeFigure 7-35).
r Some repression domains function by interacting with
co-repressorsthat are histone deacetylasecomplexes.The
subsequentdeacetylationof histone N-terminal tails in nu-
cleosomesnear the repressor-bindingsite inhibits interac-
tion betweenthe promoter DNA and generaltranscriprion
factors, thereby repressingtranscription initiation (seeFig-
ure 7-38a).
r Someactivation domains function by binding multiprotein
co-activator complexes such as histone acetylasecomplexes.
The subsequent hyperacetylation of histone N-terminal
tails in nucleosomesnear the activator-binding site facili-
tates interactions between the promoter DNA and general
transcription factors, thereby stimulating transcription ini-
tiation (seeFigure 7-38b).
r SWI/SNF chromatin-remodeling factors constitute an-
other type of co-activator. These multisubunit complexes
can transiently dissociateDNA from histone cores in an
AT?-dependentreaction and may also decondenseregions of
chromatin, thereby promoting the binding of DNA-binding
proteins neededfor initiation to occur at some promoters.
r Mediator, another type of co-activator, is an :30-
subunit complex that forms a molecular bridge between

310 C H A P T E R7 | T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S T O N
llll+ Technique
Animation:YeastTwo-HybridSystem

> EXPERIMENTAL FIGURE 7-45 The yeasttwo-hybrid (al Hybridproteins

systemprovidesa way of screeninga cDNAlibrary for
clonesencodingproteinsthat interactwith a specific
proteinof interest,Thisisa commontechnique for screening a
cDNAlibrary for clonesencoding proteins thatinteract with a
specificproteinof interest(a)Twovectorsareconstructed Bait hybrid Fish hYbrid
containing genesthatencodehybrid(chimeric) proteinsIn one
vector(/eft),the codingsequence for the DNA-binding domainof
a transcription factorisfusedto the sequences for a known (b) Transcriptional activation by hybrid proteins in yeast
protein,referred to asthe "bait"domain(lightblue)Thesecond r->
vector (right) expresses an activation domainfusedto a "fish"
domain(green) thatinteracts with the baitdomain.(b)lf yeast
cellsaretransformed with vectors expressing bothhybrids, the Transfect yeast cells
baitandfishportions of the chimeric proteins interact to produce with genes encoding
theactivator bait and fish hybrids
a functional transcriptional activator.
In thisexample,
promotes transcription of a HISgene.Oneendof thisprotein
complex bindsto the upstream activatingsequence (UAS) of the Co-activatorsand
transcriptionpre-
Hi53gene;theotherend,consisting of theactivation domain, initiationcomplex
stimulates assembly of thetranscription preinitiationcomplex
(orange) at the promoter (yellow).
(c)Toscreen a cDNAlibrary for ^€
clones encoding proteins thatinteract with a particular bait ^^^ /\^}
proteinof interest, the libraryisclonedintothevectorencoding H/S mRNA

the activation domainsothathybridproteins areexpressed. The

baitvectorandfishvectors containwild-type selectable genes
(e g , a TRPor IEUgene)Theonlytransformed cellsthatsurvive (c) Fishing for proteins that interact with bait domain
the indicated selectionscheme arethosethatexpress the bait FishcDNA from
hybridanda fishhybridthatinteracts with it Seethetextfor library
discussion [See S Fields
andO Song,1989, Nature 34O:2451
LEU
Fish vector
activation domains and RNA polymerase II by binding
directly to the polymeraseand activation domains.By bind- 1. Transfect into trp, leu, his
mutant yeast cells
ing to severaldifferent activators simultaneouslgmediator 2 . S e l e c tf o r c e l l s t h a t g r o w i n
probably helpsintegratethe effectsof multiple activatorson a b s e n c eo f t r y p t o p h a n
and leucine
a singlepromoter (seeFigure 7-42). 3 . P l a t e s e l e c t e dc e l l s o n m e d i u m
lackinghistidine
r Activators bound to a distant enhancer can interact
with transcription factors bound to a promoter because
DNA is flexible and the intervenins DNA can form a
large loop.
r The highly cooperative assembly of preinitiation com-
plexesin vivo generallyrequiresseveralactivators.A cell must
produce the specificset of activators required for transcrip-
tion of a particular genein order to expressthat gene.
r The yeast two-hybrid system is widely used to detect
cDNAs encoding protein domains that bind to a specific
*
I v
I
protein of interest(seeFigure 7-45).
Colony No colony
formation formation

lE ption-Factor
Regulation of Transcri
Activity
We have seenin the precedingdiscussionhow combinations
of activators and repressorsthat bind to specificDNA regu-
latory sequencescontrol transcription of eukaryotic genes.
Whether or not a specificgenein a multicellular organism is
expressedin a particular cell at a particular time is largely a
consequenceof the nuclear concentrations and activities of
the transcription factors that interact with the regulatory se-
quences of that gene. Which transcription factors are ex-
pressedin a particular cell type, and the amounts produced,
are determined by multiple regulatory interactions between
transcription-factor genes that occur during the develop-
ment and differentiation of a particular cell type' In

O F T R A N S C R I P T I O N . F A C TAOCRT I V I T Y
REGULATION O 311
> FIGURE 7-46 Examples of hormonesthat bind to
nuclearreceptors.Theseandrelated lipid-soluble
hormones bindto receptors
locatedin thecytosolor
nucleusTheligand-receptor
complex f unctions
asa
transcription
activator
Retinoicacid

.\-
NH
o
cH,-cH
' c''oH
"-14-)
,Y-/
Thyroxine

Chapters76 and22,we presentexamplesof suchregulatory
interactions during development and discussthe principles
of development and differentiation that have emergedfrom
theseexamples.
In addition to controlling the expressionof hundredsto
thousandsof specifictranscriptionfactors,cellsalso regulate
the activities of many of the transcription factors expressed
in a particular cell type. For example,transcriptionfactors
are often regulatedin responseto extracellularsignals.Inter-
actions between the extracellular domains of transmem-
brane receptor proteins on the surfaceof the cell and specific
protein ligandsfor thesereceptorsacrivateprotein domains
associated with the intracellulardomainsof theserransmem-
brane proteins,transducingthe signal receivedon the out-
side of the cell to a signalon the insideof the cell that even-
tually reaches transcription factors in the nucleus. In
Chapter 16, we describethe major types of cell-surfacere-
ceptors and intracellular signalingpathways that regulate
t r a n s c r i p t i o n - f a c r oarc t i v i r y .
In this section,we discussthe second major group of
extracellular signals, the small, lipid-soluble hormones-
including many different steroid hormones, retinoids, and
thyroid hormones-that can diffuse throueh plasma and
nuclear membranesand interact directly with the transcrip-
tion factors they control (Figure 7-46). As noted earlier,the
intracellular receptors for most of these lipid-soluble hor-
mones, which constitute the nuclear-receptorsuperfamily,
function as transcription activators when bound to their
ligands.
A l l N u c l e a rR e € e p t o r S
s h a r ea C o m m o n
Domain Structure
Cloning and sequencingof the genesencoding various nu-
clear receptors revealed a remarkable conservation in their
amino acid sequencesand three functional regions (Fig-
ure 7-47). All the nuclearreceptorshave a unique N-terminal
region of variablelength (100-500 amino acids).Portionsof
this variable region function as activation domains in some
nuclear receptors.The DNA-binding domain maps near the
center of the primary sequenceand has a repeat of the Ca
zinc-finger motif. The hormone-binding domain, located
near the C-terminal end, contains a hormone-dependent
activation domain. In some nuclear receptors,the hormone-
binding domain functions as a repressiondomain in the
absenceof lieand.

Estrogenreceptor(ER)

Progesteronereceptor(PR)

Glucocorticoidreceptor(GR)

Thyroxinereceptor(TR)

Retinoicacid receptor(RAR)

Ng Generalprimary structure

V a r i a b l er e g i o n DNA-binding Ligand-binding
( 1 0 0 - 5 0 0a a ) d o m a i n( 6 8a a ) domain 1225-285aal
Amino acid identity: O 42-94o/o 15-57o/o
FIGURE7-47 General design of transcription factors in the C-terminalhormone-binding domainexhibitssomewhatless
nuclear-receptorsuperfamily.The centrally locatedDNA-binding homologyThe N-terminalregionsin variousreceptorsvary in length,
domainexhibitsconsiderablesequencehomologyamong different haveuniquesequences, and may containone or more activation
receptorsand containstwo copiesof the Ca zinc-fingermotif The d o m a i n s[ S e e RM E v a n s1,9 8 8 , S c i e n c e 2 4 0 : 8 8 9 ]

312 . c H A p r E R7 | T R A N s c R t p l o N AcLo N r R o L o F G E N EE X P R E S S I O N
Nuclear-ReceptorResponseElementsContain
Invertedor DirectRepeats
The characteristicnucleotide sequencesof the DNA sites,
called response elements, that bind several nuclear recep-
tors have beendetermined.The sequences of the consensus
responseelementsfor the glucocorticoid and estrogenre-
ceptors are 6-bp inverted repeats separatedby any three
basepairs (Figure7-48a, b). This finding suggestedthat the
cognatesteroid hormone receptorswould bind to DNA as
symmetrical dimers, as was later shown from the x-ray
crystallographic analysis of the homodimeric glucocorti-
coid receptor's Ca zinc-finger DNA-binding domain (see
Figure 7-25c).
Some nuclear-receptorresponseelements,such as those
for the receptors that bind vitamin D3, thyroid hormone, and
retinoic acid, are direct repeatsof the same sequencerecog-
nized by the estrogenreceptor, separatedby three to five base
pairs (Figure 7-48c-e). The specificity for responding to these
different hormones by binding distinct receptorsis determined
by the spacingbetweenthe repeats.The receptorsthat bind to
such direct-repeatresponseelementsdo so as heterodimers
with a common nuclear-receptormonomer called RXR. The
vitamin D3 responseelement, for example, is bound by the
RXR-VDR heterodimer,and the retinoic acid responseele-
ment is bound by RXR-RAR. The monomers composing
theseheterodimersinteractwith eachother in such a way that
the two DNA-binding domains lie in the samerather than in-
verted orientation, allowing the RXR heterodimersto bind to
direct repeatsof the binding site for each monomer. In con-
(a) GRE
5'AGAACA(N)3TGTTC3
3 ' T C T T G T ( N } 3 A C A A G5A'
(bI 5 ' ,A G G T C A ( N ) 3 T G A C C3T' ,
ERE
3 ' ,T C C A G T ( N ) . A C T G GSn ' ,
(c)VDRE 5' AGZ;;A(N).nccr& s'
3 ' T C C A G T ( N ) . T C C A G5T'
5' AGGTCA(N}4AGG3 T 'C A
(d) TRE
,' taaoottr)4TccAGT 5'
(e)
RARE
;:+::Hlill,+::f+;;
A FIGURE 7-48 Consensus sequences of DNAresPonse elements
that bind three nuclearreceptors.Theresponse elements for the
glucocorticoidreceptor(GRE)andestrogen receptor(ERE) contain
invertedrepeatsthat bindthesehomodimeric proteinsTheresponse
elements receptors
for heterodimeric containa commondirectrepeat
separated bythreeto fivebasepairsfor thevitaminD3receptor
(VDRE),thyroidhormonereceptor(TRE), and retinoicacidreceptor
(RARE) Therepeatsequences areindicated by redarrows[See K
, n d A M N a a re t a l , 1 9 9 1 ,C e l l6 5 : 1 7 6 7I
U m e s o n oe t a l , 19 9 1 , C e l l6 5 : 1 2 5 5 a
trast, the monomers in homodimeric nuclear receptors(e.g.,
GRE and ERE) have an inverted orientation.
H o r m o n eB i n d i n gt o a N u c l e a rR e c e p t o r
Regulateslts Activity as a TranscriptionFactor
The mechanism whereby hormone binding controls the activ-
ity of nuclear receptors differs for heterodimeric and homod-
imeric receptors.Heterodimericnuclearreceptors(e.g.,RXR-
VDR, RXR-TR, and RXR-RAR) are located exclusively in the
nucleus.In the absenceof their hormone ligand' they repress
transcriptionwhen bound to their cognatesitesin DNA. They
do so by directing histone deacetylation at nearby nucleo-
somesby the mechanismdescribedearlier (seeFigure 7-38a).
In the ligand-bound conformation' heterodimericnuclear re-
ceptors containing RXR can direct hyperacetylation of his-
tones in nearby nucleosomes,therebyreversingthe repressing
effectsof the free ligand-binding domain. In the presenceof
ligand, ligand-bindingdomains of nuclearreceptorsalso bind
mediator,stimulating preinitiation complex assembly.
In contrast to heterodimeric nuclear receptors' homo-
dimeric receptorsare found in the cytoplasm in the absence
of their ligands.Hormone binding to thesereceptorsleadsto
their translocation to the nucleus. The hormone-dependent
translocation of the homodimeric glucocorticoid receptor
(GR) was demonstrated in the transfection experiments
shown in Figure 7-49. The GR hormone-binding domain
alone mediates this transport. Subsequentstudies showed
that, in the absenceof hormone, GR is anchored in the cyto-
plasm as a large protein aggregatecomplexed with inhibitor
proteins, including Hsp90, a protein related to Hsp70, the
major heat-shockchaperone in eukaryotic cells. As long as
T' , the receptor is confined to the cytoplasm, it cannot interact
with target genes and hence cannot activate transcription.
Hormone binding to a homodimeric nuclear receptor re-
leasesthe inhibitor proteins, allowing the receptor to enter
the nucleus, where it can bind to responseelementsassoci-
ated with target genes(Figure 7-50)' Once the receptor with
bound hormone binds to a response element, it activates
transcription by interacting with chromatin-remodeling and
histone acetylasecomplexesand mediator.
Regulation of Transcription-Factor Activity
r The activitiesof many transcription factors are indirectly
regulated by binding of extracellular proteins and peptides
to cell-surfacereceptors.These receptors activate intracel-
lular signal-transduction pathways that regulate specific
transcription factors through a variety of mechanismsdis-
cussedin Chapter 16.
r Nuclear receptorsconstitute a superfamily of dimeric Ca
ztnc-fnger transcription factors that bind lipid-soluble hor-
mones and interact with specificresponseelementsin DNA
(seeFigure 7-47).
r Hormone binding to nuclear receptors induces confor-
mational changesthat modify their interactions with other
protelns.
O F T R A N S C R I P T I O N - F A C TAOCRT I V I T Y
REGULATION 313
 Video: Hormone-Regulated
Nuclear Translocationof the GlucocorticoidReceptor

(b)

Proteins t-I-a NC
expressed: / \-r

B-Galactosidase Glucocorticoid GR ligand-binding

receptor domain
EXPERIMENTAL FTGURE 7-49 Fusionproteinsfrom expression of the glucocorticoid hormonedexamethasone (Dex).(b)ln cellsthat
vectorcdemonstratethat the hormone-binding domainof the expressed a fusionproteinconsisting of B-galactosidase
andthe
glucocorticoidreceptor(GR)mediatestranslocationto the entireglucocorticoid receptor(GR),the fusionproteinwaspresentin
nucleusin the presenceof hormone.Cultured animalcellswere the cytoplasm in the absence of hormonebut wastransported to
transfected
with expressionvectorsencoding the proteins
diagrammed the nucleus in the presence of hormone.(c)Cellsthat expressed a
at thebottom.lmmunofluorescence with a labeledantibody specific fusionproteincomposed of B-galactosidaseandjusttheGRligand-
for p-galactosidase
wasusedto detectthe exoressed oroteinsin bindingdomain(lightpurple) alsoexhibited hormone-dependent
transfectedcells(a)In cellsthatexpressed B-galactosidasealone,the transportof the fusionproteinto the nucleus. [FromD picardandK R
enzyme waslocalized to the cytoplasm in the presenceandabsence Yamamoto, 1987, EMBO J 5:3333;courtesy
of theauthors
I

r Heterodimeric nuclear receptors (e.g., those for

retinoids, vitamin D, and thyroid hormone) are found only
in the nucleus. In the absence of hormone, they repress
transcription of target genes with the corresponding re-
sponseelement.When bound to their ligands, they activate
transcription.
r Steroid hormone receptors are homodimeric nuclear re-
ceptors.In the absenceof hormone, they are trapped in the
cytoplasm by inhibitor proteins. When bound to their lig-
ands, they can translocateto the nucleusand activate tran-
scription of target genes(seeFigure 7-50).
lf, RegulatedElongationand
Termi nation of Transcri
ption
In eukaryotes,the mechanismsfor terminating transcription
differ for each of the three RNA polymerases.Transcription
of pre-rRNA genesby RNA polymeraseI is terminated by a
mechanism that requires a polymerase-specifictermination
factor. This DNA-binding protein binds to a specific DNA
sequencedownstream of the transcription unit. Efficient ter-
mination requires that the termination factor bind to the
template DNA in the correct orientation. Purified RNA

Hormone

< FIGURE 7-50 Model of hormone-dependentgene activation

by a homodimericnuclearreceptor.In the absence of hormone,
the receptoris keptin the cytoplasm by interactionbetweenits
ligand-binding domain(LBD) andinhibitor proteins.Whenhormone
ispresent,
it diffuses throughthe plasma membrane andbindsto the
Resoonse
element ligand-binding domain,causing a conformational change that
releases
the receptor fromthe inhibitorproteins. Thereceotorwith
boundligandisthentranslocated intothe nucleus, whereitsDNA-
bindingdomain(DBD) bindsto response elements,allowingthe
ligand-bindingdomainandan additional activationdomain(AD)at
the N-terminus to stimulatetranscription of tarqetqenes.

314 C H A P T E R7 | TRANSCR|PT|ONA
CLO N T R O LO F G E N EE X P R E S S I O N
polymerase III terminates after polymerizing a series of U
residues.The deoxy(A),-ribo(U),DNA-RNA hybrid that re-
sults when a stretch of U's are synthesizedis particularly un-
stable compared with all other base-pairedsequences.The
ease with which this hybrid can be melted probably
contributes to the mechanism of termination by RNA
polymeraseIII.
In most mammalian protein-coding genestranscribed by
RNA polymeraseII, once the polymerasehas transcribed be-
yond about 50 bases,further elongation is highly processive
and does not terminate until after a sequenceis transcribed
that directs cleavageand polyadenylation of the RNA at the
sequencethat forms the 3' end of the encodedmRNA. RNA
polymerase II then can terminate at multiple sites located
over a distanceof 0.5-2 kb beyond this poly(A) addition
site. Experiments with mutant genesshow that termination
is coupled to the processthat cleavesand polyadenylatesthe
3' end of a transcript, which is discussedin the next chapter.
Biochemical and chromatin immunoprecipitation experi-
ments suggestthat the protein complex that cleavesand
polyadenylatesthe nascentmRNA transcript at specific se-
quences associateswith the phosphorylated carboxyl-
terminal domain (CTD) of RNA polymeraseII following ini-
tiation (see Figure 7-31). This cleavage/polyadenylation
complex may suppresstermination by RNA polymerase II
until the sequencesignaling cleavageand polyadenylation is
transcribed by the polymerase.
Although transcription termination is unregulated for
most genes, for some specific genes, a choice is made be-
tween elongation and termination or pausing within a few
tens of basesfrom the transcription start site. This choice be-
tween elongationand termination or pausing can be regu-
lated; thus expression of the encoded protein is controlled
not only by transcription initiation but also by control of
transcription elongation early in the transcription unit. We
discusstwo examplesof such regulation next.
Transcriptionof the HIV Genomels Regulated
b y a n A n t i t e r m i n a t i o nM e c h a n i s m
Currentln transcription of the human immunodeficiency
virus (HIV) genome by RNA polymerase II provides the
best-understoodexample of regulatedtranscription termina-
tion in eukaryotes. Efficient expression of HIV genes re-
quires a small viral protein encoded atthe tat locus. Cells in-
fected with tat- mutants produce short viral transcripts that
hybridize to restriction fragments containing promoter-
proximal regions of the HIV DNA but not to restriction
fragments farther downstream from the promoter. In con-
trast, cells infected with wild-type HIV synthesizelong viral
transcripts that hybridize to restriction fragments through-
out the singleHIV transcription unit. Thus Tat protein func-
tions as an antitermination factor, permitting RNA poly-
merase II to read through a transcriptional block. Since
antitermination by Tat protein is required for HIV replica-
tion, further understanding of this gene-control mechanism
may offer possibilities for designing effective therapies for
acquired immunodeficiencysyndrome (AIDS).
ELONGATION
REGULATED
Tat is a sequence-specific RNA-binding protein. It binds
to the RNA copy of a sequencecalled TAR, which is located
near the 5' end of the HIV transcript' The TAR sequence
folds into an RNA hairpin with a bulge in the middle of the
stem (Figure 7-51). TAR contains two binding sites:one that
interacts with Tat and one that interacts with a cellular pro-
tein called cyclin T. As depicted in Figure 7-51', the HIV Tat
protein and cellular cyclin T each bind to TAR RNA and
also interact directly with each other so that they bind coop-
eratively,much like the cooperativebinding of DNA-binding
transcription factors (seeFigure 7-29).lnteruction of cyclin
T with a protein kinase called CDKS activates the kinase,
whose substrateis the CTD of RNA polymeraseII. In vitro
transcription studies using a specificinhibitor of CDK9 sug-
gest that RNA polymerase II molecules that initiate tran-
scription on the HIV promoter terminate after transcribing
-50 bases unless the CTD is hyperphosphorylatedby
CDK9. Cooperative binding of cyclin T and Tat to the TAR
sequenceat the 5' end of the HIV transcript positions CDK9
so that it can phosphorylate the CTD, thereby preventing
termination and permitting the polymerase to continue
chain elongation.
Severaladditional cellular proteins, including Spt4 and
Spt5 and the NELF complex' participate in the processby
which HIV Tat controls elongation versustermination (Fig-
ure 7-51). Experiments with the specific inhibitor of CDK9
mentioned above and with spt4 and sptS yeastmutants indi-
catethat thesecellular proteins are required for transcription
elongation beyond -50 basesfor most cellular genes.But
for most genes,these proteins appear to function constitu-
tively, that is, without being regulated.As discussedin Chap-
ter 8, RNA polymeraseII pausing instigated by Spt4/5 and
NELF is thought to delay elongation until mRNA processing
factors associatewith the phosphorylated CTD. Further
phosphorylation of the CTD by cyclin T-CDK9 (alsoknown
CTD
A FIGURE 7-51 Modelof antiterminationcomplexcomposed
of HIVTat protein and severalcellularproteins.TheTARelement
in the HIVtranscriptcontainssequences recognizedbyTatandthe
cellularprotein
cyclin T.CyclinT and
activates position
helps the
oroteinkinaseCDKgnearitssubstrate, the CTDof RNApolymerase
ll CTDphosphorylation preventsterminationandallowstranscription
to continue.CellularproteinsSpt4andSpt5andthe NELF complex
arealsoinvolved in regulatingHIV termination
transcript P weiet
[See
al , 1998, Cell92:451,T. Wada et al , 1998, GenesDev 12:357; and Y
Yamaguchiet al , 1999, Cell97:41 l
A N D T E R M I N A T I O NO F T R A N S C R I P T I O N .
315
as pTEFb) appears to reversethis pause and allow elonga-
tion to continue. Currently, it is not clear why this processis
not constitutive for the HIV promoter, where cooperative
binding of HIV Tat and cyclin T to the TAR RNA sequence
is required for CDK9 activation and efficient elongation.
Promoter-Proximal Pausingof RNA polymerasell
O c c u r si n S o m eR a p i d l yI n d u c e dG e n e s
The heat-shockgenes(e.g.,hsp70) illustrate another mecha-
nism for regulating RNA chain elongation in eukaryotes.
During transcription of these genes,RNA polymerase II
pausesafter transcribing :25 nucleotidesbut does not ter-
minate transcription (as it does when transcribing the HIV
genome in the absenceof Tat protein). The paused poly-
meraseremains associatedwith the nascent RNA and tem-
plate DNA and then, after a few minutes, continues tran-
scription of the gene. As the first polymerase transcribes
away from the promoter region, another RNA polymeraseII
binds to the promoter, initiates transcription, and pausesaf-
ter transcribing:25 nucleotides,waiting severalminutes be-
'S7hen
fore the process is repeated. heat shock occurs, the
heat-shock transcription factor (HSTF) is activated. Subse-
quent binding of activated HSTF to specificsitesin the pro-
moter-proximal region of heat-shock genes stimulates the
paused polymerase to continue chain elongation and pro-
motes rapid re-initiation by additional RNA polymerase II
molecules, leading to many transcription initiations per
mtnute.
The pausing during transcription of heat-shockgenesini-
tially was discovered in Drosophila, but a similar mecha-
nism has been shown to occur in human cells. Heat-shock
genesare induced by intracellular conditions that denature
proteins(suchas elevatedtemperature,"heat shock"). Some
encode proteins that are relatively resistant to denaturing
conditions and act to protect other proteins from denatura-
tion; others are chaperonins that refold denatured proteins
(Chapter 3). The mechanism of transcriptional control that
evolved to regulate expressionof thesegenespermits a rapid
response:these genes are always paused in a state of sus-
pended transcription and therefore, when an emergency
anses, require no time to remodel and acetylatechromatin
over the promoter and assemblea transcription preinitiation
comolex.
Regulated Elongation and Termination of Transcription
r Different mechanisms of transcription termination are
employed by each of the eukaryotic nuclear RNA poly-
merases.Transcription of most protein-coding genesis not
terminated until an RNA sequenceis synthesizedthat spec-
ifies a site of RNA cleavageand polyadenylation.
r Transcription of the HIV genome by RNA polymeraseII
is regulated by an antitermination mechanismthat requires
cooperative binding by the virus-encodedTat protein and
'114R
cyclin T to the sequencenear the 5, end of the HIV
RNA.
316 C H A P T E R7 | T R A N S C R | p T t O N ACLO N T R O LO F G E N EE X P R E S S | O N
r During transcription of Drosophila heat-shock genes,
RNA polymerase II pauses within the downstream pro-
moter-proximal region; this interruption in transcription is
releasedwhen the HSTF transcription factor is activated,
resultingin very rapid transcription of the heat-shockgenes
in responseto the accumulation of denatured proteins.
W Other Eukaryotic
Transcription
Systems
We concludethis chapterwith a brief discussionof transcrip-
tion initiation by the other two eukaryotic nuclear RNA poly-
merases,Pol I and Pol III, and by the distinct polymerasesthat
transcribe mitochondrial and chloroplast DNA. Although
thesesystems,particularly their regulation,are lessthoroughly
understoodthan transcription by RNA polymeraseII, they are
equally as fundamentalto the life of eukaryotic cells.
T r a n s c r i p t i o Inn i t i a t i o nb y P o l I a n d P o l l l l l s
A n a l o g o u st o T h a t b y P o l l l
The formation of transcription-initiation complexesinvolv-
ing Pol I and Pol III is similar in somerespecrsto assemblyof
Pol II initiation complexes (seeFigure 7-31). However, each
of the three eukaryotic nuclear RNA polymerasesrequires
its own polymerase-specificgeneraltranscription factors and
recognizesdifferent DNA control elements.Moreover, nei-
ther Pol I nor Pol III requiresATP hydrolysis to initiate tran-
scription, whereasPol II does.
Transcription initiation by Pol I, which synthesizespre-
rRNA, and by Pol III, which synthesizestRNAs, 55 rRNA,
and other short, stable RNAs (seeTabIe 7-2), has beenchar-
acterrzedmost extensively in S. cereuisiaeusing both bio-
chemical and geneticapproaches.It is clear that synthesisof
tRNAs and of rRNAs, which are incorporated into ribo-
somes,is tightly coupled to the rate of cell growth and pro-
liferation. However, much remains to be learned about how
transcription initiation by Pol I and Pol III is regulated so
that synthesisof pre-rRNA, 55 rRNA, and tRNAs is coordi-
nated with the growth and replication of cells.
Initiation by Pol I The regulatory elementsdirecting pol I
initiation are similarly located relative to the transcription
start site in both yeast and mammals. A core element span-
ning the transcription start site from -40 to *5 is essential
for Pol I transcription. An additional upstream element ex-
tending from roughly -155 to -60 stimulatesin vitro pol I
transcription tenfold.
Assemblyof a fully active Pol I initiation complex begins
with binding of a multimeric upstream activating factor (UAF)
to the upstreamelement(Figure7-52).Two of the six subunits
composing UAF are histones,which probably participate in
DNA binding. Next, a trimeric core factor binds to the core el-
ement together with TBP, which makes contact with both the
bound UAF and the core factor. Finally a preformed complex
of Pol I and Rrn3p associateswith the bound proteins, posi-
tioning Pol I near the start site. In human cells.TBP is stablv
 I
Pre-rRNApromoter DNA
Upstreamactivating
factor (UAF)
r Corefactor (CF),TPB,
and other factors
Initiation complex
A FfGURE 7-52 ln vitro assemblyof the yeastPolI
transcriptioninitiationcomplex.UAFandCF,bothmultimeric
general
transcriptionfactors,bindto the upstreamelement(UE)and
coreelement, in the promoter
respectively, DNA TBPanda
monomeric factor(Rrn3p) associatedwith RNApolymerase | (Poll)
alsoparticrpate
in formingthe initiation
complex[AdaptedfromN
Nomura,1998,in R M Paule,Transcription
of Ribosomal
RNAGenes by
pp 157-1721
RNA
Eukaryotic Polymerase1 LandesBioscience,
bound to three other polypeptides,forming an initiation fac-
tor called SLI that binds to the core promoter elementand is
functionally equivalentto yeastcore factor plus TBP.
lnitiation by Pol lll Unlike protein-codinggenesand pre-
rRNA genes,the promoter regions of IRNA and SS-rRNA
geneslie entirelywithin the transcribedsequence(Figure7-53).
Two such internal promoter elements,termed the A box and
B box, are presentin all IRNA genes.Thesehighly conserved
sequencesnot only function as promoters but also encode
two invariant portions of eukaryotic tRNAs that are re-
quired for protein synthesis.In SS-rRNA genes,a singlein-
ternal control region, the C box, acts as a promoter.
Three general transcription factors are required for Pol
III to initiate transcription of IRNA and SS-rRNA genesin
vitro. Two multimeric factors, TFIIIC and TFIIIB, partici-
pate in initiation at both IRNA and 5S-rRNA promoters;a
third factor, TFIIIA, is required for initiation at SS-rRNA
promoters. As with assemblyof Pol I and Pol II initiation
complexes, the Pol III general transcription factors bind to
promoter DNA in a definedsequence.
The N-terminal half of one TFIIIB subunit, called BRF
(for TFIIB-related /actor), is similar in sequenceto TFIIB
tRNA gene
Pollll
5S-rRNAgene
P o ll l l
A FIGURE 7-53 TranscriPtion-control elementsin genes
transcribedby RNApolymerase ll1.BothIRNAandSS-rRNA genes
containinternal promoter elements (yellow)located downstream
fromthestartsiteandnamedA, B,andC boxes, asindicated
Assembly complexes
initiation
of transcription on thesegenesbegins
general
with the bindingof Pol-lll-specific factorsTFlllA,
transcription
andTFlllC
TFlllB, to thesecontrolelementsGreenarrowsindicate
strong,sequence-specifrc protein-DNA interactions Bluearrows
indicateinteractions betweengeneral transcriptionfactorsPurple
arrowsindicate between
interactions general factors
transcription
andPollll.lFrom L Schramm andN Hernandez,2002, Genes Dev16:2593I
(a Pol II factor). This similarity suggeststhat BRF and
TFIIB perform a similar function in initiation, namely' to
direct the polymeraseto the correct start site. Once TFIIIB
has bound to either a tRNA or 5S-rRNA gene,Pol III can
bind and initiate transcription in the presenceof ribonu-
cleosidetriphosphates.The BRF subunit of TFIIIB inter-
acts specifically with one of the polymerase subunits
unique to Pol III, accounting for initiation by this specific
nuclear RNA polymerase.
Another of the three subunits composing TFIIIB is TBP'
which we can now see is a component of a general tran-
scription factor for all three eukaryotic nuclear RNA poly-
merases.The finding that TBP participates in transcription
initiation by Pol I and Pol III was surprising, since the pro-
moters recognized by these enzymes often do not contain
TATA boxes. Nonetheless,recent studies indicate that the
TBP subunit of TFIIIB interacts with DNA similarly to the
way it interacts with TATA boxes.
M i t o c h o n d r i aal n d C h l o r o p l a sDt N A sA r e
T r a n s c r i b e bd y O r g a n e l l e - S p e c i fRi cN A
Polymerases
As discussedin Chapter 6, mitochondria and chloroplasts
probably evolved from bacteria that were endocytosedinto
ancestralcells containing a eukaryotic nucleus. In modern-
day eukaryotes, both organellescontain distinct DNAs that
encode some of the proteins essentialto their specific func-
tions. Interestingly, the RNA polymerases that transcribe
T R A N S C R I P T I OSNY S T E M S . 317
O T H E RE U K A R Y O T I C
mitochondrial (mt) DNA and chloroplast DNA are similar
to polymerasesfrom bacteria and bacteriophages,reflecting
their evolutionaryorigins.
Mitochondrial Transcription The RNA polymerasethat
transcribesmtDNA is encodedin nuclear DNA. After syn-
thesisof the enzymein the cytosol, it is imported into the
mitochondrial matrix by mechanismsdescribedin Chapter
13. The mitochondrial RNA polymerases from S. cere-
uisiae and the frog Xenopus laeuis both consist of a large
subunit with ribonucleotide-polymerizingactivity and a
small B subunit (TFBM). Another matrix protein, mito-
chondrial transcription factor A (TFAM), binds to mtDNA
promoters and is essentialfor initiating transcription at the
start sitesused in the cell. The large subunit of yeastmito-
chondrial RNA polymerase clearly is related to the
monomeric RNA polymerasesof bacteriophageT7 and
similar bacteriophages. However, the mitochondrial
enzyme is functionally distinct from the bacteriophage
enzyme in its dependenceon two other polypeptides for
transcription from rhe proper start sites.
The promoter sequencesrecognizedby mitochondrial
RNA polymerases include the transcription start sire.
These promoter sequences,which are rich in A residues.
have been characterizedin the mtDNA from yeast, plants,
and animals. The circular, human mitochondrial genome
contains two related 15-bp promoter sequences,one for
the transcription of each strand. Each strand is transcribed
in its entirery; the long primary transcripts are then
processedto yield mitochondrial mRNAs, rRNAs, and
tRNAs. A secondpromoter appearsto be responsiblefor
transcribing additional copies of the rRNAs. Currently,
there is relatively little understandingof how transcription
of the mitochondrial genomeis regulatedto coordinatethe
production of the few mitochondrial proteins it encodes
with synthesis and import of the thousands of nuclear
DNA-encoded proteins that comprisethe mitochondria.
Chloroplast Transcription ChloroplastDNA is transcribed
by two types of RNA polymerases,one multisubunit protein
similar to bacterial RNA polymerasesand one similar to the
single subunit enzymesof bacteriophageand mitochondria.
The core subunits of the bacterial-typeenzyme,o., B, B,, and
o subunits, are encoded in the chloroplast DNAs of higher
plants, whereassix o7o-like o factors are encodedin the nu-
clear DNA of higher plants. This is another example of the
transfer of genes from organellar genomes to nuclear
genomesduring evolution. In this case,genesencoding the
regulatory transcription initiation factors have been trans-
ferred to the nucleus,where the control of their transcription
by nuclear RNA polymeraseII likely indirectly controls the
expression of sets of chloroplast genes. The bacterial-like
chloroplast RNA polymeraseis called the plastid polymerase
sinceits catalytic core is encodedby the chloroplast genome.
Most chloroplast genesare transcribed by theseenzymesand
have -35 and -10 control regionssimilar to promoters in
cyanobacteria, from which they evolved. The chloroplast
T7-like RNA polymerase is also encoded in the nuclear
318 C H A P T E R7 | T R A N S C R | p T t o N ACLO N T R O LO F G E N EE X P R E S S | O N
genome of higher plants. It transcribes a different set of
chloroplast genes. Curiously, this includes genes encoding
subunits of the bacterial-like multisubunit plastid poly-
merase. As for mitochondrial transcription, currently rela-
tively little is known about how transcription of chloroplast
DNA is regulated.
Other Eukaryotic TranscriptionSystems
r The processof transcription initiation by Pol I and Pol
III is similar to that by Pol II but requires different general
transcription factors, is directed by different promoter ele-
ments, and does not require AIP hydrolysis.
r Mitochondrial DNA is transcribed by a nuclear-
encoded RNA polymerase composed of two subunits.
One subunit is homologous to the monomeric RNA
polymerase from bacteriophage T7; the other resembles
bacterial o factors.
r Chloroplast DNA is transcribed by a chloroplast-
encoded RNA polymerase homologous to bacterial RNA
polymerases,except that it lacks a o factor.
A great deal has been learned in recent years about tran-
scription control in eukaryotes. Genes encoding about
2000 activatorsand repressorscan be recognizedin the hu-
man genome. We now have a glimpse of how the astro-
nomical number of possible combinations of these tran-
scription factors can generate the complexity of gene
control required to produce organisms as remarkable as
those we seearound us. But very much remains to be un,
derstood. Although we now have some understandingof
what processesturn a gene on and off, we have very little
understanding of how the frequency of transcription is
controlled in order to provide a cell with the appropriate
amounts of its various proteins. In a red blood cell precur-
sor, for example, the globin genes are transcribed at a far
greater rate than the genesencoding the enzymes of inter-
mediary metabolism (the so-called housekeepinggenes).
How are the vast differencesin the frequency of transcrip-
'S(hat
tion initiation at various genesachieved? happens to
the multiple interactions between activation domains, co-
activator complexes, general transcription factors, and
RNA polymerase II when the polymerase iniriares tran-
scription and transcribes away from the promoter region?
Do thesecompletelydissociateat promoters that are tran-
scribed infrequently, so that the combination of multiple
factors required for transcription must be reassembled
anew for each round of transcription? Do complexes of
activators with their multiple interacting co-activators
remain assembledat promoters from which re-initiation
takes place at a high rate, so that the entire assemblydoes
not have to be reconstructed each time a polymerase
initiates?
 Much remains to be learned about the structure of chro- KeyTerms
matin and how that structure influencestranscription. What
additional components besidesHP1 and methylated histone activationdomain 288 nuclear rcceptors291
H3 lysine 9 are required to direct certain regions of chro- activators 271 promoter 276
matin to form heterochromatin, where transcription is re- promoter-proximal
antitermination factor 31 5
pressed?Preciselyhow is the structure of chromatin changed elements283
carboxyl-terminal
by activators and repressors,and how does this promote or
d o m a i n( C T D ) 2 8 0 repressiondomain 290
inhibit transcription? Once chromatin-remodeling com-
plexes and histone acetylasecomplexes become associated chromatin-mediated repressors271
with a promoter region, how do they remain associated? repression299 RNA polymerasell279
Current models suggestthat certain subunits of these com- co-activator 293 silencersequences299
plexes associatewith modified histone tails so that the com- co-repressor294 specifictranscription
bination of binding to a specific histone tail modification DNase I footprintrng 286 factors 286
plus modification of neighboring histone tails in the same TATAbox 282
enhancers274
way resultsin retention of the modifying complex at an acti-
295
enhancesome TAIA box-binding
vated promoter region. In some cases,this type of assembly
generaltranscription protein (TBP) 297
mechanismcausesthe complexesto spread along the length
'What factors 296 upstream actrvatlng
of a chromatin fiber. controls when such complexes
heat-shockgenes315 sequences(UASs)285
spread and how far they will spread?
Singleactivation domains have been discoveredto inter- histone deacetylation300 yeast two-hybrid
act with several co-activator complexes. Are these interac- system 3 10
leucine zipper 291
tions transient, so that the same activation domain can in- zinc finger 291
MAT locus (inyeastl 299
teract with several co-activators sequentially?Is a specific
mediator 299
order of co-activator interaction required?How does the in-
teraction of activation domains with mediator stimulate
transcription? Do theseinteractions simply stimulate the as- Review the Concepts
sembly of a preinitiation complex, or do they also influence
the rate at which RNA polymeraseII initiates transcription 1. Describe the molecular events that occur at the lac
from an assembledpreinitiation complex? operon when E. coli cellsare shifted from a glucose-contain-
Transcriptional activation is a highly cooperative ing medium to a lactose-containingmedium.
processso that genesexpressedin a specifictype of cell are
2. The concentration of free phosphate affects transcrip-
expressedonly when the complete set of activators that
tion of some E. coli genes.Describethe mechanismfor this.
control that gene are expressedand activated. As men- '$7hat
3. types of genes are transcribed by RNA poly-
tioned earlier,someof the transcriptionfactors that control
merasesI, II, and III? Design an experiment to determine
expressionof the TTR gene in the liver are also expressed
whether a specificgeneis transcribedby RNA polymeraseII.
in intestinal and kidney cells. Yet the TTR gene is not ex-
p r e s s e di n t h e s e o t h e r t i s s u e s ,s i n c e i t s t r a n s c r i p t i o n r e - 4. The CTD of the largest subunit of RNA polymerase
'What
II
can be phosphorylated at multiple serineresidues. are
quires two additional transcription factors expressedonly
in the liver. \7hat mechanisms account for this highly co- the conditions that lead to the phosphorylated versus un-
operative action of transcription factors that is critical to phosphorylated RNA polymeraseII CTD?
cell-type-specific geneexpression? 5. \What do TAIA boxes, initiators, and CpG islands have
A thorough understanding of normal development and in common? Which was the first of these to be identified?
of abnormal processesassociatedwith diseasewill require whv?
answersto theseand many related questions.As further un- 6. Describe the methods used to identify the location of
derstandingof the principles of transcription control are dis- DNA-control elements in promoter proximal regions of
covered, applications of the knowledge will likely be made. genes.
This understandingmay allow fine control of the expression 7. lfhat is the difference between a promoter-proximal el-
of therapeutic genesintroduced by gene therapy vectors as ement and a distal enhancer?
they are developed.Detailedunderstandingof the molecular
8. Describe the methods used to identify the location of
interactionsthat regulatetranscription may provide new tar-
DNA-binding proteins in the regulatory regions of genes.
gets for the developmentof therapeutic drugs that inhibit or
9. Describe the structural features of transcriptional acti-
stimulate the expressionof specificgenes.A more complete
vator and repressorproteins.
understanding of the mechanismsof transcriptional control
may allow improved engineeringof crops with desirable 10. What happensto transcription of the EGR-I genein pa-
characteristics. Certainly, further advances in the area of tients with Wilm's tumor? Sfhy?
transcription control will help to satisfy our desireto under- 11. Using CREB and nuclear receptors as examples' com-
stand how complex organismssuch as ourselvesdevelop and pare and contrast the structural changesthat take place
function. when thesetranscription factors bind to their co-activators.

R E V I E WT H E C O N C E P T S 319
 12. \lhat general transcription factors associate with an
RNA polymeraseII promoter in addition to the polymerase?
'$fhat
In what order do they bind in vitro? structural change
occurs in the DNA when an "open" transcription-initiation
complex is formed?
L3. Expressionof recombinantproteins in yeast is an im-
portant tool for biotechnology companiesthat produce new
drugs for human use. In an attempt to get a new geneX ex-
pressedin yeast, a researcherhas integrated gene X into the
yeast genome near a telomere. Vill this strategy result in
good expression of gene X? Why or why not? \fould the
outcome of this experiment differ if the experiment had been
performed in a yeast line conraining murations in the H3 or
H4 histonetails?
14. You have isolated a new protein called STICKY. You
can predict from comparisons with other known proteins
that STICKY contains a bHLH domain and a Sin3-interact-
ing domain. Predict the function of STICKY and rationale
for the importance of thesedomains in STICKY function.
15. The yeast two-hybrid method is a powerful molecular
genetic method to identify a protein(s) that interacts with a
known protein or protein domain. You have isolatedthe glu-
cocorticoid receptor (GR) and have evidencethat it is a mod-
ular protein containing an activation domain, a DNA-
binding domain, and a second ligand-binding activation
domain. Further analysisrevealsthat in pituitary cells,the pro-
tein is anchored in the cytoplasm in the absenceof its hor-
mone ligand, a result leading you to speculatethat it binds to
other inhibitory proteins. Describehow a two-hybrid analy-
sis could be used to identify the protein(s) GR interactswith.
How would you specifically identify the domain in the GR
that binds the inhibitor(s)?
1,6. Some heat-shockgenesencodeproteins that act rapidly
to protect other proteins from harsh conditions. Describethe
mechanism that has evolved to regulate the expression of
such genes.
Analyzethe Data
In eukaryotes, the rhree RNA polymerases,pol I, II and III,
each transcribe unique genesrequired for the synthesisof ri-
b o s o m e s : 2 5 Sa n d 1 8 S r R N A s ( P o l I ) , 5 5 r R N A ( p o l I I I ) ,
and mRNAs for ribosomal proteins (Pol II). Researchers
have long speculated that the activities of the three RNA
polymerasesare coordinately regulated according to the de-
mand for ribosome synthesis:high in replicating cells in rich
nutrient conditions and low when nutrients are scarce.To
determine if the activities of the three polymerasesare coor-
dinated, Laferte and colleaguesengineereda strain of yeast
to be partially resistant to the inhibition of cell growth by
the drug rapamycin (2006, GenesDeu.2022030-2040).As
discussedin Chapter 8, rapamycininhibits a protein kinase
(called TOR for target of rapamycin) that regulates the
overall rate of protein synthesisand ribosome synthesis.
When TOR is inhibited by rapamycin, the transcription of
rRNAs by Pol I and Pol III and ribosomal protein mRNAs
by RNA polymeraseII are all rapidly repressed.part of the
C H A P T E R7 | T R A N S C R T P T T O NCAOLN T R O LO F G E N EE X P R E S S | O N
inhibition of Pol I rRNA synthesisresults from the dissoci-
ation of the Pol I transcription factor Rrn3 from Pol I (see
Figure 7-52). ln the strain constructed by Laferte and col-
leagues,the wild-type Rrn3 gene and the wild-type A43
gene, encoding the Pol I subunit to which Rrn3 binds, were
replaced with a gene encoding a fusion protein of the A43
Pol I subunit with Rrn3. The idea was that the covalent fu-
sion of the two proteins would prevent the Rrn3 dissocia-
tion from Pol I otherwise caused by rapamycin rreatment.
The resulting CARA (constitutive association of Rrn3 and
A43) strain was found to be partially resistant to ra-
pamycin. In the absenceof rapamycin, the CARA strain
grew at the same rate and had equal numbers of ribosomes
as do wild-type cells.
a. To analyzerRNA transcription by Pol I, total RNA
was isolated from rapidly growing wild-type (I7T) and
CARA cells at various times following the addition of ra-
pamycin. The concentration of the 35S rRNA precursor
transcribedby Pol I (seeFigure 8-35) was assayedby the
primer-extensionmethod. Sincethe 5' end of the 355 rRNA
precursoris degradedduring the processingof 25S and 18S
rRNA, this method measuresthe relatively short-lived pre-
rRNA precursor. This is an indirect measure of the rate of
rRNA transcription by Pol I. The results of this primer
extension assayare shown below. How does the CARA Pol
I-Rrn3 fusion affect the responseof Pol I transcriotion to ra-
pamycin?
Minutes after
rapamycln 0 20 40 60 80 100 0 20 40 60 80 100
35S rRNA
b. The concentrations of four mRNAs encoding ribo-
somal proteins,RPL30, RPS6a,RPL7a, and RPLS, and the
mRNA for actin (ACTL), a protein presentin the cytoskele-
ton, were assessed in wild-type and CARA cells by Northern
blotting at various times after addition of rapamycin to rap-
idly growing cells (upper autoradiograms). 55 rRNA tran-
scription was assayedby pulse labeling rapidly growing S7T
and CARA cellswith 3H uracil (for 20 minutes)at varioustimes
Minutesafter CARA
rapamycrn 0 20 40 60 80 100 0 20 40 60 80 100
RPL3O
RPS6a
RPLTa
RPLS
ACTl
Minutes after WT CARA
rapamycrn 0 20 40 60 80 100 0 20 40 60 80100
CJ
after addition of rapamycin to the media. Total cellular RNA
was isolatedand subjectedto gel electrophoresisand autora-
diography. The lower autoradiogram shows the region of
the gel containing 55 rRNA. Basedon these data, what can
be concluded about the influence of Pol I transcription on
the transcription of ribosomal protein genesby Pol II and 5S
rRNA by Pol III?
c. To determine if the difference in behavior of wild-
type and CARA cellscan be observedunder normal physi-
ological conditions (i.e., without drug treatment), cells
were subjectedto a shift in their food source,from nutri-
ent-rich media to nutrient-poor media. Under these condi-
tions, in wild-type cells, the TOR protein kinase becomes
inactive. ConsequentlS shifting cells from nutrient-rich
media to nutrient-poor media should result in a normal
physiological responsethat is equivalent to treating cells
with rapamycin, which inhibits TOR. To determine how
the CARA fusion protein affected the responseto this me-
dia shift, RNA was extracted from wild-type and CARA
cells and used to probe microarrays containing all yeast
open reading frames. The extent of RNA hybridization
with the arrays was quantified and is expressedin the
graphs as log2 of the ratio of CARA-cell RNA concentra-
tion to wild-type-cell RNA concentration for each open
reading frame. A value of zero indicates that the two
strains of yeast exhibit the same level of expression for
those specific RNAs. A value of 1 indicates that the CARA
cells contain twice as much of that particular RNA as do
wild-type cells. The graphs below show the number of
open reading frames (y axis) that have values for log2 of
this ratio, indicated by the x axis. The results of hybridiza-
tion to open reading frames encoding mRNAs for
ribosomal proteins are shown by black bars, those for all
other mRNAs by white bars.The graph on the left givesre-
sults for cells grown in nutrient-rich medium, the graph on
the right for cells shifted to nutrient-poor medium for
90 minutes. \fhat do thesedata suggestabout the regula-
tion of ribosomal protein genetranscription by Pol II?
Cells grown in rich media Cells grown in poor media
1
q
o Luscombe.N. M.. et al. 2000. An overview of the structuresof
o)
6
E
z genome-widecomparativeanalysisamong eukaryotes.Science
-2-1 0 1 2 3 -3-2-1 0 1 2 3
Expression
ratio ratio
Expression
(logzCARA/WT) (logzCARA/WT)
References
Control of Gene Expression in Bacteria
Borukhov,S., and J. Lee. 2005. RNA polymerasestructureand
function at lac operon. C. R. Biol.328:576-587.
Halford, S. 8., and J. F. Marko. 2004. How do site-specific
DNA-binding proteins find their targets?Nucleic Acids Res.
32:3040-3052.
Lawson, C. L., et a\.2004. Cataboliteactivator protein: DNA
binding and transcription activation. Cwrr. Opin. Struct. Biol.
14:1.0-20.
Lewis, M. 2005. The lac repressor.C. R. Biol.328:521'-548.
Muller-Hill, B. 1,998.Somerepressorsof bacterialtranscription.
Curr. C)pin.Microbiol. 1:145-151.
Murakami, K. S., and S. A. Darst. 2003. BacterialRNA poly-
merases:the whole story.Curr. Opin. Struct. Biol. 13:31'-39.
Wigneshweraraj,S. R., et al. 2005. The secondparadigm for ac-
tivation of transcription.Prog. Nucleic Acid Res.Mol. Biol.
79:339-369.
Overview of Eukaryotic Gene Control and RNA
Polymerases
Boeger,H., et al.. 2005. Structural basisof eukaryoticgenetran-
scription. FEBSLett. 579:899-903.
Regulatory Sequences in Protein-Coding Genes
Brenner,S., et al. 2002. Conservedregulation of the lympho-
cyte-specificexpressionof lck in the Fugu and mammals.Proc. Natl.
Acad. Sci.USA 99:2936-2941.
Dean, A. 2005. On a chromosomeIar, far away:LCRs and gene
expression.TrendsGenet.22:3845.
Gaszner,M., and G. Felsenfeld.2005. Insulators:exploiting tran-
scriptionaland epigeneticmechanisms.Nat. Reu.Genet.T:703-713.
Kleinian, D. A., and V. van Heyningen.2005. Long-range
control of geneexpression:emergingmechanismsand disruption in
disease.Am. J. Hum. Genet.76:8-32.
Nobrega, M. A., L Ovcharenko,Y. AfzaI,and E' M' Rubin.
2003. Scanninghuman genedesertsfor long-rangeenhancers.Scl-
ence 302:473.
Smale,S. T., and J. T. Kadonaga'2003. The RNA polymeraseII
core promoter.Annu. Reu.Biochem. T2:449479.
Activators and Repressors of Transcription
Brivanlou, A. H., and J. E. Darnell, Jr.2002. Signaltransduction
and the control of geneexpression.Science295:813-818'
Broun, P. 2004. Transcription factors as tools for metabolic en-
gineeringin plants. Curr. Opin. Plant Biol.7:202:209-
Garvie, C. W., and C. Wolberger.2001. Recognitionof specific
DNA sequences. Mol. Cell. 8:937-946.
Kadonaga,I.T. 2004. Regulation of RNA polyrneraseII transcrip-
tion by sequence-specificDNA binding factors. Cell ll6:247J57.
protein-DNA complexes.Genome Biol. t:I-37.
Marmorstein, R., and M. X. Fitzgerald.2003. Modulation of
DNA-binding domains for sequence-specific DNA recognition.
Gene 304:I-'12.
Riechmann,J. L., et al. 2000. Arabidopsistranscription factors:
290:2105J1'1.0.
Tupler,R., G. Perini, and M. R' Green.2001. Expressingthe hu-
man genome. Nature 409:832-833.
Transcription lnitiation by RNA Polymerase ll
Hahn, S. 2004. Structureand mechanismof the RNA polymerase
II transcription machinery. Nat. Struct. MoL Biol' 11:394403'
REFERENCES 321
Molecular Mechanisms of Transcription Repression and
Activation
Courey,A. J., and S.Jia. 2001. Transcriptionalrepression:rhe
long and the short of it. GenesDeu. 15:2786-2796.
Green,M. R. 2005. Eukaryotic transcription activation:right
on target. Mol. Cell 18:399402.
Horn, P.J., and C. L. Peterson.2005. Heterochromatrnassem-
bly: a new twist on an old model. ChromosomeRes.14:83-94.
Klose, R. J., and A. P. Bird. 2006. GenomicDNA methylation:
the mark and its mediators.TrendsBiochem. Sci.3l:89-97.
Kornberg, R. D. 2005. Mediator and the mechanismof tran-
scriptionalactivation. TrendsBiochem. Sci.30:235-239.
Levine,S. S., I. F. King, and R. E. Kingston.2004. Division
of labor in polycomb group repression.Trends Biochem. Sci.
29:478-485.
Lonard, D. M., and B. W. O'Malley.2006. The expandingcos-
mos of nuclearreceptorcoactivators.Cell 125:41,1414.
Lund, A. H., and M. van Lohuizen.2004. Polycombcomplexes
and silencingmechanisms.Curr. Opin. Cell Biol. 16:239-246.
Malik, S., and R. G. Roeder.2005. Dynamic regulationof pol II
transcription by the mammalian Mediator complex. Trends
Biochem.Sci.3O:256-263.
Margueron, R., P. Trojer, and D. Reinberg.2005. The key to de-
velopment:interpretingthe histone code?Curr. Opin. Genet.Deu.
15:1,63-176.
Martin, C., and Y. Zhang.2005. The diversefunctions of his-
tone lysinemethylation. Nat. Reu.Mol. Cell Biol.6:838-849.
Mohrmann, L., and C. P. Verriizer.2005. Composition and
functional specificityof S'Wi2/SNF2classchromatin remodeling
complexes.B io chim. B iophys. Acta 168l :5 9-7 3.
Roeder,R. G. 2005. Transcriptionalregulationand the role of
diversecoactivatorsin animal cells. FEBSLett.579:909-91.5.
Smith, C. L., and C. L. Pererson.2005. ATP-dependentchro-
matin remodeling.Curr. Top. Deu. Biol. 65:115-i,48.
. Spiegelman,B. M., and R. Heinrich.2004. Biologicalcontrol
through regulatedtranscriptionalcoactivators.Cetl llgir 57-167.
Struhl, K. 2005. Transcriptionalactivation:mediator can act af-
ter preinitiation complex formation. Mol. Cell 17:752-754.
Taatjes,D.J., M. T. Marr, and R. Tjian. 2004. Regulatorydiver-
srty among metazoanco-activatorcomplexes.Nat. Reu.Mol. Cell
Biol.5:403410.
Thiel, G., M. Lietz, and M. Hohl. 2004. How mammalian tran-
scriptionalrepressorswork. Eur. J. Biocbem. 27 l:28 5 5-2862.
Trojer, P.,and D. Reinberg.2005. Histone lysinedemethylases
and their impact on epigenetics.Cell 125:273-217.
Villard, J.2004. Transcriptionregulationand human diseases.
9uiss Med. l(rkly. 134:571,-579.
Wang, W. 2003. The SITVSNFfamily of ATP-dependentchro-
matin remodelers: similar mechanismsfor diverse functions. Curr.
Top. Microbiol. Immunol. 274:143-169.
Regulation of Transcription-Factor Activity
Boisvert,F. M., C. A. Chenard,and S. Richard. 2005. protein
interfacesin signalingregulatedby argininemethylation.Sci.
STKE2Tl:re2.
322 C H A P T E R7 | T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S T O N
Brahimi-Horn, C., N. Mazt;:e, and J. Pouyssegur.2005. Sig-
nalling via the hypoxia-induciblefactor-lalpha requiresmultiple
posttranslationalmodifications. Cell. Signal.17:1-9.
Gardner,K. H., and M. Montminy. 2005. Can you hear me
now? Regulatingtranscriptionalactivatorsby phosphorylation.Sci.
STKE 301:pe44.
Glozak,M. A., N. Sengupta,X. Zhang, and E. Seto.2005. Acery-
lation and deacetylationof non-histoneproteins. Gene 363:1.5-23.
Khidekel,N., and L. C. Hsieh-I7ilson.2004. A "molecular
switchboard"-covalent modificationsto proteins and their impact
on transcription. Org. Biomol. Chem. 7:1-7.
Moehren, U., M. Eckey,and A. Baniahmad.2004. Generepres-
sion by nuclearhormone receptors.EssaysBiochem. 40:89-104.
Picard,D. 2005. Chaperoningsteroidhormone action.Trends
Endocrinol. Metab. 17:229-235.
Rosenfeld,M. G., V. V. Lunyak, and C. K. Glass.2005. Sensors
and signals:a coactivator/corepressor/epigeneticcode for integrating
signal-dependent programs of transcriptionalresponse.GenesDeu.
2006 2U1405-1,428.
Regulated Elongation and Termination of Transcription
Peterlin,B. M., and D. H. Price.2005. Controlling the elonga-
tion phaseof transcriptionwith P-TEFb.Mol. Cell 23:297-305.
Saunders,A., L.J. Core, and J. T. Lis. 2005. Breakingbarriers
to transcriptionelongation.Nat. Reu.MoL Cell Biol. 7:557-567 .
Winston, F. 2001. Control of eukaryotic transcriptionelonga-
tion. GenomeBiol. 2:reviews1005.
Yamada,T., et al. 2005. P-TEFb-mediatedphosphorylationof
hSpt5 C-terminal repeatsis critical for processivetranscription elon-
gation. Mol. Cell 2I:227-237.
Other Eukaryotic Transcription Systems
Gaspari,M., N. G. Larsson,and C. M. Gustafsson.2004.The
transcriptionmachineryin mammalian mitochondria. Biochim. Bio-
phys. Acta 1659:148-'152.
Grummt, I. 2003. Life on a planet of its own: regulationof
RNA polymeraseI transcriptionin the nucleolus.GenesDeu.
17:1691-1.702.
Kanamaru,K., and K. Tanaka.2004. Rolesof chloroplastRNA
polymerasesigma factors in chloroplastdevelopmentand st.essre-
sponsein higher plants. Biosci. Biotechnol.Biochem.
68:2215-2223.
Nomura, M.2001. RibosomalRNA genes,RNA polymerases,
nucleolar structures,and synthesisof rRNA in the yeastSaccharomyces
cereuisiae.Cold Spring Harb. Symp. Quant. Biol.66:555-565.
Raska,I., et a\.2004. The nucleolusand transcriorionof riboso-
mal genes.Biol. Cell.96:579-594.
Schramm,L., and N. Hernandez.2002. Recruitmenrof RNA
polymeraseIII to its rargetpromorers.GenesDeu. 16:2593-2620.
Shiina,T., Y. Tsunoyama,Y. Nakahira, and M. S. Khan. 2005.
PlastidRNA polymerases,promoters,and transcription regulators
in higher plants.Int. Reu.Cytol.244:L-68.
'$7.
Shutt, T. E., and M. Gray. 2005. Bacteriophageorigins of
mitochondrial replication and transcriptionproteins. TrendsGenet.
22:90-95.
'White,
R. J. 2004. RNA polymerasesI and III, growth control
and cancer.Nat. Reu.Mol. Cell Biol.6:69-78.
 CHAPTER

POST-
TRANSCRIPTIONAL
GENECONTROL
Portionof a "lampbrushchromosome"from an oocyteof the
newt Nophtha/musviridescens;hnRNPproteinassociated with
nascentRNAtranscriptsfluorescesred afterstarningwith a
monoclonalantibody[Courtestyof M RothandJ Gall]

I n the previous chapter, we saw that most genesare regu- the amount of protein produced depends on the stability of
I lated at the first stepin geneexpression,namely,the initia- the corresponding mRNAs in the cytoplasm and the rate of
I tion of transcription. However, once transcription has been their translation. For example, during an immune response'
initiated, synthesisof the encoded RNA requires that RNA lymphocytes communicate by secretingpolypeptide hormones
polymerasetranscribe the entire geneand not terminate pre- called cytokines that signal neighboring lymphocytes through
maturely. Moreover, the initial primary transcripts produced cytokine receptors that span their plasma membranes (Chap-
from eukaryotic genes must undergo various processing ter 24). It is important for lymphocytes to synthesize and
reactions to yield the corresponding functional RNAs. For secretecytokines in short bursts. This is possible because
mRNAs, the 5' cap structure necessaryfor translation must cytokine mRNAs are extremely unstable. ConsequentlS the
be added (seeFigure 4-14), introns must be spliced out of concentration of the mRNA in the cytoplasm falls rapidly
pre-mRNAs, and the 3' end must be polyadenylated (see once its synthesis is stopped. In contrast' mRNAs encoding
Figure 4-15). Once formed in the nucleus, mature, func- proteins required in large amounts that function over long
tional RNAs are exported to the cytoplasm as components periods, such as ribosomal proteins, are extremely stable so
of ribonucleoproteins. Both processingof RNAs and their ihat multiple polypeptides are transcribed from each mRNA.
export from the nucleusoffer opportunities for further regu- In addition to regulation of pre-mRNA processing'nuclear
lating geneexpressionafter the initiation of transcription. export, and translation, the cellular locations of some mRNAs
Recently, the vast amount of sequencedata on human are regulated so that newly synthesizedprotein is concentrated
cDNAs has revealedthat -60 percent of human genesgive
rise to alternatively spliced mRNAs. These alternatively OUTLINE
spliced mRNAs encode related proteins with differencesin
sequenceslimited to specific functional domains. In many 8.1 of EukaryoticPre-mRNA
Processing 325
cases,alternative RNA splicing is regulatedto meet the need
for a specificprotein isoform in a specificcell type. Given the 8.2 Regulationof Pre-mRNAProcessing 337
complexity of pre-mRNA splicing, it is not surprising that
8.3 Transportof mRNAAcrossthe Nuclear
mistakes are occasionally made, giving rise to mRNA pre-
Envelope
cursors with improperly spliced exons. However, eukaryotic
cells have evolved RNA surueillance mechanismsthat pre- 8.4 I
ic Mechanismsof Post-transcriptiona
Cytoplasm
vent the transport of incorrectly processedRNAs to the cy- control 347
toplasm or lead to their degradation if they are transported.
8.5 of rRNAand IRNA
Processing 358
Additional control of gene expressioncan occur in the
cytoplasm. In the caseof protein-coding genes,for instance,

323
 Nucleolus

DNArlr polll 'lrrrrr tt pollll -t lll ll polI lll

'--'
o
Pre-rRNA
cap t r an s c r p
i tion

Ribosomal
subunit
s y n t h e s i si n o
nucleolus pre-rRNA

lmproperly
processeo Exosome
Correctly

o
processeo mRNA
mRNA

Excised
pre-tRNA
mRNA tRNA Ribosome
export Nucleus export export

Cytoplasm

AAAAA

Cytoplasmic
exosome

Cytoplasmic
polyadenylation

AAAAA

miRNA
t r a n s l a t i o ni n h i b i t i o n Translation Cytoplasmic
initiation deadenylation

FIGURE 8-1 Overviewof RNAprocessing and post- bycytoplasmic exosomes Thedegradation rateof eachmRNAis
transcriptional genecontrol.Nearly allcytoplasmicRNAs are controlled,
thereby regulatingthemRNAconcentration and,
processed fromprimary transcriptsin the nucleus beforetheyare consequently, the amountof proteintranslated SomemRNAs are
exported to the cytoplasmForproteincodinggenestranscribed by synthesizedwithoutlongpoly(A) tailsTheirtranslation
isregulatedby
RNApolymerase ll,genecontrol canbeexertedthroughI thechoice @ controlling thesynthesisof a longpoly(A) tailbya cytoplasmic
of alternative
exonsduringpre-mRNA splicingandthe Z choice of poly(A)polymerase Z Translatronisalsoregulated byother
alternativepoly(A) siteslmproperly processed mRNAs areblocked from mechanisms including miRNAs. Whenexpressed, these-22 nucleotide
exportto thecytoplasm anddegraded p bya largecomplex called the RNAs inhibittranslationof mRNAs to whichtheyhybridize,usuallyin
exosome thatcontains multiple ribonucleases Onceexported to the the3'-untranslated region.tRNAs andrRNAs arealsosynthesizedas
cytoplasm, @ translation initiation
factors bindto themRNA5,_cap precursor
RNAsthat mustbe S processed beforetheyarefunctional
cooperativelywithpoly(A)-binding proteinI boundto thepoly(A) tail Regionsof precursors cleavedfromthe matureRNAsaredegraded by
andinitiatetranslation (seeFigure4-29)E mRNAisdegraded in the nuclear exosomes I lAdaptedfrom Houseleyet.al. Nat RevMot CettBiot.
cytoplasm byde-adenylation anddecapping followedbydegradation QOO6\7:529 I

324 C H A P T E R8 | P o S T - T R A N S C R t p T t o NG
AEL N EC O N T R O L
where it is needed.Particularlystriking examplesof this occur
in the nervoussystemsof multicellularanimals.Someneurons
in the human brain make more than 1000 separatesynapses
with other neurons.During the processof learning, synapses
that fire more frequently than others increase in size many
times, even though other synapsesmade by the sameneuron
do not. This can occur becausemRNAs encoding proteins
critical for formation of an enlarged synapseare stored at
all synapses.Then the translation of these localized, stored
mRNAs is regulatedat eachsynapseindependentlyby the fre-
quency at which it initiates translation. In this way, synthesis
of synapse-associated proteinscan be regulatedindependently
at each of the many synapses made by the sameneuron.
Another type of generegulation that has recentlycome to
light involvesmicro RNAs (miRNAs), which regulatethe sta-
bility and translation of specifictarget mRNAs in multicellu-
lar animals and plants. Analysesof these short miRNAs in
various human tissuesindicatethat there are -1000 miRNAs
expressedin the multiple typesof human cells.Although some
have been recently discoveredto function through inhibition
of target geneexpressionin the appropriate tissueand at the
appropriatetime in development,the functionsof the vast ma-
jority of human miRNAs are unknown and are the subiectof
a growing new area of research.If most miRNAs do indeed
have significant functions, miRNA genesconstitute a signifi-
cant fraction of the :25,000 human genes.A closelyrelated
processcalled RNA interference(RNAi) leadsto the degrada-
tion of viral RNAs in infected cells and the degradation of
transposon-encodedRNAs in most eukaryotes. This is of
tremendoussignificanceto biological researchersbecauseit is
possibleto design short interfering RNAs (siRNA) to inhibit
the translationof specificmRNAs experimentallyby a process
called RNA knockdown. This makesit possibleto inhibit the
function of any desiredgene,even in organismsthat are not
amenableto classicgeneticmethodsfor isolating mutants.
\7e refer to all the mechanismsthat regulategeneexpres-
sion following transcription as post-transcriptionalgene con-
trol (Figure 8-1). Sincethe stability and translation rate of an
mRNA contribute to the amount of protein expressedfrom a
gene,thesepost-transcriptionalprocessesare important com-
ponents of genecontrol. Indeed,the protein output of a gene
is regulatedat every step in the life of an mRNA from the ini-
tiation of its synthesisto its degradation.Thus geneticregu-
latory processesact on RNA as well as DNA. In this chapter,
we considerthe eventsthat occur in the processingof mRNA
following transcription initiation and the various mecha-
nisms that are known to regulatetheseevents.In the last sec-
tion, we briefly discussthe processingof primary transcripts
produced from genesencodingrRNAs and tRNAs.
E[ of EukaryoticPre-mRNA
Processing
In this section,we take a closer look at how eukaryotic cells
convert the initial primary transcript synthesizedby RNA
oolvmerase II into a functional mRNA. Three maior events
occur during the process:5' capping,3' cleauage/polyadenyla'
tion, and RNA splicing (Figure 8-2). Adding these specific
modifications to the 5' and 3' ends of the pre-mRNA is
important to protect it from enzymes that quickly digest
uniapped RNAs generated by RNA processing' such as
spliced-out introns and RNA transcribed downstream from a
polyadenylationsite.The 5'-cap and 3'-poly(A) tail distinguish
pre-mRNA molecules from the many other kinds of RNAs in
ihe nucleus. Pre-mRNA molecules are bound by nuclear pro-
teins that function in mRNA export to the cytoplasm. After
mRNAs are exported to the cytoplasm, they are bound by an-
other set of cytoplasmic proteins that stimulate translation and
are critical for mRNA stability in the cytoplasm. Furthermore,
introns must be removed to generatethe correct coding region
of the mRNA. In higher eukaryotes, alternative splicing is in-
tricately regulated in order to substitute different functional
domains into proteins, producing a considerableexpansion of
the proteome of theseorganisms,which include ourselves.
The pre-mRNA processingeventsof capping, splicing, and
polyadenylation occur in the nucleus as the nascent mRNA
precursoris being transcribed.Thus pre-mRNA processingis
co-transcriptional. As the RNA emergesfrom the surface of
RNA polymeraseII, its 5' end is immediately modified by the
addition of the S'-cap structure found on all mRNAs (seeFig-
we 4-14).As the nascentpre-mRNA continuesto emergefrom
the surfaceof the polymerase,it is immediately bound by mem-
bers of a complex group of RNA-binding proteins that assistin
RNA splicing and export of the fully processed mRNA
through nuclear pore complexes into the cytoplasm. Some of
these proteins remain associatedwith the mRNA in the cyto-
plasm, but most either remain in the nucleus or shuttle back
into the nucleus shortly after the mRNA is exported to the cy-
toplasm. Cytoplasmic RNA-binding proteins are exchanged
for the nuclear ones.Consequently,mRNAs never occur as free
RNA moleculesin the cell but are always associatedwith pro-
tein as ribonucleoprotein (RNP) complexes, first as nascent
pre-mRNPs that are capped and spliced as they ate tran-
scribed. Then, following cleavage and polyadenylation, they
are referred to as nuclear zzRNPs. Following the exchangeof
proteins that accompaniesexport to the cytoplasm' they are
called cytoplasmic mRNPs. Although we frequently refer to
pre-mRNAs and mRNAs, it is important to remember that
ih.y always associatedwith proteins as RNP complexes'
"r.
The 5' Cap ls Added to NascentRNAsShortly
After TranscriptionInitiation
As the nascentRNA transcript emergesfrom the RNA chan-
-25 nu-
nel of RNA polymeraseII and reachesa length of
cleotides, a protective cap composed of 7-methylguanosine
and methylated riboses is added to the 5' end of eukaryotic
mRNAs (seeFigure 4-14).The 5' cap marks RNA molecules
as mRNA precursorsand servesto protect them from RNA-
digesting ..try-.t (5'-exoribonucleases)in the nucleus and
cyiopl"s-. This initial step in RNA processingis catalyzed
by a dimeric capping enzyme, which associateswith the
PRE-mRNA
P R O C E S S I NOGF E U K A R Y O T I C 325

Exon Intron
DNA
P r i m a r yR N A E /
transcript
enoonucteuseJcleavageat poly(A)site
polvAtrorvneras"
of;lJ p
mRNA 5'
A FIGURE 8-2 Overviewof mRNAprocessingin eukaryotes.
ShortlyafterRNApolymerase ll initiates
transcription
at thefirst
nucleotide of thefirstexonof a gene,the 5, endof thenascent
RNAiscapped with 7-methylguanylate (stepE). Transcription by
RNApolymerase ll terminates
at anyoneof multiple termtnatton
sitesdownstream fromthe poly(A)site,whichis locatedat the 3,
endof thefinalexonAfterthe primary transcript
iscleaved at the
poly(A)site(stepZ), a stringof adenosine (A)residuesisadded
phosphorylated carboxyl-terminal domain (CTD) of RNA
merasesI or III that do not have a CTD. This is important
becausepre-mRNA synthesisaccountsfor less than 10 per-
cent of the total RNA synthesizedin replicating cells.About
80 percent of RNA synthesis is preribosomal RNA tran-
scribed by RNA polymeraseI, and about 15 percent is tran-
scribed by RNA polymeraseIII, including 5S rRNA, tRNAs,
and other stable small RNAs. The two mechanisms of
(1) capping enzyme binding to initiated RNA polymerase
II specificallythrough its unique, phosphorylated CTD and
(2) activation of capping enzyme activity by binding to the
phosphorylated CTD result in specificcapping of the minor
fraction of RNAs transcribed by RNA polymeraseII.
Considerable evidence indicates that capping of the
nascent transcript is coupled to elongation by RNA poly_
meraseII so that all of its transcriptsare cappedduring the
earliest phase of elongation. In one -oJ.l, during the
326 o c H A p r E 8R | posr-TRANscRtploN
GAELN cEo N T R o L
Animation:Life Cycleof an mRNAflltl
Poly(A) Termination
site sites
E rrunr.ription,
5' cappins
I
tl
'11
polyadenylation
A roo-rso3'
E *to splicins
J
A,oo_rso3'
(stepB). Thepoly(A) -250 A residues
tailcontains in mammals,
-150 in insects,and-100 in yeastsForshortprimary transcripts
(step4) usually
withfew introns,splicing followscleavage and
polyadenylation,asshownForlargegenes with multiple introns,
introns
oftenarespliced out of the nascentRNAduringits
transcrrption,
i e.,beforetranscription
of thegeneiscomplete.
Notethatthe 5' capandsequence adjacentto the poly(A)tailare
retained
in maturemRNAs.
initial phase of transcription the polymerase elongates the
nascent transcript very slowly due to the action of elonga-
tion factors NELF (negative elongation factor) and Spt4/5,
as occursat the HIV LTR promoter (seeFigure 7-51). Once
the 5'-end of the nascentRNA is capped,phosphorylation
of the RNA polymerase CTD and Spt5 by the cyclin T-
Cdk9 protein kinase is stimulated, causing the release of
NELF. This allows RNA polymerase II to enter into a faster
mode of elongation that rapidly transcribes away from the
promoter. The net effect of this mechanism is that the poly-
merase waits for the nascent RNA to be capped before
elongating at a rapid rate. HIV appears to have exploited
this mechanism to add an additional level of transcription
control through the Tat protein and the TAR element,
which are required to bring the cyclin T-Cdk9 kinase to the
elongation complex that forms at the HIV LTR promoter
(seeFigure 7-51). Currently, ir is not understoodwhat dis-
tinguishes transcription from the HIV LTR promoter from
other promoters where Tat protein is not required to bring
in the cyclin T-Cdk9 protein kinase.
A DiverseSet of Proteinswith ConservedRNA-
Binding DomainsAssociatewith pre-mRNAs
As noted earlier,nascentRNA transcripts from protein-coding
genesand mRNA processingintermediates,collectively re-
ferred to as pre-mRNA, do not exist as free RNA molecules
in the nuclei of eukaryotic cells. From the time nascentrran-
scripts first emerge from RNA polymerase II until mature
mRNAs are transported into rhe cytoplasm, the RNA mole-
culesare associatedwith an abundant set of nuclear proteins.
 5' end of RNA been identified by constructing deletionsof hnRNP proteins
YBO and testing their ability to bind RNA.
@ru--@W
Functions of hnRNP Proteins The associationof pre-mRNAs
I
D r o . u l ' o li n r o . " s o with hnRNP proteins preventsformation of short secondary
structures dependenton base pairing of complementary re-
f, T

l\e gions, thereby making the pre-mRNAs accessiblefor inter-

s0f Pct action with other RNA molecules or proteins. Pre-mRNAs
c\___r----l +@ru--@llE associatedwith hnRNP proteins present a more uniform
crP I substrate for subsequentprocessingsteps than would free'
unbound pre-mRNAs' where each mRNA forms a unique
I
C r a r v t v tr r . r s l e a s e \ F y

+\@ secondarystructure due to its specificsequence.

Binding studies with purified hnRNP proteins indicate
c ru--trffilMl that different hnRNP proteins associatewith different re-
I gions of a newly made pre-mRNA molecule. For example'
c r a r r r ' -/ . c t r | + C H 3f r o m
" r
1 , 6 1 " r c l , . eS _ A d o _ M e t ih. httRNp proteins A1, C, and D bind preferentially to the
I pyrimidine-rich sequences at the 3' endsof introns (seeFigure
+ 8-7). Some hnRNP proteins interact with the RNA sequences
m7G w__tffi[E that specify RNA splicing or cleavage/polyadenylationand
contribute to the structure recognized by RNA-processing
2a l*cH3trom have shown that
, ,r S_Ado_Met factors. Finally, cell-fusion experiments
I localized in the nucleus,
some hnRNP proteins remain
J
whereasothers cycle in and out of the cytoplasm, suggesting
that they function in the transport of mRNA (Figure 8-4)'
FIGURE 8-3 Synthesis of 5'-capon eukaryoticmRNAs.The5'
endof a nascentRNAcontains a 5'-triphosphate fromthe initiating Conserved RNA'Binding Motifs The RNA recognition
NTPThe1-phosphate isremoved while
in thefirststepof capping, motif (RRM| also called the RNP motif and the RNA-binding
the remainingcr-
and (orange)remain associatedwith
B-phosphates domain (RBD), is the most common RNA-binding domain
the cap Thethirdphosphate of the 5-5'-triphosphate bondis
derivedfromthe a phosphate of the GTPthatdonates theguanine
Themethyldonorfor methylation of thecapguanine andthefirst
oneor two ribosesof the mRNAisS-adenosyl methioninelFrom S
Venkatesanand B Moss, 1982, Proc Natl Acad 'ci USA79:304)

These are the major protein components of heterogeneows
ribonucleoprotein particles (hnRNPs), which conrain het-
erogeneous nuclear RNA /EnRNAl, a collective term
referring to pre-mRNA and other nuclear RNAs of various
sizes.These hnRNP proteins contribute to further steps in
RNA processing,including splicing and polyadenylation and
export through nuclear pore complexesto the cytoplasm'
Researchersidentified hnRNP proteins by first exposing
cultured cells to high-doseUV irradiation, which causesco-
valent cross-linksto form betweenRNA basesand closely
associatedproteins. Chromatography of nuclear extracts
from treated cells on an oligo-dT cellulose column, which
binds RNAs with a poly(A) tail, was usedto recoverthe pro-
teins that had become cross-linkedto nuclear polyadeny-
lated RNA. Subsequenttreatment of cell extracts from unir-
radiated cells with monoclonal antibodies specific for the
major proteins identified by this cross-linking technique re-
vealed a complex set of abundant hnRNP proteins ranging
in sizefrom -30 to -120 kDa.
Like transcription factors, most hnRNP proteins have a
modular structure. They contain one or more RNA-binding
domains and at least one other domain that interacts with
other proteins. Severaldifferent RNA-binding motifs have
eukaryotic evolution.
Structural analyseshave shown that the RRM domain
consists of a four-stranded B sheet flanked on one side by
two cr helices.To interact with the negatively charged RNA
phosphates,the B sheet forms a positively.chargedsurface'
th. ionr.tu.d RNP1 and RNP2 sequenceslie side by side on
the two central p strands, and their side chains make multi-
ple contacts with a single-strandedregion of RNA that lies
acrossthe surfaceof the B sheet (Figure 8-5)'
The 45-residueKH motif is found in the hnRNP K pro-
tein and several other RNA-binding proteins' The three-
dimensionalstructureof representativeKH domains is similar
to that of the RRM domain but smaller, consisting of a
three-strandedp sheetsupported from one side by two o he-
lices.Nonetheless,the KH domain interactswith RNA much
differently than does the RRM domain' RNA binds to the
KH domain by interacting with a hydrophobic surface
formed by the a helices and one B strand' The RGG bor,
another RNA-binding motif found in hnRNP proteins,
contains five Arg-Gly-Gly (RGG) repeatswith severalinter-
spersedaromatic amino acids.Although the structure of this
motif has not yet been determined,its arginine-rich nature is
similar to the RNA-binding domains of the HIV Tat protein'
KH domains and RGG repeatsare often interspersedin two
or more setsin a single RNA-binding protein'

PRE-mRNA t 327
P R O C E S S I NOGF E U K A R Y O T I C
 Video:hnRNP41 Nucleocy c Shu

A FIGURE 8-4 HumanhnRNpA1 proteincancyclein and Hela-cell

nucleusisto therightof theoval-shaped Xenopus
out of the cytoplasm,but humanhnRNpC proteincannot. nucleus(b,c)Whenthesamepreparation wasvieweooy
CulturedHeLacellsandXenopus cellswerefusedbvtreatment with fluorescence microscopy,thestained hnRNpC proteinappeared
polyethylene glycol,producing heterokaryons containing nuclei greenandthestained hnRNP A1 proteinappeared red Notethat
fromeachcelltype Thehybridcellsweretreatedwith the unfused Xenopus cellon theleftisunstained, confirmingthat
cycloheximide immediately afterfusionto prevent protern theantibodies arespecificfor the humanproteinsln the
synthesis After2 hours,the cellswerefixedandstained with heterokaryon, hnRNP C proteinappears onlyin the Hela-cell
fluorescent-labeled antibodies specific for humanhnRNp C and nucleus (b),whereasthe41 protein appearsin bothnucleus (c)
A1 proteinsThese antibodies do not bindto the homologous Since proteinsynthesiswasblocked aftercellf usion,someof the
Xenopus proteins(a)A fixedpreparation viewedby phase- humanhnRNP A'1proteinmusthaveleftthe Hela-cell nucleus,
contrast microscopy includes unfused HeLacells(arrowhead) and movedthroughthecytoplasm, andenteredlheXenopus nucleus
Xenopuscells(dottedarrow),aswellasfusedheterokaryons in the heterokaryon[See S pinol-RomaandG Dreyfuss, 1992,
Nature
( s o l i da r r o w )l n t h e h e t e r o k a r yionnt h i sm i c r o g r a pthh,e r o u n d 355:730;courtesy
of G Dreyfussl

( a ) R N A r e c o g n i t i o nm o t i f ( R R M ) ( b ) S e x - l e t h a(lS x l )R R Md o m a r n s (c) Polypyrimidinetract binding protein (pTB)

FIGURE8-5 Structure of the RRM domain and its interaction surfaces of the positively chargedB sheets, makingmostof its
with RNA. (a) Diagramof the RRMdomainshowinqthe two cr contacts with the RNPlandRNp2regions of eachRRM.(c)Strikingly
helices(green)and four B strands(red)that characterrzethis motif d i f f e r e notr i e n t a t i oonf R R Md o m a i nisn a d i f f e r e nht n R N B
the
The conservedRNPl and RNp2regionsare locatedin the two central polypyrimidine tractbinding(PTB) protein, illustrating that RRMs are
B strands (b) Surfacerepresentationof the two RRMdomainsin oriented in different relative positions in different hnRNps; colorsare
DrosophilaSex-lethal(Sxl)protein,which bind a nine_base sequence asin (b) Polypyrimidine (p(y))singlestranded RNAis boundto the
in transformerpre-mRNA(yellow).The two RRMsare orientedlike
upward(RRM3) anddownward (RRM4) facingB-sheets RNAis
the two partsof an open pairof castanets,with the B sheetof RRMl (a)adapted
shownin yellow[Part fromK Nagai et al, 1gg5,Trends
facingupwardand the B sheetof RRM2facinqdownward.positivelv
Biochem Sci 20:735 Part(b) after N Haradaet al , 1999, Nature 399;579
chargedregionsin Sxlproteinareshown in shadesof blue;negativeli Part(c) after F C Oberstrasset al , 2006, Science309:2054 l
chargedregions,in shadesof red.The pre-mRNAis bound to the

328 C H A P T E R8 | posT-TRANSCR|pTtoNA
GLE N EC O N T R O L
 of Introns
$l ,oo."rt: Discovery
(a) < EXPERIMENTAL FIGURE 8-5 EIECTTON
Adenovirushexon gene
microscopyof mRNA-templateDNA hybrids
showsthat introns are splicedout during
s', 3', pre-mRNA processing. (a)Diagram of the
EcoRlA# EcoRlA fragment of adenovirus DNA,which
extendsfromthe left endof the genometo just
I Exons ! Introns 1 k b
beforethe endof the finalexonof the hexon
geneThegeneconsists of threeshortexons
andonelong(-3.5 kb)exonseparated bythree
intronsof -1, 2 5, and 9 kb. (b) Electron
micrograph (/eft)andschematic drawing(nght)
of hybridbetweenan EcoRl A fragmentand
hexonmRNA. TheloopsmarkedA, B,andC
correspond to the intronsindicated in (a).Since
theseintron sequences in the viralgenomtc
DNAarenot present in maturehexonmRNA,
theyloopout betweenthe exonsequences that
hybridize to theircomplementary sequences in
the mRNA.[Micrograph from 5 M Bergetet al,19]7 '
ProcNat'lAcadSciuSA74:3171;courtesyofPA
Sharpl

SplicingOccursat Short, Conserved
Sequencesin Pre-mRNAs via Two
Transesterification Reactions
During formation of a mature, functional mRNA, the introns
are removed and exons are spliced together.For short tran-
scription units, RNA splicing usually follows cleavageand
polyadenylationof the 3' end of the primary transcript' as de-
picted in Figure 8-2. However, for long transcription units
containing multiple exons, splicing of exons in the nascent
RNA beginsbefore transcription of the geneis complete.
Early evidencethat introns are removed during splicing
came from electron microscopy of RNA-DNA hybrids be-
tween adenovirusDNA and the mRNA encoding hexon, a ma-
jor virion capsid protein (Figure 8-6). Other studiesrevealed
nuclear viral RNAs that were colinear with the viral DNA
(primary transcripts) and RNAs with one or r\rvoof the introns
removed (processingintermediates).These results, together
with the findingsthat the 5' cap and 3' poly(A)tail at eachend
of long mRNA precursorsare retained in shorter mature cyto-
plasmic mRNAs, led to the realization that introns are re-
moved from primary transcripts as exons are spliced together'
The location of splice sites-that is, exon-intron iunc-
revealedmoderatelyconserved,short consensussequencesat
the splicesitesflanking introns in eukaryotic pre-mRNAs; in
higher organisms, a pyrimidine-rich region just upstream
oflhe 3' splice site also is common (Figure 8-7)' Studiesof
mutant genes with deletions introduced into introns have
shown that much of the center portion of introns can be
removed without affecting splicing; generally only 30-40

Pyrimidine-rich
5' splicesite Branchpoint r e g i o R( = 1 5b ) 3' splicesite

Pre-mRNA
95 70 80 45 80 90 80 100 80 80 100 100 60
F r e q u e n c yo f 70 60 80 100 100
o c c u r r e n c e( % )

sequencesaroundsplicesitesin adenosine, alsoinvariant, is20-50bases

usually fromthe 3' splice
FIGURE 8-7 Consensus
vertebratepre-mRNAs. bases
Theonlynearlyinvariant arethe 5' site,Thecentralregionof the intron,whichmayrangefrom40 bases
in length,generally
to 50 kilobases isunnecessary to occur.
for splicing
GUandthe 3' AG of the intron(blue),althoughtheflankingbases
arefoundat frequencies
indicated higherthanexpectedbasedon a t t a l , 1 9 8 6 , A n n R e vB i o c h e m 5 5 : 1 ' 1 9 , a n d E B K e l l e r
[ S e eR A P a d g e t e
randomdrstribution region(hatchmarked)
A pyrimidine-rich near a n d W A N o o n , 1 9 8 4 ,P r o c N a t ' l A c a d S c i U S A 8 1 : 7 4 1 71
the 3'end of the intronisfoundin mostcasesThebranch-point

PRE-mRNA
P R O C E S S I NOGF E U K A R Y O T I C 329
 Intron structure. In each transesterificationreaction) one phos-
..::, lt, phoesterbond is exchangedfor another.Sincethe number
t' , ^,n of phosphoesrerbonds in the molecule is not changed in
Z/ '
either reaction, no energy is consumed. The net result
h
-Y'n Ho
./ of these two reactions is that two exons are ligated and
I
o:[4o- o- P:o the intervening intron is released as a branched lariat
structure.
o- E x o n 2 - 3 '

O = 3' oxygen of t'rr, transesterification D u r i n gS p l i c i n gs, n R N A S

Base-pair
exon 1 f with Pre-mRNA
=
O 2'oxygenof
b r a n c hp o i n tA Splicing requires the presenceof both small nuclear RNAs
r.l'=3' oxygen of (snRNAs), important for base pairing with the pre-mRNA,
intron and associatedproteins. Five U-rich snRNAs, designated
UI,U2, U4, U5, and U6, participate in pre-mRNA splic,
5,O
I
., A
z./
ing. Ranging in length from 107 to 210 nucleotides,these
O:P-o' snRNAs are associatedwith 6 to 10 proteins in the many
I small nuclear ribonucleoprotein particles (snRNps) in rhe
O :3'
nuclei of eukaryotic cells.
P:O Definitive evidencefor the role of U1 snRNA in splicing
u'-r- l- -3' came from experiments indicating that base pairing be-
5', tween the 5' splicesite of a pre-mRNA and the 5, region of
I U1 snRNA is required for RNA splicing (Figure 8-9a). In
I Secondtransesterification vitro experiments showed that a synthetic oligonucleotide
+ that hybridizeswith the 5'-end region of U1 snRNA blocks
""" ""'
t*t"''"""""' ',..
,.o
': RNA splicing.In vivo experimentsshowed that base-pairing-
i't o- disrupting mutations in the pre-mRNA 5' splice block RNA
'_,!..!',i:iit

,, +
-I- -3', splicing. However, splicing can be restored by expressionof
s',o A a mutant U1 snRNA with a compensating mutation that
o
o:i-a/ :,,,3' S p l i c e de x o n s
restoresbasepairing to the mutant pre-mRNA 5, splice site
| (Figure 8-9b). Involvement of U2 snRNA in splicing ini-
O :;H
tially was suspectedwhen it was found to have an internal
Excisedlariat intron
sequencethat is largely complementary to the consensus
A FIGURE 8-8 Two transesterificationreactionsthat result in sequenceflanking the branch point in pre-mRNAs (see
splicingof exonsin pre-mRNA. In thefirstreaction,
the esterbond Figure 8-7). Compensatingmutation experiments,similar
between the 5' phosphorus of the intronandthe 3, oxygen(dark to those conducted with U1 snRNA and 5, splice sites,
red)of exon1 isexchanged for an esterbondwith the 2, oxygen demonstratedthat basepairing betweenU2 snRNA and the
(blue)of the branch-siteA residueInthesecondreacrton, tneester branch-point sequencein pre-mRNA also is critical to
bondbetween the 5' phosphorus of exon2 andthe 3, oxygen splicing.
(orange) of the intronisexchanged for an esterbondwiththe 3, Figure 8-9a illustrates the general strucrures of the U1
oxygenof exon1, releasing the intronasa lariatstructureandjoining and U2 snRNAs and how they base-pair with pre-mRNA
the two exons.Arrowsshowwhereactivated hydroxyl
oxygensreact
with phosphorus during splicing. Significantly,branch-point A itself, which is
atoms.
not base-pairedto U2 snRNA, "bulgesout" (Figure8-10a),
allowing its 2' hydroxyl to participate in the firsr transester-
ification reaction of RNA splicing (seeFigure 8-8).
nucleotidesat each end of an intron are necessaryfor splic_ Similar studies with other snRNAs demonstrated that
ing to occur at normal rates. base pairing between them also occurs during splicing.
Analysis of the intermediatesformed during splicing of Moreover, rearrangementsin these RNA-RNA interactions
pre-mRNAs in vitro led to the discovery that splicing of are critical in the splicing pathway, as we describenext.
exons proceedsvia two sequential transesterification reac_
tions (Figure 8-8). Inrrons are removed as a lariat_like
structure in which the 5' G of the inrron is joined in an S p l i c e o s o m eA
s ,s s e m b l e df r o m s n R N p s
u n u s u a l 2 ' , 5 ' - p h o s p h o d i e s t ebr o n d t o a n a d e n o s i n en e a r a n d a P r e - m R N AC, a r r yO u t S p l i c i n g
t h e 3 ' e n d o f t h e i n t r o n . T h i s A r e s i d u ei s c a l l e d b r a n c h The five splicing snRNPsand other proteins involved in splicing
point A because it forms an RNA branch in the lariat assembleon a pre-mRNA, forming a large ribonucleoprotein

c H A P T E R8 I p o s T - T R A N S C R | p T t O NG
ALE N EC O N T R O L
llll+ FocusAnimation:mRNA

(a)

Ul snRNA U2 snRNA
Sm
el

cap5'
Sm
J cap5'
llllll PY
||l UACUAC CAGG E x o n2 -3'
5' UAAGU
Pre-mRNA
A
\ B r a n c hp o i n t

(b)

Mutationin pre-mRNA 5' sPlicesite mutationin U1

ComPensatorY
blockssplicing sPlicing
restores

FIGURE 8-9 Basepairing between pre-mRNA,Ul snRNA, whichhavebeenusefulin characterizing components of the
and U2 snRNAearlyin the splicingprocess. (a)Inthisdiagram, splicing (b)Onlythe 5' endsof U1snRNAs
reaction. and5' splice
secondary in the snRNAs
structures that arenot alteredduring sitesin pre-mRNAs areshown.(Left)A mutation(A)in a pre-mRNA
aredepicted
splicing schematically. Theyeastbranch-point splice with basepairing
sitethatinterferes to the 5' endof U1
sequence isshownhere.Notethat U2 snRNAbase-pairs with a snRNA blocks Expression
splicing of a U1 snRNA witha
sequence thatincludesthe branch-pointA, although thisresidue is compensating mutation(U)that restoresbasepairingalsorestores
not base-pairedThepurplerectangles representsequences that bind splicingof the mutantpre-mRNA. lPart(a)adapted fromM J Moore
snRNP proteinsrecognizedby anti-Sm antibodies.Forunknown andJ Atkins,eds, Ihe RNAWorld,ColdSpring
et al,,1993,in R Gesteland
reasons,
antiserafrom patientswith the autoimmune disease systemic HarborPress, pp 303-357 Part(b)seeY ZhuangandA M Weiner,
lupuserythematosus(SLE) containantibodies to snRNP proteins, 1986.Cell45:827|

Video: Dynamic Nature of Pre-mRNASpl FactorMovementin LivingCells

(a) Self-complementary (c) Spliceosomestructure < FIGURE 8-10 Structuresof a bulged A in an RNA-RNA
sequencewith bulgingA helix and an intermediatein the splicingProcess. (a)The
A structure of an RNAduplexwith the sequence shown, containing
5 ' U A C U - - A C G UA G U A bulgedA residues (red) at position 5 in the RNA was
helix,
AUGA UGCA UCAUS' (b)ThebulgedA restdues
A determined by x-raycrystallography.
extendfromthe sideof an A-formRNA-RNA helix.Thephosphate
(b) X-ray crystallography backbone of onestrandis shownin green; the otherstrandin
structure blue Thestructure on the right is turned 90 degrees for a view
downthe axis of the helix (c) 40 A resolution structure of a
o n t a i n i nUg2 ,U 4 ,U 5 ,a n d
s p l i c e o s o msapl i c i nign t e r m e d i act e
U6 snRNPs, determined by cryoelectron mtcroscopy andimage
reconstruction TheU4/U6/U5 tri-snRNP complex has a similar
structure to the triangular body of this complex, suggesting that
thesesnRNPs areat the bottomof the structure shownhereand
that the headis composed largelyof U2snRNP lParts(a)and(b)
f r o m J . AB e r g l u n d e,t2a0l 0 1R , NAT:682 P a r t ( f
c r)o m DBoehringer
etal, 2004,Nat.Struct. Mot. Biot. 11:463 See also H Stark and R Luhrmann,
2006,Annu Rev. Biophys BiomolStruct35:435l

PRE-mRNA
P R O C E S S I NOGF E U K A R Y O T I C 331
 FocusAnimation:mRNASpli c i ng
{l l tt
< FIGURE 8-11 Modelof spliceosome-mediated splicingof
U1
pre-mRNA. Step[: AfterU1andU2snRNps associatewiththe ore-
5'Ipc**"***-A*p 3',
mRNA, viabase-pairinginteractionsshownin Flgure 8-9,a trimeric
U2 snRNP complex of U4,U5,andU6joinsthe initialcomplex to form
thespliceosome StepE: Rearrangements of base-pairing interactions
Al'u+uaus between snRNAs
conformation
convertsthespliceosome intoa catalytically
active
anddestabilizesthe U1andU4snRNps, whichare
released. StepB: Thecatalytic core,thoughtto beformedby U6
U4 andU2,thencatalyzes thefirsttransesterif
icationreaction,forming
tt'r A the intermediate containinga 2',5'-phosphodiesterbondshownin
I ta
ltA Figure 8-8.StepZl: Following furtherrearrangements between the
-I--
snRNPs, thesecond transesterification
reactionjoinsthetwo exonsby
U5 Spliceosome a standard 3',5'-phosphodiesterbondandreleases the intronasa
lariatstructure
andthe remaining snRNPs.Step5: Theexcised Iariat

EI- U 1 ,U 4
intronisconverted intoa linearRNAby a debranching
[Adaptedfrom T. Villa et al , 2002, Cell 109:1491
enzyme

r--*-:"1 to releaseof first the U1 and then the U4 snRNPs.Figure8-10b

uu
o*ol shows a cryoelectron-microscopy structure of an inter-
ul oa#,/
F----/
t'" - u2 mediate in the splicing processlacking UlsnRNP. A further
^
u5u3
rearrangementof spliceosomalcomponents occurs with the
loss of U4snRNP.This generatesa complex that catalyzesthe
I first transesterificationreaction that forms the 2,,5,-phos-
Ef f irtt transesterification phodiesterbond between the 2' hydroxyl on branch point A
and the phosphateat the 5' end of the intron (Figure8-11).
Following another rearrangementof the snRNps, the second
u2, transesterificationreaction ligates the two exons in a stan-
' GpA dard 3',5'-phosphodiesterbond, releasingthe intron as a lar-
u6 ,'
p
iat structure associatedwith the snRNPs. This final intron-
53'
snRNP complex rapidly dissociates, and the individual
snRNPs releasedcan participate in a new cycle of splicing.
The excisedintron is then rapidly degradedby a debranching
E I S " " o n Ot r a n s e s t e r i f i c a t i o n enzymeand other nuclear RNasesdiscussedlater.
+ As mentioned above, a spliceosomeis roughly the size of
a ribosome and is composed of about 100 proteins, collec-
Lariat intron
s'Io 3' + + U 2 ,U 5 ,U 6 tively referred to as splicing factors, in addition to the five
Splicedexons snRNPs.This makesRNA splicingcomparablein complexity
'ooH to initiation of transcription and protein synthesis.Some of

afru* nrng
5'pc-*,**A**- OH 3
LinearintronRNA
complex called a spliceosome(Figure 8-11). The spliceosome
is a large ribonuclear protein complex with a masssimilar to
that of a ribosome.Assemblyof a spliceosomebeginswith the
basepairing of the snRNAs of the U1 and U2 snRNps to the
After formation of the spliceosome,extensiverearrange-
ments in the pairing of snRNAs and the pre-mRNA lead
the splicing factors are associatedwith snRNPs, but others
are not. For instance,the 65-kD subunit of the U2-associated
factor (U2AF) binds to the pyrimidine-rich region near rhe 3'
end of introns and to the U2 snRNP. The 35-kD subunit of
U2AF binds to the AG dinucleotideat the 3, end of the intron
and also interacts with the larger U2AF subunit bound
nearby.Thesetwo U2AF subunitsact togetherto help specify
the 3' splice site by promoting interaction of U2 snRNp with
the branch point (seeFigure 8-9). Some splicing facors also
exhibit sequencehomologiesto known RNA helicases;these
are probably necessaryfor the base-pairingrearrangements
that occur in snRNAs during the spliceosomalsplicing cycle.
Following RNA splicing, a specific set of hnRNp pro-
teins remain bound to the spliced RNA approximately 20
nucleotides 5' to each exon-exon junction, thus forming an
exon-junction complex. One of the hnRNp proteins associ-
ated with the exon junction complex is the RNA export factor
(REF), which functions in the export of fully processed

332 C H A P T E R8 | P o S T - T R A N S C R | p T t O NGAELN EC O N T R O L
 R N A p o l y m e r a s el l
Linker
I I t l I I I I I t I I l$
A FIGURE 8-12 Schematic diagramof RNApolymerase ll with
yeastRNA
the CTDextended.Thelengthof the fullyextended
polymerase
ll carboxyl-terminaldomain(CTD)
andthe linkerregion
thatconnectsit to the polymerase to theglobular
isshownrelative
mRNPs from the nucleus to the cytoplasm, as discussedin
Section8.3. Other proteins associatedwith the exon junc-
tion complex function in a quality-control mechanism that
leads to the degradation of improperly spliced mRNAs,
known as nonsense-mediated decay(Section8.4).
A small fraction of pre-mRNAs (-1o7' in humans) con-
tain introns whose splicesitesdo not conform to the standard
consensussequence.This classof introns beginswith AU and
ends with AC rather than following the usual "GU-AG rule"
(seeFigure 8-7). Splicing of this special class of introns ap-
pearsto occur via a splicingcycle analogousto that shown in
Figure 8-11, exceptthat four novel,low-abundancesnRNPs,
together with the standardU5 snRNP,are involved.
Nearly all functional mRNAs in vertebrate, insect, and
plant cellsare derivedfrom a singlemoleculeof the correspon-
ding pre-mRNA by removal of internal introns and splicingof
exons. However, in two types of protozoans-trypanosomes
and euglenoids-mRNAs are constructedby splicingtogether
separateRNA molecules.This process,referred to as trans-
splicing, is also used in the synthesisof 10-15 percentof the
mRNAs in the nematode (roundworm\ Caenorhabditisele-
gdns, an important model organism for studying embryonic
development.Trans-splicingis carried out by snRNPs by a
processsimilar to the splicingof exonsin a singlepre-mRNA.
ChainElongationby RNAPolymerase ll ls Coupled
Factors
to the Presenceof RNA-Processing
How is RNA processingefficiently coupled with the tran-
scription of a pre-mRNA? The key lies in the long carboxyl-
terminal domain (CTD) of RNA polymeraseII, which, as dis-
cussed in Chapter 7, is composed of multiple repeats of a
seven-residue(heptapeptide)sequence.\X/henfully extended,
the CTD domain in the yeast enzyme is about 65 nm long
(Figure8-12); the CTD in human RNA polymeraseII is about
twice as long. The remarkablelength of the CTD apparently
allows multiple proteins to associatesimultaneouslywith a
single RNA polymerase II molecule. For instance' as men-
tioned earlier,the enzymesthat add the 5' cap to nascenttran-
scripts associatewith the phosphorylatedCTD shortly after
transcription initiation. In addition, RNA splicing and
polyadenylationfactors have beenfound to associatewith the
phosphorylatedCTD. As a consequence, theseprocessingfac-
tors are presentat high local concentrationswhen splicesites
and poly(A) signals are transcribed by the polymerase,en-
hancing the rate and specificityof RNA processing.
CTD
ei* s
$ s * & $ 6 $ & : q * { * $ $ $ r ' e $ a F !s x f s s s $ * 6 s l * * * * R S $ 6 * * 6 S * & & S * e
domainof the polymerase. TheCTDof mammalian RNApolymerase
ll istwiceaslong In itsextended form,the CTDcan with
associate
multipleRNA-processing factorssimultaneously
lFrom DA
P Cramer,
Bushnell,andR D Kornberg, Science
2001, 292:1863)
Recentresultsindicate that the associationof RNA splic-
ing factors with phosphorylated CTD also stimulates tran-
scription elongation. Thus chain elongation is coupled to the
binding of RNA-processing factors to the phosphorylated
CTD. This mechanismmay ensurethat a pre-mRNA is not
synthesizedunlessthe machinery for processingit is properly
positioned.
S RP r o t e i n sC o n t r i b u t et o E x o nD e f i n i t i o n
i n L o n gP r e - m R N A s
The averagelength of an exon in the human genomeis :150
bases,whereasthe averagelength of an intron is much longer
(:3500 bases).The longestintrons contain upward of 500 kb!
Becausethe sequencesof 5' and 3' splice sites and branch
points are so degenerate,multiple copies are likely to occur
iandomly in long introns. Consequently,additional sequence
information is required to define the exons that should be
splicedtogether in higher organismswith long introns.
The information for defining the splice sitesthat demar-
cate exons is encodedwithin the sequencesof exons' A fam-
ily of RNA-binding proteins, the SR proteins, interact with
sequenceswithin exons called exonic splicing enhancers.SR
proteins are a subsetof the hnRNP proteins discussedearlier
and so contain one or more RRM RNA-binding domains'
They also contain several protein-protein interaction
domains rich in serine(S) and arginine (R) residues'\fhen
bound to exonic splicing enhancers,SR proteins mediate the
cooperativebinding of U1 snRNP to a true 5' splice site and
U2 snRNP to a branch point through a network of protein-
protein interactionsthat span acrossan exon (Figure8-13)'
The comple* of SR proteins, snRNPs,and other splicing fac-
tors (e.g.,U2AF) that assembleacrossan exon' which has
been called a cross-exonrecognition complex, permits pre-
cise specificationof exons in long pre-mRNAs.
In the transcription units of higher organismswith
long introns, exons not only encodethe amino acid se-
quencesof different portions of a protein but also contain
bindi.rg sites for SR proteins. Mutations that interfere with
the binding of an SR protein to an exonic splicing enhancer,
evenif they do not changethe encodedamino acid sequence,
would prevent formation of the cross-exonrecognition com-
plex. Ai a result, the affectedexon is "skipped" during splic-
ing is not included in the final processedmRNA' The
"nd
t.Jncat.d mRNA produced in this caseis either degradedor
PRE-mRNA
P R O C E S S I NOGF E U K A R Y O T I C o 333

U2 U1
U2AF65
GU
5 ' s p l i c es i t e
Cross-exon
recognition
complex
A FIGURE 8-13 Exonrecognitionthroughcooperativebinding
of SRproteinsand splicingfactorsto pre-mRNA. Thecorrect5,
GUand3'AG splice sitesarerecognized by splicing factorson the
basisof theirproximity to exonsTheexonscontainexonicsplicing
enhancers (ESEs) thatarebindingsitesfor SRproteinsWhenbound
to ESEs, the SRproteins interact with oneanotherandpromotethe
c o o p e r a t i vbei n d i n go f t h e U 1 s n R N tpo t h e 5 , s p l i c es i t eo f t h e
d o w n s t r e aim n t r o nt,h e U 2s n R N tpo t h e b r a n c hp o i n to f t h e
upstream i n t r o nt,h e 6 5 -a n d3 5 - k Ds u b u n i tosf U 2 A Ft o t h e
pyrimidine-rich regionandAG 3, splicesiteof the upstream
andothersplicing factors(notshown)Theresulting RNA-protein
translatedinto a mutant, abnormally functioning protein. Re-
cent studieshave implicated this type of mutation in human
geneticdiseases.For example, spinal muscle atrophy is one
of the most common genetic causesof childhood mortality.
This diseaseresultsfrom mutations in a region of the genome
containing two closelyrelated genes,SMNI and SMN2, that
arose by geneduplication. SMN2 encodesa prorein identical
with SMNI. SMN2 is expressedat much lower level because
a silent mutation in one exon interfereswith the binding of an
SR protein. This leadsro exon skipping in mosr of the SMN2
mRNAs. The homologous SMN gene in the mouse, where
there is only a singlecopy, is essentialfor cell viability. Spinal
muscle atrophy in humans results from homozygous mura-
tions that inactivate SMN1. The low level of protein trans-
lated from the small fraction of SMN2 mRNAs rhar are cor-
rectly spliced is sufficient to maintain cell viability during
embryogenesisand fetal development,but it is not sufficient
to maintain viability of spinal cord motor neurons in child-
hood, resulting in their death and the associateddisease.
Approximately 15 percentof the single-base-pair muta-
tions that causehuman geneticdiseasesinterfere with proper
exon definition. Some of thesemutations occur in 5, or 3,
splice sites, often resulting in the use of nearby alternative
"cryptic" splice sitespresentin the normal genesequence.In
the absenceof the normal splice site, the cross-exonrecogni-
tion complex recognizesthesealternative sites.Other muta-
tions that causeabnormal splicing result in a new consensus
splice site sequencethat becomesrecognizedin place of the
normal splice site. Finally, some mutations can interferewith
the binding of specific SR proteins to pre-mRNAs. These
mutations inhibit splicing at normal splice sites, as in the
caseof the SMN2 gene,and thus lead ro exon skipping. I
g r o u pl l I n t r o n sp r o v i d eC l u e s
S e l f - S p l i c i nG
t o t h e E v o l u t i o no f s n R N A s
Under certain nonphysiological in vitro conditions, pure
preparationsof some RNA transcriptsslowly spliceout in_
334 o c H A p r E8R | p o s r - T R A N S c R t p I oG
NEAN
L cEo N T R o L
Spliceosome
u2
u2AF6b
// A YYYY **/l*s'
I
B r a n c hp o i n t 3 ' s p l i c es i t e 5 ' s p l i c es i t e
Cross-exon
recognition
complex
cross-exon recognition complexspansan exonandactivates the
correct splicesitesfor RNAsplicingNotethatthe U1andU2snRNps
in thisunitarenot partof thesamespliceosome TheU2snRNp on
the rightformsa spliceosome with the U1snRNp boundto the 5,
endof the sameintronTheU1snRNP shownon the rightformsa
spliceosome with the U2snRNP boundto the branchpointof the
downstream intron(notshown), andthe U2snRNp on the leftforms
a spliceosome with a U1snRNP boundto the 5, splice
siteof the
upstream intron(notshown).Double-headed arrowsindicate
Intron, protein-protein interactrons fromI Maniatis,2OO2,
[Adapted Nature
418:236; seealsoS M Berget,1995,JBiol.Chen27O:24111
trons in the absenceof any protein. This observationled to
the recognition that some introns are self-splicing. Two
types of self-splicingintrons have beendiscovered:group I
introns, presentin nuclearrRNA genesof protozoans,and
group II introns, present in protein-coding genesand some
rRNA and IRNA genesin mitochondria and chloroplasts
of plants and fungi. Discovery of the catalytic activity of self-
splicing introns revolutionized conceptsabout the functions
of RNA. As discussedin Chapter 4, RNA is now thought to
catalyzepeptide-bond formation during protein synthesisin
ribosomes. Here we discussthe probable role of group II
introns, now found only in mitochondrial and chloroplast
DNA, in the evolution of snRNAs; the functioning of group I
introns is consideredin the later sectionon rRNA processing.
Even though their precisesequencesare not highly con-
served, all group II introns fold into a conserved,complex
secondarystructure containing numerous stem loops (Figure
8-t4a). Self-splicing by a group II intron occurs vra two
transesterificationreactions, involving intermediatesand
products analogous to those found in nuclear pre-mRNA
splicing. The mechanistic similarities between group II
intron self-splicingand spliceosomalsplicing led to the hy-
pothesis that snRNAs function analogously to the stem
loops in the secondarystructure of group II introns. Accord-
ing to this hypothesis,snRNAs interact with 5, and 3, solice
sitesof pre-mRNAs and with each other to produce a three-
dimensional RNA structure functionally analogous to that
of group II self-splicingintrons (Figure8-14b).
An extension of this hypothesisis that introns in ancient
pre-mRNAs evolved from group II self-splicing introns
through the progressive loss of internal RNA structures.
which concurrently evolved into trans-acting snRNAs that
perform the same functions. Support for this type of evolu-
tionary model comesfrom experimentswith group II intron
mutants in which domain V and part of domain I are
d e l e t e dR
. N A t r a n s c r i p t cs o n t a i n i n gs u c hm u t a n ti n t r o n sa r e
defectivein self-splicing,but when RNA moleculesequivalent
( a )G r o u p l l i n t r o n ( b ) U s n R N A si n s p l i c e o s o m e
5', 3', -.-
Pre-mRNA
FIGURE 8-14 Comparison of group ll self'splicing
introns
and the spliceosome. Theschematic diagrams comparing the
secondary structures intronsand(b)U
of (a)groupll self-splicing
snRNAs present rnthe spliceosome Thefirsttransesterification
reaction is indicatedby lightgreenarrows; thesecond reaction, by
in these
bluearrows. Thebranch-point A isboldfaced Thesimilarity
structures suggests thatthe spliceosomal snRNAs evolvedf romgroup
ll introns,
with thetrans-acting snRNAs beingfunctionally analogous
to thecorresponding domarns in groupll intronsThe colored bars
flankingthe intronsin (a)and(b)represent exons.[Adapted fromPA
1991,Science 254:663
Sharp, I
to the deletedregions are added to the in vitro reaction, self-
splicing occurs. This finding demonstratesthat these do-
mains in group II introns can be trans-acting,like snRNAs.
The similarity in the mechanismsof group II intron self-
splicingand spliceosomalsplicingof pre-mRNAs also suggests
that the splicing reaction is catalyzedby the snRNA, not the
protein, components of spliceosomes.Although group II in-
trons can self-splicein vitro at elevated temperatures and
Mg2* concentrations, under in vivo conditions proteins
called matura.ses,which bind to group II intron RNA, are re-
quired for rapid splicing. Maturases are thought to stabilize
the precisethree-dimensionalinteractionsof the intron RNA
required to catalyzethe two splicing transesterificationreac-
tions. By analogg snRNP proteinsin spliceosomes are thought
to stabilize the precise geometry of snRNAs and intron nu-
cleotides required to catalyzepre-mRNA splicing.
The evolution of snRNAs may have been an important
step in the rapid evolution of higher eukaryotes'As internal
rntron sequenceswere lost and their functions in RNA splic-
ing supplantedby trans-actingsnRNAs, the remaining intron
sequenceswould be free to diverge.This in turn likely facili-
tated the evolution of new genes through exon shuffling
sincethere are few constraintson the sequenceof new introns
generatedin the process(seeFigures6-18 and 6-19). It also
permitted the increasein protein diversity that results from
alternativeRNA splicing and an additional level of genecon-
trol resulting from regulatedRNA splicing.
during the S phase. They undergo a special form of 3'-end
processingthat involves cleavagebut not polyadenylation.
SpecializedRNA-binding proteins that help to regulate his-
tone mRNA translationbind to the 3'end generatedby this
specializedsystem.) Early studies of pulse-labeledaden-
ovirus and SV40 RNA demonstratedthat the viral primary
transcripts extend beyond the site from which the poly(A)
tail extends. These results suggestedthat A residuesare
added to a 3' hydroxyl generatedby endonucleolytic cleav-
age of a longer transcript, but the predicted downstream
RNA fragments never were detectedin vivo, presumably be-
causeof their rapid degradation.However' detection of both
predicted cleavageproducts was observed in in vitro pro-
cessing reactions performed with nuclear extracts of cul-
tured human cells. The cleavage/polyadenylation process
and degradation of the RNA downstream of the cleavage
site occurs much more slowly in thesein vitro reactions' sim-
product.
-plifying detection of the downstream cleavage
Early sequencing of cDNA clones from animal cells
showed that nearly all mRNAs contain the sequence
AAUAAA 10-35 nucleotides upstream from the poly(A)
tail (Figure8-15). Polyadenylation of RNA transcriptsis vir-
tually eliminated when the corresponding sequence in the
template DNA is mutated to any other sequence except
one encoding a closely related sequence(AUUAAA). The
animal cells.This downstreamsignalis not a specificsequence
:50
but rather a GU-rich or simply a U-rich region within
site'
n u c l e o t i d eos f t h e c l e a v a g e
Identification and purification of the proteins required for
cleavageand polyadenylation of pre-mRNA have led to the
modelihown in Figure 8-15. According to this model' a 360-
kDa cleavageand polyadenylation specificity factor (CPSF)'
composed of four different polypeptides,first forms an un-
stabie complex with the upstream AAUAAA poly(A) signal'
Then at leastthree additional proteins bind to the CPSF-RNA
complex: a 200-kDa heterotrimer calledcleauagestimulatory
sequence;a
factor (CSIF),which interactswith the G/lJ-rich
150-kDa heterotrimer called cleauagefactor I (CFI); and a

3' Cleavageand Polyadenylationof Pre'mRNAs

Are Tightly Coupled
In eukaryotic cells,all mRNAs, excepthistone mRNAs, have
a 3', poly(A) tail. (The major histone mRNAs are transcribed
from repeatedgenesat prodigious levels in replicating cells

PRE-mRNA
P R O C E S S I NoGF E U K A R Y O T I C ' 335
 > FIGURE 8-15 Modelfor cleavageand polyadenylation of Poly(A) poty(A)
pre-mRNAs in mammaliancells.Cleavage andpolyadenylation signal p o l y ( A )s i t e s i g n a l
specificity factor(CPSF) bindsto the upstream AAUAAApoly(A) [I \
signalCSIFinteracts with a downstream GU-or U-richsequence s', 3'
a n dw i t h b o u n dC P S F f o, r m i n ga l o o pi n t h e R N Ab; i n d i n g
of CFI ,, I Pre-mRNA
andCFllhelpstabilize CPSF,
CSIF,
CFI,CFII
the complexBindingof poly(A)polymerase
(PAP) thenstimulates cleavage at a poly(A) site,whichusually is
10-35nucleotides
1
3' of the upstream poly(A) signal.Thecleavage
factorsarereleased, as isthe downstream RNAcleavage product,
w h i c hi s r a p i d ldy e g r a d e dB.o u n dp A pt h e na d d s: 1 2 A r e s i d u east
a slowrateto the 3'-hydroxyl groupgenerated by the creavage
reactionBindingof poly(A)-binding proteinil (pABpil) to the initial
shortpoly(A) tailaccelerates the rateof additionby pApAfter
200-250A residues havebeenadded,pABpll siqnals pApto stoo
polVmenzation

residuesoccurs slowly, followed by rapid addition of up to

200-250 more A residues.The rapid phase requires the
binding of multiple copies of a poty(A)-binding protein con-
taining the RRM motif. This protein is designatedpABpII ro
distinguish it from the poly(A)-binding protein presentin the
cytoplasm. PABPII binds to the short A tail initially added by
PAP,stimulating the rate of polymerization of additional A
residuesby PAP,resulting in rhe fast phase of polyadenyla-
J.,""'un"
tion (Figure 8-15). PABPII is also responsiblefor signaling
poly(A) polymerase to terminate polymerization when the
poly(A) tail reachesa length of 200-250 residues,although
the mechanismfor controlling the length of the tail is not yit
understood. Binding of PABP to the poly(A) tail is essential
for mRNA export into the cytoplasm.
ATP
N u c l e a rE x o n u c l e a s eDse g r a d eR N AT h a t l s o-€j-
slow
Processed ppi PolYadenYlation
Out of Pre-mRNAs + CSIF,CFl,CFll
Becausethe human genome contains long introns, only :5
percent of the nucleotides that are polymerized by RNA
polymerase II during transcription are retained in mature,
processedmRNAs. Although this process appears ineffi-
cient, it probably evolved in multicellular organismsbecause
the processof exon shuffling facilitated the evolution of new

As mentionedearlier,the 2,,5,-phosphodiesterbond in ex-

cised introns is hydrolyzed by a debranching enzyme,yielding ATP
Rapid
a linear molecule with unprotected ends that can be attacked ppi PolYadenYlation
by exonucleases(seeFigure 8-11). The predominant nuclear
decaypathway is 3' -+ 5' hydrolysis by 11 exonucleases that
associatewith one another in a large protein complex called
the exosome.Other proteins in the complex include RNR heli-
casesthat disrupt base pairing and RNA-protein interactions ou 3'
-zoo
that would otherwiseimpedethe exonucleases. Exosomesalso
function in the cytoplasm, as discussedlater. In addition, the
exosome appears to degrade pre-mRNAs that have not been
properly spliced or polyadenylated. It is not yet clear how the

336 C H A P T E R8 | posT-TRANSCR|pTtoNA
GLE N EC O N T R O L
exosomerecognizesimproperly processedpre-mRNAs. But in
yeast cells with temperature-sensitivemutant poly(A) poly-
merase(Figure8-15), pre-mRNAs are retainedat their sitesof
transcription at the nonpermissivetemperature.Theseabnor-
mally processedpre-mRNAs are releasedin cellswith a second
mutation in a subunit of the exosomefound only in nuclear
and not cytoplasmicexosomes(PM-Scl; 100 kD in humans).
Also, exosomesare found concentratedat sitesof transcription
in Drosophila polytene chromosomes,where they are associ-
ated with RNA polymeraseII elongation factors. Theseresults
suggestthat the exosomeparticipatesin an as yet poorly un-
derstood quality-control mechanismthat recognizesaberrantly
processed pre-mRNAs, preventing their export to the cyto-
plasm and ultimately leadingto their degradation.
To avoid being degraded by nuclear exonucleases,nas-
cent transcripts, pre-mRNA processing intermediates, and
mature mRNAs in the nucleus must have their ends pro-
tected.As discussedabove,the 5' end of a nascenttranscript
is protectedby addition ofthe 5'cap structureas soon as the
5' end emergesfrom the polymerase.The 5' cap is protected
becauseit is bound by a nuclear cap-binding complex, which
protects it from 5' exonucleasesand also functions in export
of mRNA to the cytoplasm. The 3' end of a nascent tran-
script lies within the RNA polymeraseand thus is inaccessi-
ble to exonucleases(seeFigure4-12). As discussedpreviously,
the free 3' end generatedby cleavageof a pre-mRNA down-
stream from the poly(A) signal is rapidly polyadenylatedby
the poly(A) polymeraseassociatedwith the other 3' process-
ing factors, and the resulting poly(A) tail is bound by PABPII
(Figure 8-15). This tight coupling of cleavageand polyadeny-
lation protects the 3' end from exonucleaseattack.
Processingof Eukaryotic Pre-mRNA
r In the nucleus of eukaryotic cells, pre-mRNAs are asso-
ciated with hnRNP proteins and processedby 5' capping,
3' cleavageand polyadenylation, and splicing before being
transported to the cytoplasm (seeFigure 8-2).
r Shortly after transcriptioninitiation, a cappingenzymeas-
sociatedwith the phosphorylatedCTD of RNA polymerase
II addsthe 5' cap to the nascenttranscript. Other RNA pro-
cessingfactors involved in RNA splicing, 3' cleavage,and
polyadenylation also associate with the phosphorylated
CTD, increasingthe rate of transcriptionelongation.Conse-
quently, transcription does not proceed at a high rate until
RNA processingfactors become associatedwith the CTD,
where they are poised to interact with the nascent pre-
mRNA as it emergesfrom the surface of the polymerase.
r Five different snRNPs interact via basepairing with one
another and with pre-mRNA to form the spliceosome(see
Figure 8-11). This very large ribonucleoproteincomplex
catalyzestwo transesterificationreactions that join two
exons and remove the intron as a lariat structure, which is
subsequentlydegraded(seeFigure 8-8).
r SR proteins that bind to exonic splicing enhancer se-
quencesin exons are critical in defining exons in the large
pre-mRNAs of higher organisms. A network of interac-
tions between SR proteins, snRNPs' and splicing factors
forms a cross-exonrecognition complex that specifiescor-
rect splicesites(seeFigure 8-13)'
r The snRNAs in the spliceosomeare thought to have an
overall tertiary structure similar to that of group II self-
splicing introns.
r For long transcription units in higher organisms,splicing
of exons usually begins as the pre-mRNA is still being
formed. Cleavageand polyadenylation to form the 3' end
of the mRNA occur after the poly(A) site is transcribed.
r In most protein-coding genes' a conserved AAUAAA
poly(A) signal lies slightly upstream from a poly(A) site
where cleavageand polyadenylation occur. A GU- or U-
rich sequence downstream from the poly(A) site con-
tributes to the efficiency of cleavageand polyadenylation.
r A multiprotein complex that includes poly(A) poly-
merase(PAP)carries out the cleavageand polyadenylation
of a pre-mRNA. A nuclear poly(A)-binding protein,
PABPII, stimulatesaddition of A residuesby PAP and stops
addition once the poly(A) tail reaches 200-250 residues
( s e eF i g u r e8 - 1 5 ) .
r Excised introns and RNA downstream from the cleavagel
poly(A) site are degradedprimarily by exosomes,multipro-
tein complexesthat contain eleven3' -> 5' exonucleasesas
well as RNA helicases.Exosomesalso degradeimproperly
processedpre-mRNAs.
fp| Regulationof Pre-mRNA
Processing
Now that we've seenhow pre-mRNAs are processedinto ma-
ture, functional mRNAs' we consider how regulation of this
processcan contribute to genecontrol. Recall from Chapter 5
that higher eukaryotescontain both simple and complex tran-
transcription units 1-69o7oof all human transcription units)
can be processedin alternative ways to yield different mRNAs
that encodedistinct proteins (seeFigure 6-3).
A l t e r n a t i v eS p l i c i n gl s t h e P r i m a r yM e c h a n i s m
for RegulatingmRNAProcessing
The discovery that a large fraction of transcription units in
higher organisms encode alternatively spliced mRNAs and
that differently splicedmRNAs are expressedin different cell
types revealedthat regulation of RNA splicing is an impor-
tun, g.n"-.ontrol mechanismin higher eukaryotes.Although
many examples of cleavageat alternative poly(A) sites in
pre-mRNAs are known, alternative splicing of different ex-
ons is the more common mechanismfor expressingdifferent
O F P R E - m R N AP R O C E S S I N G
REGULATION 337
 Pre-mRNAs mRNAs

q .- gsxt'rotein
W+
(a) sxl
z J 4

t-fl-t} -+ ?rra protein

? -/Y
(b) tra
.-\
1 2 5

(c) dsx

A FIGURE 8-16 Cascade of regulatedsplicingthat controlssex Rbpl andfra2,activates splicing betweenexons3 and4 and
determinationin Drosophilaembryos.Forclarity,onlythe exons cleavage/polyadenylation Anat the 3' endof exon4 in dsxpre-
(boxes)
andintrons(blacklines) whereregulated splicing
occursare m R N Ai n f e m a l e m b r y o sI n m a l ee m b r y o w
s ,h i c hl a c kf u n c t i o n a l
shownSplicing isindicatedby reddashed linesabove(female) and Tra,the SRproteins do not bindto exon4, andconsequently exon3
bluedashed linesbelow(male) the pre-mRNAs Vertical
redlinesin isspliced to exon5 ThedistinctDsxproteins produced in female
exonsindicateln-framestopcodons, whrchprevent synthesisof andmaleembryos asthe resultof thiscascade of regulated splicing
functional
proteinOnlyfemaleembryos produce functionalSxl repress transcription of genesrequired for sexual differentiation of
protein,
whichrepresses splicingbetweenexons2 and3 in sxlpre- the opposite sex.[Adapted fromM J Mooreet al, 1993,in R Gesteland
mRNA(a)andbetweenexons1 and2 in tra pre-mRNA (b) (c)In andJ Atkrns, eds,IheRNAWorld, ColdSpring Harbor press, pp 303-357 l
contrast,
the cooperativebindingof Traproteinandtwo SRproteins,

proteins from one complex transcription unit. In Chapter 4,
for example, we mentioned that fibroblasts produce one
type of the extracellular protein fibronectin, whereas hepa-
tocytes produce another type. Both fibronectin isoforms are
encoded by the same transcription unit, which is spliced dif-
ferendy in the two cell types to yield two different mRNAs
(seeFigure4-16).ln other cases,alternativeprocessingmay
occur simultaneouslyin the samecell type in responseto dif-
ferent developmentalor environmental signals.First we dis-
cussone of the best-undersrood examplesof regulatedRNA
processingand then briefly consider the consequencesof
RNA splicing in the developmentof rhe nervous sysrem.
A Cascade o f R e g u l a t e dR N AS p l i c i n gC o n t r o l s
D r o s o p h i l aS e x u a lD i f f e r e n t i a t i o n
One of the earliestexamplesof regulatedalternarivesplicing
of pre-mRNA came from studiesof sexual differentiaiion in
splicing in Drosophila embryos. More recent researchhas
provided insight into how theseproteins regulate RNA pro-
cessingand ultimately lead to the creation of two different
sex-specifictranscriptionalrepressorsthat suppressthe devel-
opment of characteristicsof the oppositesex.
The Sxl protein, encodedby the sex-lethalgene,isthe first
protein to act in the cascade(Figure 8-16). The Sxl protein is
present only in female embryos. Early in development,the
gene is transcribed from a promoter that functions only in
female embryos. Later in development,this female-specific
promoter is shut off and another promoter for sex-letbal
becomesactive in both male and female embryos. However,
in the absenceof early Sxl protein, the sex-lethalpre-mRNA
in male embryos is spliced to produce an mRNA that con-
tains a stop codon early in the sequence.The net result is that
male embryos produce no functional Sxl protein either early
or later in development.
In contrast, the Sxl protein expressedin early female em-
bryos directs splicing of the sex-lerEal pre-mRNA so that a
functional sex-lethalmRNA is produced (Figure 8-16a). Sxl
accomplishesthis by binding to a sequencein the pre-mRNA
near the 3' end of the intron between exon 2 and exon 3,
thereby blocking the proper association of U2AF and tJ2
snRNP.As a consequence, the U1 snRNP bound to the 3, end
of exon 2 assembles into a spliceosomewith U2 snRNp bound
to the branch point at the 3' end of the intron betweenexons
3 and 4,leading to splicing of exon 2 to 4 and skipping of
exon 3. The resulting female-specificsex-lethal mRNA is
translated into functional Sxl protein, which reinforces its
own expressionin female embryos by continuing to cause
skipping of exon 3. The absenceof Sxl protein in male em-
bryos allows the inclusion of exon 3 and, consequently,of the
stop codon that preventstranslation of functional Sxl protein.

338 . c H A p r E8R I posr-TRANscRtploN

GAELN E
coNTRoL

d
A FIGURE 8-17 Modelof splicingactivationby Traproteinand
the 5RproteinsRbpl and Tra2.In femaleDrosophila embryos,
of exons3 and4 in dsxpre-mRNA
splicing isactivatedby bindingof
fra/Tra2lRbp1complexesto sixsitesin exon4 Because Rbpl andTra2
cannotbindto the pre-mRNA in theabsence of Tra,exon4 isskipped
in maleembryos. Seethetextfor discussion A, : polyadenylation
fromT.Maniatis
lAdapted andB Tasic,2002,Nature 418:236]
Sxl protein also regulatesalternative RNA splicing of the
transformergenepre-mRNA (Figure8-16b).In male embryos,
where no Sxl is expressed,exon 1 is spliced to exon 2, which
contains a stop codon that prevents synthesisof a functional
transformer protein. In female embryos, however, binding of
Sxl protein to the 3' end of the intron between exons 1 and 2
blocks binding of U2AF at this site. The interaction of Sxl with
transformer pre-mRNA is mediated by two RRM domains in
the protein (seeFigure 8-5).Vhen Sxl is bound, U2AF binds to
a lower-affiniry site farther 3' in the pre-mRNA; as a result
exon 1 is spliced to this alternative 3' splice site, eliminating
exon2 with its stop codon. The resulting female-specifictrans-
former nRNA, which contains additional constitutively spliced
exons, is translated into functional Transformer (Tra) protein.
Finally, Tra protein regulatesthe alternativeprocessingof
pre-mRNA transcribed from the double-sex gene (Figure
8-16c). In female embryos,a complex of Tra and two consti-
tutively expressedproteins,Rbpl and Tra2, directssplicingof
exon 3 to exon 4 and also promotescleavage/polyadenylation
at the alternativepoly(A) site at the 3' end of exon 4-leading
to a short, female-specificversion of the Dsx protein. In male
embryos,which produce no Tra protein, exon 4 is skipped,so
that exon 3 is spliced to exon 5. Exon 5 is constitutively
splicedto exon 6, which is polyadenylatedat its 3' end-lead-
ing to a longer,male-specificversion of the Dsx protein. As a
result of the cascadeof regulatedRNA processingdepictedin
Figure 8-16, different Dsx proteins are expressedin male and
femaleembryos.The male Dsx protein is a transcriptionalre-
pressor that inhibits the expression of genes required for
female development.Conversely,the female Dsx protein re-
pressestranscription of genesrequired for male development.
Figure 8-17 illustrates how theTrafta2iRbpl complex
is thought to interact with double-sex(dsx)pre-mRNA. Rbpl
andTra2 are SR proteins, but they do not interact with exon
4 in the absenceof the Tra protein. Tra protein interactswith
Rbpl and Tra2, resulting in the cooperative binding of all
three proteins to six exonic splicing enhancersin exon 4. The
bound Tra2 and Rbpl proteins then promote the binding of
U2AF and U2 snRNP to the 3' end of the intron between
exons 3 and 4, just as other SR proteins do for constitutively
spliced exons (see Figure 8-13). The TralTra2lRbpl com-
plexes may also enhancebinding of the cleavage/polyadeny-
lation complex to the 3' end of exon 4.
SplicingRepressors and ActivatorsControl
Splicingat Alternative Sites
As is evident from Figure 8-15, the Drosophila Sxl protein
and Tra protein have opposite effects:Sxl prevents splicing'
causingexons to be skipped, whereasTra promotes splicing.
The action of similar proteins may explain the cell-type-
specificexpressionof fibronectin isoforms in humans. For in-
stance,an Sxl-like splicingrepressorexpressedin hepatocytes
might bind to splicesitesfor the EIIIA and EIIIB exons in the
fibronectin pre-mRNA, causing them to be skipped during
RNA splicing (seeFigure4-16). Alternatively,a Tralike splic-
ing activator expressedin fibroblastsmight activatethe splice
sites associatedwith the fibronectin EIIIA and EIIIB exons'
leading to inclusion of these exons in the mature mRNA.
Experimental examination in some systemshas revealedthat
inclusion of an exon in some cell types versusskipping of the
same exon in other cell types results from the combined
influenceof severalsplicing repressorsand enhancers.
Alternative splicing of exons is especially common in the
nervous system,generatingmultiple isoforms of many pro-
teins required for neuronal developmentand function in both
vertebratesand invertebrates.The primary transcripts from
thesegenesoften show complex splicing patterns that can gen-
erate several different mRNAs' with different spliced forms
expressedin different anatomical locationswithin the central
nervous system.We consider two remarkable examplesthat
illustrate the critical role of this processin neural function.
Expression of K*-Channel Proteins in Vertebrate Hair
Cells In the inner ear of vertebrates, individual "hair
cells," which are ciliated neurons' respond most strongly to
a specific frequency of sound. Cells tuned to low frequency
(:50 Hz) are found at one end of the tubular cochlea that
makes up the inner ear; cells responding to high frequency
(:5000 Hz\ arefound at the other end (Figure8-18a).Cells
in between the ends respond to a gradient of frequenciesbe-
tween theseextremes.One component in the tuning of hair
cells in reptiles and birds is the opening of K*ion channelsin
resDonseio increasedintracellular Ca2*concentrations.The
Ca2* concentration at which the channel opens determines
the frequency with which the membrane potential oscillates
and hencethe frequency to which the cell is tuned'
The gene encoding this Ca2+-activatedK+ channel is ex-
pressedas multiple, alternatively spliced mRNAs. The various
proteins encodedby thesealternative mRNAs open at different
Ca2* concentrations. Hair cells with different response fre-
quenciesexpressdifferent isoforms of the channel protein de-
pending on their position along the length of the cochlea (see
Figure 23-30). The sequencevariation in the protein is very
complex: there are at leasteight regions in the mRNA where al-
ternative exons are utilized, permitting the expression of 576
possibleisoforms(Figure8-18b).PCR analysisof mRNAs from
O F P R E - m R N AP R O C E S S I N G
REGULATION 339
 (a) through post-translational modifications of splicing factors
playsa significantrole in modularingneuron function.

Many examples of genessimilar to the cochlear K*-

channel neurons have been observed in vertebrate
neurons; alternatively spliced mRNAs co-expressedfrom a
Apical Auditory
h a i rc e l l Basal specific gene in one type of neuron are expressedat different
nerve
( 5 0H z ) cell body h a i rc e l l relativeconcentrationsin different regionsof the central nerv-
(5000Hz)
ous system.Expansions in the number of microsatellite re-
peatswithin the transcribedregionsof genesexpressedin neu-
rons can causean alteration in the relative concentrationsof
alternativelysplicedmRNAs transcribedfrom multiple genes.
In Chapter 6, we discussedhow backward slippage during
DNA replication can lead to expansionof a microsatellitere-
peat (seeFigure 6-5). At least 14 different types of neurologi-
Cytosol cal diseaseresult from expansion of microsatellite regions
within transcription units expressedin neurons.The resulting
long regionsof repeatedsimple sequences in nuclearRNAs of
theseneurons resultsin the abnormalitiesin the relative con-
centrationsof alternativelysplicedmRNAs. For example,the
most common of thesetypes of diseases, myotonic dystrophy,
is characterizedby paralysis,cognitive impairment, and per-
sonality and behavior disorders.Myotonic dystrophy results
from increasedcopiesof either CUG repeatsin one transcripr
<7
in some patients or CCUG repeatsin another transcript in
FIGURE 8-18 Roleof alternativesplicingin the perception other patients.When the number of theserepeatsincreasesto
of soundsof differentfrequency.(a)Thechicken cochlea, a 5- 10 or more times the normal number of repeatsin thesegenes,
mm-longtube,contains an epitheliumof auditory haircellsthatare abnormalitiesare observedin the levelof two hnRNP proteins
tunedto a gradient of vibrational
frequencies from50 Hzat the that bind to these repeatedsequences.This probably results
apicalend(/eft)to 5000Hzat the basalend(nght).(b)TheCa2*- becausethesehnRNPs are bound by the abnormally high con-
activated
K* channel containsseven transmembrane o helices (50-56), centration of this RNA sequencein the nuclei of neurons in
whichassociate to formthechannelThecytosolic domain,which such patients.The abnormal concentrationsof thesehnRNP
includes
fourhydrophobic regions (S7-S10), regulates
openingof the proteins are thought to lead to alterationsin the rate of splic-
channelin response to Ca2* lsoforms of thechannel, encoded by ing of different alternative splice sites in multiple pre-mRNAs
alternatively
splicedmRNAs produced fromthe sameprimary transcript, normally regulatedby thesehnRNP proteins.I
openat differentCa2*concentrations andthusrespond to different
frequencies Rednumbers referto regions wherealternative splicing
produces Expression of Dscam lsoforms in Drosophila Retinal
different aminoacidsequences in thevariousisoforms
Neurons The most extreme example of regulated alternative
[ A d a p t e df r o m K P R o s e n b l a tet l a l , 1 9 9 7 , N e u r o n1 9 : 1 0 6 1]
RNA processingyet uncovered occurs in expressionof the
Dscam gene in Drosophila. Mutations in this gene interfere
individual hair cellshas shown that each hair cell expressesa with the normal synaptic connectionsmade befweenaxons and
mixture of different alternative Ca2*-activated K+-channel dendrites during fly development.Analysis of the Dscam gene
mRNAs, with different forms predominating in different cells showedthat it contains 95 alternativelysplicedexonsthat could
according to their position along the cochlea.This remarkable be splicedto generateover 38,000 possibleisoforms!Recentre-
arrangementsuggeststhat splicing of the Ca2*-activatedK+- sults have shown that Drosopbila matantswith a version of the
channel pre-mRNA is regulatedin responseto extracellular sig- gene that can be spliced in only about 22,000 different ways
nals that inform the cell of its position along the cochlea. have specificdefectsin connectivity betweenneurons.Thesere-
Other studiesdemonstratedthat splicing ar one of the al- sults indicate that expressionof most of the possibleDscam tso-
ternative splice sites in the Ca2+-activatedK+-channel pre- forms through regulatedRNA splicing helps to specify the tens
mRNA in the rat is suppressedwhen a specificprotein kinase of millions of different specific synaptic connections between
is activatedby neuron depolarizationin responseto synaptic neurons in the Drosophila brain. In other words, the correct
activity from interactingneurons.This observationraisesthe wiring of neurons in the brain requiresregulatedRNA splicing.
possibilitythat a splicingrepressorspecificfor this site may be
activated when it is phosphorylated by this protein kinase,
whose activity in turn is regulatedby synaptic activity. Since R N AE d i t i n gA l t e r st h e S e q u e n c e s
hnRNP and SR proteinsare extensivelymodified by phospho- of SomePre-mRNAs
rylation and other post-translationalmodificarions, it seems In the mid-1980s,sequencing
of numerouscDNA clonesand
likely that complex regulation of alternative RNA splicing corresponding genomic DNAs from multiple organisms led

340 C H A P T E RI I p o s T - T R A N S C R t p T t O NG
AEL N EC O N T R O L

- t
apoB
gene
apoB
mRNA
Proteins NH,
ApoB-100
FfGURE8-19 RNAeditingof apo-Bpre-mRNA.
mRNAoroduced in the liverhasthesameseouence
t h ep r i m a rtyr a n s c r i pTth i sm R N Ai st r a n s l a t ei ndt oa p o B - 1 0w 0 ,h i c h
h a st w o f u n c t i o n adlo m a i n sa:n N - t e r m i n d ao l m a i n( g r e e nt )h a t
associates with lipidsanda C-terminal domain(orange)
to the unexpected discovery of another type of pre-mRNA
processing.In this type of processing,called RNA editing,
the sequenceof a pre-mRNA is altered;as a result, the se-
quenceof the correspondingmature mRNA differs from the
exons encoding it in genomic DNA.
RNA editingis widespreadin the mitochondriaof proto-
zoansand plants and also in chloroplasts.In the mitochon-
dria of certainpathogenictrypanosomes,more than half the
sequenceof some mRNAs is altered from the sequenceof the
corresponding primary transcripts. Additions and deletions
of specificnumbers of U's follows templatesprovided by
base-pairedshort "guide" RNAs. TheseRNAs are encoded
by thousandsof mini-mitochondrialDNA circlescatenated
to many fewer large mitochondrial DNA molecules.The rea-
son for this baroquemechanismfor encodingmitochondrial
proteinsin suchprotozoansis not clear.But this systemdoes
rcpresenta potential target for drugs to inhibit the complex
processingenzymesessentialto the microbe that do not ex-
ist in the cellsof their human or other vertebratehosts.
In higher eukaryotes,RNA editing is much rarer,and thus
far, only single-basechangeshave beenobserved.Such minor
editing, however,turns out to have significantfunctional con-
sequences in some cases.An important exampleof RNA edit-
ing in mammals involves the apoB gene. This gene encodes
two alternativeforms of a serumprotein central to the uptake
and transport of cholesterol.Consequently,it is important in
the pathogenicprocesses that lead to atherosclerosls,
the arte-
rial diseasethat is the major causeof death in the developed
world. The apoB gene expressesboth the serum protein
apolipoproteinB-100 (apoB-100)in hepatocytes, the major
cell type in the liver, and apoB-48, expressedin intestinalep-
ithelial cells.The :240-kDa apoB-48 correspondsto the N-
terminal region of the :500-kDa apoB-100. As we detail in
Chapter 10, both apoB proteins are componentsof large
lipoprotein complexesthat transport lipids in the serum.How-
ever, only low-density lipoprotein (LDL) complexes,which
contain apoB-100on their surface,delivercholesterolto body
tissuesby binding to the LDL receptorpresenton all cells.
The cell-type-specificexpressionof the two forms of apoB
results from editing of apoB pre-mRNA so as to changethe
CAA
CAA+UAA UAA
v
5', A
.t,
2152
cooH NH, cooH
APoB-48
TheapoB LDLreceptors on cellmembranes In theapo-BmRNAproduced in
asthe exonsin the
the intestine. CAA codonin exon 26 iseditedto a UAAstop
codonAsa result, cellsproduce
intestinal apoB-48, whichcorresponds
domainof apoB-100lAdapted
to the N-terminal fromP Hodgesand
thatbindsto J Scott, 1992, TrendsBiochem Sci 17:77 l
nucleotideat position 6666 in the sequencefrom a C to a U.
This alteration, which occurs only in intestinalcells,converts
a CAA codon for glutamine to a UAA stop codon, leadingto
synthesisof the shorter apoB-48 (Figure 8-19)' Studieswith
the partially purified enzymethat performs the post-transcrip-
tional deaminationof C6666to U shows that it can recognize
and edit an RNA as short as 26 nucleotideswith the sequence
surrounding C5566in the apoB primary transcript.
Regulation of Pre-mRNAProcessing
r Becauseof alternativesplicing of primary transcripts,the
use of alternative promoters, and cleavageat different
poly(A) sites,different mRNAs may be expressedfrom the
same gene in different cell types or at different develop-
mental stages(seeFigure 6-3 and Figure 8-16).
r Alternative splicing can be regulated by RNA-binding
proteins that bind to specific sequencesnear regulated
splice sites. Splicing repressorsmay sterically block the
binding of splicing factors to specific sites in pre-mRNAs
or inhibit their function. Splicing activators enhancesplic-
ing by interacting with splicing factors, thus promoting
their associationwith a regulated splice site.
r In RNA editing the nucleotide sequenceof a pre-mRNA
is altered in the nucleus.In vertebrates,this processis fairly
rare and entails deamination of a single basein the mRNA
sequence,resulting in a change in the amino acid specified
by the correspondingcodon and production of a function-
ally differentprotein (seeFigure 8-19).
Ifil of mRNAAcross
Transport
the NuclearEnvelope
Fully processedmRNAs in the nucleus remain bound by
hnRNP proteins in complexes now referred to as nuclear
zRNPs. Before an mRNA can be translated into its en-
coded protein. it must be exported out of the nucleusinto
ENVELOPE
THENUCLEAR
T R A N S P O ROTF m R N A A C R O S S 341
the cytoplasm. The nuclear envelopeis a double membrane macromolecules including tRNAs and ribosomal subunits
that separatesthe nucleusfrom the cytoplasm(seeFigure9-1). traverse the nuclear envelope through nwclear pores. This
Like the plasma membrane surrounding cells, each nuclear section will focus on the export of mRNPs through the
membrane consistsof a water-impermeablephospholipid nuclear pore and the mechanismsthat allow some level of
bilayer and multiple associatedproteins. mRNPs and other regulation of this step.

(al NuclearPoreComplexesControl lmport

Nucleoplasm
a n d E x p o r tf r o m t h e N u c l e u s
I n n e rn u c l e a r Nuclearbasket
Nuclear pore complexes (NPCs) are large, complicated
membrane
structurescomposedof multiple copiesof approximately 30
,,, r.Ii I
different proteins called nucleoporins. Embedded in the nu-
t IIIt:t,:IIt.:, .,.
clear envelope, NPCs are roughly octagonal in shape with
eight filaments extending into the nucleoplasm and another
eight filaments extending into the cytoplasm. (Figure 8-20a;
LUMEN
Outter nuclear seealso Figure 13-32 for a more detailed discussionof NPC
rlir:a:i.t:rjr ,
membrane structure). The filaments extending from the nuclear face of
the NPC are joined at their ends by a terminal ring forming
Cytoplasm
a structure called the nuclear basket. A special class of nu-
Central
Iransporter cleoporins called FG-nucleoporins line the central channel
through the NPC. FG-nucleoporins contain long stretches
of hydrophilic amino acid sequencespunctuated by hy-
Cytoplasmic drophobic FG-repeats,short sequencesrich in hydrophobic
filaments
phenylalanine(F) and glycine(G).
'Water,
ions, metabolites,and small globular proteins up
to =40 kDa can diffuse through the water-filled channel in
the nuclear pore complex. However, FG-nucleoporinsin the
central channel form a barrier restricting the diffusion of
macromoleculesbetweenthe cytoplasm and nucleus. These
larger molecules must be selectivelytransported across the
nuclear envelope with the assistanceof soluble transporter
proteins that bind them and also interact with FG-repeatsof
(c) (d) Transporter
FG-nucleoporins.
In one current model of the nuclear pore complex central
channel, FG-repeat domains of FG-nucleoporins form ran-
dom coil regions of polypeptide that extend into the channel
(Figure 8-20b). The FG-repeat domains of different FG-
nucleoporin molecules are proposed to associatewith each
other reversibly,forming a constantly rearranging molecular
meshwork that acts like a sieve(Figure8-20c). Small mole-
cules can diffuse through the spaces between FG-repeat
domains. But large proteins and ribonucleoproteins, in-
cluding mRNPs, are too large to pass betweenthe protein
filaments that form the molecular sieve and consequently
cannot diffuse through the nuclear pore complex.
Nuclear transport proteins bind reversibly to the hy-
A FIGURE8-20 Model of transporter passagethrough an NpC.
drophobic FG-regionsof FG-nucleoporins.These interac-
(a) Diagramof NPCstructure(b)The FG-domarns of FG-nucleoporrns tions are thought to involve multiple surfacesof the trans-
arethoughtto havean extendedconformationwith long hydrophilic
porters, allowing the proteins to diffuse through the central
regionsof polypeptide separatedby shortFG-hydrophobic domains
(c) FG-repeats channel(Figure8-20d).
arethoughtto associate with eachother reversiblyand
rapidly,forminga constantlyre-arranging mRNPs are transported through the NPC by the zRNP
molecularsievethat allows
diffusionof smallwater-soluble molecules throughit However,
exporter. The mRNP exporter is a heterodimer consisting
macromolecules aretoo big to fit throughthe channelsin the of a large subunit, called nuclearexport factot | (NXF1) or
molecularsieve (d) Nucleartransporters havehydrophobicregionson TAP,and a small subunit, ntclear export transporter1 (Nxt1).
their surface(darkbluedots)that bind reversibly to the FG-domains in TAP binds nuclear mRNPs through associationswith both
the FG-nucleoporins As a consequence, they can penetratethe RNA and other proteins in the mRNP complex. One of the
molecularsievein the NPCcentralchanneland diffusein and out of most important of these is REF (RNA export factor), a
the nucleus[FromK Ribbeck andD Gorlich, zOOj,EMBO I 20:1320] component of the exon junction complexes discussed

342 . c H A p r E R8 I p o s r - T R A N s c R t p l o N AGLE N Ec o N T R o L

r4*1g*!s:ii!llttl!irlb4*e*'.**,,,r,,uuu*,r,.,r,rt,*.r**u,*,r,urril
Gytoplasm
earlier, bound approximately 20 nucleotides 5' to each
exon-exon junction (Figure 8-21). The TAP/Nxt1 nRNP
exporter also associateswith SR proteins bound to exonic
splicingenhancers.Thus SR proteins associatedwith exons
function to direct both the splicing of pre-mRNAs and the
export of fully processedmRNAs through NPCs to the cy-
toplasm. mRNPs are probably bound along their length by
several TAPiNxtl mRNP exporters, which both interact
with the FG-domains of FG-nucleoporins to facilitate
export of mRNPs through the NPC central channel (Figure
8-20). The filamentsthat extend from the nuclearand cyto-
plasmic faces of the NPC also assist in mRNP export.
Gle2, an adapter protein that reversibly binds both TAP and
a nucleoporinin the nuclear basket,brings nuclearmRNPs
to the pore in preparationfor export. A nucleoporin in the
cytoplasmicfilaments of the NPC binds an RNA helicase
that is proposed to function in the dissociationof TAPA{xt1
and other hnRNP proteins from the mRNP as it reachesthe
cytoplasm.
In a processcalled zRNP remodeling,the proteins as-
sociated with an mRNA in the nuclear mRNP complex are
exchanged for a different set of proteins as the mRNP is
transported through the NPC. Some nuclear mRNP pro-
teins dissociateearly in tranport, remaining in the nucleus
to bind to newly synthesizednascentpre-mRNA. Other nu-
clear mRNP proteins remain with the mRNP complex as it
traversesthe pore and do not dissociatefrom the mRNP un-
til the complex reachesthe cytoplasm.Proteinsin this cat-
egory include the TAP/Nxt1 mRNP exporter, CBC bound to
the 5' cap, and PABPII bound to the poly(A) tail. They dis-
sociate from the mRNP on the cytoplasmic side of the NPC
throush the action of the RNA helicasethat associateswith
< FIGURE 8-21 Remodeling of mRNPs
during nuclearexport.SomemRNP proteins
(rectangles) dissociate fromnuclear mRNP
complexes beforeexportthroughan NPC
Some(ovals) areexported throughthe NPC
associated with the mRNP but dissociate in the
cytoplasm andareshuttledbackintothe
n u c l e utsh r o u g ha n N P Cl n t h ec y t o p l a s m ,
translation initiation factorelF4E replaces CBC
boundto the 5' capandPABPI replaces
PABPII.
cytoplasmicNPC filaments,as discussedabove.Thesepro-
teins are then imported back into the nucleusas discussed
for other nuclear proteins in Chapter 13' where they can
function in the export of another mRNP. In the cytoplasm,
the cap-binding translation initiation factor eIF4E replaces
CBC bound to the 5' cap of nuclearmRNPs. In vertebrates,
the nuclearpoly(A)-bindingprotein PABPIIis replacedwith
the cytoplasmicpoly(A)-binding protein PABPI (so named
becauseit was discoveredbefore PABPII). (Only a single
PABP is found in budding yeast' in both the nucleus and the
cytoplasm.)
Yeast SR Protein Recentresultssuggestthat the direction of
mRNP export from the nucleus into the cytoplasm is
controlled by phosphorylation and dephosphorylation of
mRNP adapterproteins suchas REF that assistin the binding
of the TAPNxtl mRNP exporter to mRNPs. In one case,a
yeast SR protein (Npl3) functions as an adapter protein that
promotes the binding of the yeastmRNP exporter (Figure 8-
22).The SR-protein initially binds to nascentpre-mRNA in
its phosphorylatedform.'When 3'-cleavageand polyadenyla-
tion are completed,the adapter protein is dephosphorylated
by a specificnuclear protein phosphataseessentialfor mRNP
export. Only the dephosphorylatedadapter protein can bind
the mRNP exporter, thereby coupling mRNP export to cor-
rect polyadenylation. This is one form of mRNA "qualify
control. " If the nascentmRNP is not correctly processed,it is
not recognized by the phosphatasethat dephosphorylates
Npl3. Consequently,it is not bound by the mRNA exporter
and not exported from the nucleus.Insteadit is degradedby
exosomes,the multiprotein complexesthat degrade unpro-
tected RNAs in the nucleusand cytoplasm (seeFigure 8-1)'
' 343
T F m R N A A C R O S ST H E N U C L E A RE N V E L O P E
T R A N S P O RO
 RNApol ll

R N Ap o l l l R N Ap o l l l

AAAAAAA

Nucleoplsam E
:ti

AAAAAAA ,a ..'ri:
NPC
z
r:ll llllr''

TAP/Nxt1
Cytoplasm

AAAAAAA
g
AAAAAAA

TAP/Nxt1

Translation

A FIGURE 8-22 Reversible phosphorylation and directionof Step4 Thecytoplasmic proteinkinaseSkyl phosphorylatesNpl3in
mRNPnuclearexport.StepIl: TheyeastSRproteinNpl3binds the cytoplasm,
causingE dissociation
of the mRNP exporter,
and
nascent pre-mRNAs in itsphosphorylated form StepA When phosphorylatedNpl3probably
throughtheactionof an RNAhelicase
p o l y a d e n y l a thi oanso c c u r r esdu c c e susl fl yt,h e G l c Tn u c l e a r associated
with NPCcytoplasmicfilaments6 ThemRNAtransporter
p h o s p h a t aesses e n t ifaolr m R N pe x p o rdt e p h o s p h o r y l aNt e p sl 3 , andphosphorylatedNpl3aretransported backintothe nucleus
promoting the bindingof the yeastmRNpexporter, TAp/Nxt1. Step throughNPCsZ Transported mRNAisavailable for translation
in
B. the mRNP e x p o r t earl l o w sd i f f u s i oonf t h e m R N pc o m p l e x thecytoplasm E lzaurralde,2004,Nat
fFrom StructMol Biol11:Z1O-Z\2
t h r o u g ht h ec e n t r acl h a n n eolf t h e n u c l e acro r ec o m p l e(xN p C ) seew Gilbert
andc Guthrie,
2004,Mol Cell13:201-2121

Following exporr to the cytoplasm, the Npl3 SR protein
is phosphorylated by a specific protein kinase resrricted to
the cytoplasm.This causesit to dissociatefrom the mRNp,
along with the mRNP exporter. In this wa5 dephosphoryla-
tion of adapter mRNP proteins in the nucleus once RNA
processingis complete and their phosphorylation in the
cytoplasm results in a higher concentration of mRNp
exporter-mRNP complexesin the nucleus,where they form,
and a lower concentration of these complexes in the cyto-
plasm, where they dissociate.As a result, the direction of
mRNP export may be driven by simple diffusion down a
concentration gradient of the transport-competent mRNP
exporter-mRNP complex across the NPC from high in the
nucleusto low in the cytoplasm.
Nuclear Export of Balbiani Ring mRNPs The larval sali-
vary glands of the insect Chironomows tentans provide a
good model systemfor electron microscopy studiesof the

344 CHAPTER
8 | P o S T - T R A N S C R t p T t OG
NEAN
L EC O N T R O L
 < FIGURE 8-23 Formationof heterogeneousribonucleoprotein
particles(hnRNPs) and exportof mRNPs from the nucleus'
(a)Modelof a singlechromatin transcription loopandassembly of
Balbiani ring(BR)mRNP in Chironomous tentansNascent RNA
transcripts produced fromthetemplate DNArapidly associate with
proteins, forminghnRNPs Thegradual increase in sizeof the hnRNPs
reflects the increasing lengthof RNAtranscripts at greater distances
fromthetranscription startsite.Themodelwasreconstructed from
electron micrographs of serialthinsections of salivary glandcells.
o f t h e b i o g e n e soi sf h n R N P F
( b )S c h e m a tdi ci a g r a m s .o l l o w i n g
processing of the pre-mRNA, the resulting ribonucleoprotein particle
is referred to as an mRNP (c) Model for the transport of BR mRNPs
throughthe nuclear porecomplex (NPC) based on electron microscopic
studres, Notethatthe curvedmRNPs appearto uncoilastheypass
throughnuclear pores. Asthe mRNAentersthecytoplasm, it rapidly
associates with ribosomes, indicating that the 5' end passes through
the NPCfirst [Part (a)fromC Erricson etal, 1989, Ceil55:631; courtesy of
B Daneholt Parts(b) and (c) adaptedfrom B Daneholt,1997, Ce//88:585
\
Seealso B Daneholt,2001, Proc Nat'l Acad Sci USA 98:7012 l
Template mRNP
DNA

(c) N u c l e a re n v e l o P e

i :i Cytoplasm
Nucleoplasm

formation of hnRNPs and their export through NPCs. In
these larvae, genesin large chromosomal puffs called
Balbiani rings are abundantly transcribedinto nascentpre-
mRNAs that associate with hnRNP proteins and are
processedinto coiled mRNPs with an mRNA of =75 kb
(Figure 8-23a, b). These giant mRNAs encode large glue
proteins that adhere the developinglarvae to a leaf. After
processingof the pre-mRNA in Balbiani ring hnRNPs, the
resulting mRNPs move through nuclear pores to the cyto-
p l a s m . E l e c t r o n m i c r o g r a p h s o f s e c t i o n so f t h e s e c e l l s
show mRNPs that appear to uncoil during their passage
through nuclear pores and then bind to ribosomesas they
enter the cytoplasm. This uncoiling is probably a conse-
quenceof the remodeling of mRNPs as the result of phos-
phorylation of mRNP proteins by cytoplasmickinasesand
the action of an RNA helicaseassociatedwith NPC cyto-
plasmic filaments,as discussedin the previous section.The
o b s e r v a t i o n t h a t m R N P s b e c o m e a s s o c i a t e dw i t h r i b o -
somesduring transport indicatesthat the 5' end leads the
way through the nuclear pore complex. Detailed electron
microscopic studies of the transport of Balbiani ring
mRNPs through nuclear pore complexesled to the model
depictedin Figure 8-23c.
Are Not Exported
in Spliceosomes
Pre-mRNAs
from the Nucleus
It is critical that only fully processedmature mRNAs be ex-
ported from the nucleusbecausetranslation of incompletely
processedpre-mRNAs containing introns would produce
defectiveproteins that might interfere with the functioning
of the cell. To prevent this, pre-mRNAs associatedwith
snRNPs in spliceosomesusually are prevented from being
transportedto the cytoplasm.
In one type of experiment demonstratingthis restriction'
a gene encoding a pre-mRNA with a single intron that nor-
mally is splicedout was mutated to introduce deviationsfrom
the consensussplice-sitesequences. Mutation of either the 5'
or the 3'invariant splice-sitebasesat the ends of the intron
resultedin pre-mRNAs that were bound by snRNPs to form
spliceosomes;however, RNA splicing was blocked, and the
ore-mRNA was retainedin the nucleus.In contrast, mutation

T F M R N A A C R O S ST H E N U C L E A RE N V E L O P E
T R A N S P O RO 345
of both the 5'and 3'splice sitesin the samepre-mRNA re-
sulted in export of the unspliced pre-mRNA, although less
efficiently than for the splicedmRNA.'fi/hen both splicesites
were mutated, the pre-mRNAs were not efficiently bound by
snRNPs,and, consequently,their export was not blocked.
Recentstudiesin yeasthave shown that a nuclear protein
that associates with a nucleoporinin the NPC nucleaibasket
is requiredto retain pre-mRNAs associatedwith snRNPsin
the nucleus.If either this protein or the nucleoporin to which
it binds is deleted,unsplicedpre-mRNAs are exported.
Many casesof thalassemia,an inherited diseasethat
resultsin abnormallylow levelsof globin proterns,are
due to mutationsin globin-genesplicesitesthat decreasethe
efficiency of splicing but do not prevent association of the
pre-mRNA with snRNPs. The resulting unspliced globin
pre-mRNAs are retained in reticulocyte nuclei and are rap-
idly degraded.I
HIV Rev ProteinRegulatesthe Transport
o f U n s p l i c e dV i r a l m R N A s
As discussedearlier,transport of mRNPs containing marure,
functional mRNAs from the nucleusto the cytoplasm entails
a complex mechanismthat is crucial to gene expression(see
Figures8-21, 8-22, and 8-23). Regulationof this transport
theoreticallycould provide another meansof genecontrol, al-
though it appearsto be relativelyrare. Indeed,the only known
examplesof regulatedmRNA export occur during the cellular
H I Vp r o v i r u s
**'
|
Transcription, splicing
N U C L E A Rm R N A s I CYTOPLASMIC
*
9-kb
Unspliced
4-kb
S i n g l ys p l i c e d
2-kb
M u l t i p l ys p l i c e d
Nucleoplasm
A FIGURE8-24 Transport of HIV mRNAs from the nucleus to
the cytoplasm. The HIVgenome,which containsseveralcoding
regions,is transcribedinto a single9-kb primarytranscriptSeveral
=4-kb mRNAsresultfrom alternativesplicingout of any one of
severalintrons(dashedlines)and several=2-kb mRNAsfrom splicing
out of two or more alternativeintrons After transportto the
346 c H A p T E R8 | P o S T - T R A N S C R t p T t O NGAELN EC O N T R O L
responseto conditions (e.g.,heat shock)that causeprotein de-
naturation or during viral infection when virus-inducedalter-
ations in nuclear transport maximize viral replication. Here
we describethe regulation of mRNP export mediated by a
protein encodedby human immunodeficiencyvirus (HIV).
A retrovirus, HIV integratesa DNA copy of its RNA
genomeinto the host-cellDNA (seeFigure4-49).The
integrated viral DNA, or provirus, contains a single tran-
scription unit, which is transcribedinto a singleprimary tran-
script by cellular RNA polymeraseII. The HIV transcript can
be spliced in alternative ways to yield three classesof mR-
NAs: a 9-kb unsplicedmRNA; :4-kb mRNAs formed by re-
moval of one intron; and:2-kb mRNAs formed by removal
of two or more introns (Figure 8-24). After their synthesisin
the host-cell nucleus, all three classesof HIV mRNAs are
transported to the cytoplasm and translated into viral pro-
teins; some of the 9-kb unspliced RNA is used as the viral
genomein progeny virions that bud from the cell surface.
Sincethe 9-kb and 4-kb HIV mRNAs contain splicesites,
they can be viewed as incompletely spliced mRNAs. However,
as discussedearlier,associationof such incompletely spliced
mRNAs with snRNPs in spliceosomesnormally blocks their
export from the nucleus.Thus HIV, as well as other retro-
viruses, must have some mechanism for overcoming this
block, permitting export of the longer viral mRNAs. Some
retroviruseshave evolved a sequencecalled the constitutiue
trdnsport element (CTE), which binds to the TAP-Nxt1
mRNP exporter with high affintty,thereby permitting export
mRNAs
9kb
-+
$ Rev protein
2 kb Translation
Cytoplasm
cytoplasm,the variousRNAspeciesare translatedinto differentviral
proteins Revprotein,encodedby a 2-kb mRNA,interactswith the
Rev-response element(RRE) in the unspliced
and singlysplicedmRNAs,
stimulatingtheir transportto the cytoplasm[Adapred fromB R Cullen
andM H Malim,1991,Trends BiochemScl 16:346I
of unsplicedretroviral RNA into the cytoplasm. HIV solved
the problem differently.
Studies with HIV mutants showed that transport of
unspliced 9-kb and singly spliced4-kb viral mRNAs from
the nucleus to the cytoplasm requires the virus-encoded
Rev protein. Subsequentbiochemicalexperimentsdemon-
strated that Rev binds to a specificRev-responseelement
(RRE) present in HIV RNA. In cells infected with HIV
mutants lacking the RRE, unspliced and singly spliced
viral mRNAs remain in the nucleus, demonstrating that
the RRE is required for Rev-mediated stimulation of
nuclear export. Rev contains a leucine-richnuclear export
signal that interacts with transporter exportinl. As discussed
in Chapter 13, this results in export of the unspliced and
singly spliced HIV mRNAs through the nuclear pore
complex. I
Transport of mRNA Across the Nuclear Envelope
r Most mRNPs are exported from the nucleus by a het-
erodimeric mRNP exporter that interacts with FG-repeats
of FG-nucleoporins(see Figure 8-20). The direction of
transport (nucleus-+ cytoplasm) may result from dissocia-
tion of the exporter-mRNP complex in the cytoplasm by
phosphorylation of mRNP proteins by cytoplasmic kinases
and the action of an RNA helicase associatedwith the
cytoplasmic filaments of the nuclear pore complexes (see
Figure8-20).
r The mRNP exporter binds to most mRNAs coopera-
tively with SR proteins bound to exons and REF associated
with exon-junction complexesthat bind to mRNAs follow-
ing RNA splicing, and to additional mRNP proteins.
r Pre-mRNAs bound by a spliceosomenormally are not
exported from the nucleus, ensuring that only fully
processed,functional mRNAs reach the cytoplasm for
translation.
f[ CytoplasmicMechanisms
of Post-transcriptional
Control
Before proceeding,let's quickly review the stepsin gene ex-
'We
pression at which control is exerted. saw in the previous
chapter that regulation of transcription initiation is the prin-
cipal mechanism for controlling the expressionof genes.In
preceding sections of this chapter, we also learned that the
expression of protein isoforms is controlled by regulating
alternative RNA splicing. Although nuclear export of fully
and correctly processedmRNPs to the cytoplasm is rarely
regulated, the export of improperly processedor aberrantly
remodeled pre-mRNPs is prevented, and such abnormal
transcripts are degraded by the exosome. However, retro-
viruses, including HIV, have evolved mechanismsthat per-
mit pre-mRNAs that retain splice sites to be exported and
translated(seeFigure 8-24).
CYTOPLASMIM
In this section we consider other mechanismsof post-
transcriptional control that contribute to regulating the ex-
pression of some genes.Most of these mechanismsoperate
localization
in the cytoplasm, controlling the stability or.!7e
of mRNA or its translation into protein. begin by
discussingtwo recently discoveredand related mechanisms
of gene control that provide powerful new techniques for
manipulating the expressionof specificgenesfor experimen-
tal and therapeutic purposes. These mechanisms are con-
trolled by short, -21 nucleotide, single-strandedRNAs
called micro RNAs (miRNAs) and short interfering
(siRNAs). Both base-pairwith specifictarget mRNAs, either
inhibiting their translation (miRNAs) or causingtheir degra-
dation (siRNAs). Humans express=1,000 miRNAs. Most
of theseare expressedin specificcell types at particular times
during embryogenesisand after birth. Many miRNAs can
target more than one mRNA. Consequently,thesenewly dis-
covered mechanismscontribute significantly to the regula-
tion of gene expression. siRNAs, involved in the process
called RNA interference, are also an important cellular
defenseagainstviral infection and excessivetransposition by
transposons.
Micro RNAsRepressTranslation
of SpecificmRNAs
Micro RNAs (miRNAs) were first discoveredduring analy-
sis of mutations in the lin-4 and let-7 genesof the nematode
C. elegans,which influence development of the organism.
Cloning and analysis of wild-type lin-4 and let-7 revealed
that they encode no protein products but rather RNAs
only 2t and 22 nucleotides long, respectively.The RNAs
hybridize to the 3' untranslated regions of specific target
mRNAs. For example, the lin-4 miRNA, which is expressed
early in embryogenesis,hybridizes to the 3' untranslatedre-
gions of both the lin-14 and /lz-28 mRNAs in the cytoplasm,
thereby repressingtheir translation by a mechanism dis-
cussedbelow. Expressionof lin-4 miRNA ceaseslater in de-
velopment, allowing translation of newly synthesizedlin-14
and lin-28 mRNAs at that time. Expressionof /er-7 miRNA
occurs at comparable times during embryogenesisof all
bilaterally symmetric animals. The role of lin-4 and let-7
miRNAs in coordinating the timing of early developmental
events in C. elegans is discussedin Chapter 22. Hete we
focus on what is currently understood about how miRNAs
represstranslation.
miRNA regulation of translation appears to be wide-
spreadin all multicellular plants and animals. In the past few
years, small RNAs of 20-26 nucleotideshave been isolated,
cloned, and sequenced from various tissues of multiple
model organisms.Recentestimatessuggestthe expressionof
one-third of all human genesis regulated by -1,000 human
miRNAs isolated from various tissues.The potential for reg-
ulation of multiple mRNAs by one miRNA is great because
base pairing between the miRNA and the sequencein the
3'-ends of mRNAs that they regulate need not be perfect
(Figure 8-25). In fact, considerableexperimentationwith
CA
OF POST-TRANSCRIPTION
C ECHANISMS OLN T R O L 347
 ( a ) m i R N A- + t r a n s l a t i o ni n h i b i t i o n
p
OH
tlllllllllllltttt!rttNt'tttrrii
TargetRNA
UC
CA
CA
5,-UCCCUGAGA GuGuGR-g,
||r|
3' -UCCAGGGACUCAACCAACACUCAA-
5'
|||l
/in-4miRNAandlin-l4 mRNA(C.e/egans)
b'-ucuuAccuGGAucnnnAcrr-
3,
- lIlll| |r||t
3'-GCCACAAUCGAAACACUUUUGAAGGC-5'
miRNAandtargetmRNA(H. sapiensl
CXCR4
FIGURE 8-25 Basepairingwith target RNAsdistinguishes
miRNAand siRNA.(a)miRNAs hybridize imperfectly
withtheir
targetmRNAs, repressing
translation
of the mRNANucleotides 2 to
7 of an miRNA(highlighted
blue)arethe mostcritical
for targeting
syntheticmiRNAs has shown that complementarity between
the six or seven5' nucleotides of an miRNA and its target
mRNA 3' untranslated region are most critical for target
mRNA selection.
Most miRNAs are processedfrom RNA polymerase II
transcriptsof severalhundred to thousandsof nucleotides
in length called pri (for primary transcript)-miRNAs (Fig-
we 8-26). Pri-miRNAs can contain the sequenceof one or
more miRNAs. miRNAs are also processedout of some
excisedintrons and from 3' untranslatedregions of some
pre-mRNAs. Within these long transcripts are sequences
that fold into hairpin structures of -70 nucleotides in
length with imperfect base pairing in rhe stem. A nuclear
RNase specific for double-strandedRNA called Drosha
acts with a nuclear double-stranded RNA-binding protein
called DGCRS in humans (Pasha in Drosophila) and
cleavesthe hairpin region out of the long precursor RNA,
generating a pre-miRNA. Pre-miRNAs are recognized and
bound by a specific nuclear transporter, ExportinS, which
interacts with the FG-domains of nucleoporins, allowing
the complex to diffuse through the inner channel of the
nuclearpore complex, as discussedabove (seeFigure 8-20),
and in Chapter 13. Once in the cytoplasm, a cytoplasmic
double-strandedRNA-specific RNase calledDicer acs with
a cytoplasmic double-strandedRNA-binding protein called
TRBP in humans (for Tar binding protein; called Loquo-
cious in Drosopbila) to further processthe pre-miRNA into
a double-strandedmiRNA. The double-strandedmiRNA
is approximately two turns of an A-form RNA helix in
length, with strands 21--23 nucleotides long and two un-
paired 3'-nucleotides at each end. Finally, one of the two
strands is selectedfor assemblyinto a mature RNA-lnduced
silencing complex (RISC) containing a single-stranded
348 C H A P T E R8 I POST.TRANSCRIPTION
GAELN EC O N T R O L
(b) siRNA-+ RNA cleavage
TargetRNA
5' -UAGGUAGUUUCAUGUUGUUGGG- 3'
l I l l l l I l | | | |
3' -CUUAUCCGUCAAAGUACAACAACCUUCU-
| |
5'
miR-196a and HOXBSmRNA (H. sapiensl
s,-ucGGAccAGGcuucAUUcccc-
3,
|||||||||t
3'-UUAGGCCUGGUCCGAAGUAGGGUUAGU-
mRNA(A.thalianal
miR-166andPHAVOLUIA
mRNA(b)siRNAhybridizes
it to a specific perfectly
with itstarget
mRNA, causingcleavageof the mRNAat the position indicatedby
the redarrow,triggering itsrapiddegradation fndaptedfromp D
Zamore andB Haley,2005,Sclence309:1
519 l
mature miRNA bound by a multidomain Argonaute pro-
tein, a member of a protein family with a recognizablecon-
served sequence.SeveralArgonaute proteins are expressed
in some organisms,especiallyplants, and are found in dis-
tinct RISC complexeswith different functions.
The miRNA-RISC complexesassociatewith target mRNPs
by basepairing betweenthe Argonaute-bound mature miRNA
and complementary regions in the 3'-untranslated regions
(3'-UTRs) of target mRNAs (seeFigure 8-25). Inhibition of
target mRNA translation requires the binding of two or
more RISC complexesto distinct complementary regions in
the target mRNA 3'-UTR. It has been suggestedthat this
may allow combinatorial regulation of mRNA translation
by separately regulating the transcription of two or more
different pri-miRNAs, which are processedto miRNAs that
are required in combination to suppressthe translation of a
specifictarget mRNA.
The binding of several RISC complexes to an mRNA
inhibit translation initiation by a mechanismcurrently being
analyzed.Recent discoveriesshowed that binding of RISC
complexescausesthe bound mRNPs to associatewith dense
cytoplasmicdomains many times the sizeof a ribosomecalled
cytoplasmicRNA-processingbodies,or simply P bodies.P
bodies, which will be describedin greater detail below, are
sites of RNA degradation that contain no ribosomes or
translation factors, potentially explaining the inhibition of
translation. The associationwith P bodies may also explain
why expressionof an miRNA often decreasesthe stability of
a targeted mRNA.
As mentioned earlier, approximately 1000 different hu-
man miRNAs have beenobserved,many of them expressed
only in specific cell types. Determining the function of
these miRNAs is currently a highly active area of research.
 Figure 8-16). It binds to the 3' splicesite region in the pre-
mRNAs of many genes,leading to exon skipping or use of
alternative 3' splice sites.I7hen miR-133 is expressedin
differentiating myoblasts, the PTB concentration falls
without a significant decreasein the concentrationof PTB
mRNA. As a result, alternativeisoforms of multiple pro-
teins important for muscle-cellfunction are expressedin
the differentiatedcells.
Other examplesof miRNA regulation in various organ-
isms are being discoveredat a rapid pace' Knocking out the
dicer geneeliminatesthe generationof miRNA in mammals.
This causesembryonic death early in mouse development.
However, when dicer is knocked out only in limb primordia,
the influence of miRNA on the development of the
nonessentiallimbs can be observed(Figure8-27). Although
Drosha
Pasha all major cell types differentiated and fundamental aspects
of limb patterning were maintained, development was
pre-miR-1-1
abnormal-demonstrating the importance of miRNAs in
regulatingthe proper level of translation of multiple mRNAs.
Of the :1,000 human miRNAs, 53 appearto be unique to
primates. It seems likely that new miRNAs arose readily
during evolution by the duplication of a pri-miRNA gene
followed by mutation of basesencodingthe mature miRNA.
miRNAs are particularly abundant in plants-more than
1.5 million distinct miRNAs have been characterizedin
Arabidopsis tbaliana!

R N AI n t e r f e r e n c eI n d u c e sD e g r a d a t i o n
y o m p l e m e n t a rm
o f P r e c i s e lC Y RNAs
s'-pQAUAQUUQVuuaunuGccc4un-
a'
RNA interference(RNAi) was discoveredunexpectedlydur-
mrH-l-l |iltIt|||t Il
3'-oUGUAUGAAGAAAUGUA 5'
O GGUp- ing attempts to experimentally manipulate the expression

M a t u r em i R - 1 - 1
I of specificgenes.Researcherstried to inhibit the expression
of a gene in C. elegansby microiniecting a single-stranded,
complementary RNA that would hybridize to the encoded
b o u n dt o a n
RISC mRNA and prevent its translation, a method called antisense
Argonaute
protein
inhibition. But in control experiments,perfectly base-paired
double-strandedRNA a few hundred base pairs long was

FIGURE 8-26 miRNAprocessing. Thisdiagram shows

transcriptionandprocessingof themiR-1-1 miRNATheprtmary Wild type Dicer mutant
miRNA transcript(pri-miRNA)istranscribedby RNApolymerase ll The
nucleardouble-stranded specific
endoribonuclease Droshawithits
partnerdouble-stranded RNA-binding proteinDGCRB (Pasha in
Drosophila) maketheinitialcleavagesin thepri-miRNA, generating
a =70nucleotide pre-miRNA thatisexported to thecytoplasm by
nucleartransporter 5 Thepre-miRNA
Exportin isfurtherprocessed in
thecytoplasm to a double-strandedmiRNA witha two-base single-
stranded 3' endby Dicerinconlunction withtheDSRNA-binding protein
TRBP (Loquatiousin Drosophila)Finally,
oneof the two strands is
incorporated intoan RISC complex, whereit isboundbyanArgonaute
A EXPERIMENTAL FIGURE 8-27 miRNAfunctionin limb
protein[Adapted fromP D Zamoreand B Haley,2005,Sclence309:1519 ]
development.Micrographs comparing normal(/eft)andDicer
knockout (nghf)limbsof embryonic developmentday-13mouse
embryos immunostained for the Gd5 protein,
a marker of joint
In one example, a specific miRNA called miR-133 is in- formation Diceris knocked out in mouse
developing embryos by
duced when myoblastsdifferentiateinto musclecells.miR- conditionalexpression of Creto inducedeletronof the Dicergene
133 suppresses the translationof PTB, a regulatory splicing onlyin thesecells(seeFigure B D Harfe
5-42)[From etal,2005,Proc
factor that functions similarly to Sxl in Drosophila (see Natl Acad Sci IJSA102:10898l

CAOLN T R O L . 349
C E C H A N I s M SO F P O S T - T R A N S C R I P T I O N
CYTOPLASMIM
 much more effectiveat inhibiting expressionof the genethan
the antisensestrand alone. Similar inhibition of geneexpres-
sion by an introduced double-strandedRNA soon was
observed in plants. In each case, the double-stranded
RNA induced degradation of all cellular RNAs containing
a sequencethat was exactly the same as one strand of the
double-stranded RNA. Becauseof the specificity of RNA
interference in targeting mRNAs for destruction, it has
become a powerful experimental tool for studying gene
function (seeFigure 5-45).
Subsequent biochemical studies with extracts of
Drosophila embryos showed that a long double-stranded
RNA that mediatesinterferenceis initially processedinto a
double-stranded short interfering RNA (siRNA). The
strands in siRNA contain 21-23 nucleotides hybridized to
each other so that the two basesat the 3' end of each strand
are single-stranded.Further studies revealed that the cyto-
plasmic double-strandedRNA-specific ribonucleasethat
cleaveslong double-strandedRNA into siRNAs is the same
Dicer enzymeinvolved in processingpre-miRNAs after their
nuclear export to the cytoplasm (seeFigure 8-27). This dis-
covery led to the realization that RNA interference and
miRNA-mediated translational repression are related
processes.In both cases,the mature short single-stranded
RNA, either mature siRNA or mature miRNA, is assembled
into RISC complexesin which the short RNAs are bound by
an Argonaute protein. I7hat distinguishesa RISC complex
containing an siRNA from one containing an miRNA is that
the siRNA base-pairsextensivelywith its target RNA and in-
ducesits cleavage,whereas a RISC complex associatedwith
an miRNA recognizes its target through imperfect base-
pairing and results in inhibition of translation.
The Argonaute protein appears to be responsiblefor
cleavageof target RNA; one domain of the Argonaure pro-
tein is homologous to RNase H enzymesthat degrade the
RNA of an RNA-DNA hybrid (seeFigure 5-14). When the
5' end of the short RNA of a RISC complex base-pairspre-
cisely with a target mRNA over a distanceof one turn of an
RNA helix (10-12 base pairs), this domain of Argonaute
cleavesthe phosphodiester bond of the target RNA across
from nucleotides10 and 1.1of the siRNA (seeFigure 8-25).
The cleaved RNAs are releasedand subsequentlydegraded
by cytoplasmic exosomesand 5' exoribonucleases.If base
pairing is not perfect, the Argonaute domain doesnot cleave
or releasethe target mRNA. Instead,if severalmiRNA-RISC
complexes associatewith a target mRNA, its translation is
inhibited and the mRNA becomesassociatedwith p bodies,
where, as mentioned earlier, it is probably degraded by a
different and slower mechanismthan the degradation path-
way initiated by RISC cleavageof a perfectly complementary
target RNA.
When double-strandedRNA is introduced into the cyto-
plasm of eukaryotic cells, it entersthe pathway for assembly
of siRNAs into a RISC complex becauseit is recognizedby
the cytoplasmic Dicer enzyme and TRBp double-stranded
RNA binding protein that processpre-miRNAs (seeFigure
8-25). This processof RNA interferenceis believedto be an
ancient cellular defenseagainst certain viruses and mobile
350 . c H A p r E R8 | p o s r - T R A N s c R t p l o N AGLE N Ec o N T R o L
genetic elements in both plants and animals. Plants with
mutations in the genes encoding Dicer and RISC proteins
exhibit increasedsensitivityto infection by RNA virusesand
increasedmovement of transposons within their genomes.
The double-stranded RNA intermediates generated during
replication of RNA viruses are thought to be recognizedby
the Dicer ribonuclease,inducing a RNAi responsethat
ultimately degradesviral mRNAs. During transposition,
transposonsare inserted into cellular genesin a random ori-
entation, and their transcription from different promoters
produces complementary RNAs that can hybridize with
each other, initiating the RNAi system that then interferes
with the expressionof transposon proteins required for ad-
ditional transpositions.
In plants and C. elegans the RNAi response can be
induced in all cells of the organism by introduction of
double-strandedRNA into just a few cells. Such organism-
wide induction requires production of a protein that is ho-
mologous to the RNA replicasesof RNA viruses.This find-
ing suggeststhat double-strandedsiRNAs are replicated and
then transferred to other cells in theseorganisms.In plants,
transfer of siRNAs might occur through plasmodesmata,the
cytoplasmicconnectionsbetweenplant cellsthat traversethe
cell walls between them (seeFigure 1,9-38).Organism-wide
induction of RNA interferencedoesnot occur in Drosophila
or mammals, presumably becausetheir genomes do not
encodeRNA replicasehomologs.
In mammalian cells,the introduction of long RNA-RNA
duplex moleculesinto the cytoplasm results in the general-
ized inhibition of protein synthesisvia the PKR pathway,
discussedfurther below. This greatly limits the use of long
double-strandedRNAs to experimentally induce an RNAi
response against a specific targeted mRNA. FortunatelS
researchersdiscovered that one strand of double-stranded
siRNAs of 21-23 nucleotidesin length with two-base 3' single-
stranded regions leads to the generation of mature siRNA
RISC complexeswithout inducing the generalizedinhibition
of protein synthesis. This has allowed researchersto use
synthetic double-strandedsiRNAs to "knock down" the
expressionof specificgenesin human cellsas well as in other
mammals. This method of siRNA knockdown is now widely
used in studies of diverse processes,including the RNAi
pathway itself.
RNA| Inhibition of Transcription In plants and the fis-
sion yeast Schizosaccharomyces pombe, double-stranded
RNA also induces the formation of heterochromatin on
geneswith the same sequenceas the double-strandedRNA,
inhibiting their transcription. Nuclear proteins homologous
to cytoplasmic Dicer and Argonaute proteins generatenu-
clear siRNA complexescomposedof different proteins from
the cytoplasmicRISC complexes.Thesenuclear siRNA com-
plexes are thought to be targeted to specific genesby base
pairing with nascentpre-mRNAs during their transcription.
This interaction induces the methylation of histone H3 at
lysine 9, generating a binding site for HP1 proteins and the
subsequentassemblyof heterochromatin,as discussedin
Chapter 6 (seeFigure 6-34).In plants, the DNA in these
heterochromatic regions also is methylated, contributing
to the formation of heterochromatin. Components of the
RNAi system are also required for the formation of hete-
rochromatin at centromeresand the proper function of cen-
tromeres in S. pombe, plants, and cultured mammalian
cells. Centromeres from most organisms contain highly
repetitive DNA sequences.Consequently,the RNAi system
that leads to heterochromatizationof repeated genes in
S. pombe and plants may be exploited generallyby most
eukaryotes for the proper formation of the DNA-protein
kinetochore complex formed at centromeresand critical for
cell division (Chapter20).
CytoplasmicPolyadenylationPromotes
Translationof SomemRNAs
In addition to repressionof translation by miRNAs, other
protein-mediatedtranslational controls help regulateexpres-
sion of some genes.Regulatory sequences,or elements,in
mRNAs that interact with specificproteinsto control transla-
tion generallyare presentin the untranslatedregion (UTR) at
the 3' or 5' end of an mRNA. Here we discussa type of pro-
tein-mediatedtranslational control involving 3' regulatory ele-
ments. A different mechanisminvolving RNA-binding proteins
that interact with 5' regulatory elementsis discussedlater.
Translation of many eukaryotic mRNAs is regulated by
sequence-specific RNA-binding proteins that bind coopera-
tively to neighboring sites in 3' UTRs. This allows them to
function in a combinatorial manner,similar to the cooperative
binding of transcription factors to regulatory sites in an en-
hancer or promoter region. In most casesstudied,translation
is repressedby protein binding to 3' regulatory elementsand
regulationresultsfrom derepressionat the appropriatetime or
place in a cell or developingembryo. The mechanismof such
repressionis best understoodfor mRNAs that must undergo
cytoplasmic polyadenylation before they can be translated.
Cytoplasmic polyadenylation is a critical aspectof gene
expression in the early embryo of animals. The egg cells
(oocytes) of multicellular animals contain many mRNAs,
encoding numerous different proteins, that are not trans-
lated until after the egg is fertilized by a sperm cell. Some of
these "stored" mRNAs have a short poly(A) tail, consisting
of only -20-40 A residues,to which just a few moleculesof
cytoplasmic poly(A)-binding protein (PABPI) can bind. As
discussedin Chapter 4, multiple PABPI moleculesbound to
the long poly(A) tail of an mRNA interact with the eIF4G
initiation factor, thereby stabilizing the interaction of the
mRNA 5' cap with eIF4E, which is required for translation
initiation (seeFigure 4-28b). Becausethis stabilization can-
not occur with mRNAs that have short poly(A) tails, such
mRNAs stored in oocytes are not translated efficiently. At
the appropriate time during oocyte maturation or after fer-
tilization of an egg cell, usually in responseto an external
signal, approximately 150 A residuesare added to the short
poly(A) tails on thesemRNAs in the cytoplasm, stimulating
their translation.
Recent studies with mRNAs stored in Xenopus oocytes
have helped elucidatethe mechanismof this type of transla-
tional control. Experiments in which short-tailed mRNAs
are injected into oocytes have shown that two sequencesin
their 3' UTR are required for their polyadenylation in the
cytoplasm: the AAUAAA poly(A) signal that is also required
for the nuclear polyadenylation of pre-mRNAs and one or
more copiesof an upstreamU-rich cytoplasmicpolyadenyla-
tion element (CPE). This regulatory element is bound by a
highly conservedCPE-binding protein (CPEB) that contains
an RRM domain and a zinc-finger domain.
According to the current model, in the absenceof a stim-
ulatory signal, CPEB bound to the U-rich CPE interactswith
the protein Maskin, which in turn binds to eIF4E associated
with the mRNA 5' cap (Figure 8-28,left). As a result' eIF4E
cannot interact with other initiation factors and the 40S

T r a n s l a t i o n a l dl yo r m a n t ly active
Translational

TAAUAAA-A
UUUUAU
t-
A FIGURE 8-28 Model for control of cytoplasmic cleavage andpolyadenylation specificity factor(CPSF) thenbinds
pofyadenylationand translationinitiation.(Left)ln immature a n dt h e
t o t h e p o l y ( As)i t e ,i n t e r a c t i nwgi t h b o t hb o u n dC P E B
mRNAs
oocytes, containingthe U-richcytoplasmic polyadenylation cytoplasmic form of poly(A)polymerase (PAP). Afterthe poly(A)tail
element(CPE)haveshortpoly(A) tails CPE-binding protein(CPEB) islengthened, multiple copies of the cytoplasmic poly(A)-binding
mediatesrepression
of translationthroughthe interactions depicted, protein| (PABPI) canbindto it andinteract with elF4G, which
whichpreventassemblyof an initiationcomplex at the 5' endof the functions with otherinitiation factorsto bindthe40Sribosome
mRNA(R/EIht) Hormone stimulationof oocytes activatesa protein subunitandinitiate translation lAdapted fromR Mendez andJ D
thatphosphorylates
kinase CPEB, causrng it to releaseMaskinThe Richter. 2001.Nature Rev. Mol Cell Biol 2:521 I

C Y T O P L A S M IM CA
OF POST-TRANSCRIPTION
C ECHANISMS OLN T R O L 351
ribosomal subunit, so translation initiation is blocked. Dur-
ing oocyte maturation, a specific CPEB serineis phosphory-
lated, causing Maskin to dissociatefrom the complex. This
allows cytoplasmic forms of the cleavageand polyadenyla-
tion specificity factor (CPSF)and poly(A) polymeraseto
bind to the mRNA cooperatively with CPEB. Once the
poly(A) polymerase catalyzesthe addition of A residues,
PABPI can bind to the lengthenedpoly(A) tail, leading to the
stabilizedinteraction of all the participants neededto initiate
translation (Figure 8-28, right; seealso Figure 4-28). In the
case of Xenopus oocyte maturation, the protein kinase that
phosphorylatesCPEBis activatedin responseto the hormone
progesterone.Thus timing of the translationof storedmRNAs
encoding proteins neededfor oocyte maturation is regulated
by this external signal.
Considerableevidenceindicatesthat a similar mecha-
nism of translational control plays a role in learning and
memory. In the central nervous system, the axons from a
thousand or so neurons can make connections(synapses)
with the dendrites of a single postsynaptic neuron (Figure
23-23). When one of these axons is stimulated, the postsy-
naptic neuron "remembers" which one of thesethousandsof
synapseswas stimulated. The next time that synapseis stim-
ulated, the strengthof the responsetriggeredin ihe postsy-
naptic cell differs from the first time. This changein response
has been shown to result largely from the translational acti-
vation of mRNAs stored in the region of the synapse,lead-
ing to the local synthesisof new proteins that increasethe
size and alter the neurophysiological characteristicsof the
synapse.The finding that CPEB is present in neuronal den-
drites has led to the proposal that cytoplasmic polyadenyla-
tion stimulates translation of specific mRNAs in dendrites,
much as it does in oocytes.In this case,presumably,synaptic
activity (rather than a hormone) is the signal that induces
phosphorylation of CPEB and subsequentactivation of
translation.
Decapping pathway Deadenylation-dependent
(deadenylation-independent)
AAAAAA
A FIGURE 8-29 Pathwaysfor degradationof eukaryotic
mRNAs.ln the deadenylation-dependenl(niddle) pathways, the
poly(A)tailis progressively
shortened by a deadenylase(orange) until
it reaches
a lengthof 20 or fewerA residues,at whichpointthe
interaction
with PABPIisdestabilized,
leadingto weakened interactions
betweenthe 5' capandtranslation-initiation
factors.
Thedeadenylated
mRNAthenmayeither(1)be decapped anddegraded by a 5, -+ 3,
352 C H A P T E R8 I POST-TRANSCRIPTION
GAELN EC O N T R O L
Degradationof mRNAsin the Cytoplasm
Occursby SeveralMechanisms
The concentration of an mRNA is a function of both its rate
of synthesisand its rate of degradation. For this reason, if
two genesare transcribed at the same rate, the steady-state
concentration of the corresponding mRNA that is more
stablewill be higher than the concentration of the other. The
stability of an mRNA also determineshow rapidly synthesis
of the encoded protein can be shut down. For a stable
mRNA, synthesisof the encoded protein persistslong after
transcription of the geneis repressed.Most bacterialmRNAs
are unstable, decaying exponentially with a typical half-life
of a few minutes. For this reason,a bacterial cell can rapidly
adjust the synthesisof proteins to accommodatechangesin
the cellular environment. Most cells in multicellular organ-
isms, on the other hand, exist in a fairly constant environ-
ment and carry out a specificset of functions over periods of
days to months or even the lifetime of the organism (nerve
cells, for example). Accordingly, most mRNAs of higher
eukaryoteshave half-lives of many hours.
However, some proteins in eukaryotic cells are required
only for short periods and must be expressedin bursts. For
example, as discussedin the chapter introduction, certain
signaling moleculescalled cytokines, which are involved in
the immune responseof mammals, are synthesizedand se-
creted in short bursts. Similarly, many of the transcriprion
factors that regulatethe onset of the S phaseof the cell cycle,
such as c-Fos and c-Jun, are synthesizedfor brief periods
only (Chapter 20). Expression of such proteins occurs in
short bursts becausetranscription of their genescan be rap-
idly turned on and off, and their mRNAs have unusually
short half-lives, on the order of 30 minutes or less.
Cytoplasmic mRNAs are degraded by one of the three
pathways shown in Figure 8-29. For most mRNAs, the
deadenylation-dependentpatbway is followed: the length
Endonucleolytic
pathways pathway
AAAAAA AAAAAA
e.oonu"leotytic
I
creavase
I
exonuclease or (2) be degraded by a 3' -+ 5' exonuclease in
cytoplasmic
exosomes. SomemRNAs (right)arecleaved internally
by
an endonuclease andthefragments degraded by an exosome. Other
mRNAs (/eft)aredecapped beforetheyaredeadenylated andthen
degradedby a 5' + 3' exonuclease. lAdapted fromM Tucker and
R parker,
2ooo,Ann Rev. Biochem69:571I
of the poly(A) tail gradually decreaseswith time through
the action of a deadenylatingnuclease.Sfhen it is short-
ened sufficiently, PABPI molecules can no longer bind and
stabilizethe interaction of the 5' cap and translation initi-
ation factors (see Figure 4-28b). The exposed cap then is
removed by a decapping enzyme (Dcp1/Dcp2 in S. cere-
uisiae),and the unprotectedmRNA is degradedby a 5' -+
3' exonuclease(Xrn1 in S. cereuisiae).Removal of the
poly(A) tail also makes mRNAs susceptibleto degradation
by cytoplasmicexosomescontaining 3' -+ 5'exonucleases.
The 5' J 3' exonucleasespredominate in yeast, and the
3' -+ 5' exosome predominatesin mammalian cells. The
decappingenzymesand 5' -+ 3' exonucleaseare concen-
trated in the P bodies,regions of the cytoplasm of unusu-
ally high density (seeFigure 8-31).
Some mRNAs are degraded primarily by a deadenyla-
tion-independentdecappingpathway (seeFigure 8-29). This
is becausecertain sequencesat the 5' end of an mRNA seem
to make the cap sensitiveto the decappingenzyme.For these
mRNAs, the rate at which they are decapped controls the
rate at which they are degradedbecauseonce the 5' cap is re-
moved, the RNA is rapidly hydrolyzed by the 5' -+ 3' ex-
onuclease.
The rate of mRNA deadenylation varies inversely with
the frequency of translation initiation for an mRNA: the
higher the frequency of initiation, the slower the rate of
deadenylation.This relation probably is due to the recipro-
cal interactions between translation initiation factors bound
at the 5' cap and PABPI bound to the poly(A) tail. For an
mRNA that is translated at a high rate, initiation factors are
bound to the cap much of the time, stabilizing the binding of
PABPI and thereby protecting the poly(A) tail from the
deadenylationexonuclease.
Many short-lived mRNAs in mammalian cellscontain mul-
tiple, sometimesoverlapping copiesof the sequenceAUUUA
in their 3'-untranslated region. Specific RNA-binding pro-
teins have been found that both bind to these 3' AU-rich
sequencesand also interact with a deadenylating enzyme
and with the exosome.This causesrapid deadenylationand
subsequent3' --> 5' degradationof these mRNAs. In this
mechanism, the rate of mRNA degradation is uncoupled
from the frequency of translation. Thus mRNAs containing
the AUUUA sequencecan be translatedat high frequencyyet
also be degraded rapidly, allowing the encoded proteins to
be expressedin short bursts.
As shown in Figure 8-29, some mRNAs are degraded
by an endonwcleolyticpathuay that does not involve de-
capping or significant deadenylation.One example of this
type of pathway is the RNAi pathway discussedabove (see
Figure 8-25). Each siRNA-RISC complex can degrade
thousands of targeted RNA molecules. The fragments
generated by internal cleavage then are degraded by
exonucreases.
P Bodies As mentioned above, P bodies are sitesof transla-
tional repressionof mRNAs bound by miRNA-RISC com-
plexes. They are also the major sites of mRNA degradation
in the cytoplasm. These denseregions of cytoplasm contain
C Y T O P L A S M IM
the decapping enzyme (Dcp1/Dcp2 in yeast), activators of
decapping(Dhh, Pat1, Lsml-7 in yeast),the major 5' -+ 3'
exonuclease(Xrn1), as well as denselyassociatedmRNAs.
P bodies are dynamic structuresthat grow and shrink in size
dependingon the rate at which mRNPs associatewith them,
the rate at which mRNAs are degraded, and the rate at
which mRNPs exit P bodies and reenter the pool of trans-
lated mRNPs.
ProteinSynthesisCan Be GloballyRegulated
Like proteins involved in other processes,translation initia-
tion factors and ribosomal proteins can be regulatedby post-
translational modifications such as phosphorylation. Such
mechanismsaffect the translation rate of most mRNAs and
hencethe overall rate of cellular protein synthesis.
TOR Pathway The TOR pathway was discoveredthrough
researchinto the mechanismof action of rapamycin, an an-
tibiotic produced by a strain of Streptomycesbacteria,useful
for suppressingthe immune response in organ transplant
patients. The target of rupamycin (TOR)was identified by
isolating yeast mutants resistant to rapamycin inhibition of
cell growth. TOR is a large (-2400 amino acid residue)
protein kinase that regulatesseveralcellular processesin
yeast cells in responseto nutritional status. In multicellular
eukaryotes,metazoan TOR (wTOR) also respondsto mul-
tiple signals from cell-surface-signalingproteins to coordi-
nate cell growth with developmentalprograms as well as nu-
tritional status.
Current understandingof the mTOR pathway is summa-
rized in Figure 8-30. Active mTOR stimulates the overall
rate of protein synthesisby phosphorylating two critical pro-
teins that regulate translation directly. mTOR also activates
transcription factors that control expression of ribosomal
components,tRNAs, and translation factors, further activat-
ing protein synthesisand cell growth.
Recall that the first step in translation of a eukaryotic
mRNA is binding of the eIF4 initiation complex to the 5'
cap via its eIF4E cap-binding subunit (see Figure 4-24).
The concentrationof active eIF4E is regulated by a small
family of homologous elF4E-binding proteins (4E-BPs)
that inhibit the interaction of eIF4E with mRNA 5' caps.
4E-BPs are direct targets of mTOR. !(hen phosphorylated
by mTOR, 4E-BPs releaseeIF4E, stimulating translation
initiation. mTOR also phosphorylates and acttvatesan-
other protein kinase that phosphorylatesthe small riboso-
mal subunit protein S6 (S6K) and probably additional sub-
strates, leading to a further increase in the rate of protein
synthesis.
Translation of a specific subset of mRNAs that have a
string of pyrimidines in their 5' untranslated regions (called
TOP mRNAs for tract of oligopyrimidine) is stimulated par-
ticularly strongly by mTOR. The 5'TOP mRNAs encoderi-
bosomal proteins and translation elongation factors. mTOR
also activates the RNA polymerase I transcription factor
TIF1A, stimulating transcription of the large rRNA precursor
(seeSection 8.5 below). mTOR also activatestranscription
C ECHANISMS CAOLN T R O L
OF POST.TRANSCRIPTION
 Growth factor oo o
receptor Nutrients
o o
o
Exterior Stress o
hypoxia Low
o
o o
Cytoplasm o

Low
nutrients

Rapamycin

Ribosome Transcription Macroautophagy

biogenesis

FIGURE 8-30 mTORpathway.mTORisan activeproteinkinase activates mTORproteinkinase activity. Lownutrientconcentratton

whenboundby a complex of Rhebandan associated GTP(/ower alsoregulates RhebGTPase activity by a mechanism thatdoesnot
/eff) ln contrast,
mTORisinactive whenboundby a complex of Rheb require TSCl/TSC2 ActivemTORphosphorylates 4E-BP, causing it to
associated with GDP(lowerright).Whenactive, theTSCl/TSC2 release elF4E, stimulating translation initiationlt alsophosphorylates
Rheb-GTPase protein(Rheb-GAP)
activating causeshydrolysis
of andactivates 56 kinase (S6K), whichin turnphosphorylates ribosomal
Rheb-bound GTPto GDBtherebyinactivating mTORTheTSCl/TSC2 proteins, stimulating translation. Activated mTORalsoactivates
Rheb-GAP (arrows)
isactivated by phosphorylation
by AMPkinase t r a n s c r i p t ifoanc t o r fso r R N Ap o l y m e r a sl e, lsl ,a n dl l l ,l e a d i ntgo
(AMPK) whencellular energychargeis lowandby othercellular synthesis andassembly of ribosomes, tRNAs, andtranslation factors
stress responses.Signal-transduction
pathways activated
by cell- Intheabsence of mTORactivity, allof theseprocesses areinhibited
surface groMhfactorreceptors leadto phosphorylation
of inactivating I n c o n t r a sat ,c t i v a t em d T O Ri n h i b i tm
s a c r o a u t o p h awg hy i, c hi s
siteson TSCl/TSC2, inhibiting
itsGAPactivityConsequently,they stimulated in cellswith inactive mTOR[Adapted fromS Wullschleger
leavea higherfractionof cellularRhebin the GTPconformation that et al, 2006, Cell 124:471 l

by RNA polymerase III, although the mechanism is not
clear. In addition, mTOR activatestwo RNA polymeraseII
activators that stimulate transcription of ribosomal protein
and translation factor genes. Finally, mTOR stimulates
processingof the rRNA precursor(Section8.5). As a conse-
quence of phosphorylation of these several mTOR sub-
strates,the synthesisand assemblyof ribosomes as well as
the synthesisof translation factors and tRNAs are greatly in-
creased.Alternatively, when mTOR kinase activity is inhib-
ited, these substratesbecome dephosphorylated,greatly
decreasingthe rate of protein synthesisand the production
of ribosomes, translation factors, and tRNAs, thus halting
cell growth.
mTOR activity is regulated by a monomeric small G
protein in the Ras protein family called Rheb. Like other
small G proteins, Rheb is in its active conformation when it
is bound to GTP. Rheb .GTP binds the mTOR complex.
activating mTOR kinase activity, probably by inducing a
conformation changein its kinase domain. Rheb is in turn
regulatedby a heterodimercomposedof subunitsTSCl and
TSC2, named for their involvement in the medical syn-
drome /uberous sclerosis complex, as discussedbelow. In
the active conformation, the TSC1/TSC2 heterodimer func-
tions as a GTPaseactivatingprotein for Rheb (Rheb-GAP),
causing hydrolysis of the Rheb-bound GTP to GDP. This
converts Rheb to its GDP-bound conformation, which
binds to the mTOR complex and inhibits its kinase activiry.
Finally, the activity of the TSCI 1TSC2Rheb-GAP is regu-
lated by severalinputs, allowing the cell to integrate differ-
ent cellular signaling pathways to control the overall rate of
protein synthesis.Signaling from cell-surfacegrowth factor
receptors leads to phosphorylation of TSCIiTSC2 at in-
hibitory sites,causingan increasein Rheb'GTP and activa-
tion of mTOR kinase activity. This type of regulation

354 C H A P T E R8 I POST-TRANSCRIPTION
GAELN EC O N T R O L
through cell-surface receptors links the control of cell
growth to developmental processescontrolled by cell-cell
rnteractrons.
mTOR activity also is regulated in response to nutri-
tional status. When energy from nutrients is not sufficient
for cell growth, the resulting fall in the ratio of ATP to AMP
concentrationsis detectedby the AMP kinase. The activated
AMP kinase phosphorylatesTSCIiTSC2 at activating sites,
stimulating its Rheb-GAP activity and consequentlyinhibit-
ing mTOR kinase activity and the global rate of translation.
Hypoxia and other cellular stressesalso activate the TSCI/
TSC2 Rheb-GAP. Finally, the concentration of nutrients
in the extracellular space also regulatesRheb by an un-
known mechanismthat does not require the TSC1/TSC2
complex.
In addition to regulating the global rate of cellular pro-
tein synthesisand the production of ribosomes,tRNAs, and
translation factors, mTOR regulates at least one other
processinvolved in the responseto low levels of nutrients:
macroautophagy. Starved cells degrade cytoplasmic con-
stituents, including whole organelles,to supply energy and
precursors for essential cellular processes.During this
process alarge, double-membranestructure engulfs a region
of cytoplasm to form an autophagosome,which then fuses
with a lysosome where the entrapped proteins, lipids, and
other macromoleculesare degraded,completing the process
of macroautophagy.Activated mTOR inhibits macroau-
tophagy in growing cells when nutrients are plentiful.
Macroautophagy is stimulated when mTOR activity falls in
nutrient-deprived cells.
Genesencoding componentsof the mTOR pathway
ffi
3l a.e mutated in many human cancers,resulting in cell
growth in the absenceof normal growth signals.TSCl and
TSC2 (Figure 8-30) were initially identified becauseone or
the other of the proteins is mutant in a rare human genetic
syndrome: tuberous sclerosiscomplex. Patients with this
disorder develop benign tumors in multiple tissues. The
diseaseresultsbecauseinactivation of either TSCl or TSC2
eliminates the Rheb-GAP activity of the TSC1/TSC2 het-
erodimer,resulting in an abnormally high and unregulated
'
level of Rheb GTP and the resulting high, unregulated ac-
tivity of mTOR. Mutations in components of cell-surface
receptor signal-transduction pathways that lead to inhibi-
TSC1/TSCZ Rheb-GAP activity are also common
tion of
in human tumors and contribute to cell growth and repli-
cation in the absenceof normal signals for growth and
proliferation.
High mTOR protein kinase activity in tumors corre-
lates with a poor clinical prognosis.Consequently,mTOR
inhibitors are currently in clinical trials to test their effec-
tiveness for treating cancers in coniunction with other
modes of therapy. Rapamycin and other structurally related
mTOR inhibitors are potent suppressorsof the immune re-
sponsebecausethey inhibit activation and replication of
T lymphocytesin responseto foreign antigens(Chapter24).
C Y T O P L A S M IM
Severalviruses encode proteins that activate mTOR early
after viral infection. The resulting stimulation of transla-
tion has an obvious selective advantagefor these cellular
parasites.I
elF2 Kinases eIF2 kinasesalso regulatethe global rate of
cellular protein synthesis. Figure 4-24 summarizes the
steps in translation initiation. Translation initiation fac-
toi eIF2 brings the charged initiator tRNA to the small
ribosome subunit P site. eIF2 is a trimeric G protein and
consequently exists in either a GTP-bound or a GDP-
boundconformation. Only the GTP-bound form of eIF2
is able to bind the charged initiator IRNA and associate
with the small ribosomal subunit. The small subunit with
bound initiation factors and charged initiator IRNA then
interactswith the eIF4 complex bound to the 5' cap of an
mRNA via its eIF4E subunit. The small ribosomal sub-
unit then scansdown the mRNA in the 3'direction until
it reaches an AUG initiation codon that can base-pair
with the initiator IRNA in its P site. When this occurs,the
GTP bound by eIFZis hydrolyzed to GDP and the result-
ing eIF2'GDP complex is released.GTP hydrolysis results
in an irreversible "proofreading" step that prepares the
small ribosomal subunit to associatewith the large sub-
unit only when an initiator IRNA is properly bound in
the P site and is properly base-pairedwith the AUG start
codon. Before eIF2 can participate in another round of
initiation, its bound GDP must be replaced with a GTP.
This process is catalyzed by the translation initiation fac-
tor eIF2B, a guanine nucleotide exchange factor (GEF)
specific for eIF2.
A mechanism for inhibiting general protein synthesisin
stressedcells involves phosphorylation of the eIF2 ct subunit
at a specificserine.Phosphorylation at this site does not in-
terfere with eIF2 function in protein synthesis directly.
Rather, phosphorylated eIF2 has very high affinity for the
eIF2 guanine nucleotide exchange factor, eIF2B' which can-
not rilease the phosphorylated eIF2 and consequently is
blocked from catalyzing GTP exchange of additional eIF2
factors. Since there is an excessof eIF2 over eIF2B, phos-
phorylation of a fraction of elF2 results in inhibition of all
ihe cellular eIF2B. The remaining eIF2 accumulatesin its
GDP-bound form, which cannot participate in protein syn-
thesis,thereby inhibiting nearly all cellular protein synthesis'
Howeve! some mRNAs have 5' regions that allow transla-
tion initiation at the low eIF2-GTP concentration that re-
thesestress-induced Proteins.
Human cells contain four eIF2 kinasesthat phosphory-
late the same inhibitory eIF2a serine' Each of these is regu-
lated by a different type of cellular stress,inhibiting protein
synthesisand allowing cells to divert the large fraction of
CA
OF POST-TRANSCRIPTION
C ECHANISMS OLN T R O L
 cellular resourcesdevoted to protein synthesisin growing
cells for use in responding to the stress.
The GCN2 (general control non-derepressible2) eIF2-
k i n a s ei s a c t i v a t e db y b i n d i n g u n c h a r g e d
iRNRr. The con-
centration of uncharged tRNAs increaseswhen cells are
starved for amino acids,activating GCN2 elF2-kinaseactiv-
ity and greatly inhibiting protein synthesis.
PEK (pancreaticeIF2a kinase) is activatedwhen proteins
translocatedinto the endoplasmicreticulum (ER) do not
fold properly becauseof abnormalities in the ER lumen en-
vironment. Inducersinclude abnormal carbohydrateconcen-
tration becausethis inhibits the glycosylation of many ER
proteins and inactivating mutations in an ER chaperonere-
quired for proper folding of many ER proteins (Chapters 13
and 14).
Heme-regulatedinhibitor (HRI) is activatedin develoo-
ing red blood cells when the supply of heme prostheric
group is too low to accommodatethe rate of globin protein
synthesis. This negative feedback loop lowers the rate of
globin protein synthesis until ir matches the rate of heme
synthesis.HR1 is also activated in other types of cells in
responseto oxidative stressor heat shock.
Finally, protein kinase RNA activated (pKR) is acivated
by double-strandedRNAs longer than -30 basepairs. Un-
der normal circumstancesin mammalian cells, such double-
stranded RNAs are produced only during a viral infection.
Long regions of double-strandedRNA are generated in
replication intermediates of RNA viruses or from hy-
bridization of complementary regions of RNA transcribed
from both strands of DNA virus genomes.Inhibition of
protein synthesisprevents the production of progeny viri-
ons, protecting neighboring cells from infection. Inrerest-
inglS adenovirusesevolved a defenseagainst pKR: they
express prodigious amounts of an -1,60-nucleotide virus-
associated(VA) RNA with long double-strandedhairpin
regions. VA RNA is transcribed by RNA polymeraseIII and
exported from the nucleus by Exportin5, the exportin for
pre-miRNAs (seeFigure 8-27).VA RNA binds to pKR with
high affinity, inhibiting its protein kinase activity and pre-
venting the inhibition of protein synthesisobservedin iells
infected with a mutant adenovirus from which the VA gene
wasdeleted.
RNA-BindingproteinsControl
Sequence-Specific
SpecificmRNATranslation
In contrast to global mRNA regulation, mechanismshave
also evolved for conrolling the translation of certain specific
mRNAs. This is usually done by sequence-specificRNA-
binding proteins that bind to a particular sequenceor RNA
Control of intracellular iron concentration by the iron
response element-binding protein (IRE-BP) is an elegant
example of a singleprotein that regulatesthe translation of
356 . c H A p r E 8R | p o s r - T R A N S c R t p I oG
NEAN
LEcoNTRoL
one mRNA and the degradation of another. precise regula-
tion of cellular iron ion concentrationis critical to the cell.
Multiple enzymesand proteins contain Fe2* as a cofactor,
such as enzymesof the Krebs cycle (seeFigure 12-10) and
electron-carryingproteins involved in the generation of
ATP by mitochondria and chloroplasts (Chapter 12). On
the other hand, excessFe2* generatesfree radicalsthat re-
act with and damagecellular macromolecules.Ifhen intra-
cellular iron sroresare low, a dual-control systemoperates
to increasethe level of cellular iron; when iron is in excess,
the system operatesto prevent accumulation of toxic levels
of free ions.
One component in this system is the regulation of the
production of ferritin, an intracellular iron-binding protein
that binds and storesexcesscellular iron. The 5' untranslated
region of ferritin mRNA contains iron-response elements
(IREs)that have a stemJoop structure.The IRE-binding pro-
tein (IRE-BP) recognizesfive specific basesin the IRE loop
and the duplex nature of the stem. At low iron concentra-
tions, IRE-BP is in an active conformation that binds to the
IREs (Figure 8-31a). The bound IRE-BP blocks the small
ribosomal subunit from scanning for the AUG start codon
(see Figure 4-24), thereby inhibiting translation initiation.
The resulting decreasein ferritin means less iron is com-
plexed with the ferritin and is therefore available to iron-
requiring enzymes.At high iron concentrations,IRE-BP is in
an inactive conformation that does not bind to the 5, IREs,
so translation initiation can proceed.The newly synthesized
ferritin then binds free iron ions, preventing their accumula-
tion to harmful levels.
The other part of this regulatory system controls the
import of iron into cells. In vertebrates,ingested iron is
carried through the circulatory systembound to a protein
called transferrin. After binding to the transferrin receptor
(TfR) in the plasma membrane, the transferrin-iron com-
plex is brought into cells by receptor-mediatedendocyto-
sis (Chapter 14). The 3' untranslated region of TfR
mRNA contains IREs whose stemshave AU-rich destabi-
lizing sequences(Figure 8-31b). At high iron concentra-
tions, when the IRE-BP is in the inactive, nonbinding con-
formation, these AU-rich sequencespromote degradation
of TfR mRNA by the same mechanismthat leads to rapid
degradation of other short-lived mRNAs, as described
previously. The resulting decreasein production of the
transferrin receptor quickly reducesiron import, thus pro-
tecting the cell from excessiron. At low iron concenrra-
tions, however, IRE-BP can bind to the 3, IREs in TfR
mRNA. The bound IRE-BP blocks recognition of the
destabilizing AU-rich sequences by the proteins that
would otherwise rapidly degradethe mRNAs. As a result,
production of the transferrin receptor increasesand more
iron is brought into the cell.
Other regulated RNA-binding proteins may also func-
tion to control mRNA translation or degradation,much like
the dual-actingIRE-BP.For example,a heme-sensitiveRNA-
binding protein controls translation of the mRNA encoding
aminolevulinate (ALA) synthase,a key enzyme in the syn-
thesisof heme. In vitro studieshave shown that the mRNA
( a ) F e r r i t i nm R N A Another mechanism called nonsense-mediateddecay
lREs C o d i n gr e g i o n cooH causesdegradation of mRNAs in which one or more exons
have been incorrectly skipped during splicing. Such exon skip-
ping often will alter the open reading frame of the mRNA 3' to
+ ih.i-ptop"t exon junction, resulting in introduction of out-of-
An
frame miJsensemutations and an incorrect stop codon' For
nearly all properly splicedmRNAs' the stop codon is in the last
Translated
ferritin ."otr. Th. p.ocessof nonsense-mediateddecay (NMD) results
in the rapid degradation of mRNAs with stop codons that oc-
cur before the last splice junction in the mRNA since in most
cases,such mRNAs arise from errors in RNA splicing
A search for possible molecular signals that might indi-
cate the positions of splice junctions in a processedmRNA
5' A^ JF No translation
initiation led to thi discovery of exon-junction complexes. As noted

( b ) T f Rm R N A lREs
AU-rich region

\/ t t/-/---
t ' - - ar ) Ir r t
'r't)l? tants indicate that one of the proteins in exon-junction com-
plexes (Upf3) functions in nonsense-mediateddecay' In the
Degraded iytoplasm, this component of exon-junction complexes in-
mononucleotides
teractswith a protein (Upf 1) that causesthe mRNA to asso-
ciate with P bodies,repressingtranslation of the mRNA' An

5' A" aF Little

degradation

A FIGURE 8-31 lron-dependentregulationof mRNAtranslation

and degradation.Theironresponse element-bindingprotein
(lRE-BP)controls translationof ferritinmRNA(a)anddegradatton
of transferrin-receptor (TfR)mRNA(b) At low intracellular
iron
concentrations IRE-BP bindsto iron-responseelements (lREs)
in
the 5' or 3' untranslated regionof thesemRNAs. At highiron
concentrations, IRE-BP undergoesa conformationalchange and
precisely with the mRNA, resulting in nonsense-mediateddecay'
cannotbrndeithermRNAThedualcontrolby IRE-BP
regulates the levelof f reeironionswithincells.Seethetextfor
discussion. Localizationof mRNAsPermitsProduction
of Proteinsat SpecificRegionsWithin
the Cytoplasm
encoding the milk protein casein is stabilized by the hor-
mone prolactin and rapidly degradedin its absence.

SurveillanceMechanismsPreventTranslation
mRNAs
of lmproperlyProcessed
Translation of an improperly processedmRNA could lead to

the 3' untranslatedregion of the mRNA'

As mentioned earlier, in neurons, localization of specific

the translation of improperly processedmRNA molecules.We

have previously mentioned two such surveillancemechanisms:
the recognition of improperly processedpre-mRNAs in the
nucleus and their degradation by the exosome and the general
restriction against nuclear export of incompletely spliced
pre-mRNAs that remain associatedwith a spliceosome.

C Y T O P L A S M IM CA
OF POST-TRANSCRIPTION
C ECHANISMS OLN T R O L 357
 r Both miRNAs and siRNAs contain 21-23 nucleotides,
are generated from longer precursor molecules, and are
assembled into a multiprotein RNA-induced silencing
complex (RISC) that either repressestranslation of target
mRNAs or cleavesthem (seeFigures 8-26 and 8-27).
r Cytoplasmic polyadenylation is required for translation of
mRNAs with a short poly(A) tail. Binding of a specificpro-
tein to regulatoryelementsin their 3' UTRs represses transla-
tion of thesemRNAs. Phosphorylation of rh[ RNA-binding
protein, induced by an external signal, leadsto lengtheningof
EXPERIMENTAL FTGURE 8-32 A specificneuronalmRNA the 3' poly(A) tail and translation(seeFigure 8-29).
focafizesto synapses.Sensory neuronsfromthe seaslugAplysia Most mRNAs are degradedas the result of the gradual
californica-among the largest neurons in theanimalkingdom_ ortening of their poly(A) tail (deadenylation)followed by
werecultured with targetmotorneurons sothat processes fromthe exosome-mediated3' -+ 5' digestion or removal of the 5'
sensory neuronformedsynapses with processes fromthe motor cap and digestionby a 5' -+ 3' exonuclease(seeFigure 8-30).
neuron. Themicrograph at the leftshowsmotorneuronprocesses
visualized with a bluefluorescent dye GFp-VAMp (green) was Eukaryotic mRNAs encoding proteins that are expressed
expressed In sensory neurons andmarksthe location of synapses short burstsgenerallyhave repeatedcopiesof an AU-rich
formedbetween sensory andmotorneuronprocesses (arrows). The sequencein their 3' UTR. Specificproteinsthat bind to these
micrograph on the rightshowsredfluorescence from in situ elementsalso interactwith the deadenylatingenzymeand cy-
h y b r i d i z a t i o fna n a n t i s e n s o rmi nR N Ap r o b eS e n s o r r n
sa toplasmicexosomes,promoting rapid RNA degradation.
neurotransmrtter expressed by thesensory neurononly;sensory
neuronprocesses r Binding of various proteins to regulatory elementsin the
arenot otherwise visualized in thispreparation, but
theylieadjacent to the motorneuronprocesses. 3' or 5' UTRs of mRNAs regulatesthe translation or degra-
Thein situ
hybridization results indicate thatsensorin mRNAislocalized dation of many mRNAs in the cytoplasm.
to
synapses [From V Lyles, y Zhao, andK C Martin. 2006,Neuron 49:3231 r Translation of ferritin mRNA and degradation of trans-
ferrin receptor (TfR) mRNA are both regulatedby the same
properties of this one synapseout of hundreds to thousands
iron-sensitiveRNA-binding protein. At low iron concentra-
of synapsesmade by a neuron. tions, this protein is in a conformation that binds to specific
elementsin the mRNAs, inhibiting ferritin mRNA transla-
tion or degradation of TfR mRNA (seeFigure 8-32). This
dual control preciselyregulatesthe iron level within cells.
r Nonsense-mediateddecay and other mRNA surveillance
mechanismsprevent the translation of improperly processed
mRNAs encoding abnormal proteins that might interfere
with functioning of the correspondingnormal proteins.
r Some mRNAs are directed to specific subcellular loca-
tions by sequencesusually found in the 3, UTR, leading to
localization of the encodedproteins.

example of this mechanism of mRNA localization occurs dur_
ing cell division in S. cereuisiap-as we describein Chapter 21.
Cytoplasmic Mechanismsof post-transcriptional
Control
r Translation can be repressedby micro-RNAs (miRNAs),
which form imperfect hybrids with sequencesin the 3, un_
translated region (UTR) of specifictarget mRNAs.
r The related phenomenon of RNA interference, which
probably evolved as an early defensesystemagainstviruses
and transposons,Ieads to degradation of mRNAs that
form perfecthybrids with short interferins RNAs (siRNAs).
ff,l Processing
of rRNAandIRNA
Approximately 80 percent of the total RNA in rapidly grow-
ing mammalian cells (e.g.,cultured HeLa cells)is rRNA, and
15 percent is IRNA; protein-coding mRNA rhus consrrtutes
only a small portion of the total RNA. The primary tran-
scripts produced from most rRNA genes and from IRNA
genes, like pre-mRNAs, are extensively processedto yield
the mature, functional forms of theseRNAs.
dination of all three nuclear RNA polymerases.The 2gS and
5.8S rRNAs associatedwith the large ribosomal subunit and
the single 18S rRNA of the small subunit are transcribedby

358 c H A p T E R8 | P o S T _ T R A N S C R I p T t o NGAELN EC O N T R O L
RNA polymeraseI. The 55 rRNA of the large subunit is tran-
scribedby RNA polymeraseIII, and the mRNAs encodingthe
ribosomal proteins are transcribedby RNA polymeraseII. In
addition to the four rRNAs and :70 ribosomal proteins' at
least 150 other RNAs and proteins interact transiently with
the two ribosomal subunits during their assemblythrough a
series of coordinated steps. Furthermore, multiple specific
basesand ribosesof the mature rRNAs are modified to opti-
mize their function in protein synthesis.Although most of the
stepsin ribosomal subunit synthesisand assemblyoccur in the
nucleolus(a subcompartmentof the nucleusnot boundedby a +
.I
membrane), some occur in the nucleoplasmduring passage Nucleolar
from the nucleolusto nuclearpore complexes.A quality-control chromatin
E
step occursbefore nuclearexport so that only fully functional
subunits are exported to the cytoplasm,where the final steps a
(!
of ribosome subunit maturation occur. tRNAs also are
processedfrom precursor primary transcripts in the nucleus o
c
and modified extensivelybeforethey are exportedto the cyto- {;
plasm and usedin protein synthesis.First we'll discussthe pro- 0)
,=
cessing and modification of rRNA and the assembly and
nuclearexport of ribosomes.Then we'll considerthe process-
ing and modification of tRNAs.

Pre-rRNA G e n e sF u n c t i o na s N u c l e o l a r
O r g a n i z e r sa n d A r e S i m i l a ri n A l l E u k a r y o t e s
The 28S and 5.8S rRNAs associatedwith the large (605) a EXPERIMENTAL FIGURE 8-33 Electronmicrographof pre'
ribosomal subunit and the 18S rRNA associatedwith the rRNAtranscriptionunits from the nucleolusof a frog oocyte.
small (40S)ribosomal subunit in higher eukaryotes(and the Each"feather"represents multiplepre-rRNAmolecules associated
with proteinin a complex
pre-ribonucleoprotein (pre-RNP) emerging
functionally equivalent rRNAs in all other eukaryotes) are
from a transcrrptionunit.Notethe dense"knob" at the 5'-endof each
encoded by a single type of pre-rRNA transcription unit. In
nascent pre-RNP thoughtto be a processome Pre-rRNA transcription
human cells,transcriptionby RNA polymeraseI yieldsa 45S
unitsarearranged in tandem,separatedby nontranscribed spacer
(:13.7 kb) primary transcript (pre-rRNA), which is
regions of nucleolar chromatin. of Y osheim
[Courtesy andO J Miller,
Jr]
processedinto the mature 285, 18S,and 5.8S rRNAs found
in cytoplasmic ribosomes.Sequencingof the DNA encoding
pre-rRNA from many speciesshowed that this DNA shares
several properties in all eukaryotes.First, the pre-rRNA
genesare arranged in long tandem arrays separatedby non-
transcribed spacer regions ranging in length from :2 kb in 185 5.8S 28S
frogs to :30 kb in humans (Figure 8-33). Second, the
H u m a n ,= 1 3 . 7K b
genomic regions corresponding to the three mature rRNAs s', 3',
are alwaysarrangedin the same5' -> 3' order: 18S,5'8S' and
X' laevis (frog)' =7'9 Kb
28S. Third, in all eukaryoticcells(and evenin bacteria),the
pre-rRNA gene codes for regions that are removed during
processingand rapidly degraded.These regions probably D. melanogasfer(fruit tlyl, =7.7 g6
contribute to proper folding of the rRNAs but are not re-
quired once the folding has occurred. The general structure s' cerevisiae(Yeast)'=6'6 Kb
of pre-rRNAs is diagrammedin Figure 8-34.
The synthesisand most of the processingof pre-rRNA T r a n s c r i b e ds P a c e r
occurs in the nucleolus. Sfhen pre-rRNA genes initially R e g i o np r e s e r v e di n r R N A
were identified in the nucleolusby in situ hybridization, it
was not known whether any other DNA was required to A FIGURE 8-34 Generalstructureof eukaryoticpre-rRNA
form the nucleolus.Subsequentexperimentswith transgenic transcriptionunits.Thethreecodingregions (red)encode the 185,
Drosophila strains demonstrated that a single complete 5 8S,and28SrRNAs found in of
ribosomes higher or
eukaryotes
pre-rRNA transcription unit induces formation of a small in
theirequivalents otherspecies Theorder of these codingregions
nucleolus.Thus a singlepre-rRNA geneis sufficienttobe a nu- in the genomeisalways5' -+ 3' Variations of the
in the lengths
cleoldr organizer, and all the other components of the ribo- spacer
transcribed regions(blue)accountfor the majordifference in
some diffuse to the newly formed pre-rRNA. The structure of pre-rRNA
the lengths units
transcription in organisms'
different

P R O C E S S I NOGF r R N A A N D I R N A ' 359

 of the nucleolus observed by light and electron microscopy
results from the processingof pre-RNA and the assemblyof
ribosomal subunits.
r N A sA s s i s ti n p r o c e s s i n g
S m a l lN u c l e o l a R
Pre-rRNAs
Ribosomal subunit assemblg maturation, and export to the
cytoplasm are best understood in the yeast S. cereuisiae.
However, nearly all the proteins and RNAs involved are
highly conservedin multicellulareukaryotes,where the funda-
mental aspectsof ribosome biosynthesis are likely to be the
same. As for pre-mRNAs, nascent pre-rRNA uanscnprs are
immediatelybound by proteins,forming preribosomalribonu-
cleoproteinparticles(pre-rRNPs).For reasonsnot yet known,
cleavageof the pre-rRNA doesnot beginuntil transcription of
the pre-rRNA is nearly complete. In yeast,it takes approxi-
mately six minutes for a pre-rRNA to be transcribed. Once
transcription is complete, the rRNA is cleaved, and bases
and riboses are modified in about 10 seconds.In a rapidly
growing yeastcell, -40 pairs of ribosomal subunits are syn-
thesized,processed,and transported to the cytoplasm every
second.This extremely high rate of ribosome synthesisin the
face of the seemingly long period required to transcribe a
pre-rRNA is possible becausepre-rRNA genes are packed
with RNA polymerase I molecules transcribing the same
gene simultaneously(seeFigure 8-33) and becausethere are
100-200 such geneson chromosomeXII, the yeastnucleolar
otganrzeL
The primary transcript of -7 kb is cut in a seriesof cleav-
age and exonucleolytic steps that ultimately yield the mature
rRNAs found in ribosomes(Figure 8-35). During processing,

35S

Exosome

20s - 275A2
I Xrnl
r.vrc'ea' Ratl
exporr
I
I

-
185 5.8Ss 25S or S.8SL 2SS
FIGURE 8-35 rRNAprocessing. Endoribonucleases
that make rRNAsoccursfollowing the initialcleavageat the 3, end, before
internalcleavagesarerepresented
asscissors.
Exoribonucleases
that the initialcleavageat the 5' end Proteinsand snoRNpsknown to
digestfromoneend,either5, or 3,, areshownaspac-Men. Most2,_ participatein thesestepsare indicated.lFromJVenemaandD Tollervev.
O-ribose (CH:)andgeneration
methylation of pseudouridines
in the 1999,Ann ReyGenefics 33:261I

350 C H A P T E R8 I P o S T - T R A N S C R | p T t o NG
AEL N EC O N T R O L
 OH OH
Uridine

o
ll
tl
HN",\NH
NNon rl
\cAo

OH OH
Pseudouridine

modificationof pre-rRNA. bulgesin thestems.Pre-rRNA

single-stranded hybridizesto the
A FIGURE 8-36 SnoRNP-directed
(a)A snoRNA isinvolvedin ribose2'-O- bulges,
single-stranded demarcating a siteof pseudouridylation'
calledboxC+D snoRNA
in thissnoRNA to two different
hybridize fromuridineto pseudouridine
(c)Conversion directed by the box
methylation.Sequences
regionsin the pre-rRNA,directing at the indicated
methylation sites H+ACA snoRNAs of part(b).lPart(a)f romr' Kiss,2001, EMBO J
(b)BoxH+ACAsnoRNAs fold intotwo stemloopswith internal (b)
20:3617Part fromU I Meier,2005,Chromosoma | 114:1

pre-rRNA also is extensivelymodified, mostly by methylation of the helical double-strandedregion, like the branch point
of the 2'-hydroxyl group of specificribosesand conversionof A bulges out in pre-mRNA spliceosomalsplicing (seeFigure
specific uridine residuesto pseudouridine.These post-tran- 8-10). Other modifications of pre-rRNA nucleotides'such as
scriptionalmodificationsof rRNA are probably important for adenine dimethylation, are carried out by specific proteins
protein synthesisbecausethey are highly conserved.Mrtually without the assistanceof guiding snoRNAs.
all of these modifications occur in the most conserved core The U3snoRNA is assembledinto a large snoRNP con-
structure of the ribosome, which is directly involved in protein
synthesis.The positions of the specificsitesof 2'-O-methyla-
tion and pseudouridineformation are determinedby approx-
imately 150 different small nucleolus-restricted RNA species,
called small nucleolar RNAs (snoRNAs), which hybridize
transiently to pre-rRNA molecules. Like the snRNAs that
function in pre-mRNA processing,snoRNAs associatewith
proteins,forming ribonucleoproteinparticlescalledsnoRNPs.
One class of more than 40 snoRNPs (box C+D snoRNAs)
positions a methyltransferaseenzymenear methylation sitesin
the pre-mRNA. The multiple different box C+D snoRNAs
direct methylation at multiple sitesthrough a similar mecha-
nism. They share common sequenceand structural features
and are bound by a common set of proteins. One or two re- from pre-mRNAs. Nuclear 5' -+ 3' exoribonucleases(Rat1;
gions of each of thesesnoRNAs are preciselycomplementary Xrnl) also remove someregionsof 5' spacer'
to sites on the pre-rRNA and direct the methyltransferaseto SomesnoRNAs are expressedfrom their own promoters
specificribosesin the hybrid region (Figure 8-36a). A second
maior class of snoRNPs (box H+ACA snoRNAs) positions
the enzyme that converts uridine to pseudouridine' This
conversion involves rotation of the pyrimidine ring (Figure
8-36c). Baseson either sideof the modified uridine in the pre-
rRNA base-pair with basesin the bulge of a stem in the
H+ACA snoRNAs, leaving the modified uridine bulged out exist onlv to expresssnoRNAs from excisedintrons'

O 361
P R O C E S S I NOGF T R N AA N D I R N A
 Nucleolus Nucleoplasm Cytoplasm
Early Intermediate Late i,.'.,, Mature

_
; t , "''
""

rDNA

Cleavage , , ' ,. ,

rRNA Helicases
R N A p o l y m e r a s e, I n t r a n u c l e at r a n s p o r t( N o cp r o t e i n s )
S
3Q US-associatedfactors L-l (J I rases

,,j: U3-snoRNP AAA-type ATPase

O rRNA processing/modification
factors Exportfactors(Nmd3, Nxt1, Ran_GTp)
$

FIGURE 8-37 Ribosomalsubunitassembly. Ribosomal transientlywith the maturing

subunits
aredepicted
in different
protetns
andRNAsin the maturing smallandlargeribosomal colors,asshownin the key fFromH Tschochner
andE Hurt,2OO3.Trends
subunits
aredeptctedin blue,wrtha shapesimilar
to the iconsfor CellBiol 13:2551
the maturesubunits
in the cytoplasmOtherfactorsthatassociate

dimethylationof two adjacentadeninesnear the 3, end of

malns associatedwith the region that is cleavedinto the pre_
cursor of the largeribosomal subunit.
Most of the ribosomal proteinsof the small 40S riboso_
subunit occurs in the cytoplasm: exonucleolyric processing
of the 20S rRNA into mature small subunit 1gS rRNA by
the cytoplasmic 5' -+ 3, exoribonucleaseXrnl and the
18SrRNA by the cytoplasmicenzymeDim1.
In contrast to the pre-40S particle, the precursor of the
large subunit requiresconsiderablymore remodelingthrough
many more transient interactions with nonribosomal pro-
teins before it is sufficiently mature for export to the cyto-
plasm. Consequently,it takes a considerably longer period
for the maturing 605 subunit to exit the nucleus than for
the 40S subunit (30 minutescomparedto 5 minutesfor ex-
port of the small subunit in cultured human cells). Multiole
presumptive RNA helicasesand small G proteins urro.i-
ated with the maturing pre-60Ssubunits.Some RNA "r.
heli-
casesare necessaryto dislodgethe snoRNps that base-pair
perfectly with pre-rRNA over up ro 30 base pairs. Oiher
RNA helicasesmay function in the disruption of protein-
RNA interactions. The requirement for so many GTpases
suggeststhat there are many quality-control checkpoints in
the assembly and remodeling of the large subunit RNp,
where one step must be completed before a GTpase is

362 . c H A p r E8R | p o s r - T R A N S c R t p I oG
NEAN
L cEo N T R o L
activated to allow the next step to proceed.Members of the The splicing mechanismsused by group I introns, group
AAA ATPasefamily are also bound transiently.This classof II introns, and spliceosomesare generally similar, involving
proteins is often involved in large molecular movementsand two transesterificationreactions, which require no input of
may be required to fold the complex, large rRNA into the
proper conformation. Somestepsin 605 subunit maturation
occur in the nucleoplasm,during passagefrom the nucleolus
to nuclear pore complexes (seeFigure 8-37). Much remains
to be learned about the complex, fascinating, and essential
remodeling processesthat occur during formation of the ri-
bosomalsubunits. catalysis.The group I intron functions like a metalloenzyme
The large ribosomal subunit is one of the largest struc- to piecisely orient the atoms that participate in the two
tures to passthrough nuclear pore complexes.Maturation of transesterificationreactionsadjacentto catalytic Mg2* ions'
the large subunit in the nucleoplasmleads to the generation Considerableevidencenow indicates that splicing by group
of binding sites for a nuclear export adapter called Nmd3. II introns and by snRNAs in the spliceosomealso involves
Nmd3 is bound by the nuclear tranporter Exportinl (also bound catalytic Mg2* ions. In both the group I and II self-
called Crml). This is another quality-control step since only splicing introns and probably in the spliceosome,RNA func-
correctly assembled subunits can bind Nmd3 and be ex- tions a ribozyme, an RNA sequencewith catalytic ability'
"s
ported. The small subunit of the mRNP exporter (Nxt1) also
b..o-.t associatedwith the nearly mature large ribosomal Pre-tRNAsUndergoExtensiveModification
subunit. Thesenucleartransportersinteractwith FG-domains
in the Nucleus
of FG-nucleoporins.This mechanism allows penetration of
the molecular meshwork that makes up the central channel Mature cytosolictRNAs, which average75-80 nucleotidesin
of the NPC (seeFigure 8-20). Severalspecific nucleoporins length, aie producedfrom larger precursors-(pre-tRNAs)syn-
without FG-domains are also required for ribosomal subunit thesizedfy nNe polymeraseIII in the nucleoplasm'Mature
export and may have additional functions specific for this tRNAs also contain numerous modified basesthat are not
task. The dimensionsof ribosomal subunits(-25-30 nm in
diameter) and the central channel of the NPC are compara-
ble, so passagemay not require distortion of either the ribo-
somal subunit or the channel. Final maturation of the large
subunit in the cytoplasm includes removal of these export
factors. As for the export of most macromoleculesfrom the
nucleus, including tRNAs and pre-miRNAs (but not most
mRNPs), ribosome subunit export requiresthe function of a an endonucleolytic cleavage specified by the IRNA three-
small G protein called Ran, as discussedin Chapter 13. dimensional structure rather than the start site of transcrip-

g r o u pI I n t r o n sW e r et h e F i r s t
S e l f - S p l i c i nG
Examplesof CatalyticRNA
During the 1970s, the pre-rRNA genes of the protozoan
Tetrahymena thermophila were discovered to contain an
intron. Careful searchesfailed to uncover even one pre-
rRNA genewithout the extra sequence'indicating that splic-
ing is required to produce mature rRNA in theseorganisms'
In 1,982, in vitro studies showing that the pre-rRNA was
spliced at the correct sitesin the absenceof any protein pro-
vided the first indication that RNA can function as a cata-
lyst. like enzymes.
A whole raft of self-splicingsequencessubsequentlywere
found in pre-rRNAs from other single-celledorganisms, in
mitochondrial and chloroplast pre-rRNAs, in several pre-
mRNAs from certain E. coli bacteriophages'and in some
bacterial tRNA primary transcripts. The self-splicingse-

self-splicingintron, designatedgroup II'

' 363
P R O C E S S I NOGF r R N A A N D I R N A
 Self-splicing introns Spliceosome-catalyzedsplicing
G r o u pI of pre-mRNA

Spliceosome

HO-*9
-^ r
b' '-p,f-.,
3, =,,.- z,
I
I
*

->P

FIGURE8-38 Splicing mechanismsin group I and group ll

thatinvolving the 2'-hydroxyl groupsof branch-site As in groupll
self-splicingintrons and spliceosome-catalyzedsplicing of pre_
introns andpre-mRNA introns spliced
in spliceosomes (seeFigure g_g).
mRNA. The intron is shown in gray,the exonsto be joined in red. In
Thesubsequent transesterification that linksthe 5, and3, exonsis
group I Introns,a guanosinecofactor(G)that is not part
of the RNA s i m i l ai rn a l lt h r e es p l i c i nm
g echanism Nso t et h a ts p l i c e d _ ogur ot u pI
chajnassociates with the activesite.The 3,_hydroxylgroup of this intronsarelinearstructures, unlikethe branched intronproducts in
guanosineparticrpates in a transesterificatlon
reactionwith the theothertwo cases. [Adapted fromp A. Sharp, 1987,Science235:769.1
phosphateat the 5' end of the intron;this reactionrsanatogous
to

As shown in Figure 8-39, the pre-tRNA expressed from

. tRNAs generally are associatedwith proteins and spend
the yeast tyrosine IRNA (tRNATt.; gene contains a 14_base
intron that is not present in mature tRNATy.. Some
other
eukaryotic IRNA genes and some archaeal IRNA genes also
contain introns. The introns in nuclear pre_tRNAs
are
shorter than those in pre-mRNAs and lack the consensus
little time free in the cell, as is also the case for mRNAs
and rRNAs.
N u c l e a rB o d i e sA r e F u n c t i o n a l t S
y pecialized
N u c l e a rD o m a i n s
High-resolution visualization of plant- and animal_cellnu_
clei by electron microscopy and subsequentstaining with
fluorescentlylabeled antibodies has revealed domains in
nuclei in addition to chromosome territories and nucleoli.
Thesespecializednuclear domains, called nuclear bodies.are
not surrounded by membranes but are nonethelessregions
of high concentrarions of specific proteins and RNAs that
form distinct, roughly spherical structures within the nu_
cleus. The most prominent nuclear bodies are nucleoli, the
sites of ribosomal subunit synthesisand assemblydiscussed
earlier. Severalorher types of nuclear bodies also have been
describedin structuralstudies.
Experiments with fluorescently labeled nuclear proteins
have shown that the nucleus is a highly dynamic environ_
ment, with rapid diffusion of proteins through the nucleo_
plasm. Proteins associatedwith nuclear bodies are often also
observedat lower concentration in the nucleoplasm outside

364 . posr-TRANScRtpIoNA
c H A p r E R8 | GLE N Ec o N T R o L
 OH OH

Mature tRNATY'

Pre-tRNATYT
in the stemloopsareconverted to characteristic modifiedbases
FIGURE 8-39 Changesthat occurduringthe processing of
(yellow). Notall pre-tRNAs contain introns that are out during
spliced
tyrosinepre-tRNA. A 14-nucleotide intron(blue)in theanticodon
(green)
at the processing, but theyallundergo theothertypesof changes shown
loopisremoved by splicingA 16-nucleotide sequence
5' endiscleavedby RNase P U residuesat the 3' endarereplacedby h e r eD : d i h y d r o u r i d iinl ,e=; p s e u d o u r i d i n e
the CCAsequence (red)foundin all maturetRNAsNumerous bases

the nuclear bodies, and fluorescencestudies indicate that U6 snRNPs releasedduring the removal of each intron (see
they diffusein and out of the nuclearbodies.Basedon these Figure 8-11). SinceCajal bodiesalso contain a high concen-
measurementsof molecularmobility in living cells,nuclear ,ri io., ol the uTsnRNP involved in the specialized3'-end
bodies can be mathematically modeled as the expected processingof the major histone mRNAs, it is likely that this
steady state for diffusing proteins that interact with suffi- pro..r, o..nr. in Calal bodies, as may the assemblyof
"l'ro RNP.
cient affinity to form self-organizedregions of high concen- the telomerase
trations of specificproteins but with low enough affinity for
each other to be able to diffuse in and out of the structure. 3 kDa
Electron micrographsshow thesestructuresappear to be a
heterogeneous,spongelikenetwork of interacting compo- ,l::l:ll:
'We
nents. discussa few of these nuclear bodies here as ex-
amplesof thesenucleardomains.

Cajaf Bodies Calal bodies are :0'2-L pm sphericalstruc-

tures that have been observedin large nuclei for more than a
A FIGURE 8-40 Nuclearbodiesare differentiallypermeableto
century (Figure 8-40). Current research indicates that like showsa
moleculesin the bulk nucleoplasm' Eachpairof panels
nucleoli,Cajal bodiesare centersof RNP-complexassembly oocyte nucleus, whichwas
singleareathrougha livingXenopus
for spliceosomalsnRNPs and other RNPs. Like rRNAs' dextran of the indicatedmolecular
previouslyinjectedwithfluorescent
snRNAs undergo specificmodifications, such as the conver- of the upperpanelisa confocal
mass(3-2000kDa),Eachsection
sion of specificuridine residuesto pseudouridineand addi- rmagein whichthe intensity of fluorescence isa measure of dextran
tion of methyl groups to the 2'-hydroxyl groups of specific concentration (i.e.,darkerareas showregions wheredextran hasbeen
riboses.Thesepost-transcriptionalmodificationsare impor- excluded). Eachsection of the lower panel is a interference
differential
tant for the proper assemblyand function of snRNPs in pre- contrastimageof thesamefield.openarrowheads nucleoli,
indicate
mRNA splicing. These modifications occur in Cajal bodies, closedarrowheads cajalbodies (CBs) with attached nuclearspeckles
where they are directed by a class of snoRNA-like guide whicharemuchlargerin Xenopus oocytes thanin mostsomatic cells'
RNA molecules called scaRNAs (small Caial body-associ- Dextrans of low molecular mass (e g., 3 kDa) almost completely
CBsbutwereexcluded morefromnuclear and
speckles
ated RNAs). There is also evidencethat the Caial body is the penetrated
Exclusionof dextranincreased with molecular mass Bar'
site of reassemblyof the U4N6NS tri-snRNP complexes nucleoli.
1Opm, [From K E Handwerger,2OO5, Mol BiolCell15:2021
required for pre-mRNA splicing from the free U4, U5, and
' 365
P R O C E S S I NOGF r R N A A N D I R N A
 Nuclear Speckles Nuclear speckleswere observed with r Synthesisand processingof pre-rRNA occur in the nu-
fluorescently labeled antibodies to snRNp proteins and cleolus. The 55 rRNA component of the large ribosomal
other proteins involved in pre-mRNA splicing as -25-50 ir- subunit is synthesizedin the nucleoplasm by RNA poly-
regular,amorphous structures0.5-2 pm in diameter that are meraseIIL
distributed through the nucleoplasm of vertebrate cells.
r :150 snoRNAs associatedwith proteins in snoRNps
base-pair with specific sites in pre-RNA, directing ribose
methylation, modification of uridine to pseudouridine,and
cleavage at specific sites during rRNA processing in the
nucleolus.

Promyelocytic Leukemia (pML) Nuclear Bodies The

PML gene was originally discovered when chromosomal
translocations within the gene were observed in the
leukemic cells of patients with the rare diseasepromyelol_
cytic leukemia (PML). When antibodies speci?icfor the r Pre-tRNAs synthesizedby RNA polymerase III in the
PML protein were used in immunofluorescence ml_ nucleoplasm are processedby removal of the S,-end se-
croscopy studies, the protein was found to localize to quence,addition of CCA to the 3' end, and modification of
-10-30 roughly sphericalregions0.3-1 pm
in diameterin multiple internal bases(seeFigure 8-39).
the nuclei of mammalian cells. Multiple functions have
been proposed for PML nuclear bodies,but a consensusis r Some pre-tRNAs contain a short intron that is removed
by a protein-catalyzedmechanism distinct from the solic-
ing of pre-mRNA and self-splicingintrons.
pecies of RNA molecules are associatedwith pro-
various types of ribonucleoprotein particles both in
leus and after export to the cytoplasm.
r Nuclear bodies are functionally specializedregionsin the
nucleus where interacting proteins form self-or ganized,
responsegenes.
structures.Many of these,like the nucleolus,are regions of
Recent results indicate that pML nuclear bodies are assemblyof RNP complexes.
also sites of protein post-translational modification
through the addition of a small, ubiquitin-like protein
called SUMOl (small zbiquitin-like miiety-1), whlch can
control the activity and subcellular localization of the
modified protein. Many transcriptional rcpressors are
sumoylated,and mutation of their site of sumoylation re_ In this and the previous chapter, we have seen that in eu_
duces their repressionactivity. These results suggestthat
PML nuclear bodies may be involved in a mechanism of
transcriptional repressionthat remains to be studied and
understood.
In addition to PML nuclear bodies,the first nuclear bod_
ies to be observed,the nucleoli may have specializedregions
of substructurethat are dedicatedto functironsother thin ri_
bosome biogenesis.There is evidencethat immature SRp ri_
bonucleoprotein complexes involved in protein secretion
and ER membraneinsertion (Chapter 13) are assembledin RNA sequencesand provides various avenuesfor regulating
nucleoli and then exported to the cytoplasm, where their fi_ synthesis of a polypeptide chain. Much remains to be
nal maturation takes place. learned about the structure, operation, and regulation of
such complex machines as spliceosomesand the cleavage/
polyadenylation apparatus.
Recent examples of the regulation of pre-mRNA splic_
.
Processing ing raise the question of how extracellular signals might
of rRNAand IRNA
control such events, especiallyin the nervous system of
r. A.l.arg1precursorpre-rRNA (45Sin humans)synthe- vertebrates.A case in point is the remarkable situation in
sizedby RNA polymerase I undergoescleavage, exonucle_
olytic digestion,and basemodificationsto yi.ld -"t.rr.
28S,185,and5.8SrRNAs,whichassociate with ribosomal
proteinsinto ribosomalsubunits.

365 c H A p T E R8 | P o S T _ T R A N S C R t p T t o NGAELN EC O N T R O L
of Slo pre-mRNA. The challengingtask facing researchersis KeyTerms
to discoverhow such cell-cellinteractions regulatethe activ-
ity of RNA-processingfactors. 5' cap325 nuclearpore complex
The mechanism of mRNP transport through nuclear (NPC)342
alternativesplicing337
pore complexesposesmany intriguing questions.Future re- cleavage/polyadenylation poly(A)tail335
searchwill likely reveal additional activities of hnRNP and complex335 pre-mRNA326
nuclear mRNP proteins and clarify their mechanisms of pre-rRNA359
cross-exon recognrtlon
action. For instance, there is a small gene family encoding
complex 333 rrbozyme363
proteins homologous to the large subunit of the mRNA
exporter. What are the functions of these related proteins? Dicer348 RNA editing341
Do they participate in the transport of overlapping sets of Drosha348 RNA-inducedsilencing
mRNPs? Some hnRNP proteins contain nuclear-retention exosome 335 complex(RISC)348
signals that prevent nuclear export when fused to hnRNP expoftin 347 RNA interference
proteins with nuclear-export signals (NESs).How are these FG-nucleoporins 342 (RNAi)34e
hnRNP proteins selectivelyremoved from processedmRNAs RNA splicing329
groupI introns353
in the nucleus,allowing the mRNAs to be transported to the short interferingRNAs
cytoplasm? groupII introns334
(siRNA)347
The localization of certain mRNAs to specificsubcellular importin 344
siRNA knockdown351
locations is fundamental to the developmentof multicellular iron-response element-
organisms.As discussedin Chapter 22, during development bindingprotein smallnuclearRNAs
(snRNAs)330
an individual cell frequently divides into daughter cells that (rRE-BP)356
function differently from each other. In the languageof de- micro RNAs (miRNAs) smallnucleolarRNAs
velopmental biology, the two daughter cells are said to have (snoRNAs)361
347
different developmentalfates. In many cases'this difference mRNP expofiet343 spliceosome 332
in developmental fate results from the localization of an SRproteins333
mRNA surveillance 357
mRNA to one region of the cell before mitosis so that after
cell division, it is present in one daughter cell and not the
other. Much exciting work remains to be done to fully un-
derstand the molecular mechanisms controlling mRNA Reviewthe ConcePts
localization that are critical for the normal development of
multicellular organisms. l. Describe three types of post-transcriptional regulation
Someof the most exciting and unanticipated discoveries of protein-coding genes.
in molecular cell biology in recent years concern the exis- 2. You are investigatingthe transcriptional regulation of
tence and function of miRNAs and the processof RNA in-
terference.RNA interference(RNAi) provides molecular cell
biologists with a powerful method for studying gene func-
tion. The discoveryof -1000 miRNAs in humans and other
organisms suggeststhat multiple significant examples of
translational control by this mechanismawait to be charac-
terized. Recent studies in S. pombe and plants link similar not?
'S(hat
DNA methylation and
short nuclear RNAs to the control of 'Will 3. is the evidencethat transcription termination by
the formation of heterochromatin. similar processes RNA polymeraseII is coupled to polyadenylation?
control geneexpressionthrough the assemblyof heterochro- 4. It has been suggestedthat manipulation of HIV antiter-
matin in humans and other animals?'What other regulatory mination might provide for effectivetherapiesin combating
processesmight be directed by other kinds of small RNAs? 'S(hat
AIDS. effect would a mutation in the TAR sequence
Sincecontrol by these mechanismsdependson base pairing that abolishesTat binding have on HIV transcription after
between miRNAs and target mRNAs or genes'genomic and HIV infection and why? A mutation in Cdkg that abolishes
suggestgenesthat may
bioinformatic methods will probably'What activity?
be controlled by thesemechanisms. other processesin
5. Describe how the discovery that introns are removed
addition to translation control' mRNA degradation, and
during splicing was made. How are the locations of exon-
heterochromatin assemblymight be controlled by miRNAs?
intron junctions Predicted?
These are iust a few of the fascinating questions con-
6. \7hat is the difference between hnRNAs' snRNAs,
cerning RNA processing,post-transcriptional control, and
miRNAs. siRNAs, and snoRNAs?
nuclear transport that will challenge molecular cell biolo-
gistsin the coming decades.The astoundingdiscoveriesof 7. \7hat are the mechanisticsimilarities between group II
entirely unanticipated mechanisms of gene control by intron self-splicing and spliceosomal splicing? \fhat is the
miRNAs remind us that many more surprisesare likely in evidencethat there may be an evolutionary relationship be-
the future. tween the two?

T H EC O N C E P T S .
REVIEW 367
 8. rWheredo researchersbelieve most transcription and
RNA-processing eventsoccur? \fhat is the evidenceto suD-
port this?
9. You obtain the sequenceof a gene containing 10 exons,
9 introns, and a 3' UTR containing a polyadenylation con-
sensussequence.The fifth intron also contains a polyadeny-
lation site. To test whether both polyadenylation sites are
used, you isolate mRNA and find a longer transcript from
muscle tissueand a shorter mRNA transcript from all other
tissues.Speculateabout the mechanisminvolrred in the oro-
duction of thesetwo transcrlDrs.
11. A protein complex in the nucleus is responsiblefor
transporting mRNA moleculesinto the cytoplaim. Describe
the proteins that form this exporter and whar two protern
groups are likely behind the mechanism involved in the
directional movement of the mRNp and complex into the
cytosol.
12. RNA knockdown has become a powerful tool in the
arsenal of methods to deregulat. g.n. expression. Briefly
describehow gene expressioncan be knocked down. What
effect would introducing siRNAs to TSC1 have on human
cells?
L3. Speculateabout why plants deficient in Dicer activity
show increasedsensitivity to infection by RNA viruses.
14. lVhat is the evidencethat some mRNAs are directed to
accumulatein specificsubcellular locations?
Analyze the Data
Most humans are infected with herpes simplex virus_1
(HSV-1), the causative agenr of cold sores. The HSV_1
genomeencodesabout 100 genes,most of which are expressed
in infected host cells at the site of oral sores. The infectious
These latently infected neurons are the source of active
type HSV-1. To determineif LAT functions to block apopto_
sis by encoding a miRNA, the following studies *.r. dtn.
(seeGupta er al., 2006, Nature C+2:52-g5).
a. A cell line was rransfectedwith an expressionvector
that expressesa Pst-Msu (part b, diagram belowl fragment
of the LAT gene. The percentageof these transfecteJ cells
368 . c H A p r E8R | posr-TRANscRtploN
GAELN E
coNTRoL
that then undergo drug-induced cell death was compared to
that of control cells. The experiment was repeatedin cells in
which Dicer expression was knocked down using Dicer
siRNA. The data obtained are shown in the graph below.
\fhy did the scientistswho conductedthis study examine the
effects of silencing Dicer? Under what conditions does the
LAT geneprotect the cells from apoptosis?
! cetts transfectedwith
o control expressionvector
o6
;9
6i1 @ celts transfectedwith LAr
expresstonvector
n'G
ffi cetts transfectedwith LAr
e x p r e s s i o nv e c t o ra n d
99) e x p r e s s i n gc o n t r o ls i R N A
LC)
^.o ! celts transfectedwith LAr
!
e x p r e s s i o nv e c t o ra n d
e x p r e s s i n gD i c e rs i R N A
b. Cells were transfected with an expression vector
expressing the Pst-Msu fragment of the LAT gene from
which the region between the two Sty restriction sites was
deleted (ASty; diagram below). Ifhen these cells were in-
duced to undergo apoptosis, they died at the same rate as
did non-transfected cells. In additional studies, cells were
transfectedwith an expressionvector expressingthe Sty-Sty
region of the LAT gene. These cells exhibited the same re-
sistance to apoptosis as did cells transfected with the pst-
Msu fragment. What can be deduced from these findings
about the region of the LAT gene required to protect cels
from apoptosis?
|, P s t / M l uf r a g m e n t
-
F>l Regiondeletedin ASty
LAT gene
Exon 1 Exon2
5'-crcGccGccccGcccccGccccccccGGAcccAAGGGGcccccGcccccGeccctli_
g,
I s t . r n( b ' a r m ) LOOP S t e m( 3 ' a r m ) |
c. RNA encodedwithin the Sty-Styregion is predicted
to form a stem loop (part b, diagram above). Northern blot
analysiswas performed on total-cell RNA isolatedfrom con-
trol cells (mock), cells infected with wild-type HSV-1, cells
infected with an HSV-1 deletion mutant from which the se-
quence between the two Sty sites in the LAT gene was
deleted (ASty), and cells infected with a rescuedASty virus
into which the deleted region was re-insertedinto the viral
genome (StyR). The probe used for the Northern blot was
the labeled 3' stem region of the LAT RNA in the Sty-Sty
region, as diagrammed in part (b). The RNAs recognizedby
this probe were either -55 nucleotidesor 20 nucleotides,as
shown in the Northern blot below. -il/hy were two different-
size RNAs detected?\7hen a second probe was used that
was the labeled 5' stem region of the RNA sequenceshown
in part (b), only the -S5-nucleotide RNA was detected. Shatkin,A. J., and J. L. Manley. 2000. The endsof the affair:
capping and polyadenylation. Nature Struct. Biol. 7 2838-842'
What can you deduce about the processingof RNA ex-
'What Soller,M. 2006. Pre-messenger RNA processingand its regula-
pressedfrom the LAT gene? enzyme likely produced
tion: a genomicperspective.Cell Mol. Life Sci.63:796-81'9'
the -55 nucleotide RNA? In what part of the cell? What en- Valadkhan,S. 2005. snRNAs as the catalystsof pre-mRNA
zyme likely produced the 20-nucleotide RNA? In what part splicing.Curr. Opin. Chem.Biol.9:603-608.
of the cell? Villa, T., J. A. Pleiss,and C. Guthrie.2002. Spliceosomal snRNAs:
Mg(2*)-dependent chemistry at the catalytic core?Cell lO9:149-1'52'
Control wt HSV-1 A Sty StyR
Regulation of Pre-mRNA Processing
6
q)
Black, D. L.2003' Mechanismsof alternativepre-mRNA splic-
ing. Ann. Reu.Biocbem. 72:291-336.
950 Blencowe,B. J. 2005. Alternative splicing:new insightsfrom
giobal analyses.Cell 126:37-47.
Buratti, E., M. Baralle,and F. E. Baralle.2006. De{ectivesplic-
o
N ing, disease and therapy:searchingfor mastercheckpointsin exon
d"Jinition.NucleicAcidsRes.34:3494-3510-
z
820 t fll Lee, C., and Q. \7ang. 2005. Bioinformaticsanalysisof alterna-
tive splicing.Brief Bioinform. 6:23-33.
Licatalosi,D. D., and R. B. Darnell' 2006. Splicingregulationin
neurologicdisease.Neuron 52293-101'.
d. TGF-B mRNA encodes a protein, transforming
Maniatis, T., and B. Tasic' 2002. Alternative pre-mRNA splicing
growth factor B, that inhibits cell growth and inducesapop-
and proteomeexpansionin metazoans.Nature 418:236-243'
tosis.The 3' untranslatedregion (3'-UTR) of TGF-B mRNA
Sanford,J. R., J. Ellis, and J. F. Caceres.2005. Multiple roles of
can form an imperfect duplex with RNA encoded by the 5' arginine/serine-rich splicingfactors in RNA processing'Biochem'
stem region of the LAT Sty-Sty domain (miR-LAT), as Soc.Trans.33:443446.
shown below. In what way might the expression levels of Xing, Y., and C. Lee. 2006. Alternative-splicingand RNA selec-
tion pressure-evolutionary consequences tor eukaryottcgenomes'
TGF-p differ in cells infected with wild-type HSV-1 com-
'What N at.-Reu.G enet.7 :499-5 09 .
pared to uninfected cells? can you infer about latent
HSV-1 infections from thesestudies? Transport of mRNA Across the Nuclear Envelope
Cole, C. N., and J. J. Scarcelli.2006. Transport of messenger
miR-LAr A RNA from the nucleusio the cytoplasm.Curr. Opin' Cell Biol'
78:299-306.
Darzacg,X., R. H. Singer,and Y. Shav-Tal.2.005'Dynamicsof-
transcriptionand mRNA eiport. Curr. Opin- Cell Biol' 17:332-339'
| | rGF-B-3,urR
CAG Dimaano,C., and K. S.Ullman' 2004. Nucleocytoplasmic trans-.
oort, intes.atine mRNA production and turnover with export through
ih. .ru.l.i, po... Moi. cail B iol. 24:3069-3076.
Fried, H., and U. Kutay. 2003. Nucleocytoplasmictransport:
References taking an inventory.Cell Mol. Life Sci. 60t1'659-1688'
Huang, Y., and J' A. Steitz.2005. SRprisesalong a messenger's
of EukaryoticPre-mRNA
Processing journey.Mol. Cell. 77:613-615.

Bentley,D. L. 2005. Rules of engagement:co-transcriptional
recruitmentof pre-mRNA processingfactors. Curr. Opin. Cell Biol.
17251.-256.
Buratowski, S. 2005. ConnectionsbetweenmRNA 3' end pro-
cessingand transcriptiontermination. Curr. Opin. Cell Biol.
17:257-261..
Gu, M., and C. D. Lima. 2005. Processing the message:structural
insights into capping and decappingmRNA. Curr' Opin. Struct. Biol-
15:99-106.
Houseley,J., J. LaCava,and D. Tollervey'2006. RNA-quality
control by the exosome.Nat. Reu.Mol. Cell Biol.7:29-539.
Hsieh, J., A. J. Andrews, and C. A. Fierke.2004. Roles of pro-
tein subunitsin RNA-protein complexes:lessonsfrom ribonuclease
P. Biopolymers73:79-89.
Lambowitz, A. M., and S. Zimmerly. 2004' Mobile group II
introns. Annu. Reu.Genet.3821.-35.
Lehmann,K., and U. Schmidt.2003. Group II introns: structure
and catalytic versatilityof large natural ribozymes.Crit. Reu.
Biochem.Mol. Biol. 38:249-303.
Moore, M. J. 2005. From birth to death:the complex lives of
eukaryotic mRNAs. Science309:15 14-1 5 18'
Sharp,P.A. 2005. The discoveryof split genesand RNA splicing.
TrendsB io chem. Sci. 3O279 -28 t.
Izaurralde,8.2004. Directing mRNA export. Nat' Struct' MoL
Biol. ll:21.0-272.
Kuersten.S.. and E. B' Goodwin. 2005. Linking nuclearmRNP
assemblyand cytoplasmicdestiny.Biol. Cell. 97:469-478'
Lim, R. Y., and B. Fahrenkrog.2006.The nuclearpore complex
up close.Curr. Opin. Cell Biol. 18:342-347.
Maco, B., B. Fahrenkrog,N. P. Huang, and U' Aebi' 2005' Nu-
clear pore-complexstructuri and plasti:ity r9ve115{by electronand
atomfo force -^i.to..opy. Methods Mol. Biol.322:273-288'
Reed,R., and H. Cheng.2005. TREX, SR proteins and export
of mRNA. Curr. Opin. Cell Biol. 17:269-273.
Ribbeck, K., and D. Gorlich. 2001. Kinetic analysisof tra-nslo-
cation through nuclearpore complexes.EMBO J ' 2O:L320-1'330'
Rodriguez,M. S., C. Dargemont,and F. Stutz' 2004' Nuclear
export of RNA. Blol. Cell 96:639-655.
Saguez,C., J. R. Olesen,and T. H. Jensen'2005' Formation of
.*pori.o-p.t.nt mRNP: escapingnucleardestruction' Cun' Opin'
Cell Biol. 17:287-293.
Sommer.P..and U. Nehrbass.2005. Quality control of messen-
ger ribonucleoproteinparticlesin the nucleusand at the pore' Curr'
Opin. Cell Biol. l7:294-30t.
Tran, E. J., and S' R. Wente.2006. Dynamic nuclearpore com-
plexes: life on the edge' Cell 125:t041'-1'053 '

REFERENCES 359
Cytoplasmic Mechanisms of post-transcriptional Control
Almeida, R., and R. C. Allshire. 2005. RNA silencrnsand
genomeregulation.Trends Cell Biol. 1,5:25j,-25g.
Ambros, V.2004. The functions of animal microRNAs. Natwre
431:350-355.
. . Anant,,S., V. Blanc, and N. O. Davidson.2003. Molecular regu_
lation, evolutionary,and functional adaptationsassociatedwith ito
U editing of mammalianapolipoproteinBmRNA. prog. Nucleic Acid
Res.Mol. Biol. 75:1.41..
Baulcombe,D. 2005. RNA silencing.TrendsBiochem.Sci.
302290-293.
_ Chan, S. \7., and L R. Henderson,and S. E. Jacobsen. 2005.
G_ardening the genome:DNA methylation in Aribidopsis thaliana.
Nat. Reu.Genet.6:351-360.
- Chen,C.2., andH. F. Lodish.2005. MicroRNAs as regulators
of mammalianhematopoiesis . Semin.Immunol. 17:IS5-165.
Engels,B. M., and G. Hutvagner.2005. principlesand effectsof
T].!91f4_T:diated post-transcriptionalgeneregulation. Oncogene
25:61.53-6169.
Filipowicz, W 2005. RNAi: the nuts and bolts of the RISC ma-
chine. Cell 122:1.7-20.
Grewal, S. I., and J. C. Rice. 2004. Reeulationof heterochro-
T3tjn by \iltone merhylarionand small RNAs. Carr. Opin. Ceil
Biol. 16:230-238.
. Hirokawa, N. 2005. mRNA rransport in dendrites:RNA gran_
ules,motors, and tracks./. Neurosci.2elB9-2t42.
Huang, Y. S., and J. D. Richter.2004. Regulationof local mRNA
translarion.Curr. Opin. Cell Biol.l6:308-3 iJ.
_ Kidner, C. A., and R. A. Martienssen.200j. The developmental
role of microRNA in plants. Curr. Opin. plant Biol. g:3g44.
Lejeune,F., and L. E. Maquar. 2005. Mechanisticlinks between
nonsense-mediated mRNA decayand pre-mRNA splicingin mam_
maliancells.Curr. Opin. Cell Biol.lT;j09-J 15.
Maas, S., A Rich, and K. Nishikura. 2003. A-to-I RNA editing:
recentnews and residualmysteries.J. Biol. Chem.27g:I39I_1394".
. . .Mangus,D. A., M. C. Evans,and A. Jacobson.2003. poly(A)-
binding proteins: multifunctional scaffoldsfor rhe post-transcripiional
control of geneexpression.GenomeBiol.4:223.
Matzke, M., et al. 2004. Genericanalysisof RNA-mediated
transcriptionalgenesilencing.Biochim. Biophys.Acta 1677:729_14L
_ - Mello,,C. C., and D. Conte lr. 2004. Revealingthe world of
RNA interference.Nature 431:338-342. C. C. Me"llo,sNobel prize
lecturecan be viewed at http://nobelprize.org/nobel_prizes/medicine/
laur e atesI 20 0 6/announcement.html
Pqk, J., and A. Fire. 2006. Distinct populations of primary and
::.o"gr.ty..ffg.tors during RNAi in C. elegins.ScienceNo".2j,2006
(E-pub.).A. Z. Fire'sNobel Prize lectureian be viewed athtt1ll
nobelprize.org/nobel_prizes/medicine/laureate sl200 6I announcemenr.
html
_ Parker,R., and H. Song. 2004.The enzymesand control of eu_
karyotic mRNA turnover. Nat. Struct. Mol. Biol. ll:121-I27.
. Richter,J. D., and N. Sonenberg.2005. Regulationof cap_
dependenttranslation by eIF4E inhibitory pror;ns. Nature
433:477480.
370 C H A P T E R8 | P o S T - T R A N S C R | P T | O NGAELN EC O N T R O L
Ruvkun, G. B. 2004. The tiny RNA world. HarueyLect.99:1.-21..
Schmauss,C. 2003. Serotonin2C receptors:suicide,serotonin,
and runaway RNA editing. Neuroscientist9:237J42.
Shikanai,T.2006. RNA editing in plant organelles:machinery,
physiologicalfunction and evolurion.Cell Mol. Life Sci.63$98-708.
Simpson,L., S. Sbicego,and R. Aphasizhev.2003.Uridine inser-
tion/deletionRNA editing in trypanosomemitochondria: a complex
business.RNA 92265-276.
Sontheimer,E. J., and R. W. Carthew.2005. Silencefrom within:
endogenoussiRNAs and miRNAs. Cell 122:9-12.
St. Johnston,D. 2005. Moving messages: the intracellularlocal-
ization of mRNAs. Nat. Reu.MoL Cell Biol.6:363-375.
Tang, G. 2005. siRNA and miRNA: an insight into RISCs.
TrendsBiochem.Sci.30:106-114.
Ule, J., and R. B. Darnell. 2006. RNA binding proreinsand the
regulationof neuronal synapticplasticity.Curr. Opln. Neurobiol.
L6:102-11.0.
Valencia-Sanchez, M. A., J. Liu, G. J. Hannon, and R. parker.
2006. Control of translationand mRNA degradationby miRNAs
and siRNAs. GenesDeu. 20:51.5-524.
Verdel,A., and D. Moazed. 2005. RNAi-directed assemblyof
heterochromatinin fission yeast.FEBS Lett. 579:5872-5878.
.
$Tullschleger,S., R. Loewith, and M. N. Hall. 2006. TOR sig-
naling in growth and metabolism.Cell 124:471484.
. Zamore, P.D., and B. Haley. 2005. Ribo-gnome:the big world
of small RNAs. Sclezce309:1.519-1,524.
Processing of rRNA and IRNA
_. Anderson,J. T. 2005. RNA turnover: unexpectedconsequences
of being tailed. Curr. Biol. l5:R635-R638.
Decatur,W. A., and M. J. Fournier.2003. RNA-euided nu-
cleotidemodification of ribosomal and other RNAs. /. Biol. Chem.
278:695-698.
Dez, C., and D. Tollervey.2004.Ribosomesynthesismeetsthe
cell cycle. Curr. Opin. Miuobiol. T:631-637.
Evans,D., S. M. Marquez,and N. R. Pace.2006. RNasep: in-
terfaceof the RNA and protein worlds. TrendsBiochem. Sci.
3l:333-347.
Fatica,A., and D. Tollervey.2002.Making ribosomes.Curr. Opin.
Cell Biol. 14:313-318.
Handwerger,K. E., and J. G. Gall. 2006. Subnuclearorqanelles:
new insights into form and function. Trends Cell Biot. 16:15-26.
Hopper, A. K., and E. M. Phizicky.2003. IRNA rransfersto rhe
limelight. GenesDeu. 17i.62-780.
Mayer, C., and I. Grummt. 2006. Ribosomebiogenesisand cell
growth: mTOR coordinatestranscription by all threi classesof nu-
clear RNA polymerases.Oncogene2006 25:6384-6391..
Stahley,M. R., and S. A. Strobel.2006. RNA splicing:group I
intron crystal structuresrevealthe basisof splicesite selecti,cnand
metal ion catalysis.Curr. Opin. Struct. Biol. 16:319-326.
Tschochner,H., and E. Hurt. 2003. Pre-ribosomeson the road
from the nucleolusro rhe cytoplasm.TrendsCettBiot. L3:255-263.
 Microtubules Golgi Actinfibers Mitochondria CHAPTER

VISUALIZING,
FRACTIONATING,
AND CULTURING
CELLS
microscopy
Fluorescence shows thelocationof DNAandmultiple
proteins
withinthesamecellHere, fluorescenttaggingand
techniques
staining usingdifferentfluorescentmoleculesreveal
proteins
thecytoskeletal (green)
ct-tubulin andactin(red),DNA
(blue),
theGolgicomplex (yellow),
andmitochondria (purple)The
imagesalongthetoparefalse-colored images of eachstructure
Thelarger
individually
stained imagemerges theseseparate
imagesto depictthefullcell.Scalebars,20 pm [FromBNG
Giepmans,etal, 2006,Science312:217 |

A lmost 200 years ago Matthias Schleiden and of organelle contain a unique group of proteins that are es-
/ \ theodore Schwannused a primitive light microscope sential for the organelleto carry out its unique functions. In
/1ro show that individual cells constitute the funda- addition, we see how certain chimeric proteins-consisting
mental unit of life, and light microscopy has been an in- of a protein of interest covalently linked to a naturally fluo-
creasingly important researchtool for biologists ever since. ,.r.*t protein-enable biologists to image movements of
Sophisticated light microscopes developed in the last two individual proteins in live cells. Introduction of digital sys-
decades now enable cell biologists to reveal the myriad tems also has resulted in the improved quality of micro-
movements of cells ranging from the translocation of chro- scopic images,as well as digital storageand retrieval. Digital
mosomesand vesiclesto cell crawling and swimming. algorithms also permit three-dimensionalreconstructionsof
Electron microscopy provides a much higher resolution ..il .o-pottents from two-dimensional images, and allow
of cell ultrastructure than light microscopy,but the technol- visualization and quantification of specific proteins and
ogy requires that the cell be fixed and sectionedand thus all other moleculesin cells.
cell movements are frozen in time. Electron microscopy re-
vealed that all eukaryotic cells-be they of fungal, plant' or
animal origin-are divided into similar multiple membrane- OUTLIN E
limited compartments termed organelles.In the first section
of this chapter we describethe basic structuresand functions 9.1 Organellesof the EukarYoticCell
of the major organellesin animal and plant cells.
In the second and third sectionsof this chapter we dis- 9.2 Light Microscopy:VisualizingCellStructure
a n d L o c a l i z i n gP r o t e i n sW i t h i n C e l l s 380
cuss many modern techniques in light and electron
microscopy that are suitable for detecting and imaging par- Methods
9.3 ElectronMicroscoPY:
ticular structural features of the cell. Developmentsin both 388
andApplications
light and electron microscopS togetherwith those for gener-
ating monoclonal antibodies,have enabledmodern cell biol- 9.4 Purificationof Cell Organelles 391
ogiststo detect specificproteins in fixed cells' thus providing
a static image of their location within cells, as illustrated in 9.5 lsolation,Culture,and Differentiation
the chapter opening figure. Such studiesled to the important of MetazoanCells
concept that the membranesand interior spacesof each type

371
 Parallel developmentsin subcellular fractionation have
enabled cell biologists to isolate individual organellesto a
high degree of purity. These techniques,detailed in the
fourth section of this chapter,continue to provide important
information about the protein composition and biochemical
function of organelles.For example, the use of proteomic
approaches including mass spectrometry to determine the
identity of all of the major proteins present in preparatrons
of purified mitochondria has revealedmany novel functions
for this organelle.
Many technicalconstraints hamper studieson cells in in-
tact animals and plants. One alternativeis the use of intact
organs that are removed from animals and treated to main-
tain their physiologic integrity and function. However, the
organization of organs, even isolated ones, is sufficiently
complex to posenumerousproblemsfor research.Thus mo-
lecular cell biologists often conduct experimental studieson
cells isolated from an organism. tn the fifth secion of this
chapter we learn how to isolate certain types of cells to high
purity from a complex mixture of cells.
In many cases,isolated cells can be maintained in the
laboratory under conditions that permit their survival and
growth, a procedure known as culturing. Cultured cells have
severaladvantagesover intact organismsfor cell biology re-
search.Cells of a singlespecifictype can be grown in culture,
experimental conditions can be better controlled, and in
many casesa single cell can be readily grown into a colony
of many identicalcells.The resultingsrrain of cells,which is
genetically homogeneous,is called a clone. In culture, cer-
tain lines of undifferentiated mammalian cells can be in-
duced to differentiate over a period of days to a specifictype
of cell such as muscle or nerve when switched to a different
culture medium. As we learn in the last section of this chao-
ter, such lines of cellsprovide powerful tools for understand-
ing how specific types of differentiated cells are formed in
the body.
fl| Organelles
of the EukaryoticCell
The major organellesin animal and plant cells are depicted
in Figure9-1. Unique proteinsin the interior and membranes
of each type of organelle largely determine its specific func-
tional characteristics,which are examined in more detail in
later chapters. Those organellesbounded by a single mem-
brane are coveredfirst, followed by the three types that have
a double membrane-the nucleus, mitochondrion, and
chloroplast. The internal organizationof organelles,and the
structure of the plasma membrane, is organized by the fi-
brous cytoskeleton; Chapters 1,7 and 18 discussthesefibers
in detail.
T h e P l a s m aM e m b r a n eH a sM a n y C o m m o n
F u n c t i o n si n A l l C e l l s
Although the lipid composirion of a membrane largely de-
termines its physical characteristics,its complement of pro_
teins is primarily responsiblefor a membrane.sfunctional
372 . c H A p r E R9 | v t s u A l t z t N c ,F R A C ' ' . N A T ' N GA,N D c u L T U R ' N G
properties. In all cells, the plasma membrane acts as a per-
meability barrier that prevents the entry of unwanted mate-
rials from the extracellular milieu and the exit of needed
metabolites. Specific membrane transport proteins in the
plasma membrane permit the passageof nutrients into the
cell and metabolic wastes out of it; others function to
maintain the proper ionic composition and pH (=7.2) of the
cytosol, the aqueous portion of the cytoplasm excluding
organelles,membranes, and insoluble cytoskeletal compo-
nents. The structure and function of proteins that make the
plasma membrane selectivelypermeable to different mole-
culesare discussedin Chapters10 and 11.
Unlike animal cells, most bacterial, and all fungal and
plant cells are surrounded by a rigid cell wall and lack the
extracellular matrix found in animal tissues. The plasma
membrane is intimately engagedin the assembly of cell
walls, which in plants are built primarily of cellulose.The
cell wall prevents the swelling or shrinking of a cell that
would otherwise occur when it is placed in a medium that is
less concentrated (hypotonic) or more concentrated (hyper-
tonic), respectively,than the cell interior. For this reason,
cells surrounded by a wall can grow in media having an
osmotic strength much less than that of the cytosol. The
properties,function, and formation of the plant cell wall are
coveredin Chapter 19.
In addition to these universal functions, the plasma
membranehas other crucial roles in multicellular organisms.
Few of the cells in multicellular plants and animals exist as
isolated entities; rather, groups of cells with related special-
izations combine to form tissues.In animal cells, specialized
areasof the plasma membrane, called cell junctions, contain
proteins and glycolipids that form specific structures be-
tween cells that strengthentissuesand allow the exchangeof
metabolites between cells. Certain plasma membrane pro-
teins anchor cells to componentsof the extracellular matrix,
the mixture of fibrous proteins and polysaccharidesthat
provides a bedding on which sheets of epithelial cells or
small glands lie. \Weexamine both of thesemembrane func-
tions in Chapter 19. Still other proteins in the plasma mem-
brane act as anchoring points for many of the cytoskeletal
fibers that permeate the cytosol, imparting shape and
strength to cells (Chapters 17 and 18).
The plasma membranesof many types of eukaryotic cells
also contain proteins that function as receptors by binding
specific signaling molecules, such as hormones, growth
factors, and neurotransmitters, leading to various cellular
responses.These proteins, which are critical for cell devel-
opment and functioning, are describedin severallater chap-
ters. Finally, peripheral cytosolic proteins that are recruited
to the membrane surface function as enzymes,intracellular
signal transducers,and structural proteins for stabilizing the
membrane.
EndosomesTakeUp SolubleMacromolecules
from the Cell Exterior
Although transport proteins in the plasma membrane medi-
ate the movement of ions and small moleculesinto and out
cELLs
a n i m a lc e l l
plantcell
FIGURE 9-1 Schematic overviewof a "typical"animalcell
(top) and plant cell(bottom)and their majorsubstructures.
n l lt h eo r g a n e l l egsr,a n u l eas n
l i l lc o n t a i a
N o te v e r yc e l w
of the cell across the lipid bilayer, proteins and some other
soluble macromoleculesin the extracellular milieu are inter-
nalized by endocytosis.In this process,a segmentof the
plasma membraneinvaginatesinto a "coated pit," whose cy-
tosolic face is lined by a specificset of proteins; severaltypes
of endocytosis,each involving different sets of proteins, are
known. In receptor-mediatedendocytosis, for example,
certain receptor proteins in the plasma membrane bind
macromoleculesin the cell exterior and then becomeincor-
porated into the invaginating coated pit. The pit pinches
from the membrane into a small membrane-boundedvesicle
that contains extracellular material-both soluble and
bound to receptors-and is deliveredto an early endosome,
a sorting station of membrane-limited tubules and vesicles
(Figure 9-2a,b). From this compartment' some membrane
proteins are recycled back to the plasma membrane; other
membrane proteins are transported to a late endosome
E P l a s m am e m b r a n e c o n t r o l s m o v e m e n t o f m o l e c u l e si n a n d
o u t o f t h e c e l l a n d f u n c t i o n s i n c e l l - c e l ls i g n a l i n ga n d c e l l
adhesion.
z M i t o c h o n d r i a ,w h i c h a r e s u r r o u n d e db y a d o u b l e m e m b r a n e ,
g e n e r a t eA T P b y o x i d a t i o n o f g l u c o s ea n d f a t t y a c i d s '
E L v s o s o m e s ,w h i c h h a v e a n a c i d i c l u m e n , d e g r a d e m a t e r i a l
i n t e r n a l i z e db v t h e c e l l a n d w o r n - o u t c e l l u l a rm e m b r a n e sa n d
o r g a n elle s .
E N u c l e a re n v e l o p e ,a d o u b l e m e m b r a n e ,e n c l o s e st h e c o n t e n t s
o f t h e n u c l e u s ;t h e o u t e r n u c l e a rm e m b r a n e i s c o n t i n u o u s
with the rough ER.
E N u c l e o l u si s a n u c l e a rs u b c o m p a r t m e n tw h e r e m o s t o f t h e
c e l l ' sr R N A i s s y n t h e s i z e d .
tr N u c l e u si s f i l l e d w i t h c h r o m a t i n c o m p o s e d o f D N A a n d
p r o t e i n s ;s i t e o f m R N A a n d t R N A s y n t h e s i s .
z S m o o t h e n d o p l a s m i cr e t i c u l u m ( E R )s y n t h e s i z e sl i p i d s a n d
d e t o x i f i e sc e r t a i nh y d r o p h o b i cc o m p o u n d s .
E R o u g h e n d o p l a s m i cr e t i c u l u m( E R )f u n c t i o n s i n t h e s y n t h e s i s ,
processinga , n d s o r t i n g o f s e c r e t e dp r o t e i n s ,l y s o s o m a l
p r o t e i n s ,a n d c e r t a i nm e m b r a n e p r o t e i n s .
p
- C o t g i c o m p l e x p r o c e s s e sa n d s o r t s s e c r e t e dp r o t e i n s ,
l v s o s o m a lp r o t e i n s ,a n d m e m b r a n e p r o t e i n ss y n t h e s i z e do n
t h e r o u g hE R .
-@ S e c r e t o r yv e s i c l e ss t o r e s e c r e t e dp r o t e i n sa n d f u s e w i t h t h e
o l a s m a m e m b r a n et o r e l e a s et h e i r c o n t e n t s .
-I P e r o x i s o m e sd e t o x i f yv a r i o u s m o l e c u l e sa n d a l s o b r e a kd o w n
f a t t v a c i d st o p r o d u c ea c e t y lg r o u p s f o r b i o s y n t h e s i s .
-@ C y t o s k e l e t afli b e r s f o r m n e t w o r k sa n d b u n d l e st h a t s u p p o r t
c e l l u l a rm e m b r a n e s ,h e l p o r g a n i z eo r g a n e l l e s ,a n d p a r t i c i p a t e
in cell movement.
@ M i c r o v i l l ii n c r e ; s e s u r f a c ea r e a f o r a b s o r p t i o no f n u t r i e n t s
f r o m s u r r o u n d i n gm e d i u m .
s a i n t a i nt h e
C g l l w a l l , c o m p o s e d l a r g e l yo f c e l l u l o s e , . h e l p m
-E
c e l l ' s s h a p e a n d p r o v i d e sp r o t e c t i o na g a i n s t m e c h a n i c a l
stress.
@
- V a c u o l es t o r e sw a t e r , i o n s , a n d n u t r i e n t s ,d e g r a d e s
m a c r o m o l e c u l e sa, n d f u n c t i o n s i n c e l l e l o n g a t i o nd u r i n g
growth.
@
- , h i c h c a r r y o u t p h o t o s y n t h e s i sa, r e s u r r o u n d e d
C h l o r o p l a s t sw
b v a d o u b l e m e m b r a n e a n d c o n t a i na n e t w o r k o f i n t e r n a l
m e m b r a n e - b o u n d e ds a c s .
structures shownhere,andothersubstructures canbe present in
c
s o m eC e l l sa l s od i f f e r o n s i d e r a i
b nl s
y h a p a
e n di n t h e p r o m i n ence
, df i b r o u s of various organelles and substructures
where further sorting takes place' The endocytic pathway
ends when a late endosomedeliversits membrane and inter-
nal contents-including material from the extracellular solu-
tion-to lysosomes for degradation. The entire endocytic
pathway is describedin somedetail in Chapter 14.
Lysosomes Are Acidic OrganellesThat Contain
a Battery of DegradativeEnzymes
Lysosomesprovide an excellentexample of the ability of in-
tracellular membranes to form closed compartments in
which the composition of the lumen (the aqueousinterior of
the compartment) differs substantially from that of the sur-
rounding cytosol. Found exclusively in animal cells, lyso-
somesare responsiblefor degradingcertain componentsthat
have becomeobsoletefor the cell or organism' (The vacuoles
in plant and fungal cells have many of the same functions as
SF T H E E U K A R Y O T I C E L L
O R G A N E L L EO . 373
 (a) .i l,:
t\ uh/l

plasma phagosome
meKne ',.
Bacterium _*[)
\ -\L_,-,y primary
is 7._ , E
rvsosome
\/! q
E primarylysosome
__l
,r \ ,_
w"/'l ,{t
\(\ 'secondary
\ - t
- 0 . 1p m t
\,/ \ lysosome
I (c) 'r "
: Early Late \-/-
endosome end,
"I
ii '' t,
Mitochonr
,\
-+-
/,.:J-=- R" A,,t^^r,"
P
M

\lZ@
--) @))^"oonusosome
--7
a-
Autophagy
1!m

A FIGURE 9-2 Cellularstructures that participatein delivering hydrolytic enzymes degrade proteins, nucleic acids,andotherlarge
materialsto lysosomes.(a)Schematic overview of threepathways m o l e c u l e(sb )A n e l e c t r om
n i c r o g r a pohf a s e c t i o o
nf a c u l t u r e d
bywhichmaterials aremovedto lysosomes Soluble macromotecutes m a m m a l i acne l lt h a th a dt a k e nu p s m a lgl o l dp a r t i c l ecso a t e dw i t h
aretakenintothecellby invagination of coatedpitsin the plasma the eggproteinovalbumin. Gold-labeled ovalbumin (black spots) is
membrane anddehvered to lysosomes throughtheendocytic pathway foundin earlyendosomes (EE) andlateendosomes (LE), butverylittle
I l . W h o l ec e l l sa n do t h e rl a r g ei,n s o l u b lpea r t i c l ems o v ef r o mt h e is presentin autophagosomes (AV) (c)Electron micrograph of a
cellsurface to lysosomes throughthe phagocytic pathwayE Worn-out section of a ratlivercellshowing a secondary lysosome containing
o r g a n e l l easn db u l kc y t o p l a s amr ed e l i v e r et d
o l y s o s o m tehsr o u g h fragments of a mrtochondrion (M)anda peroxisome (p).lpart(b)from
t h ea u t o p h a g p i ca t h w a yB W i t h i nt h ea c i d i cl u m e no f l v s o s o m e s , T.E Tjelle et al, 1996,J CellSci109:2905 Part(c)courtesy of D Friend l

animal lysosomes.)The processby which an agedorganelle
is degradedin a lysosomeis called autophagy (,,eating one-
self"). Materials are taken into a cell not only by endocytosis
but also by phagocytosis,a processin which large, insoluble
particles (e.g., bacteria) are envelopedby the plasma mem-
brane and internalized(seeFigure 9-2a).ln borh cases,the
internalizedmaterial may be degradedin lysosomes.
Lysosomescontain a group of enzymesthat degradepoly-
mers, releasingtheir monomeric subunits. For example, nu-
cleases degrade RNA and DNA to mononucleotides;
proteasesdegradea variety of proteins and peptidesto amino
acids;phosphatasesremovephosphategroups from mononu-
cleotides, phospholipids, and other compounds; still other
enzymes degrade complex polysaccharidesand glycolipids
into smaller units. All the lysosomalenzymeswork most effi-
Lysosomesvary in size and shape, and severalhundred
may be presentin a typical animal cell (Figure 9-3). In effect,
they function as siteswhere various materialsto be degraded
collect. Primary lysosomesare roughly sphericaland do not
contain obvious particulate or membrane debris. Secondary
lysosomes,which are larger and irregularly shaped, result
from the fusion of primary lysosomeswith late endosomes
and other membrane-boundedorganellesand vesicles.They
contain particles or membranes in the process of being di-
gested(Figure9-2c).
Severalhuman diseasesare causedby defectsin specific
lysosomalenzymesbecausetheir substratesaccumulate
insidethe organelle.For example, Tay-Sachsdiseaseis caused
by a defect in one enzyme catalyzing a step in the lysosomal
breakdown of gangliosides.The resulting accumulation of
these glycolipids, especially in nerve cells, has devastating
consequences.The symptoms of this inherited diseaseare
usually evident before the age of 1. Affected children com-
monly becomedementedand blind by age 2 and die before
their third birthday. Nerve cells from such children are
greatly enlargedwith swollen lipid-filled lysosomes.I

PeroxisomesDegradeFatty Acids
a n d T o x i cC o m p o u n d s
All animal cells (except erythrocytes) and many plant cells
contain peroxisomes,a classof roughly sphericalorganelles,
protein complexesin the cytosol (seeFigure 3-29). 0.2-1.0 pr.min diameter (Figure 9-4). Peroxisomescontain

374 . cHAprER
9 vtsuALtztNG
FR, Ac'oNATtNG
A ,N D c u L T U R t NcGE L L s
|
 S m o o t h E R R o u g hE R

#,.

Glycogen pm

FIGURE 9-4 Electronmicrographshowing variousorganelles

in a rat liver cell.Twoperoxisomes(P)lie in closeproximity to
mitochondria (M)andthe roughandsmoothendoplasmic reticulum
(ER)Alsovisible of glycogen,
areaccumulations a polysaccharidethat is
the primary molecule
glucose-storage in animals of
[courtesy P Lazarow]

A FIGURE 9-3 Locationof lysosomes and mitochondriain a many of the sametypes of enzymesas well as additional ones
culturedliving bovinepulmonaryarteryendothelialcell.The usedto convert fatty acids into glucoseprecursors.I
cellwasstained with a green-f luorescing dyethat isspecifically
boundto mitochondria, anda red-fluorescing dyethat isspecifically
incorporated into lysosomes. The image was sharpened usinga c e t i c u l u ml s a N e t w o r k
T h e E n d o p l a s m iR
deconvolution computerprogramdiscussed laterin the chapter of InterconnectedInternal Membranes
N : nucleus[Courtesy Invitrogen/ Probes
Molecular Inc]
Generally,the largestmembrane in a eukaryotic cell encloses
the endoplasmic reticulum (ER)-an extensive network of
closed, flattened membrane-bounded sacs called cisternae
severalox.idases-enzymesthat use molecular oxygen to ox- (seeFigure9-1). The endoplasmicreticulumhas a number of
idize organic substances,in the process forming hydrogen
functions in the cell but is particularly important in the syn-
peroxide (HzOz), a corrosive substance.Peroxisomesalso
thesis of lipids, membrane proteins, and secretedproteins'
contain copious amounts of the enzyme catdlase,which de-
The smooth endoplasmicreticulwm is smooth becauseit
gradeshydrogen peroxide to yield water and oxygen:
lacks ribosomes.In contrast' the cytosolic face of the rowgh
ca ta lase endoplasmicreticulum is studded with ribosomes'
ZH2O2 ----+ 2H2O + 02
The Smooth Endoplasmic Reticulum The synthesisof
In contrast with the oxidation of fatty acids in mito- fatty acids and phospholipidstakes place in the smooth ER'
chondria, which producesCO2 and is coupled to the gen- Although many cellshavevery little smooth ER, this organelle
eration of ATP, peroxisomal oxidation of fatty acids yields is abundant in hepatocytes'Enzymesin the smooth ER of the
acetyl groups and is not linked to ATP formation (see Fig- liver also modify or detoxify hydrophobic chemicalssuch as
ure 1.2-L2).The energy releasedduring peroxisomal oxi- pesticidesand carcinogensby chemicallyconvertingthem into
dation is converted into heat, and the acetyl groups are more water-soluble,conjugatedproducts that can be excreted
transported into the cytosol, where they are used in the from the body. High dosesof such compounds result in alarge
synthesis of cholesterol and other metabolites. In most proliferation of the smooth ER in liver cells.
eukaryotic cells, the peroxisome is the principal organelle
in which fatty acids are oxidized, thereby generating pre- The Rough Endoplasmic Reticulum Cytoplasmic ribo-
cursors for important biosyntheticpathways. Particularly somes bound to the rough ER synthesizecertain membrane
in liver and kidney cells,various toxic moleculesthat enter and organelle proteins and virtually all proteins to be secreted
the bloodstreamalso are degradedin peroxisomes,produc- from the cell (Chapter 13). Sequences in the nascentpolypeptide
ing harmlessproducts.

Plant seedscontain glyoxisomes,small organellesthat

oxidize stored lipids as a source of carbon and energy
for growth. They are similar to peroxisomes and contain

SF T H E E U K A R Y O T I C E L L
O R G A N E L L EO . 375
 overviewAnimation:proteinSecretion
{lllt
Mitochondrion

(b)
Secretedprotein

Secretorvvesicle

G o l g iv e s i c l e s

R o u g hE R

Golgi Endoplasmic plasma Intercellular

vesicles reticulum membrane soace

FIGURE 9-5 Characteristic featuresof cellsspecializedto ribosomes (smallblackdots)of the roughER,secreted proteins arefound
secretelargeamountsof particularproteins(e.g.,hormones, inthelumenof theroughERTransport vesicles budoff andcarrythese
antibodies). (a)Electron micrograph of a thinsection
of a hormone- proteins to the Golgicomplex (lf ), wherethe proteins areconcentrated
secretingcellfromthe rat pituitaryThebasalendof thecell(botfom) andpackaged intoimmature secretory vesicles ([) These vesicles then
isfilledwithabundant roughERandGolgisacs, wherepolypeptide coalesce to form largermaturesecretory vesicles that losewaterto
hormones aresynthesized andpackaged At theopposite apical
end the cytosol, leaving an almostcrystalline mixtureof secreted proteins
of thecell(top)arenumerous secretoryvesicles,
whichcontain i n t h e l u m e n( B ) A f t e rt h e s ev e s i c l easc c u m u l aut en d e tr h ea p i c a l
recentlymadehormones eventually
to besecreted (b)Diagram of a surface, theyfusewiththeplasma membrane andrelease theircontents
typicalsecretorycelltracingthe pathwayfollowedbya protein(small (exocytosis) in response to appropriate hormonal or nervestimulation
reddots)to besecreted, lmmediatelvaftertheirsvnthesison (4) IPaft(a)courtesy of Biophoto Associates l

interior space,or lumen. There the protein folds, assistedby
folding catalysts termed chaperones.Secretedproteins are
modified in severalways by enzymeslocalizedin the ER lu-
men, including covalentaddition of sugars(glycosylation)and
formation of disulfide bonds. Newly made membrane pro-
teins remain associatedwith the rough ER membrane, and
proteins to be secretedaccumulate in the lumen of the or-
ganelle.
AII eukaryotic cells contain a discernible amounr of
rough ER becauseit is neededfor the synthesisof plasma
membraneproteins and secretedproteins that compiise the
extracellularmatrix. Phospholipidsare also synthesizedin
associationwith the rough ER. Rough ER is particularly
abundant in specializedcellsthat produce an abundanceof
specificproteins to be secrered.For example, plasma cells
produce antibodies, pancreatic acinar cells synthesizedi-
gestiveenzymes,and cellsin the pancreaticisletsof Langer-
hans produce the polypeptide hormones insulin and
glucagon.In thesesecretorycellsand others, alarge part of
the cytosol is filled with rough ER and secretoryvesicles
( F i g u r e9 - 5 ) .
The Golgi ComplexProcesses and SortsSecreted
a n d M e m b r a n eP r o t e i n s
Severalminutes after proteins are synthesizedin the rough
ER, most of them leave the organelle within small mem-
brane-boundedtransportvesicles.Thesevesicles,which bud
from regions of the rough ER not coated with ribosomes,
carry the proteins to another membrane-limited organelle,
the Golgi complex (seeFigure 9-5), named after the Italian
microscopistCamillio Golgi.
Three-dimensional reconstructions from serial sections
of a Golgi complex revealthis organelleto be a seriesof flat-
tenedmembranevesiclesor sacs(cisternae), surroundedby a
number of more or lesssphericalmembrane-limitedvesicles
(Figure 9-6). The stack of Golgi cisrernaehas three defined
regions-the cis, the medial, and the trans. Translort vesi-
cles from the rough ER fuse with the cls region of ihe Golgi
complex, where they deposit their protein contents. As de-
tailed in Chapter 14, these proreins then progress from the
cis to the medial to the trons region. Within each region are
different luminal enzymesthat modify proteins to be secreted

376 CHAPTER
9 I v t s u A l t z t N G ,F R A C T I O N A T I NAGN, D C U L T U R T NCGE L L S
 Model of a Golgi Complex
&) vlA"o: Three-Dimensional
> FIGURE 9-6 Modelof the Golgicomplexbasedon three-
dimensionalreconstruction of electronmicroscopyimages.
Transport (whitespheres)
vesicles that havebuddedoff the rough
ERfusewith the crsmembranes (lightblue)of the Golgicomplex
Bymechanisms described in Chapter14,proteins movefromthe
crsregionto the medralregionandfinallyto the transregionof
the GolgicomplexEventually, vesiclesbudoff the trans-Golgi
membranes (orange andred);somemoveto the cellsurface and
othersmoveto lysosomes. TheGolgicomplex, likethe rough
endoplasmic reticulum, prominent
isespecially in secretorycells
[FromB J Marshet al , 2001,ProcNat'lAcadSciUSA98:2399 I

and membrane proteins differentln depending on their small moleculesstored within it. Becausethe solute concen-
structuresand their final destinations. tration is much higher in the vacuole lumen than in the cy-
After proteins to be secretedand membrane proteins are tosol or extracellular fluids, water tends to move by osmotic
modified in the Golgi complex, they are transported out of flow into vacuoles. This influx of water, which causesthe
the complex by a secondset of vesicles,which bud from the vacuole to expand, createshydrostatic pressure' or turgor,
trans stde of the Golgi complex. Some vesiclescarry mem- inside the cell. This pressureis balanced by the mechanical
brane proteins destinedfor the plasma membrane or soluble
proteins to be releasedinto the extracellular medium; others
carry soluble or membrane proteins to lysosomesor other
organelles.Continuous budding of vesiclesin this manner
would resultin accumulationof phospholipidsat the plasma
membrane, but endocytic vesicles(seeFigure 9-2) return
plasma membrane phospholipids to lysosomesor to the
Golgi. Yet other vesiclesbud from Golgi vesiclesand fuse
with earlier Golgi vesiclesor with the rough ER. How intra-
cellular transport vesicles"know" with which membranesto
fuse and where to deliver their contents is also discussedin
Chapter 14.

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e s
a n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y
Most plant cells contain at least one membrane-lim-
ited internal vacuole.The number and sizeof vacuoles
dependon both the type of cell and its stageof development;
a single vacuole may occupy as much as 80 percent of a ma-
ture plant cell (Figure9-7). A varietyof transportproteinsin
the vacuolar membrane allow plant cells to accumulateand
store water, ions, and nutrients (e.g., sucrose,amino acids)
within vacuoles(Chapter11). Like a lysosome,the lumen of
a vacuole contains a battery of degradativeenzymesand has
, 2trt ,
an acidic pH, which is maintained by similar transport pro-
teins in the vacuolar membrane. Thus plant vacuoles may A FIGURE 9-7 Electronmicrographof a thin sectionof a
also have a degradativefunction similar to that of lysosomes leaf cellshowinga prominentvacuole.In thiscell,the single
in animal cells. Similar storage vacuolesare found in green largevacuole occupies muchof the cellvolumePartsof f ive
algae and many microorganismssuch as fung^. chloroplastsa n dt h ec e l a l la l s oa r ev i s i b l eN o t et h e i n t e r n a l
lw
Like most cellular membranes,the vacuolar mem- subcompartments in the chloroplasts. [Courtesy of Biophoto
brane is permeableto water but is poorly permeableto the Associates/MyronC Ledbetter/Brookhaven National Laboratory I

CELL
SF T H E E U K A R Y O T I C
O R G A N E L L EO . 377
 Video:Three-Dimensional
Model Most of the cell'sribosomal RNA is synthesizedin the nu-
of a Mitochondrion cleolus,a subcompartmentof the nucleusthat is not bounded
by a phospholipid membrane. Ribosomal proteins, like all
Outer nuclear-encoded proteins,are made in the cytosol.Most enter
I n n e rm e m b r a n e Cristae membrane
the nucleus via nuclear pores and are added to ribosomal
RNAs within the nucleolus. The finished or partly finished ri-
bosomalsubunits,exit through a nuclearpore into the cytosol
for use in protein synthesis(Chapter 4). In mature erythro-
cytes from nonmammalian vertebratesand in other types of
"resting" cells,the nucleusis inactiveor dormant and minimal
synthesisof DNA and RNA takesplace.Similarly,mRNA and
IRNA is synthesizedin the nucleus;following extensivepro-
cessingwithin the nucleus, particles containing these RNAs
also exit the nucleusinto the cytosol through nuclearpores.
The packagingof nuclear DNA into chromosomesis de-
scribedin Chapter 6. Only during cell division are individual
chromosomesvisible by light microscopy.In the electronmi-
croscope,the nonnucleolarregions of the nucleus,called the
nucleoplasm, can be seen to have dark- and light-staining
areas.The dark areas,which are often closelyassociatedwith
the nuclearmembrane,contain condensedconcentratedDNA,
called heterochromatin (seeFigure 6-33a). Fibrous proteins
Intermembrane Matrix Matrix
space gran utes called lamins form a two-dimensional network along the inner
surface of the inner membrane, giving it shapeand apparently
A FIGURE 9-8 Electronmicrographof a mitochondrion.
binding DNA to it. The breakdown of this network occurs
M o s tA T Pp r o d u c t i oin n o n p h o t o s y n t h ecterlcl st a k e sp r a c er n
early in cell division, as we detail in Chapter 20.
m i t o c h o n d rT
i ah ei n n e rm e m b r a n w
e ,h i c hs u r r o u n dt hs e m a t r i x
space, hasmanyinfoldings, calledcristaeSmallcalcium-containing
matrixgranules alsoareevident[From D W Fawcett, 1981 , TheCell, MitochondriaAre the PrincipalSitesof ATP
2ded, Saunders, p a21l
Productionin Aerobic NonphotosyntheticCells
Most eukaryotic cells contain many mitochondria (sing.,
mitochondrion), which occupyup to 25 percentof the volume
resistanceof the cellulose-containingcell walls thar surround of the cytoplasm (seeFigure 9-3). Thesecomplex organelles,
plant cells. Most plant cells have a rurgor of 5-20 atmos- the main sitesof ATP production during aerobic metabolism,
pheres (atm); their cell walls must be strong enough to react are generally exceededin size only by the nucleus,vacuoles,
to this pressurein a controlled way. Unlike animal cells, and chloroplasts (which also produce ATP).
plant cells can elongate extremely rapidly, at rates of 20-75 The two membranesthat bound a mitochondrion differ in
prmlh. This elongation, which usually accompaniesplant compositionand function. The outer membrane,composedof
growth, occurs when a segmentof the somewhat elasticcell about half lipid and half protein, containsporin proteins (see
wall stretchesunder the pressurecreated by water taken into Figure 10-18) that render the membranepermeableto mole-
the vacuole.I cules having molecular weights as high as 10,000. The inner
membraneis much lesspermeable;its surfacearea is greatly
T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A increasedby alarge number of infoldings, or cristae, that pro-
trude into the matrix, or central space(Figure9-8).
SyntheticApparatus,and a FibrousMatrix
In nonphotosynthetic cells, the principal fuels for ATp
The nucleus, the largest organelle in animal cells, is sur- synthesisare fatty acids and glucose.The complete aerobic
rounded by two membranes,each containing many different degradation of glucose to CO2 and H2O is coupled to the
types of proteins. The inner nuclear membrane defines the synthesisof about 30 moleculesof AIP (the exact number is
nucleus itself. In most cells, the outer nuclear membrane is in question). In eukaryotic cells, the initial stagesof glucose
continuous with the rough endoplasmic reticulum, and the degradation take place in the cytosol, where 2 ATP mole-
space between the inner and outer nuclear membranes is cules per glucose molecule are generated. The terminal
stagesof oxidation and the coupled synthesisof ATP are car-
ried out by enzymesin the mitochondrial matrix and inner
membrane (Chapter 12). As many as 28 ATP moleculesper
glucose molecule are generated in mitochondria. Similarly
virtually all the AIP formed in the oxidation of fatty acidsto
ture of nuclear pores and the regulatedtransport of material CO2 is generatedin mitochondria. Thus mitochondria can
through them are detailed in Chapter 8. be regardedas the "power plants" of the cell.

378 . c H A p r E R9 | v t s u A l t z t N c ,F R A c I o N A ' N G ,A N D c u L T U R I N cGE L L s

ChloroplastsContainInternal Compartments
in Which Photosynthesis
TakesPlace
Except for vacuoles, chloroplasts are the largest and
the most characteristic organelles in the cells of
plants and green algae.They can be as long as 10 pcmand
are typically 0.5-2 p"n thick, but they vary in size and
shape in different cells, especially among the algae. In ad-
dition to the double membranethat bounds a chloroplast,
this organellealso contains an extensiveinternal systemof
interconnectedmembrane-limited sacs called thylakoids,
which are flattened to form disks (Figure 9-9). Thylakoids
often form stacks called grana and are embedded in a ma-
trix space, the stroma. The thylakoid membranes contain
green pigments (chlorophylls) and other pigments that ab-
sorb light, as well as enzymes that generate ATP during
photosynthesis.Some of the ATP is used to convert CO2
into three-carbon intermediates by enzymeslocated in the
stroma; the intermediates are then exported to the cytosol
and convertedinto sugars.I
The molecular mechanismsby which ATP is formed in
mitochondria and chloroplastsare very similar, as explained
in Chapter 12. Chloroplasts and mitochondria have other
featuresin common: both often migrate from place to place
within cells, and they contain their own DNA, which en-
codes some of the key organellar proteins (Chapter 6). The
proteins encoded by mitochondrial or chloroplast DNA are
synthesizedon ribosomes within the organelles.However,
most of the proteins in each organelleare encodedin nuclear
DNA and are synthesizedin the cytosol; these proteins are
P l a s m am e m b r a n e 'i.: i., 1:
{.{'.f
Grana
Thylakoid
memorane
Starch
granule
llpm
FIGURE9-9 Electronmicrographof a plant chloroplast. The
internalmembrane (thylakoids)
vesicles arefusedintostacks (grana),
whichresidein a matrix(thestroma)All the chlorophyll
in the cell
in the thylakoidmembranes,
is contained wherethe light-induced
productionof ATPtakesplaceduringphotosynthesis [Courtesyof
BiophotoAssociates/M National
C Ledbetter/Brookhaven Laboratoryl
then incorporated into the organellesby processesdescribed
in Chaoter 13.
Organelles of the Eukaryotic Cell
r AII eukaryotic cells contain a nucleus as well as numer-
ous other organellesin their cytoplasm (seeFigure 9-1).
r The nucleus, mitochondrion, and chloroplast are
bounded by two bilayer membranesseparatedby an inter-
membrane space.All other organellesare surrounded by
single membranes.
I The plasma membrane acts as a permeability barrier. It
contains a multitude of proteins that transport nutrients
and waste molecules or that bind components of the ex-
tracellular matrix or, in plants, the cell wall. The plasma
membranesof many eukaryotic cells also contain receptor
proteins that bind specificsignaling molecules.
r Endosomesinternalize plasma membrane proteins and
soluble materials from the extracellular medium, and sort
the internalizedmaterials back to the membrane or to lyso-
somesfor degradation.
r Lysosomeshave an acidic interior and contain various
hydrolasesthat degrade worn-out or unneeded cellular
components and some ingestedmaterials (seeFigure 9-2).
r Peroxisomes are small organelles containing enzymes
that oxidize various organic compounds without the pro-
duction of ATP. By-products of oxidation are used in
biosynthetic reactions.
r Secretedproteins and membraneproteins are synthesized
on the rough endoplasmicreticulum' a network of flattened
membrane-boundedsacsstuddedwith ribosomes.
r Proteins synthesizedon the rough ER first move to the
Golgi complex, where they are processedand sorted for trans-
port to the cell surfaceor other destinations(seeFigure 9-5).
r Plant cellscontain one or more large vacuoles,which are
storagesitesfor ions and nutrients. Osmotic flow of water
into vacuoles generates turgor pressure that pushes the
plasma membrane againstthe cell wall.
r The nucleus housesthe genome of a cell. The inner and
outer nuclear membranes are fused at numerous nuclear
pores, through which materials pass between the nucleus
and the cytosol. The outer nuclear membrane is continuous
with that of the rough endoplasmicreticulum.
r Mitochondria have a highly permeableouter membrane
and an inner membranethat is extensivelyfolded. Enzymes
in the inner mitochondrial membrane and central matrix
I spacecarry out the terminal stagesof sugar and lipid oxi-
dation coupled to AIP synthesis'
r Chloroplasts contain a complex system of thylakoid
membranesin their interiors. Thesemembranescontain the
pigments and enzymesthat absorb light and produce ATP
during photosynthesis.
CELL
SF T H E E U K A R Y O T I C
O R G A N E L L EO 379
 (a) Opticalmicroscope (b) Bright-field
lightpath (c) Phase-contrast
lightpath

Detector B---
.......E - lmageplane
:'
t:
:'
- Refractedor
--!
Projection :, Excitation deffractedlight
lens ,.: filter
Lamp raea nl4lo

Dichroic U - Unobstructedlighl
mtrror {l 0 - Objectivelens

Objectivs ---i

Specimen
srage

condenser_ ,:.. (,, Condenser

Collector , ...; l- lens
lens

Lamp @

ffi tI I
'i:'--
\,.:l:.

Lar I rp

(d) Epifluorescence
lightpath < EXPERIMENTAL FIGURE 9-10 Opticalmicroscopes are commonlyconfiguredfor both
bright-field(transmitted),phase-contrast, and epifluorescence microscopy. (a)In a typical
lightmicroscope, the specimen is usuallymountedon a transparent glassslideandpositioned on the
movable specimen stageThethreeimagingmethods requiredifferent illumination systems and
condensers; brightfieldandepifluorescence microscopy usethe samelight-gathering anddetection
systems, (b)In bright-fieldlightmicroscopy, lightfroma tungsten lamptsfocused on thespecimen
by a condenser lensbelowthestage;the lighttravels the pathway shownin yellow(c)ln phase-
Dichroicmirror contrast microscopy, incident lightpasses throughan annular diaphragm, whichfocuses a circular
annulus (ring)of lighton thesampleLightthatpasses unobstructed throughthespecimen is
focused by the oblective lens onto the thickgray ring of the phase plate, which absorbs some of the
W
| il directlightandaltersitsphaseby one-quarter of a wavelength. lf a specimen refracts (bends) or
diffractsthe light,the phaseof somelightwavesisaltered(greenlines) andthe lightwavesare
redirected throughthethin,clearregionof the phaseplateTherefracted andunrefracted lightare
recombined at the imageplaneto formthe image(d)In epifluorescence microscopy, ultraviolet light
(greenline)froma mercury lamppositioned abovethestageisfocused on thespecimen bythe
Specimen
objective lensFilters in the lightpathselect a particular wavelength of ultravioletlightfor
illumination andarematched to capture onlylightemittedbythe specimen of a predetermined
wavelength that islongerthanthatof the incident light(redline)

@ LightMicroscopy:Visualizing Using techniquesof recombinant DNA, researcherscan ex-

CellStructureand Localizingproteins
Within Cells
For many years, light microscopy has been an essentialpart
of virtually all researchon eukaryotic cells. Basic light mi-
croscopesare used to enumeratethe number of cells under
study, and simple staining procedures enable the study of
live cells. More specializedtechniques in lighr mrcroscopy
enable the investigator to visualize movements such as the
crawling of cells along a substrate,extensionof nerve axons,
and movementsof chromosomesand organelleswithin cells.
press,in a cell or in an organism, a chimera of any desired
protein of interest linked to a naturally fluorescentprotein.
Usually a chimera exhibits the normal function of the de-
sired protein, and its location in living cells can be observed
over time. Using immunofluorescence,researcherscan deter-
mine the location of specificproteins in fixed cells, as well as
any localization changesin responseto changesin the cell's
environment. Finally, many microscopic images of the same
cell can be stored in a computer data base;digital reconstruc-
tions permits three-dimensionalreconstructionsof cell com-
ponents from two-dimensional images,and a determination

380 C H A P T E R9 | vtsuAltztNG, FRACTIONAT|NG

A ,N D C U L T U R T NC
GE L L S
of whether two or more oroteins are found in the same sub-
cellular compartment.
The Resolutionof the Light Microscope
fs About O.2p,m
All microscopesproduce a magnified image of a small object,
but the nature of the imagedependson the type of microscope
employedand on the way in which the specimenis prepared.
The compound microscope,used in conventionalbright-field
light microscopy,contains severallensesthat magnify the im-
age of a specimenunder study (Figure 9-10a, b). The total
magnificationis a product of the magnificationof the individ-
ual lenses:if the objectiuelens,the lensclosestto the specimen,
magnifies 100-fold (a 100x lens, the maximum usually em-
ployed) and the proiection lens, sometimescalled the ocular or
eyepiece,magnifies 10-fold, the final magnification recorded
by the human eye or on film will be 1000-fold.
However, the most important property of any micro-
scope is not its magnification but its resolving power, or
resolution-the ability to distinguish between two very
closely positioned objects. Merely enlarging the image of a
specimenaccomplishesnothing if the image is blurred. The
resolution of a microscope lens is numerically equivalent to
D, the minimum distance between two distinguishable
objects.The smallerthe value of D, the betterthe resolution.
The value of D is given by the equation
0.61i
u: * (e-1) L i v i n gC e l l s
/\ slnd
where a is the angular aperture,or half-angle,of the cone of
light entering the objective lens from the specimen;N is the
refractive index of the medium between the specimen and
the objective lens (i.e., the relative velocity of light in the
medium compared with the velocity in air); and \ is the
wavelength of the incident light. Resolution is improved by
using shorter wavelengthsof light (decreasingthe value of \)
or gathering more light (increasingeither N or a). Note that
the magnification is not part of this equation.
EXPERIMENTAL FIGURE 9-11 Livecellscanbe visualized by
microscopy techniquesthat generatecontrastby interference.
These micrographs showlive,cultured macrophage cellsviewedby
bright-fieldmicroscopy(/eft),differential contrast
interference (DlC)
microscopy (middle),
andphase-contrast microscopy(righ\ Ina phase-
L I G H TM I C R O S C O P YV:I S U A L I Z I N G
Owing to limitations on the values of a, )", and N based
on the physical properties of light, the limit of resolution
of a light microscope using visible light is about 0.2 trr.m
(200 nm). No matter how many times the image is magni-
fied, a conventional light microscope can never resolve
objects that are less than =0.2 p'm apart or reveal details
smaller than =0.2 pr,min size. But exotic new light micro-
scopesdescribedbelow can resolve two fluorescent obiects
even if they are as close together as =20 nm.
A conventional light microscopecan be usedto track the
location of a small bead of known sizeto a precision of only
a few nanometers.If we know the precise size and shapeof
an obiect-say, a 5-nm sphereof gold that is attached to an
antibody in turn bound to a cell-surfaceprotein-and if we
use a video camera to record the microscopic image as a
digital image, then a computer can calculate the position of
the center of the object to within a few nanometers.In this
way, computer algorithms can be used to make observa-
tions at a more preciselevel-in this casethe movement of
a cell-surface protein labeled with the gold-tagged anti-
body-than would be possible based on the light micro-
scope'sresolution alone. This technique has also been used
to measure nanometer-sizesteps as molecules and vesicles
move along cytoskeletalfilaments(seeFigures1'7-21,17-26,
and 17-27).
Phase-Contrast and DifferentialInterference
ContrastMicroscopyVisualizeUnstained
Two common methods for imaging live cells and unstained
tissuesgeneratecontrast by taking advantageof differences
in the refractive index and thickness of cellular materials'
These methods, called phase-contrastmicroscopy and dif'
(or No-
ferential interference contrast (DIC) microscopy
marski interference microscopy), produce images that dif-
fer in appe arance and reveal different features of cell
architecture. Figure 9-11 compares images of live, cultured
cells obtained with thesetwo methods and standard bright-
field microscopy.
contrastimage,cellsaresurrounded byalternatingdarkandlight
bands;in-focus andout-of-focus imagedin a
aresimultaneously
details
phase-contrast Ina DICimage,cellsappearin pseudorelief
microscope.
Because onlya narrowin-focusregionisimaged, a DICimageisan
opticalslicethroughthe object.
[Courtesyof N andJ Evans
Watson ]
S ITHINCELLS
PROTEINW
C E L LS T R U C T U RAEN D L O C A L I Z I N G
381
 Phase-contrastmicroscopy (Figure 9-10c) generatesan Expression of Fluorescent Proteins in Live Cells and
image in which the degreeof darknessor brightnessof a re- Organisms A naturally fluorescentprotein found in the
gion of the sample depends on the refractiue index of that jellyfish Aequorea uictoria can be exploited to visualize live
region. Light moves more slowly in a medium of higher cells and specific proteins within them. This 238-residue
refractive index. Thus, a beam of light is refracted (bent) protein, called green fluorescent protein (GFP), contains a
once as it passesfrom air into a transparentobject and again serine, tyrosine, and glycine sequencewhose side chains
when it departs. Consequently, part of a light wave that spontaneously cyclize to form a green-fluorescing chro-
passesthrough a specimenwill be refracted and will be out mophore. rWith the use of recombinant DNA techniques
of phase (out of synchrony) with the part of the wave that discussedin Chapter 5, the GFP gene can be introduced
does not passthrough the specimen.How much their phases into living cultured cells or into specific cells of an entire
will differ dependson the differencein refractive index along animal. Cells containing the introduced gene will produce
the two paths and on the thickness of the specimen.If the GFP and thus emit a green fluorescencewhen irradiated;
two parts of the light wave are recombined, the resultant this GFP fluorescence can be used to localize the cells
light will be brighter if they are in phase and less bright if within a tissue or even a whole organism, as illustrated in
they are out of phase.The refractedand unrefractedlight are Figure 9-12 for a primitive metazoan organism, the
recombined at the image plane to form the image. Phase- Cnidairian Hydra uulgans.
contrast microscopy is suitable for observing single cells or In a particularly useful application of GFP, a cellular
thin cell layers, but not thick tissues.It is particularly useful protein of interest is "tagged" with GFP to localize it. In
for examining the location and movement of larger or- this technique, the gene for GFP is fused to the gene for a
ganellesin live cells. particular cellular protein, producing a recombinant DNA
DIC microscopy is based on interferencebetween po- encoding one long chimeric protein that contains the
larized light and is the method of choice for visualizing entirety of both proteins. GFP fusion proteins frequently
extremely small details and thick objects.Contrast is gen- retain normal function, even with the large GFP polypep-
erated by differences in the index of refraction of the ob- tide appended. Thus GFP fusions are exrremely useful
ject and its surrounding medium. In DIC images, objects
appear to cast a shadow to one side. The "shadow" pri-
marily representsa differencein the refractive index of a
specimenrather than its topography.DIC microscopy eas-
ily definesthe outlines of large organelles,such as the nu-
cleus and vacuole.In addition to having a "relief"-like ap-
pearance,a DIC image is a thin optical section, or slice,
through the object. Thus details of the nucleus in thick
specimens (e.9., an intact Caenorhabditis elegans round-
worm; seeFigure 21.-4)can be observedin a seriesof such
optical sections, and the three-dimensionalstructure of
the object can be reconstructedby combining the individ-
ual DIC images.
Both phase-contrastand DIC microscopy can be used in
time-lapse microscopy, in which the same cell is pho-
tographed at regular intervals over periods of severalhours.
This procedure allows the observerto study cell movemenr,
provided the microscope'sstagecan control the temperature
of the specimenand the gas environmenr.

Fluorescence MicroscopyCan Localize

A EXPERf MENTAT FIGURE 9-12 Transgenichydra H. vulgaris
a n d Q u a n t i f yS p e c i f i cM o l e c u l e si n L i v eC e l l s expressinggreenfluorescentprotein (GFP).A recombinant
Perhapsthe most versatileand powerful technique for local- plasmidin whichthe promoterof the H vulgaris B-actingene
izing proteins within a cell by light microscopy is fluorescent drivesexpression of the GFPgenewasmicroinjected intoembryos
staining of cellsand observationby fluorescencemtcroscopy. at the two- to eight-cell stage.In certainhydralines,the plasmid
A chemical is said to be fluorescentif it absorbslight at one integrated intothe genomeof oneor a few adjacent cellsthat
wavelength (the excitation wavelength) and emits light (flu- t h e nu n d e r w e ndti v i s i o nT.h eG F P - p r o d u cci negl l s( g r e e nw) e r e
visualized by fluorescence microscopy In the specimen shownhere,
oresces)at a specific and longer wavelength. In modern flu-
GFPwasconfinedto certaininternalendodermal epithelial cells
orescencemicroscopes,only fluorescentlight emitted by the
(/nset)A patchof GFP*endodermal epithelial cellsvisualized by
sample is used to form an image; light of the exciting wave-
confocalmicroscopy Scalebar,20 pm With both imaging
length induces the fluorescencebut is then not allowed to
techniques, the GFP-producing cellscanbe followedduringgrowth
passthe filters placed betweenthe objective lens and the eye anddifferentiation of theorganism[From J Wittlieb eral,2006,proc
or camera (Figure 9-10d). Nat'l Acad Sci USA 103:6208 l

382 C H A P T E R9 | vtsuAltztNG, FRACT|ONAT|NG

A,N D C U L T U R T NC
GE L L S
tools for looking at the normal function and trafficking of Podcast;Light and ElectronMicroscopy
native proteins. Cells in which this recombinant DNA has
b e e n i n t r o d u c e d w i l l s y n t h e s i z et h e c h i m e r i c p r o t e i n
whose green fluorescencerevealsthe subcellular location
of the protein of interest.This GFP-taggingtechnique,for
example, has been used to visualize the expression and
distribution of lamin A, a protein that lines the inner, or
intranucleaq surface of the inner nuclear membrane; it
also forms tubule-like structuresthat protrude into the nu-
c l e u s( F i g u r e9 - 1 3 ) .
In a variation of this technique,a cDNA encoding a pro-
tein of interest is modified at its C-terminus by the addition
of a segmentthat encodesfour cysteineresiduesin a defined
sequence(Cys-Cys-Xaa-Xaa-Cys-Cys). Following expres-
sion of the recombinant protein in cultured cells, a chemi-
cally modified red-fluorescingdye (ReAsH) is added to the
culture; this dye forms stable covalent bonds with the four 9-14 Detectionof tetracysteine'
^| EXPERIMENTAL FIGURE
cysteinesin the tetracysteinesequence.Becausethis sequence a gap-junctionprotein,by both light and
taggedConnexin43,
is not found in natural proteins, the dye binds to no other electronmicroscopy. ThecDNAencoding Connexin43 (Cx43)was
cellular proteins. After the cells are washed in buffers to re- extended at the C-terminus with a sequence that encodes a short
move unattached dye, the protein of interest can be visual- peptidecontaining the Cys-Cys-Xaa-Xaa-Cys-Cys sequence. The
ized by fluorescencemicroscopy on either live or fixed cells. recombinant cDNAwasexpressed in HeLacells,whichwerethen
The subcellularlocalizationof the taggedprotein in the same stained with ReAsH, a red-fluorescing dyethatcovalently bindsonly
cellscan also be visualizedat a higherresolutionby electron to proteins with thisspecific tetracysteine (TC) sequence. (a)Confocal
microscopy. Figure 9-14 shows the use of this fluorescent- image reveals two segments of the plasma membranes of adjacent
tagging technique to localize connexin, a plasma membrane cells thatareconnected by multiple gapjunctions thatcontain the
protein that is found in gap junctions. These cell-surface Cx43-TCprotein(brightlinesindicated bywhitearrows)Thecells
weresubsequently fixedwith2% glutaraldehyde, treatedwith the
structurespermit the rapid diffusion of small, water-soluble
dyediaminobenzidine, and subjected to intense illumination Under
molecules between the cytoplasms of two adjacent cells
theseconditions, the ReAsH dyeundergoes a photoconversion that
(Chapter 19). Another exampleof this taggingtechniqueis
results in formationof a denseprecipitate, whichcanbe seen,after
shown in the chapter opening figure. In this case,HeLa cells (b)Inthiselectron
thecellsaresectioned, in theelectron microscope
were transfectedwith a cDNAs encoding a B actin with a shown in (a) afterphotoconversion,
micrograph of thesamearea
tetracysteinetag and then stained with ReAsH dye to label thedarklines(blackarrows) represent the gapiunctions(c)High-
the B actin. magnification viewof thesection depicted in panel(b)reveals many
Cx43-containing gapjunction proteins in theplasma membranes of
thetwo adjacent cells(darkstain)Thisimagehighlights the high
resolutron obtainable with the tetracysteine labeling methodBar
i n ( a )1, O U mi n; ( b )1, p m ;a n d i n ( c 1) ,0 0 n ml F r o m G G a i e t t ,a e t a l
296:503; see also B N G Giepmanset al , 2006, Sclence
2OO2,Science
V o l 3 1 2 , p a g e2 0 7 l

Determination of Intracellular Ca2* and H+ Levels with

lon-sensitive Fluorescent Dyes The concentration of
Ca2* or H+ within live cellscan be measuredwith the aid of
fluorescent dyes, or fluorochromes, whose fluorescencede-
oends on the concentration of these ions. As discussedin
i"te, .hapters, intracellular Ca2* and H* concentrations
have pronounced effectson many cellular processes.For in-
stance,many hormones and other stimuli causea rise in cy-
EXPERIMENTAL FIGURE 9-13An opticalsectionof a living
tosolic Ca2* from the restinglevelof about 10-' M to 10-"
CHO-K1cellexpressinga recombinantlaminA-GFPchimeric
protein.Shownhereistheoval-shaped nucleus with laminA (white) M, which induces various cellular responsesincluding the
A i sa l s of o u n di n i n t r aa- n d
r u c l e amr e m b r a nLea m i n
l i n i n gt h ei n n e n c o n t r a c t i o no f m u s c l e .
transnuclear tubule-like structures Therestof thecelldoesnotcontarn The fluorescentdye fwra-2, which is sensitiveto Ca"-,
anylamin-GFP fusionprotein andthusisblack[rromJ L V Broers et al, contains five carboxylate groups that form ester linkages
1999,J CellSci112:3463 l with ethanol.The resultingfura-Zesteris lipophilic and can

S ITHINCELLS
PROTEINW
C E L LS T R U C T U RAEN D L O C A L I Z I N G
L I G H TM I C R O S C O P YV:I S U A L I Z I N G
383
 l m a g i n gS u b c e l l u l aD
r e t a i l sO f t e n R e q u i r e st h a t
the SamplesBe Fixed,Sectioned,and Stained
Live cells and tissuesgenerally lack compounds that ab-
sorb light and are thus nearly invisible in a light micro-
scope.Although such specimenscan be visualized by the
specialtechniqueswe just discussed,thesemethods do not
reveal the fine details of structure.Microscopy of live cells
also requires thar cells be housed in special glass-faced
chambers,called culture chambers,that can be mounted
on a microscope stage. For these reasons, cells are
often fixed, sectioned,and stained to reveal subcellular
structures.
Specimensfor light and electron microscopy are com-
EXPERIMENTAL FIGURE 9-15 Fura-2,a Ca2*-sensitive monly fixed with a solution containing chemicals that
fluorochrome,can be usedto monitorthe relativeconcentrations cross-linkmost proteinsand nucleicacids.Formaldehyde,a
of cytosolicCa2*in differentregionsof live cells.(left)Ina moving common fixative, cross-links amino groups on adjacent
leukocyte, a Ca2*gradientisestablished. Thehighestlevels (green) molecules; these covalent bonds stabilize protein-protein
areat the rearof thecell,wherecortical contractions
takeplace, and and protein-nucleic acid interactions and render the mole-
the lowestlevels (blue)areat the cellfront,whereactinundergoes cules insoluble and stable for subsequentprocedures.After
polymerization. (Right)Whena pipettefilledwith chemotactic fixation, a sample used for light microscopy is usually
molecules placed to thesideof thecellinduces thecellto turn,the embeddedin paraffin and cut into sections0.5-50 ir,m thick
Ca2*concentration momentarily increases
throughout thecytoplasm (Figure9-16).For electronmicroscopysamplesare imbedded
anda newgradient isestablished Thegradientisoriented suchthat in liquid plastic and, after hardening, sections 50-100 nm
the regionof lowestCa2*(blue)liesin thedirection thatthecellwill thick are cut. Alternativeln the samplecan be frozenwithout
turn,whereas a regionof highCa2*(yellow) always
formsat thesite
thatwillbecome the rearof thecell [FromR A Brundage etal, 1991,
Science254:703;courtesy of F.Fayl

Block Specimen
diffuse from the medium across the plasma membrane into
cells. \Tithin the cytosol, esteraseshydrolyze fura-2 ester,
yielding fura-Z whose free carboxylate groups render the
molecule nonlipophilic and thus unable to cross cellular
membranes, so it remains in the cytosol. Inside cells, each
fura-2 molecule can bind a single Ca2* ion but no other cel-
Iular cation. This binding, which is proportional to the cy-
tosolic Ca2* concentration over a certain range, rncreases
the fluorescenceof fura-2 at one particular wavelength.At a
second wavelength, the fluorescenceof fura-2 js the same Microtome
whether or not Ca2+ is bound and provides a measureof the
total amount of fura-Z in a region of the cell. By examining
cells continuously in the fluorescencemicroscope and mea-
suring rapid changes in the ratio of fura-2 fluorcscence at
these two wavelengths, one can quantify rapid changes in Copper
the fraction of fura-2 that has a bound Ca2* ion and thus in Microscopeslide mesh
the concentration of cytosolic Ca2+ (Figure 9-15). grid
Fluorescentdyes (e.g., SNARF-1)-th^t urc sensitiveto A EXPERIMENTAL FIGURE9-16 Tissuesfor microscopyare
the H* concentrationcan similarly be used to monitor the commonlyfixed,embeddedin a solidmedium,and cut into
thin sections. A fixedtissueisdehydrated by soakingin a seriesof
alcohol-water solutions,endingwith an organic solventcompatible
with the embedding mediumToembedthetissuefor sectioning,
thetissueis placedin liquidparaffinfor lightmicroscopyor in liquid
plastic
for electron microscopy.
Afterthe blockcontainingthespecimen
hashardened, it ismounted on thearmof a microtome andslices are
recrossthe organelle membrane, they accumulate in the Iu-
cutwith a knife.Typicalsectionscutfor lightmicroscopy are0 5-50
men in concentrations many fold greater than in the cy-
pm thick.Thosecutfor electron microscopy aregenerally50-100nm
tosol. Thus this type of fluorescent dye can be used to
thick Thesections arecollectedeitheron microscope slides (light
specifically stain lysosomesin living cells, as Figure 9-3 microscopy) or coppermeshgrids(electron microscopy)andstalned
snows, with an appropriate agent

384 C H A P T E9R | v t s u A l t z t N GF, R A C T | O N A T | N

AGN ,D C U L T U R T N
CGELLS
prior fixation and then sectioned;such treatment preserves
the activity of enzymesfor later detection by cytochemical
reagents. Cultured cells growing on glass coverslips, as
describedlater, are thin enough so they can be fixed in situ
and visualized by light microscopy without the need for
sectioning.
A final step in preparing a specimen for light mi-
croscopy ls to starn rt so as to visualize the main struc-
tural features of the cell or tissue. Many chemical stains
bind to molecules that have specific features.For exam-
ple, hematoxylin binds to basic amino acids (lysine and
arginine) on many different kinds of proteins, whereas
eosin binds to acidic molecules (such as DNA and side
chains of aspartate and glutamate). Becauseof their dif-
ferent binding properties, these dyes stain various cell
types sufficiently differently that they are distinguishable
visually. lf an enzymecatalyzesa reaction that producesa
colored or otherwise visible precipitate from a colorless
precursor,the enzyme may be detectedin cell sectionsby
their colored reaction products. Such staining techniques,
although once quite common, have been largely replaced
by other techniquesfor visualizing particular proteins, as
discussednext.
lmmunofluorescence MicroscopyCan Detect
SpecificProteinsin FixedCells
The common chemical dyes just mentioned stain nucleic
acids or broad classesof proteins, but investigators often
want to detect the presenceand location of specificproteins.
A widely used method for this purpose employs specific
antibodies that are detectedby fluorescence.In one version
of this technology, the antibody-generally a monoclonal
antibody-is covalently linked to a fluorochrome. Com-
monly used fluorochromes include rhodamine and Texas
red, which emit red light; Cy3, which emits orange light; and
fluorescein, which emits green light. These fluorochromes
can be chemically coupled to purified antibodies specificfor
almost any desiredmacromolecule.'Whena fluorochrome--
antibody complex is added to a permeabilizedcell or tissue
section,the complex will bind to the correspondingantigens,
which then light up when illuminated by the exciting wave-
Iength, a technique called immunofluorescencemicroscopy
(Figure 9-17). Staining a specimenwith different dyes that
fluoresce at different wavelengths allows multiple proteins
as well as DNA to be localized within the same cell (see
chapter opening figure).
In a variation of the immunofluorescence technique, a
monoclonal or polyclonal antibody is applied to the fixed
tissue section, followed by a second fluorochrome-tagged
antibody that binds to the common (Fc) segment of the
first antibody. For example, a "second" antibody (called
"goat anti-rabbit") is preparedby immunizing a goat with
the Fc segmentthat is common to all rabbit IgG antibodies;
when coupled to a fluorochrome, this second antibody
preparation will detect any rabbit antibody used to stain a
tissue or cell. Becauseseveral goat anti-rabbit antibody
moleculescan bind to a singlerabbit antibody moleculein
L I G H TM I C R O S C O P YV:I S U A L I Z I N G
, 2oP'm ,
A EXPERIMENTAL FIGURE 9-17 Aspecificprotein can be
localizedin fixed tissuesectionsby immunofluorescence
wallwasstainedwith
microscopy. A sectionof the rat intestinal
blue,whichgenerates a nonspecific redfluorescence, andwith
Evans
a yellow green-fluorescingantibody specificfor GLUT2, a glucose
transport protein.As evidentfromthisfluorescence micrograph,
GLUT2 ispresent in the basalandlateral cells
sidesof the intestinal
but isabsentfromthe brushborder, composed of closelypacked
on theapicalsurface
microvilli facingthe intestinal lumen.Capillaries
runthroughthe laminapropria, a looseconnective tissuebeneath
theepithelial lseeB Thorenset
layer. al, 1990,Am.J Physio 259:C279'
courtesyof B,Thorensl
a section, the fluorescenceis often brighter than if just a
protein-specific antibody directly coupled to a fluo-
rochrome is used.
In another widely used version of this technolog5 cells
are transfected with a cDNA encoding a recombinant
protein that has appended,generally to one end' a short
sequenceof amino acids called an epitope tag. Two com-
monly used epitope tags are called FLAG' encoding the
amino acid sequenceDYKDDDDK (single-letter code),
and myc, encoding the sequenceEQKLISEEDL. Commer-
cial fluorochrome-coupled monoclonal anti-epitope anti-
bodies can then be used to detect the recombinant protein
in the cell.
New types of fluorescencemicroscopy, such as one
with the exotic name of STED (stimulatedemissiondeple-
tion). enabletwo fluorescentobiectsto be resolvedeven if
they are as closetogether as =20 nm, well below the =200-
nm resolution limit for standard light microscopy. For
example, nerve cells contain a class of membrane-lined
vesicles, termed synaptic vesicles, that ate too small (=40
nm in diameter) and too densely packed to be resolved by
available fluorescence microscopes. However, STED can
resolve individual vesiclesin nerve cells. This technique
should also enable investigators to detect single fluores-
cent protein molecules in the membranes of purified
orqanelles.
S ITHINCELL5
PROTEINW
C E L LS T R U C T U RAEN D L O C A L I Z I N G
385
 C o n f o c aal n d D e c o n v o l u t i o nM i c r o s c o p yE n a b l e
Visualization o f T h r e e - D i m e n s i o nO a lb j e c t s
C o n v e n t i o n a l f l u o r e s c e n c em i c r o s c o p y h a s r w o m a j o r
limitations. First, the fluorescent light emitted by a sam-
ple comes from molecules above and below the plane of
focus; thus the observer seesa blurred image caused by
the superpositionof fluorescentimagesfrom moleculesat
many depths in the cell. The blurring effect makes it diffi-
cult to determine the actual molecular arrangements(Fig-
u r e 9 - 1 8 a ) .S e c o n d t, o v i s u a l i z et h i c k s p e c i m e n sc, o n s e c u - able can be obtained by deconvolution,a computationally
tive (serial) secrions must be prepared, imaged, and
aligned to reconstruct structuresin thick tissues.Two ap-
p r o a c h e sc a n b e u s e d t o a v o i d t h e p r e p a r a t i o n o f s e r i a l
s e c t i o n sa n d t o o b t a i n h i g h - r e s o l u t i o nt h r e e - d i m e n s i o n a l croscopy,a seriesof images of an object are taken at differ-
information.
One approach, called confocal microcopy differs from
conventional fluorescencemicroscopy by the use of a pin-
hole located in front of the detector that blocks light not
originating from that focal plane. The resulting imagesdo
not contain blurs from structures above and below the
c u r r e n tp o s i t i o no f t h e f o c a l p l a n e ( F i g u r e9 - 1 8 b ) .T h e m a -
jority of confocal microscopesuse a laser as the source of
illumination; lasers provide a defined excitation wave-
length and becauseof their focused energy are often well
suited to penetrating thick specimens.Since a laser is fo-
c u s e d o n a s i n g l e p o i n t o n t h e s p e c i m e n ,i t m u s t b e
scannedacrossand down to build an image. The intensity
of light from these in-focus areas is recor"dedby a photo-
multiplier tube, and the image is stored in a comouter.The
( a )C o n v e n t i o n af l u o r e s c e n cm
e icroscopy
lmaged
volume
A EXPERIMENTAL FIGURE 9-18 Confocalmicroscopyproducesan
in-focus optical section through thick cells.A mitoticfertilizedegg
from a seaurchin(Psammechinus) was lysedwith a detergenr,exposeo
to an anti-tubulinantibody,and then exposedto a fluorescein-tagged
antibodythat bindsto the anti-tubulinantibody.(a)When viewedby
conventional f luorescencemicroscopy,the mitoticspindleis blurred
386 C H A P T E R9 | V t S U A L t Z t N GF, R A C T | O N A T I N G
A ,N D C U L T U R T NC
scanning processcan often take a few minutes and so is
not well-suited to imaging fast biological processesin liv,
ing samples.A revolutionary newly developedvariation of
laser-scanningconfocal microscopy is Nipkow confocal
microscopy.This method usesa spinning disk with multi-
ple pinholes that split the laser into hundreds of beamlets,
increasingthe rate at which the image is collectedby a fac-
tor of 10 or more.
Imagesof the highest possibleresolution currently avail-
intensive mathematical processwhereby blurred objects are
sharpened.Similar algorithms are used by astronomersto
sharpen images of distant stars. In deconuolution mi-
ent focal planes with a conventional fluorescencemicro-
scope or confocal microscope. A separateseriesof images
are made from a test slide containing tiny fluorescentbeads
smaller (e.g.,0.2 trrmin diameter)than the resolutionof the
microscope.Each bead representsa pinpoint of light that be-
comesa blurred object becauseof the imperfect optics of the
microscope;from theseimagesa point spreadfunction is de-
termined that enablesthe investigator to calculatethe distri-
bution of fluorescentpoint sourcesthat generatedthe "blur"
in the object image. In other words, the light generaringthe
blurred sample image from adjacent focal planes is reas-
signed to the correct focal plane via iterative comparlson
with the point spreadfunction. Imagesrestored by deconvo,
lution display impressivedetail without any blurring as illus-
trated in Figure9-19.
(b) Confocalfluorescencemicroscopy
' 4o P'm
i'*7
Focal plane ---+/ l__,
\-r tmaged
, __*\:...J \-
votume
Thisblurringoccursbecausebackground fluorescence is detectedfrom
tubulinaboveand belowthe focal planeas depictedin the sketch
(b) Theconfocalmicroscopic imageis sharp,particularly in the center
of the mitoticspindleln thiscase,fluorescence is detectedonlyfrom
molecules in the focalplane,generatinga verythin opticalsection
[MicrographsfromJ G Whiteet al , 1987,J CellBiol 104:41]
GE L L S
 Graphicsand InformaticsHaveTransformed
Modern Microscopy
In the past decade,digital cameras have largely replaced
optical camerasto record microscopy images.Digital im-
agescan be stored in a computer and manipulated by con-
ventional photographic software as well as by specialized
algorithms. As mentioned above, deconvolution algo-
rithms can sharpen an image by restoring out-of-focus
photons to their origin. Other computer algorithms,
which previously required supercomputing facilities' en-
able visualizationof intricate three-dimensionalstructures
reconstructed on desktop computers (see Figure 9-1'9).
Informatics including image analysis algorithms and sta-
tistical approachesallow quantitation of shapes,move-
ments and signal intensitieswithin objects such as cells or
organelles.

Light Microscopy:Visualizing Cell Structure

and LocalizingProteins Within Cells
r The limit of resolution of a light microscope is about
200 nm.
r Becausecells and tissuesare almost transparent' various
types of stains and optical techniquesare used to generate
sufficientcontrastfor imaging'
r Phase-contrastand differential interference contrast
(DIC) microscopy are used to view the details of live'
unstained cells and to monitor cell movement (seeFig-
ure 9-11).
'Sfhen
r proteins tagged with naturally occurring green
fluorescent protein (GFP) or its variants are expressedin
live cells, they can be visualized in a fluorescencemicro-
scope(seeFigure9-12).
r \fith the use of dyes whose fluorescenceis proportional
to the concentration of Ca2* or H* ions' fluorescencemi-
croscopycan measurethe local concentration of Ca2* ions
and intracellular pH in living cells.
r In immunofluorescencemicroscopy, specific proteins
and organelles in fixed cells are stained with fluo-
rochrome-labeledmonoclonal antibodies. Multiple pro-
teins can be localized in the same sample by staining with
EXPERIMENTAL FIGURE 9-19 Deconvolutionfluorescence antibodies labeled with different fluorochromes (see
microscopy yieldshigh-resolution opticalsectionsthat can be chapter opening figure).
reconstructed into one three-dimensional image.A macrophage r Confocal microscopy and deconvolution microscopy
cellwasstained with fluorochrome-labeled reagents specific for DNA use different methods to optically section a specimen,
( b l u e )m, i c r o t u b u l(egsr e e n a) ,n da c t i nm i c r o f i l a m e n( rtes d )T. h e thereby reducing the blurring due to out-of-focus fluores-
series of f luorescent imagesobtainedat consecutive focalplanes cence light (see Figures 9-18 and 9-19). Both methods
(optical sections) through thecellwererecombined in threedimensions
provide much sharper images, particularly of thick speci-
(a)In thisthree-dimensional reconstruction of the rawimages, the
mens, than does standard fluorescence or light mi-
D N A .m i c r o t u b u l easn.da c t i na o p e aar sd i f f u s ez o n e si n t h ec e l l .
(b)Afterapplication croscopy.
of thedeconvolution algorithm to the images,
t h ef i b r i l l aor r g a n i z a t i oonf m i c r o t u b u laens dt h e l o c a l i z a t i o n f r Advances in computation and graphics enable measure-
a c t i nt o a d h e s i o nbse c o m e r e a d i lvyi s i b l ien t h e r e c o n s t r u c t i o n ments to be extracted from microscopeimages.
[Courtesy of J Evans, Whitehead Institute ]

S ITHINCELLS
PROTEINW
C E L LS T R U C T U RAEN D L O C A L I Z I N G
L I G H TM I C R O S C O P YV:I S U A L I Z I N G
387
 !f,l Electron
Microscopy:
Methods
andApplications
Electron microscopy of cells and tissues provides a much
higher resolution of cell ultrastrucrure than can be obtained
by light microscopy. However, the technology requires that
the cell be fixed and sectioned,and thus living cells cannot
be imaged. Researchersweigh these kinds of trade-offs and
selectfrom a variety of methods to produce imagesthat an-
swer their questions. For example, immunoelectron mi-
croscopycan be usedto localize specificproreins at high res-
olution within cells. Two proteins, but generally not more,
can be detected simultaneously,though the procedure is
technically challenging. By comparison, fluorescencemi-
croscopy can be used to detect four or more proteins at the
same time (seechapter opening figure) albeit at a lower res-
olution. Preparations of certain purified particles, such as
viruses and ribosomes,can be visualized by electron mi-
croscopy without prior fixation or staining if the sample is
frozen in liquid nitrogen and maintained in the frozen state
while being observed.
Figure 9-16). The generationof the image dependson dif-
ferentialscatteringof the incident electronsby moleculesin
the preparation.
'Without
staining, the beam of electrons
passesthrough a specimen uniformlS and so the entire
sample appears uniformly bright with little differentiation
of components.To obtain useful imagesby TEM, samples
are commonly stainedwith heavy metals such as lead and
uranium, during or just after the fixation step. Metal-
stained areas appear dark on a micrograph becausethe
metalsscatter(diffract) most of the incident electronslscat-
tered electrons are not focused by the electromasnetic
TEM SEM
Tungstenfilament
(cathode)
- C o n d e n s elre n s

Resolutionof Transmission ElectronMicroscopy Beam of electrons

ls VastlyGreaterThan That of Light Microscopy
Scanning
The fundamental principles of electron microscopy are slm- coils
ilar to those of light microscopy; the major differenceis that Specimen
electromagneticlensesfocus a high-velocityelectron beam
instead of the visible light used by optical lenses.In the Electromagnetic-
transmission electron microscope (TEM), electrons are objectivelens
emitted from a filament and acceleratedin an electric field.
A condenser lens focusesthe electron beam onto the sam-
ple; objectiveand projector lensesfocus the electronsthat
passthrough the specimenand project them onto a viewing Projectorlens
screenor other detector (Figure 9-20, left). Becauseatoms
in air absorb electrons,the entire tube between the electron
source and the detector is maintained under an ultrahigh
vacuum.
Detector
The short wavelength of electronsmeansthat the limit of
resolution for a transmissionelectron microscopeis theoret-
Specimen
ically 0.005 nm (lessthan the diameterof a singleatom), or
40,000 times better than the resolution of a light micro- EXPERIMENTAL FIGURE 9-20 In electronmicroscopy, images
scope, and 2 million times better than that of the unaided are formed from electronsthat passthrough a specimenor are
human eye. However, the effective resolution of the trans- scatteredfrom a metal-coatedspecimen,In a transmlssron
mission electron microscope in the study of biological sys- electron microscope (TEM), electrons areextracted froma heated
tems is considerablylessthan this ideal.Under optimal con- filament, accelerated by an electric field,andfocused on the
ditions, a resolution of 0.10 nm can be obtained with specimen by a magnetic condenser lens Electrons that passthrough
transmissionelectron microscopes,about 2000 times better the specrmen arefocused by a series of magnetic objective and
than the best resolution of light microscopes.Severalexam- projector lenses to forma magnified imageof the specimen on a
ples of cells and subcellularstructuresimaeed bv TEM were detector, whichmaybe a fluorescent viewingscreen, photographic
includedin Section9.1. film,or a charged-couple-device (CCD)cameraIn a scanning
electron microscope (5EM), electrons arefocused by condensor and
BecauseTEM requiresvery thin, fixed sections(about
objective lenses on a metal-coated specimen Scanning coilsmove
50 nm), only a small part of a cell can be observedin any
the beamacross the specimen, andelectrons scattered fromthe
one section.Sectionedspecimensare preparedin a manner
metalarecollected by a photomultiplier tubedetectorIn bothtypes
similar to that used for light microscopy,by using a knife of microscopes, because electrons areeasily scattered by air
capableof producing sections50-100 nm in thickness(see m o l e c u l et h
s ,ee n t i r e
c o l u m ni sm a i n t a i n eadt a v e r yh i g hv a c u u m ,

388 CHAPTER
9 I v t s u A l t z t N G ,F R A C T | O N A T | NAGN, D C U L T U R T NCGE L L S
 (al < EXPERIMENTAL FIGURE 9-21 Gold particlescoatedwith
proteinA are usedto detectan antibody-boundprotein by
transmissionelectronmicroscopy. (a)Firstantibodies are
allowedto interact with theirspecific antigen(e g , catalase) in a
sectionof fixedtissueThenthe sectionistreatedwith electron-
Antigen densegoldparticles coatedwith proteinA fromthe bacterium
(catalase) of the
S aureusBindingof the boundproteinA to the Fcdomains
antibodymolecules makes the location of the target protein,
n t h i sc a s ev, i s i b l ien t h ee l e c t r om
c a t a l a si e n i c r o s c o p( eb .)A s l i c e
of livertissuewasfixedwith glutaraldehyde, sectioned, andthen
(b) Peroxisomes treatedasdescribed in part(a)to localize catalaseThegold
particles (blackdots)indicating the presence of catalase arelocated
exclusively in peroxisomes [From H J Geuze etal, 1981, J CellBiol
89:653 Reproducedfrom lhe Journalof Cell Biologyby copyright
p e r m i s s i o no f T h e R o c k e f e l l eUr n i v e r s i t P
y r e s sl

of cryoelectron microscopy, an aqueous suspenslonol a

'o'5Pm'
lensesand do not contribute to the image. Areas that take
up less stain appear lighter. Osmium tetroxide preferen-
tially stains certain cellular components, such as mem-
b r a n e s( s e eF i g u r e 1 0 - 6 ) .
Specific proteins can be detected in thin sections using
protein-specificantibodies.In this technique the cells are
lightly fixed (to avoid denaturing epitopes on the desired
protein) and then hozen at the temperature of liquid nitro-
gen and sectioned.After thawing, an antibody is applied to
the section, which then is treated with electron-densegold
particles coated with protein A, a bacterial protein that
binds the Fc segmentof all antibodymolecules(Figure9-21).
Becausethe gold particles diffract incident electrons, they
appearas dark dots.
CryoelectronMicroscopyAllows Visualization
of ParticlesWithout Fixationor Staining
Standard transmissionelectron microscopy cannot be
used to study live cells becausethey are generallytoo vul-
nerable to the required conditions and preparatory tech-
niques. [n particular, the absenceof water causesmacro-
molecules to become denatured and nonfunctional.
However, hydrated, unfixed, and unstained biological
specimenscan be viewed directly in a transmission elec-
tron microscopeif the sample is frozen. In this technique
sample is applied to a grid in an extremely thin film,
frozen in liquid nitrogen, and maintained in this state by
means of a special mount. The frozen sample then is
placed in the electron microscope.The very low tempera-
' C ) k e e p sw a t e r f r o m e v a p o r a t i n g ,e v e n i n a
ture (- 196
vacuum. Thus, the sample can be observedin detail in its
native, hydrated statewithout fixing or heavy metal stain-
ing, although cells and some viruses subjected to this
tr*t-.ttt will be killed. By computer-basedaveraging of
hundreds of images, a three-dimensional model can be
generatedalmost to atomic resolution. For example, this
method has been used to generatemodels of ribosomes
(seeFigure 4-26), the muscle calcium pump discussedin
Chapter 11, and other large proteins that are difficult to
crystallize.
Many virusescontain coats' or capsids,that contain mul-
tiple copiesof one or a few proteins arrangedin a symmetric
^ir^y.lia cryoelectronmicroscope,imagesof theseparticles
can be viewed from a number of angles.A computer analy-
sis of multiple images can make use of the symmetry of the
particle to ihe calculate three-dimensionalstructure of the
iapsid to about 5-nm resolution. Examples of such images
are shown in Figure 4-44.
An extension of this technique,cryoelectrontomogra-
phy, allows researchersto determine the three-dimen-
iional architecture of organellesor even whole cells em-
bedded in ice, that is, in a state close to life' In this
technique, the specimen holder is tilted in small incre-
-.nt, the axis perpendicularto the electron beam;
"rotr.td
thus images the object viewed from different directions
of
are obtained (Figure 9-22a, b). The images are then
merged computationally into a three-dimensionalrecon-
struition termed a tomogram (Figure 9-22c)' A disadvan-
tage of cryoelectrontomography is that the samplesmust
be relativelythin, about 200 nm; this is much thinner than
the samples(200 p'm thick) that can be studied by confo-
cal light microscopy.

M I C R O S C O P YM: E T H O D SA N D A P P L I C A T I O N S
ELECTRON 389
 (a) < EXPERIMENTAL FIGURE 9-22 Structure
I of the nuclearporecomplex(NPC)by
\* / cryoelectrontomography.(a)In electron
\tt tomography, a semicircular series of two-
dimensional projection imagesis recorded
fromthe three-dimensional specimen that is
@
located at the center;the specimen istilted
whilethe electron opticsanddetector
.h'/t remainstationary Thethree-dimensional
\
qb / \
structure iscomputed fromthe individual
t two-dimensional images
whenthe objectis imagedby electrons
that areobtained

comingfromdifferent directions (arrows in

leftpanel) Theseindividual images areused
to generate a 3-dimensional imageof the
object(arrows, rightpanel)(b)lsolated
n u c l ef ir o mt h ec e l l u l asrl i m em o l d
Dictyostelium drscoideum werequick-frozen
i n l i q u i dn i t r o g eann dm a i n t a i n ei ndt h i s
stateasthe sample wasobserved in the
electron microscope. Thepanelshowsthree
s e q u e n t i tai l t e di m a g e sD. i f f e r e n t
orientations of NPCs(arrows) areshownin
top-view(leftand center) andside-view
(nghf).Ribosomes connected to theouter
nuclear membrane arevisible, asisa patch
of roughER(arrowheads) (c)Computer-
generated surface-rendered representation
of a segment of the nuclear envelope
membrane (yellow) studded with NPCs
(blue)lpart(a)after5 Nickell etal. 2006.
Nature Rev.Mol Cell Biol 7.225 Parts(b) and (c)
f r o m M B e c ke I a l , 2 0 0 4 , S c i e n c e
3 0 6 : 1 3 8 7l

ElectronMicroscopyof Metal-Coated
SpecimensCan RevealSurfaceFeatures
o f C e l l sa n d T h e i rC o m p o n e n t s
Information about the shapesof purified viruses,fibers, en-
zymes, and other subcellular particles can be obtained with
TEM by using metal shadowing. In this preparative tech-
nique,,a thin layer of metal, such as platinum, is evaporated
on a fixed or rapidly frozen biological sample (Figure9-23a).
teatment with acid and bleachdissolvesaway thecell, leaving
a metal replica that is viewed in a transmissionelectron mi_
croscope(Figure9-23b).
AlternativelS the scanning electron microscope (SEM)
_.
allows investigators to view the surfacesof unsectioned
metal-coatedspecimens.An intense electron beam inside
the microscope scansrapidly over rhe sample. Molecules
in the coating are excited and releasesecondaryelectrons
that are focusedonto a scintillation detector;the resulting
signal is displayedon a cathode-raytube much like , .orl
ventional television (seeFigure 9-20, right). The resulting
scanning electron micrograph has a three-dimensional
a p p e a r a n c eb e c a u s et h e n u m b e r o f s e c o n d a r ye l e c t r o n s
produced by any one point on the sample dependson the
angle of the electron beam in relation to the surface
(Figure 9-24). The resolving power of scanning electron
microscopes,which is limited by the thicknessof the metal
coating, is only about 10 nm, much lessthan that of trans-
mission lnstruments.
Electron Microscopy:Methods and Applications
r Specimens for transmission electron mrcroscopy
(TEM) generally must be fixed, dehydrated, embedded,
sectioned, and then stained with electron-denseheavy
metals.
r Cryoelectron microscopy allows examination of hydrated,
unfixed, and unstained biological specimensdirectly in a
transmission electron microscope; samples are frozen in
liquid nitrogen and maintained in this state.
r Surfacedetails of objectscan be revealedby transmission
electron microscopy of metal-coatedspecimens.
r Scanning elecrron microscopy (SEM) of metal-coated
unsectionedcells or rissuesproducesimagesthat appear to
be three-dimensional.

390 C H A P T E R9 I vtsuAltztNG, FRACT|ONAT|NG

A ,N D C U L T U R T NC
GE L L S
 (a)
Mica surface
I
Absorptive
epithelial
Evaporatedplatinum cells

iii+ Metal replica

Evaporatedcarbon

;;;; 5pm
Carbonfilm

A EXPERIMENTAL FIGURE 9-24 Scanningelectronmicroscopy

(SEM)producesa three-dimensional imageof the surface
Metal replicaready of an unsectionedspecimen.Shownhereis an SEMimage
lization l i n i n gt h e l u m e no f t h e i n t e s t i n e
o f t h ee p i t h e l i u m A.b u n d a n t
fingerlike microvilli extend from the lumen-facing surface of each
g c e l lT
. he b a s alla m i n b
a e n e a t t
h h e e p i t h e l i u h
m e l p s
s u p p o rat n d
a n c h oirt t o t h e u n d e r l y i ncgo n n e c t i vt ies s u eC. o m p a rteh i s
i m a g eo f i n t e s t i n cael l l sw i t h t h a ti n F i g u r 9e - 1 7 ,a f l u o r e s c e n c e
micrographlFrom R Kessel andR Kardon, 1979, Tissues andOrgans: A
Text-Atlas of Scanning Electron Microscopy, W H Freeman and Company,
o t/b I

@ Purificationof CellOrganelles
Many studies on cell structure and function require sam-
ples of a particular type of subcellular organelle' As one
i*ample, a recent proteomic study on mitochondria puri-
fied fiom mouse brain, heart, kidney' and liver revealed
591 mitochondrial proteins, including 163 proteins not
previously associatedwith this organelle.Severalproteins
were found in mitochondria only in specificcell types' De-
termining the functions associatedwith these newly iden-
, 1gm , tified mitochondrial proteins is a maior objective of cur-
A EXPERIMENTAL FIGURE 9-23 Metalshadowingmakessurface rent researchon this organelle.In this section'we describe
detailson very smallobjectsvisible by transmissionelectron severalcommonly used techniquesfor separatingdifferent
microscopy. (a)Thesampleis spreadon a micasurtace andthen organelles.
d r i e di n a v a c u u m evaporato ( trr ) T h es a m p l g e r i di sc o a t e dw i t h
a t h i nf i l mo f a h e a v ym e t a ls, u c ha sp l a t i n u m o r g o l d ,e v a p o r a t e d
D i s r u p t i o no f C e l l sR e l e a s eTs h e i r O r g a n e l l e s
f r o ma n e l e c t r i c a h y a t e dm e t afl i l a m e n(tE ) T os t a b i l i zt e
l le he
r e p l i c at h, e s p e c i m e n i st h e nc o a t e w
d i t h a c a r b o f
n i l me v a porated and Other Contents
f r o ma n o v e r h e aedl e c t r o d(eB ) T h eb i o l o g i c m a l a t e r i ai sl t h e n The initial step in purifying subcellular structures is to re-
d i s s o l v ebdy a c i da n db l e a c h ( E l ) ,w h i c hi sv i e w e di n a T E M I n leasethe cell's contents by rupturing the plasma membrane
electronmrcrographs of suchpreparations, the carbon-coated
and the cell wall, if present.First, the cells are suspendedin
areaa s p p e alri g h t - t h er e v e r soef m i c r o g r a p o h fss i m p l em e t a l -
a solution of appropriate pH and salt content, usually iso-
stained p r e p a r a t i o n
i n sw h i c ht h e a r e a o
s f h e a v i e smt e t a sl t a i n i n g
tonic sucrose(0.25 M) or a combination of salts similar in
a p p e atrh e d a r k e s (t b )A p l a t i n u m - s h a d o wr eepdl i coaf t h e
s u b s t r u c t u fr iabl e r so f c a l f s k icno l l a g e nt h, e m a j o rs t r u c t u r a l
p r o t e i no f t e n d o n sb, o n e a , n ds i m i l atri s s u e sT h ef i b e r sa r ea b o u t
200 nm thick; a characteristic 64-nmrepeated pattern(white
p a r a l l el iln e si)sv i s i b l a e l o n gt h e l e n g t ho f e a c hf i b e r[.c o u r t e os fy
K BTUNS
I

NF C E L LO R G A N E L L E S
P U R I F I C A T I OO 391
 through a very narrow spacebetween a plunger and the ves-
sel wall; the pressureof being forced betweenthe wall of the
vesseland the plunger ruptures the cell.
Recall that water flows into cellswhen they are placed in
a hypotonic solution, that is, one with a lower concenrration
of ions and small moleculesthan found inside the cell. This
Disrupting the cell produces a mix of suspendedcellular
components, the homogenate, from which the desired or-
ganellescan be retrieved.Becauserat liver containsan abun-
dnnce of a single cell type, this tissuehas been used in many
classicstudiesof cell organelles.However,the sameisolation
principles apply to virtually all cells and tissues,and modifi-
cations of these cell-fractionation techniquescan be used to
separateand purify any desiredcomponents.
CentrifugationCan SeparateMany Types
of Organelles
In Chapter 3, we consideredthe principles of centrifugation
and the usesof centrifugation techniquesfor separatingpro-
teins and nucleic acids. Similar approachesare used foriep-
arating and purifying the various organelles,which differ in
both sizeand density and thus undergo sedimentationat dif-
ferentrates.
Most cell-fractionation procedures begin with differ-
ential centrifugation of a filtered cell homogenate ar in-
creasingly higher speeds(Figure 9-25). After centrifuga-
tion at each speedfor an appropriare time, the liquid that
remains at the top of the vessel,called the supernaranr,ls
poured off and centrifuged at higher speed.The pelleted
fractions obtained by differential centrifugation generally
contain a mixture of organelles,although nuclei and viral
particles can sometimes be purified completely by this
procedure.
An impure organelle fraction obtained by differential
centrifugation can be further purified by equilibrium
density-gradient centrifugation, which separates cellular
components according to their density.After the fraction is
resuspended,it is layered on top of a solution that contains
a gradient of a dense nonionic substance(e.g., sucrose or
glycerol). The tube is centrifuged at a high speed (about
40,000 rpm) for severalhours, allowing each particle to mi-
grate to an equilibrium position where the density of the sur-
rounding liquid is equal to the density of the particle (Figure
9-26). The different layers of liquid are then recovered by
pumping out the contents of the centrifuge tube through a
narrow piece of tubing.
Becauseeach organelle has unique morphological fea-
tures, the purity of organellepreparationscan be assessed by
examination in an electron microscope. Alternatively, or-
ganelle-specificmarker moleculescan be quantified. For ex-
ample, the protein cytochrome c is present only in mito-
chondria; so the presenceof this protein in a fraction of
lysosomeswould indicate irs conramination by mitochon-
dria. Similarly, catalaseis present only in peroxisomes;acid
phosphatase,only in lysosomes;and ribosomes, only in the
rough endoplasmicreticulum or the cyrosol.

> EXPERIMENTAL FTGURE 9-25 Differentiat

centrifugationis a commonfirst step in
fractionatinga cell homogenate.The
h o m o g e n a rt e s u l t i nfgr o md i s r u p t i ncge l l si s
usually filteredto removeunbroken cellsandthen
centrifuged at a fairlylowspeedto selectively
pelletthe nucleus-thelargest organelle The
undeposited materiai (thesupernatant) isnext
centrifuged at a higherspeedto sediment the Centrifuge
m i t o c h o n d r icah, l o r o p l a sltyss, o s o m easn, d
peroxisomes. Subsequent centrifugation in the
u l t r a c e n t r i f uagte1 0 0 , 0 0 0f9o r 6 0 m i n u t e s
resultsin deposition of theplasma membrane,
fragments of theendoplasmic reticulum, and
largepolyribosomes Therecovery of ribosomal
subunits, smallpolyribosomes, andparticles such
a sc o m p l e x eosf e n z y m erse q u i r easd d i t i o n a l
c e n t r i f u g a t iao tns t i l lh i g h e sr p e e d sO. n l yt h e
c y t o s o l - t h es o l u b l a e q u e o upsa r to f t h e
c y t o p l a s m - r e m a i n st h es u p e r n a t aanftt e r
centrifugation at 300,0009 for 2 hours

peroxisomes (fragmentsof somes

endoplasmic
reticulum),
and large
polyribosomes

392 c H A p T E R9 | vtsuAltztNG, FRACT|ONAT|NG

A ,N D C U L T U R T NC
GE L L S
 < EXPERIMENTAL FIGURE 9-26 A mixed organellefraction
can be further separated by equilibriumdensity-gradient
centrifugation.In thisexample, utilizingrat liver,materialin
( s e eF i g u r 9
o tn1 5 , 0 0 0 9
t h e p e l l eftr o mc e n t r i f u g a t i a e - 2 5 )i s
resuspended and layered on a gradient of increasingly more
] organelle
densesucrose solutions in a centrifuge tube. During centrifugation
J fraction
for several hours,eachorganelle migrates to itsappropriate
Lysosomes e q u i l i b r i udme n s i tay n dr e m a i ntsh e r eT oo b t a i na g o o ds e p a r a t t o n
2; ( 1 . 1 2g / c m ' )
of lysosomes from mitochondria, the liveris perfused with a
solutioncontaining a smallamount of detergent before the tissue
Mitochondria
( 1 . 1 8g / c m 3 ) is disrupted. During this perfusion period, detergent istaken intothe
cellsbyendocytosis andtransferred to the lysosomes, maktngthem
: L

lessdensethantheywouldnormally be andpermitting a "clean"

Peroxisomesf
( t . 2 3g / c m 3 )\ separatron of lysosomes frommitochondria.

Before After rification of vesicles whose outer surface is covered with

centrifugation centrifugation

Organelle-Specific AntibodiesAre Useful

i n P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s
Cell fractions remaining after differential and equilibrium
density-gradientcentrifugation usually contain more than
one type of organelle.Monoclonal antibodiesfor various
organelle-specificmembrane proteins are a powerful tool
for further purifying such fractions. One example is the pu-
isolated by low-speed centrifugation (Figure 9-27)' A te-
lated technique uses tiny metallic beads coated with spe-
cific antibodies. Organellesthat bind to the antibodies, and
are thus linked to the metallic beads, are recovered from

(a) (b)
Coated
vesicles
Clathrin Bacterialcell
Antibodv to clathrin

ProteinA

Coatedvesicle

0.1pum
, ,

bindsto the Fcconstantregionof antibodies. (a)Interaction of

EXPERIMENTAL FIGURE 9-27 Smallcoatedvesiclescan be
proteinA with antibodies boundto clathrin-coated vesicleslinks
purified by binding of antibody specificfor a vesiclesurface
a suspension thevesicles cells.Thevesicle-bacteria
to the bacterial complexes
proteinand linkageto bacterialcells.Inthisexample,
canthenbe recovered by low-speed ugation
centrif (b)A thin-section
of membranes from rat is
liver with
incubated an antibodyspecific
electronmicrograph revealsclathrin-coatedvesiclesbound to an
a proteinthatcoatsthe outersurface
for clathrin. of certaincytosolic
of Staphylococcus S aureus cell [SeeE Merisko I CellBiol93:846Micrograph
etal, 1982,
Tothismrxtureis addeda suspension
vesicles.
aureusbacteria whosesurface membrane contains proteinA, which c o u r t e s yo f G P a l a d el

. 393
N F C E L LO R G A N E L L E S
P U R I F I C A T I OO
 the preparation by adhesion to a small magnet on the side
of the test tube.
tain a particular glucosetransporter(GLLT4) that is localized
to the membraneof one of theserypesof vesicle.\Wheninsulin
is added to the cells, these vesiclesfuse with the cell-surface
membrane and increasethe number of glucoserransporters
able to take up glucose from the blood. As we will see in
Chapter 15, this processis critical to maintaining the appro-
priate concentration of sugar in the blood. The GLUIa-
Purification of Cell Organelles
r Disruption of cells by vigorous homogenization, sonica-
tion,.or other techniquesreleasestheir organelles.Swelling
of cells in a hypotonic solution weakens the plasma mem-
brane,making it easierro ruprure.
r Sequentialdifferential centrifugation of a cell homogenate
yields fractions of partly purified organellesthat diifer in
mass and density (seeFigure 9-25).
r Equilibrium density-gradientcentrifugation, which sepa_
rates cellular components according to their densities,ian
further purify cell fractions obtained by differential cen-
trifugation (seeFigure9-26).
r Immunological techniques using antibodies against or-
ganelle-specificmembrane proteins are particulaily useful
in purifying organellesand vesiclesof similar sizesand den_
sities (seeFigure 9-27).
![ lsolation,Culture,
and Differentiationof MetazoanCells
Most animal and plant rissuescontain multiple types of cells,
but biochemical and molecular investigationi b.rt u..o-_
plished on homogenouscell populationi. In the"r.
first part of this
section we describe a powerful instrument, the flulrescence_
394 . C H A P T E R g, V I S U A L I Z I N GF, R A C T I O N A T I N G
A ,N D C U L T U R I N G
animal in the laboratory under conditions that permit their
survival and growth for at least severaldivisions.
Certain types of primary cells, especially those from em-
bryos, can undergo differentiation in culture. As an example,
we will describehow muscle cell precursors grown in culture
can differentiate and form apparently normal musclecells,pro-
viding a good systemfor studying this developmentalprocess.
Although many types of primary cells undergo only a limited
number of divisions in culture, some accumulate cancer-
causing (oncogenic)mutations that allow them to be cultured
indefinitely. In many casesa single cell can be readily grown
into a colony of identical cells, a process called cell cloning.
Becausethesecells are genetically homogeneous,they are pa:.
ticularly suitable for many types of biochemical and genetic
studies. Certain cloned cells can undergo differentiation into
specificcellstypes such as adipocytes(fat-storingcells),nerve,
or muscle, allowing studieson the mechanism of cell differen-
tiation to be conducted on homogenous cell populations.
Many times in this chapter we have shown how monoclonal
antibodies facilitate cell biological experiments; at the end of
this section we describehow special cultured cells are used to
generatetheseantibodres.
Flow CytometrySeparatesDifferent CellTypes
Some cell types differ sufficiently in density that they can be
separatedon the basisofthis physical property. Nfhite blood
cells (leukocytes)and red blood cells (erythrocytes),for in-
stance, have very different densities becauseerythrocvtes
have no nucleus;thus thesecellscan be separatedfy .quitiU-
rium density centrifugation (describedabove). Becausemost
cell types cannot be differentiated so easilS other techniques
such as flow cytometry must be used to separatethem.
A flow cytometer identifies different celli by measuringthe
light that they scatterand the fluorescencethat they emit as-they
flow through a laser beam; thus it can quantify the numbers of
cellsof a particular type from a mixture. Indeed,a fluorescence-
activated cell sorter (FACS),which is basedon flow cytometry
can selectone or a few cells from thousands of other cells and
sort them into a separateculture dish (Figure 9-28). For exam-
ple, if an antibody specificto a certain cell-surfacemolecule is
linked to a fluorescent dye, any cell bearing this molecule will
bind the antibody and will then be separaredfrom other cells
when it fluorescesin the FACS. Having been sorted from other
cells,the selectedcellscan be grown in culture.
The FACS procedure is commonly usedto purify the dif-
ferent types of white blood cells, each of which bears on its
surface one or more distinctive proteins and will thus bind
monoclonal antibodies specific for that protein. Only the
T cells of the immune system,for instance,have both CD3 and
Thyl .2 proteins on their surfaces.The presenceof thesesur-
face proteins allows T cells to be separatedeasily from other
CELLS
 C e l ls u s p e n s i o n < EXPERIMENTAL FIGURE 9-28 Fluorescence-
activatedcell sorter (FACS)separatescells
that are labeleddifferentiallywith a
fluorescentreagent.StepE: A concentrated
suspension of labeled cellsis mixedwith a buffer
(thesheathf luid)sothatthe cellspasssingle-file
t h r o u g ha l a s e lri g h tb e a m S . t e pA : B o t ht h e
fluorescent lightemittedandthe lightscattered
by eachcellaremeasured; frommeasurements
of the scattered light,the sizeandshapeof the
cellcanbe determined StepS: Thesuspension
isthenforcedthrougha nozzle, whichformstiny
*E droplets containing at most a single cell At the
timeof formation, each droplet isgiven a negative
e l e c t r icch a r g ep r o p o r t i o ntaol t h e a m o u not f
fluorescence of itscell.Step4: Droplets wrthno
chargeandthosewith different electric charges
areseparated by an electric f ieldandcollected
Because i t t a k e o
s n l ym i l l i s e c o ntdoss o r te a c h
d r o p l e ta,sm a n ya s 1 0 m i l l i o n c e l l sp e rh o u rc a n
passthroughthe machinelAdapted fromD R Parks
andL A Herzenberg, 1982, Meth Cell Biol26:2831

F l u o r e s c e ncte l l d r o p l e t s S o r t e dc h a r g e d
,
d r o P l e t sc o n t a i n i n g
'fluorescent cells
Nonfluorescentcell droplet :

typesof blood cellsor spleencells(Figure9-29).Smallmagnetic
beadsmay be usedin a variation of this processthat doesnot
involve flow cytometry. The beadsare coated with a mono-
clonal antibody specificfor a surfaceprotein such as CD3 or
Thyl.2. Only cells with theseproteins will stick to the beads
and can be recoveredfrom the preparation by adhesionto a
small magneton the side of the test tube.
Other uses of flow cytometry include the measurement
of a cell's DNA and RNA content and the determination of
its generalshapeand size.The FACS can make simultaneous
measurements of the sizeof a cell (from the amount of scat-
104
tered light) and the amount of DNA that it contains (from
the amlunt of fluorescenceemitted from a DNA-binding
dye). Measurementsof the DNA content of individual cells
.rs.d to follow replication of DNA as the cells progress
"ie
through the cell cycle (Chapter 20).
C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h
M e d i a a n d S p e c i aS l o l i dS u r f a c e s
In contrast with most bacterial cells, which can be cultured
quite easily,animal cells require many specializednutrients
and often speciallycoated dishesfor successfulculturing' To
permit the iurvival and normal function of cultured tissues
Lr cells, the temperature, pH, ionic strength, and accessto

< EXPERIMENTAL FIGURE 9-29 T cellsboundto fluorescence-

A
II
to3 tagged antibodiesto two cell-surfaceproteinsare separated
0)
trom ottrerwhite blood cellsby FACS.Spleencellsfroma mouse
weretreatedwith a redfluorescent monoclonal antibodyspecific for
ON
theCD3 cell-surfaceproteinand with a green fluorescent monoclonal
J?J
p _e10' antibody for a second
specific cel|-surfaceprotein, Ihyl '2. Asthece||s
OF
J *ere passed througha FACS machine, the intensityof thegreenand
c redfluorescence emittedby each cellwas recorded Eachdot
0) (vertical
a) representsa singlecell.Thisplotof the green fluorescence
(9 10' N o n - Tc e l l s (horizontal axis)for thousands of spleen
axis)versusredfluorescence
cellsshowsthatabouthalfof them-the T cells-express bothCD3
andThyl.2proteins on theirsurfaces (upper-right quadrant) The
,.,,,11,'rr remaining which
cells, exhibitlow fluorescence (lower-leftquadrant)'
100 andareothertypes
100 101 102 103 104 onlybackground of theseproteins
levels
"*pr"r,
of whitebloodcells.Notethe logarithmic scaleon bothaxes. lcourtesy
cD3
Redfluorescence---> g h a n g ,W h i t e h e a dI n s t i t u t eI
o { C h e n g c h e nZ

I S O L A T I O NC. U L T U R EA, N D D I F F E R E N T I A T I OONF M E T A Z O A N

CELLS 395
 essentialnutrients must simulate as closely as possible the
conditions within an intact organism. Isolated animal cells
are typically placed in a nurrient-rich liquid, called the cul_
ture medium, within speciallytreated plastic dishesor flasks.
The cultures are kept in incubators in which the tempera_
ture, atmosphere,and humidity can be controlled. To reduce
ganismsand other airborne contaminants.
fa91o1sinclude the polypeptide hormone insulin; rransferrin,
which supplies iron in a bioaccessibleform; and numerous
growth factors. In addition, certain cell types requlre spe_
cializedprotein growth factors not present in serum. For in_
can be maintained or grown in suspensionas singlecells.
PrimaryCell CulturesCan Be Used
to Study Cell Differentiation
Normal animal tissues(e.g.,skin, kidneg liver) or whole em_
bryos are commonly used to establishprimary cell cwbures.
To prepare tissuecellsfor a primary .ulrr.., rhe cell_celland
cell-matrix interactions must be broken. To do so, tissue
396 C H A P T E R9 | vtSuALtZtNGF
, RACT|ONAT|NG
A,N D c u L T U R t N GC E L L S
when calcium is removed; other cell-adhesionmoleculesthat
are not calcium dependent need to be proteolyzed for the
cells to separate.The releasedcells are then placed in dishes
in a nutrient-rich, serum-supplementedmedium, where they
can adhere to the surface and one another. The same pro-
tease/chelatorsolution is usedto remove adherentcells from
a culture dish for biochemicalstudiesor subculturing (trans-
fer to another dish).
Fibroblasts are the predominant cells in connectivetissue
a_ndnormally produce ECM components such as collagen
that bind to cell-adhesionmolecules,thereby anchoring cells
to a surface. In culture, fibroblasts usually divide more
rapidly than other cells in a tissue,eventually becoming the
predominant cell type in a primary culture unless special
precautions are taken to remove them when isolatinq other
typesof cells.
sion, the cells induce synthesisof dozens of muscle,specific
proteins necessaryfor further muscle development furr.-
tion. Similar mononucleated cells, termed ,itrllit, "nJ
cells, are
found in adult muscle and can fuse to form multinucleated
myotubes and simultaneously induce synthesis of muscle-
ling muscle development.
Certain cells from blood, spleen,or bone marrow adhere
poorly, if at all, to a culture dish but nonethelessgrow well
in culture. In the bodg such nonadherent cells are held in
P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v e
a F i n i t eL i f e S p a n
ril/hen cells removed
from an embryo or an adult animal
are cultured, most of the adherentones will divide a finite
number of times and then ceasegrowing (cell senescence).
For instance,human fetal fibroblastsdivide about 50 times
 Undifferentiatedcells Differentiatedcells

Undifferentiatedcells Differentiatedcells

syncytiaduringmuscledifferentiation (c,d)Stainingwith a red-

A EXPERIMENTAL FIGURE 9-30 Differentiationin cultureof
specif
antibody
f iuoiescing ic for myosin heavy chainrevealsthatthis
primarymousesatellite (myoblast) cellsinto musclecells.
protein
muscle-specific is induced during Staining
differentiation
Primary mousesatellite cellswerecultured undernondifferentiating
with Hoechst dye(blue)detects nucleiBar: 100pm [Courtesy
o n d i t i o n( sa , c o) r d i f f e r e n t i a t icoonn d i t i o n( sb , d )
p r o l i f e r a t i oc n
( a , b )P h a s e - c o n t riamsat g es h o wf o r m a t i o on f m u l t i n u c l e a t e d CharlesEmersonandJenniferChen,BostonBiomedicaIResearc

pended animation and stored for extended periods at liquid

before they ceasegrowth (Figure 9-3lal. Starting with 106
nltrogen temperature' provided that a preservativethat pre-
cells, 50 doublings can produce 106 x 2)u, or more than
vents the foimation of damagittg ice crystals is used' Al-
1020cells, which is equivalent to the weight of about 1000
people.Normally, only a very small fraction of thesecellsare though some cells do not survive thawing, many do survive
and resumegrowth.
used in any one experiment.Thus, eventhough its lifetime is
limited, a singleculture, if carefully maintained, can be stud-
ied through many generations.Such a lineage of cells origi- TransformedCellsCan Grow Indefinitely
nating from one initial primary culture is called a cell strain.
in Culture
Researchwith cell strains is simplified by the ability to
freeze and successfullythaw them at a latet time for experi- To be able to clone individual cells, modify cell behavior,or
mental analysis.Cell strains can be frozen in a state of sus- select mutants, biologists often want to maintain cell

CELLS 397
I S O L A T I O NC, U L T U R EA, N D D I F F E R E N T I A T I OONF M E T A Z O A N
 (a) Human cells
arisesspontaneouslyand takes over, or overgrows, the cul-
Phase
ture. A cell line derived from such a transformed variant will
grow indefinitely if provided with the necessarynutrienrs.
Regardlessof the source, cells in immortalized lines often
Growth
have chromosomes with abnormal DNA sequences.In addi-
rate of tion, the number of chromosomesin suchcellsis usually greater
culture than that in the normal cell from which they arose. and the
chromosomenumber expandsand contractsas the cells con-
tinue to divide in culture. A noteworrhy exception is the Chi-
nesehamster ovary (CHO) line and its derivatives,which have
25 50 fewer chromosomesthan their hamster progenitors. Cells with
Cellgenerations an abnormal number of chromosomesare said tobe aneuploid.

SomeCell LinesUndergoDifferentiation
Initialloss Emergenceof in Culture
,z of growlh immortal
variant Most cell lines have lost some or many of the functions char-
Growth acteristicof the differentiatedcells from which they were de-
rate of rived. Such relatively undifferentiated cells are poor models
culture for investigating the normal functions of specific cell types.
Better in this regard are severalmore-differentiatedcell'lines
that exhibit many properties of normal nontransformed
cells. Theselines include the human liver tumor (hepatoma)
HepG2 line, which synthesizesmost of the serum proteins
l.V, #", initiation
ot made by normal liver cells (hepatocytes)and has been em_
"rft3?" ployed in studiesidentifying transcription factors that regu-
FIGURE 9-31 Stagesin the establishment of a cellculture.
(a)Whencellsisolated fromhumantissue late synthesisof liver proteins.
areinitiallv cultured.some
cellsdieandothers(mainly fibroblasts)startto grow;overall, More useful for cell and developmentalbiologistsare cell
the
growthrateincreases (phasel) lf the remaining cellsareharvested, lines that grow without acquiring characteristicsof a differ-
diluted,andreplated intodishes againandagain,thecellstrain entiated cell, yet can undergo differentiation into a particu-
continues to divideat a constant ratefor about50 cellgenerations lar cell type when placed in a different culrure medium.
(phase ll),afterwhichthegrowthratefallsrapidly. Intheensuing Becauselarge numbers of such cells can be induced to un-
period(phase lll),allthecellsin theculturestopgrowing(senescence) dergo synchronized differentiation, they are often used in
(b)Ina culture prepared frommouseor otherrodentcelrs, initiar biochemicaland molecularsrudies.
cerl
death(notshown)jscoupled withtheemergence of healthy growing One example is a line of transformed mouse myoblasts
cells.Asthesedividing cellsaredilutedandallowedto conttnue termed C2C1,2 cells. Derived from adult mouse muscle,
growth,theysoonbeginto losegrowthpotential, andmoststop these_cellsdivide rapidly and induce none of the principai
growing(i e , theculturegoesintosenescence) Veryrarecels muscle-specificproteins when grown in media rich in
undergooncogenic mutations thatallowthemto survlve ano
continue growth factors. When the culture is placedin a medium with
dividing untiltheirprogeny overgrow theculture, These cells
constitute a cellline,whichwillgrowindefinitely if it isappropriately
dilutedandfedwithnutrients. Suchcellsaresaidtobe immortal
cultures for many more rhan 100 doublings. Suchprolonged
growth is exhibited by cells derived from some tumors. In
addition, rare cells in a population of primary cells may un_
dergo spontaneous oncogenic mutations, leading to onco_ tion, these cells have been particularly valuable in uncover-
genic transformation (Chapter 25). Such cells,iaid to be
ing the roles of many rranscription factors in muscle devel-
oncogenically transformed or simply transformed, are able opment (Chapter 22).
to grow indefinitely. A culture of cells with an indefinite life Similarl5 the murine 3T3-L1 preadipocytecell line grows
span is consideredimmortal and is called a cell line. with fibroblastJike morphology in culture. Vhen switchedto
The HeLa cell line, the first human cell line, was origi_ a medium containing insulin and dexamethasone(a glucocor_
nally obtained in 1952 from a malignant tumor (carcinoma
) ticoid steroid hormone), 3T3-L1 cells undergo synchronized
of the uterine cervix. Although primary cell cultures of nor_ differentiation into adipocytes, as shown both by accumula-
mal human cells rarely undergo transformation into a cell
tion of intracellular lipids (Figure 9-33a-d) and induction of
line, rodent cells commonly do. After rodent cells are grown
adipocyte-specificmRNAs (Figure 9-33e). Becausepnmary
in culture for severalgenerations,the culture goesinto senes_
adipocytesdo nor divide in culture, thesecell lines ari widely
cence (Figure 9-31b). During rhis period, mlst of the cells used in biochemical,molecular,and cell bioloeical studieson
stop growing, but often a rapidly dividing transformed cell the developmenrand function of adiposecells.

398 o c H A p r E R9 v t s u A l t z t N c ,F R A C ' . N A T ' N GA, N D c u L T U R , N cGE L L s

|
(c)
10000

c)
0) 100

< g E
ze
E
Zc.
Eq 1oo
-O

A Glut4 y>
;> PX
PX O Glutl o
q)
A Myogenin
10
O HKI1
O MyoD N G3PDH
10
I Myf5
O o-Actin
n B-nctin
'012345678
I

Days Days
(majorcomponent filaments),
of contractile andthe GLUT4 glucose
A EXPERIMENTAL FIGURE 9-32 Differentiationin cultureof
transporter (found only in muscle and fat increased
cells) 5 to
C2C12cells,a line of transformedmousemyoblasts'(a)C2C12
50 fold.In contrast, mRNAs encodrng proteins specificfor growing
cellsproliferate
but do not differentiatein a mediumcontaining fetal
cells,suchasthe GLUT1 glucose transporter and B-actin, were
calfserum,whichcontains highconcentrations of mitogenic growth
downregulated OthermRNAs, such as the glycolytic
enzyme
factors(b)Afterplacing C2C12cellsin a mediumwith horseserum,
glyceraldehyde 3 phosphate dehydrogenase (G3PDH), were
whichcontains lower concentrations of suchgrowthfactors, manyof
unaffected. IndividualmRNAs were measured by using reverse
thecellsfusedto formmultinucleate syncytia thatexpress the
with a green- transcriptase to copytotalcellular mRNAinto DNA,followedby
muscle-specificmyosinheavychain,detected
quantitative polymerase chainreaction (PCR) amplificationof specific
fluorescingantibodyspecificfor thisproteinStaining of DNAwith
(c) cDNAs. Theresults were normalized to the amount present in
blueDAPIdyereveals the nuclei Bardenotes 20 pm. During
growingcellslParts (a)and(b)courtesyJames EvansandPrakash Rao'
of cultured
differentiation C2C'12 cellsthe levelsof certainmRNAs
Whitehead InstitutePart(c)adaptedfromT.Shimokawa et al, 1998'
rncreasemarkedly, asshownin thesegraphsForinstance, the
mRNAs for myogenin (a muscle-specifictranscription o-actin
factor), Biochem BioPhYs ResComm246:287 I

As detailed in Chapter 19, many cell types function only
when closelylinked to other cells.Key examplesare the sheet-
like layers of epithelial tissue, called epithelia (sing.,
epithelium),which cover the external and internal surfacesof
organs.Typically,the distinct surfacesof a polarizedepithelial
cell are called the apical (top), basal (base or bottom), and
lateral (side) surfaces(seeFigure 19-8). The basal surface
usually contactsan underlying extracellularmatrix called the
basal iamina, whose composition and function are discussed
in Section 19.3. Severaltypes of cell junctions interconnect
adjacentepithelialcells and anchor them to the basal lamina'
Cultured iells called Madin-Darby canine kidney (MDCK)
cells arc often used to study the formation and function of
epithelial cells. \flhen grown in specializedcontainers,these

. 399
I S O L A T I O NC, U L T U R EA, N D D I F F E R E N T I A T I OONF M E T A Z O A NC E L L S
 Undifferentiatedcells Differentiatedcells < E X P E R I M E N TFA IG L U R9E- 3 3 A
line of 3T311preadipocytes can
differentiateinto adipocytesand
expressadipocyte-specific mRNAsin
culture.Fibroblast-like 3T3-11
preadtpocytes growingin standard
serum-containing proliferation medium
(a,c)wereswitched to a differentiation
m e d i u mc,o n t a i n i ni n g s u l i nt h, es t e r o i d
h o r m o ndee x a m e t h a s oanned,
i s o b u t y l m e t h y l x a n ta hni ni n
eh, ibitor
o f c A M Pp h o s p h o d i e s t e r faosre ,
B d a y s( b , d )(.a , b )D i f f e r e n t r a r
i n t e r f e r e nc o e n t r a s( tD l C m ) icroscopy
r e v e a lt sh e c o n s i d e r a b l e
m o r p h o l o g i ccahla n g eisn t h ec e l l s
d u r i n gd i f f e r e n t i a t i o( cn, d )S t a i n i n g
with Oil RedO reveals droplets of
triglycerides (red)in differentiated but
not undifferentiated cells,(e)Northern
blotanalysis demonstrates expression of
two keyadipocyte genes, encoding the
transcription factorPPARI andthe
insulin-responsive glucose transporter
GLUT4 in thedifferentiated 3T3-11
cells(ngrht /ane)but not in the
PPARl
undifferentiated 3T3-11preadipocytes
(leftlane)B-Actinwasuseoasa conrrol
to showloading of equalamounts of
RNAin thetwo gellanes[part a-d
GLUT4
c o u r t e s yJ a m e sE v a n sa n d H u a n g m i n gX i e ,
WhiteheadInstitute Part(e)from N L Harvey
e t a l , 2 0 0 5 , N a t u r eG e n 3 7 : 1 0 7 2 1
B-Actin

ties of cells in complex rissuesand organismsonly on the

basisof experimentswith isolated, cultured cells.

A major disadvantageof cultured cells is that they are
not in their normal environment and hence their activities
are not regulatedby the other cellsand rissuesas rhey would
be in an intact organism.For example,insulin producedby
the pancreashas an enormous effect on liver glucosemetab_
olism; however, this normal regulatory mechanismdoes not
operatein a purified population of liver cells(calledhepato_
cytes)grown in culture.In addition, as alreadydescribed,the
three-dimensionaldistribution of cells and extracellular
matrix around a cell influences its shape and behavior.
Becausethe immediateenvironmentof coitured cellsdiffers
radically from this "normal', environment,their properties
may be affected in various ways. Thus care must always be
exercisedin drawing conclusionsabout the normal proper_
H y b r i dC e l l sC a l l e dH y b r i d o m a sp r o d u c e
A b u n d a n tM o n o c l o n aA l ntibodies
In addition to serving as researchmodels for studies on cell
function, cultured cells can be convertedinto ,,factories" for
producing specificproreins. In Chapter 5, we describedhow
this is done by introducing genesencoding insulin, growth
factors, and other therapeutically useful proteins into bacte-
rial or eukaryoticcells(seeFigures5-31 and 5-32). Here we
consider the use of special cultured cells to generatemono-
clonal antibodies,which we have seenare experimentaltools
widely used in many aspectsof cell biological research.In-

400 CHAPTER
9 | v t S u A L t Z t N GF, R A C T | O N A T | NAGN, D C U L T U R T NCGE L L S
 Apical surface
C u l t u r ed i s h
I producing viable hybrid cells wtth a single nucleusthat con-
Apicam
l edium
Basal Porous
lamina filter
A EXPERIMENTAL FIGURE 9-34 Madin-Darbycaninekidney
(MDCK)cellsgrown in specialized containersprovidea useful
experimentalsystemfor studyingepithelialcells.MDCKcellsform
a polarized
epithelium whengrownon a porous membrane filtercoated
on onesidewithcollagen andothercomponents of thebasallamina
Withtheuseof thespecial culturedishshownhere,themedium on
eachsideof thefilter(apical
andbasal sidesof themonolayer) canbe
manipulated
experimentally andthemovement across
of molecules the
layermonitoredSeveral celljunctions
thatinterconnect thecellsform
onlyif thegrowthmedium contains Ca2'
sufficient
normal antibody-producingB lymphocyte in a mammal is
capableof producinga singletype of antibody that can bind
to a specific chemical structure (called a determinant or
epitope) on an antigenmolecule.If an animal is injectedwith
an antigen,many B lymphocytesthat make antibodiesrecog-
nizing that antigen are stimulated to grow and secretethe
antibodies.Each antigen-activatedB lymphocyte forms a
clone of cells in the spleenor lymph nodes,with each cell of
the clone producing the identical antibody-that is, a mono-
clonal antibody.Becausemost natural antigenscontain mul-
tiple epitopes,exposureof an animal to an antigen usually
stimulates the formation of multiple different B-lymphocyte
clones, each producing a different antibody. The resulting
mixture of antibodiesthat recognizedifferent epitopeson the
same antigen is said to be polyclonal. Stch polyclonal anti-
bodiescirculatein the blood and can be isolatedas a group.
Although polyclonal antibodiesare useful for a variety of
experiments,monoclonal antibodiesare required for many
types of experimentsand medical applications. Unfortu-
nately,the biochemicalpurification of any one type of mon-
oclonal antibody from blood is not feasiblefor two main
reasons:the concentrationof any given antibody is quite
low, and all antibodieshave the samebasicmoleculararchi-
tecture(seeFigure3-19).
Becauseof their limited life span, primary culturesof nor-
mal B lymphocytesare of limited usefulnessfor the produc-
tion of monoclonal antibodies.Thus the first step in produc-
ing a monoclonal antibody is to generate immortal,
antibody-producingcells.This immortality is achievedby fus-
ing normal B lymphocytesfrom an immunized animal with
transformed, immortal lymphocytes calledmyeloma cells that
themselvessynthesizeneither the heavy (H) nor the light (L)
polypeptidesthat constituteall antibodies(seeFigure 3-19).
During cell fusion, the plasmamembranesof two cellsfuseto-
gether,allowing their cytosols and organellesto intermingle.
Treatment with certain viral glycoproteins or the chemical
I S O L A T I O NC, U L T U R EA, N D D I F F E R E N T I A T I OONF M E T A Z O A NC E L L S
polyethyleneglycol promotes cell fusion. Some of the fused
cells undergo division, and their nuclei eventually coalesce,
iains chromosomesfrom both "parents." The fusion of two
cells that are geneticallydifferent can yield a hybrid cell with
novel characteristics.For instance,the fusion of a myeloma
cell with a normal antibody-producing cell from a rat or
mouse spleen yields a hybrid that proliferates into a clone
calleda hybridoma. Like myeloma cells,hybridoma cellsgrow
rapidly and are immortal. Each hybridoma producesthe mon-
oclonal antibody encodedby its BJymphocyteparent.
The second step in this procedure for producing mono-
clonal antibody is to separate'or select,the hybridoma cells
from the unfused parental cells and the self-fusedcells gen-
erated by the fusion reaction. This selection is usually per-
formed by incubating the mixture of cells in a specialculture
medium, calledselectionmedium, that permits the growth of
only the hybridoma cells becauseof their novel characteris-
tics. If the myeloma cellsusedfor the fusion cany a mutation
that blocks a metabolic pathway, a selectionmedium can be
used that is lethal to them and not their lymphocyte fusion
Dartnersthat do not have the mutation. In the immortal hy-
trid cells,the functional genefrom the lymphocyte can sup-
ply the missing gene product, and thus the hybridoma cells
will be able to grow in the selection medium. Becausethe
lymphocytesused in the fusion are not immortalized and do
not divide rapidly, only the hybridoma cells will proliferate
rapidly in the selectionmedium and can thus be readily iso-
lated from the initial mixture of cells.
Figure 9-35 depicts the general procedure for generating
and selectinghybridomas.In this case' normal B lympho-
cytesin a sampleof spleencells are fused with myeloma cells
that cannot grow in HAT mediwm' a common selection
medium. Only the myeloma-lymphocyte hybrids can survive
and grow for an extended period in HAT medium for rea-
sons describedshortly' Thus, this selectionmedium permits
chromatographyto isolate and purify proteins from complex
mixtures (seeFigure 3-37c).They can also be usedto label and
thus iocatea particular protein in specificcellsof an organ and
within cultured cellswith the use of immunofluorescence mi-
croscopytechniques or in specificcell fractionswith the use of
immunoblotting (seeFigure 3-38). Monoclonal antibodies also
have become important diagnostic and therapeutic tools in
medicine. For example, monoclonal antibodiesthat bind to
and inactivatetoxins secretedby bacterialpathogensare used
to treat diseases.Other monoclonal antibodiesare specificfor
cell-surfaceproteins expressedby certain types of tumor cells'
Severalof theseanti-tumor antibodiesare widely usedin can-
cer therapy,including monoclonal antibody againsta mutant
form of the Her2 receptorthat is overexpressed in somebreast
cancers(seeChapter16, Figure16-18).
O 401
 Animation:preparingMonoclonalAntibodi", {lltl
Technique
Injectmouse < EXPERIMENTAL FIGURE 9-35 Useof cellfusionand
w i t h a n t i g e nX selectionto obtain hybridomasproducingmonoclonal
antibodyto a specificprotein.Step[: lmmortalmyeloma cells
thatlackHGPRT, an enzyme required for growthon HATmedium,
arefusedwith normalantibody-producing spleen cellsfroman
animalthatwasimmunized with antigenX. Thespleencellscan
makeHGPRT. Step[: Whenplatedon HATmedium, unfused and
self-fusedcellsdo not grow:the mutantmyeloma cellsbecause
theycannotmakepurines throughan HGPRT-dependent metabolic
"salvage" pathway(seeFigure 9-36),andthespleen cellsbecause
theyhavea limitedlifespanin cultureThusonlyfusedcellsformed
froma myeloma cellanda spleen cellsurvive
on HATmedium,
proliferating
intoclones calledhybridomas Eachhybridoma
M u t a n tm o u s e M o u s es p l e e nc e l l s ; produces a singleantibodyStepB: Testing of individualclones
m y e l o m ac e l l s s o m e c e l l s( r e d )m a k e identifies
thosethat recognize antigenX Aftera hybridoma that
u n a b l et o g r o w
i n H A Tm e d i u m
E antibodyto antigenX produces a desiredantibody hasbeenidentified, theclonecanbe
culturedto yieldlargeamounts of thatantibody.

o
tifolates becausethey block reactionsof tetrahydrofolate, an
active form of folic acid.
Many cells, however, are resistant to antifolates be-
causethey contain enzymesthat can synthesizethe neces-
U n f u s e dc e l l s sary nucleotides by a different route from purine bases
(OOO)die
and thymidine (Figure 9-36, bottom). Two key enzymesrn
Fusedcells these nucleotide saluage pathways are thymidine kinase
(O O ) grow
(TK) and hypoxanthine-guanine phosphoribosyl trans-
ferase (HGPRT). Cells that produce these enzymes can
El C u l t u r es i n g l ec e l l s
in separatewells grow on HAT medium, which supplies a salvageable
V purine and thymidine, whereas those lacking either of
tneseenzymescannot.
/C C Cells with a TK mutation that prevents the production
z----\
\) OO O of the functional TK enzymecan be isolated becausesuch
cells are resistant to the otherwise toxic thymidine analog
O O S-bromodeoxyuridine. Cells containing TK convert this
compound into 5-bromodeoxyuridine monophosphate,
Testeachwell for antibodyto antigenX which is then converted into a nucleoside triphosphate by
other enzymes.The triphosphate analog is incorporated by
DNA polymeraseinto DNA, where it exerts its toxic effects.
This pathway is blocked in TK- mutanrs, and thus they are
HATMedium ls CommonlyUsedto lsolate resistant to the toxic effects of S-bromodeoxyuridine. Simi-
larly, cells lacking the HGPRT enzyme,such as the HGpRT
H y b r i dC e l l s
myeloma cell lines used in producing hybridomas, can be
The principles underlying HAT selectionare important not isolated becausethey are resistant to the otherwise toxic
only for understandinghow hybridoma cells are isolated but guanine analog 6-thioguanrne.
also for understanding several other frequently used selec- Normal cells can grow in HAI medium becauseeven
tion methods, including selectionof the embryonic stem (ES) though the aminopterin in the medium blocks de novo synthe-
cells used in generatingknockout mice (seeFigure 5-40). sis of purines and TMP, the thymidine in the medium is trans-
HAT medium containsDypoxanthine(a purine), 4minopterin, ported into the cell and converted into TMp by TK and the
and rhymidine. Most animal cells can synthesizethe purine hypoxanthine is transported and converted into usableDurines
and pyrimidine nucleotidesfrom simpler carbon and nitro- by HGPRT. On the other hand, neither TK- nor HGPRT-
gen compounds (Figure 9-36, top). The folic acid antago_ ceLlscan grow in HAI medium becauseeach lacks an enzyme
nists amethopterin and aminopterin block thesebiochemiial of the salvagepathway. However, hybrids formed by the fusion
pathways; they interfere with the donation of methyl and of these two mutants will carry a normal TI( gene from the
formyl groups by tetrahydrofolic acid in the early stagesof HGPRT- parent and a normal HGPRT gene from the TK-
the_synthesisof glycine, purine nucleosidemonophosphates, parent. The hybrids will thus produce both functional salvage-
and thymidine monophosphate. These drugs are calledan_ pathway enzymesand will grow on HAT medium.

402 . c H A p r E R9 v t s u A l t z t N c ,F R A . ' . N A T ' N G ,A N D c u L T U R . N cGE L L s

|
 De novo
synthesisof
p u n n en u c l e o t i d e s
PRPP(5-Phosphoribosyl-1 -pyrophosphate)

I
Y lockedby antifolates De novo
De novo cHo from tetrahydrofolate synthesisof
pathways h '
Y TMP
Deoxyuridylate(dUMP)
\ I
locked bY antifolates
I
H cH"from
\ Y tetiahydrofolate
€ Thymidylate(TMP)

Guanine Hypoxanthine Adenine Thymidine

Salvagepathways

A FIGURE 9-36 De novo and salvagepathwaysfor nucleotide andamethopterin, blockthe reactivation of tetrahydrofolate,
synthesis.Animalcellscansynthesize purinenucleotides (AMP, preventing purineandthymidylate synthesis.Manyanimalcellscanalso
) n dt h y m i d y l a (t T
c M P ,I M P a e M Pf)r o ms i m p l ecro m p o u n dbsy d e usesalvage pathways (red)to incorporatepurinebasesor nucleosides
novopathways (blue)Several of thesereactions requirethe transfer andthymidine. lf theseprecursorsarepresent in the medium,normal
of a methylor formyl("CHO")groupfroman activated form of cellswill grow even in the presenceof antifolates cultured
However,
tetrahydrofolate (e g., tvs,N10-methylenetetrahydrofolate), asshown cellslackingoneof the enzymes-HGPRL APRLoTTK--rcfthe salvage
in the upperpartof the diagram. Antifolates, suchasaminopterin pathways will not survivein mediacontaining antifolates.

r The fusion of an immortal myeloma cell and a single B

lsolation, Culture, and Differentiation
of Metazoan Cells
r Flow cytometry can identify different cells on the basis
of the light that they scatter and the fluorescencethat they
cell sorter (FACS)is useful
emit. The fluorescence-activated
in separatingdifferent types of cells (seeFigures 9-28 and
9-29).
r Growth of vertebratecells in culture requires rich media
containing essentialamino acids, vitamins, fatty acids, and
peptide or protein growth factors; the last are frequently
provided by serum.
r Most cultured vertebrate cells will grow only when at-
tached to a negatively charged substratum that is coated
with components of the extracellular matrix.
r Primary cells, which are derived directly from animal
tissue, have limited growth potential in culture and may
give rise to a cell strain (seeFigure 9-31). Some primary
cells can undergo differentiation into a specific type of
cell.
r Transformed cells, which are derived from animal tu-
mors or arise spontaneouslyby transformation of primary
cells,grow indefinitely in culture, forming cell lines.
r Certain cell lines can be induced to undergo differentia-
tion in culture to generatemuscle, adipose, epithelial, and
other types of cells and are widely used in cell biological
studies(seeFigures9-30,9-32, and 9-33).
lymphocyte yields a hybrid cell that can proliferate indefi-
nitely, forming a clone called a hybridoma (seeFigure 9-35).
Becauseeach individual B lymphocyte produces antibodies
specificfor one antigenic determinant (epitope),a hybridoma
produces only the monoclonal antibody synthesizedby its
original B-lymphocyte parental cell.
r HAT medium is commonly used to isolate hybridoma
cells and other types of hybrid cells.
Light microscopy will continue to be a major tool in cell bi-
ologS providing images that relate to both the interactions
proteins and the movements' or mechanics,involved
"-o.g
various cell processes.The use of more fluorescentlabels
in
and tags will allow visualization of five or six different types
of molecules simultaneously. Vith more labeledproteins, the
complex interactions among proteins and organellesinside
cells will becomebetter understood.
Very recent advancesin light microscopy are opening entire
new areasfor investigation.As an example, the two-photon
fluorescencemicroscope enablesvisualization of fluorescent
proteins (e.g., GFP expressedfrom a reporter gene) in thick
iissuesamples.With this technologythe propertiesof individ-
ual cellscan assessed in tissuesinside living animals.
Although the limit of resolution of a light microscope
using visible light is about 0.2 pm (200 nm), new types of

I S O L A T I O NC, U L T U R EA, N D D I F F E R E N T I A T I OONF M E T A Z O A NC E L L S 403

 fluorescencemicroscopy,such as STED (stimulated emission differential centrifugation immunofluorescence
depletion),can resolvetwo fluorescentobiects as close to- 392 microscopy 385
gether as =20 nm. For example, we noted that synaptic vesi-
differential interference lumen 373
cles are too small (=40 nm in diameter) and too densely conrrast(DIC) Iysosome373
packed to be resolvedby availablefluorescencemicroscopes.
microscopy381
Howeveq STED can resolve these individual vesicles.This membrane transport
endoplasmicreticulum protern 372
technique will also enable investigatorsto detect single fluo-
(ER)325
rescent protein molecules in the membranes of purified metal shadowing 390
organelles.Another type of fluorescencemicroscopy under endosome373
mitochondrion 378
development,termed total internal reflection (TIR) fluores- equilibrium density-gradient
monoclonal antrbody 394
cencemicroscopy, enablesdetection of single fluorochrome- centiftgation 392
nucleolus378
tagged proteins on the surfaceof living cells. extracellular matrix
Improvements in cell culture technology will allow both (ECM)372 organelles371
primary cells and cultured lines to be studied in more natu- peroxisome324
fluorescence-activated cell
ral contexts, not on glass cover slips but in three-dimen- sorter (FACS)394 phase-contrast
sional gelsof extracellularmatrix molecules.This technique microscopy381
fluorescentstaining 3 82
will permit cultured cells such as liver and severalhormone- resolution 381
gap junctions383
producing cells to achieveand maintain their differentiated scanningelectron
state for days, enabling many types of experimentsto be per- Golgi complex 376
microscope(SEM)390
formed. Bioengineersalso are fabricating artificial tissues HAT medium 401
transmissionelectron
basedon a syntheticthree-dimensionalarchitecrurelncorDo- hybridoma 401
m i c r o s c o p e( T E M ) 3 8 8
rating layers of different cells. Eventually such artificial iis-
sueswill provide replacementsfor defective tissuesin sick,
injured, or aging individuals. Review the Concepts
In parallel, investigatorswill use advancedmicrofabri-
cation techniques to culture minute numbers of cells in l. One of the defining features of eukaryotic cells is the
microliter volumes on a glassslide consistingof microfab- presenceof organelles.I7hat are the major organellesof eu-
ricated wells and channels.With this technique, reagenrs karyotic cells, and what is the function of each?What is the
in nanoliter volumes can be introduced and exposedto se- cytosol? Sfhat cellular processesoccur within the cytosol?
lected parts of individual cells; rhe responsesof the cells
2. The internalization of proteins, soluble macromolecules
can then be detected by light microscopy and,analyzed by
and large particles is a fundamental processrequired by all
powerful image-processingsoftware. In these types of
cells.The sizeof the moleculeoften dictateshow ir getsinter-
studies cells can be screenedrapidly with millions of dif- nalized. Describehow phagocytosisdiffers from endocytosis
ferent chemicalcompounds, thus facilitarine the discovery
and what generallyhappensto the internalizedmaterial.
o f n e w d r u g s , d e r e c t i o no f s u b t l e p h e n o r y l e s o f m u r a n r
3. Cell organellessuch as mitochondria, chloroplasts, and
cells (e.g., tumor cells), and development of comprehen-
the Golgi apparatuseach have unique structures.How is the
sive models of cellularprocesses. Thus advancesin bioengi-
structure of each organellerelated to its function?
neering will make major contributions not only to our un-
derstandingof cell and tissuefunction but also to the qualitv 4. Much of what we know about cellular function deoends
o f h u m a nh e a l t h . on experimentsutilizing specificcells and specificprrrc 1..g.,
'$fhat
Finally the electron microscope will become the domi- organelles)of cells. techniquesdo scientistscommonly
nant insrrument for studying the structure of multiprotein use to isolate cells and organellesfrom complex mixtures,
machinesin vitro and in situ. Tomographic methods applied and how do thesetechniqueswork?
to single cells and molecules combined with automated re- 5. Both light and electron microscopy are commonly used
construction methods will generatemodels of protein-based to visualize cells, cell structures,and the location of specific
structures that cannot be determined by x-ray crystallogra- molecules. Explain why a scientist may choose one or the
phy. High resolution three-dimensionalmodels of molecules other microscopy technique for use in research.
in cells will help explain the intricate biochemical interac- 6. The magnification possiblewith any type of microscope
trons among proteins. is an important property, but its resolution, the ability to dis-
tinguish between two very closely apposed objects, is even
KeyTerms more critical. Describewhy the resolving power of a micro-
scopeis more important for seeingfiner details than its mag-
autophagy 374 chimericproteins3Zl nification. \fhat is the formula to describethe resolution of
a microscopelens and what are the limitations placed on the
bright-field light chloroplast 379
values in the formula?
mrcroscopy381 cryoelectron microscopy
389 7. Sfhy are chemical srains required for visualizing cells
cell line 398 culturing 372 and tissueswith the basic light microscope?What advantage
cell strain 397 cytosol372 do fluorescent dyes and fluorescencemicroscopy provide in

404 . cHAprER
9 V I S U A L I Z I N GF, R A C T I O N A T I N G
A ,N D C U L T U R I N G
CELLS
comparison to the chemical dyes used to stain specimensfor
light microscopy? What advantagesdo confocal scanning
microscopy and deconvolution microscopy provide in com-
parison to conventional fluorescencemicroscopy?
8. In certain electron microscopy methods, the specimenis
not directly imaged. How do these methods provide infor-
mation about cellular structure, and what types of structures
do they visualize?
9. Vhat is the difference between a cell strain, a cell line,
and a clone?
10. Explain why certain cell lines are used to study the dif-
ferentiation and function of muscle or fat cells.
1L. Explain why the process of cell fusion is necessaryto
produce monoclonal antibodies used for research.
Analyze the Data
Mouse liver cells were homogenizedand the homogenate
subjected to equilibrium density-gradient centrifugation
with sucrosegradients.Fractions obtained from thesegradi-
ents were assayedfor marker molecules(i.e., moleculesthat
are limited to specificorganelles).The results of theseassays
are shown in the figure. The marker moleculeshave the
following functions: Cytochrome oxidase is an enzyme
involved in the processby which ATP is formed in the com-
plete aerobic degradation of glucose or fatty acids;
ribosomal RNA forms part of the protein-synthesizing
ribosomes;catalase catalyzesdecomposition of hydrogen
peroxide; acid phosphatasehydrolyzes monophosphoric
estersat acid pH; cytidylyl transferaseis involved in phos-
pholipid biosynthesis;and amino acid permeaseaids in
transDort of amino acids acrossmembranes.
E
tr
=ou
E Egner,A., and S. Hell. 2005. Fluorescence
x super-iesolvedoptical sections.Trends Cell Biol- 15:207-215.
o
E Gaietta.G.. et al. 2002. Multicolor and electronmicroscopic
x9 + o
10 15 20
Fractionnumber
50% Sucrose0%
CurveA = cytochromeoxidase Curve D = acid phosphatase
C u r v e B = r i b o s o m aRl N A Curve E = cytidylyltransferase
Curve C = catalase C u r v eF = a m i n o a c i d p e r m e a s e
a. Name the marker molecule and give the number of
the fraction that is most emiched for each of the following
cell components: lysosomes;peroxisomes;mitochondria;
plasma membrane; rough endoplasmic reticulum; smooth
endoplasmicreticulum.
b. Is the rough endoplasmicreticulum more or less
densethan the smooth endoplasmicreticulum? lfhy?
c. Describean alternativeapproachby which you could
identify which fraction was enrichedfor which organelle.
d. How would addition of a detergent to the ho-
mogenate, which disrupts membranes by solubilizing their
lipid and protein components, affectthe equilibrium density-
gradientresults?
References
Organelles of the EukarYotic Cell
Bainton, D. 1981. The discoveryof lysosomes.J. Cell Biol'
9lz66s-76s.
Cuervo, A. M., and J. F. Dice. 1998. Lysosomes:a meetingpoint
of proteins,chaperones,and proteases.J. Mol. Med. 76:6-1'2.
de Duve, C. 1'996.The peroxisomein retrospect.Ann. NY
Acad. Sci.804:1-10.
Foster,L. J., et al. 2006. A mammalian organellemap by pro-
tein correlationprofiling. Cell t25 :187-1,99.
Holtzman, E. 1989. Lysosomes.Plenum Press.
Lamond, A., and \0. Earnshaw.1998. Structureand function in
the nucleus.Science2802547-553'
Mootha, V.K., et al. 2003. Integratedanalysisof protein compo-
sition, tissuediversity,and generegulationin mousemitochondria.
Cell tl5z629-640.
Palade,G. 197S.Intracellular aspectsof the processof protein
synthesis.Science1.89:347-358.The Nobel Prizelectureof a pioneer
in the study of cellular organelles'
'Wanders.
R.. and H. R. Waterham.2006. Biochemistryof mam-
malian peroxisomesrevisited.Ann' Reu.Biochem. 7 52295-332'
Light Microscopy: Visualizing Cell Structure and Localizing
Proteins Within Cells
Chen, X., M. Velliste,and R. F Murphy. 2006' Automated in-
terpretation of subcellular patterns in fluorescencemicroscope
imagesfor location proteomics. Cytometry (Part A) 69,4.:
631.-640.
microscopywith
imaging of connexin trafficking' Science296:503-507.
Giepmans,B. N. G., et aI.2006- The fluorescenttoolbox for
assessing protein location and function. Science3t2z2l7-224'
Gilroy, S. 1997. Fluorescence microscopyof living plant cells'
Ann. Reu.Plant Physiol. Plant Mol. Biol- 48:165-190.
'l'997.
Inou6, S., and K. Spring. Video Microscopy,2d ed'
Plenum Press.
Matsumoto. B.. ed. 2002. Methods in Cell Biology.Yol. T0: Cell
Biological Applications of Confocal Microscopy.AcademicPress'
Misteli, T., and D. L. Spector. 1997' Applications of the green
fluorescentprotein in cell biology and biotechnology.Nature
Biotech.15:961-964.
Pepperkok,R., and J. Ellenberg.2006. High-throughput fluo-
..r...ri. microicopy for systemsbiology. Nature Reu.Mol' Cell
Biol.7:690-696.
R E F E R E N C E 5. 405
 Sako,Y., S. Minoguchi, and T. Yanagida.2000.Single-molecule
imaging of EGFR signalling on the surface of living celli. Nature
Cell Btol.2:168-172.
Simon,S.,and J. Jaiswal.2004. Potentialsand pitfallsof fluorescent
quantum dots for biologicalimaging.TrendsCell Biol. 14:497-504.
Sluder,G., and D. Wolf, eds. 1998. Methods in Cell Biology.
Vol. 55: Video Microscopy.AcademicPress.
So, P.T.C.,et al. 2000. Two-photon excitation fluorescencemi-
croscopy.Ann. Reu.Biomed.tng. Z:3VO-42T.
\fillig, K. I., et al. 2005. STED microscopyrevealsthat synapro-
tagmin remainsclusteredafter synapticvesicleexocytosis.Nature
440:935-939.
Electron Microscopy: Methods and Applications
Beck,M., et al. 2004. Nuclear pore complex structure and dynam-
ics revealedby cryoelectrontomography. Science306:j.387-1390.
FreS T. G., G. A. Perkins,and M. H. Ellisman.2006. Electron
tomography of membrane-boundcellular organelles.Ann. Reu.
Biophy. Biomol. Struc. 352199-224.
. . Hlatq M. A. Principlesand Techniquesof Electron Microscopy,
4th ed. 2000. CambridgeUniversity Press.
.. Koster,A., and J. Klumperman. 2003. Electron microscopyin
cell biology: integratingsrructureand function. Nature Reu.Mol.
Cell Biol.4:SS6-5510.
Medalia, O., et al. 2002. Macromoleculararchitecturein
eukaryotic cellsvisualizedby cryoelectrontomography.Science
298:1209-1213.
Nickell, S., et al. 2006. A visual approachto proteomics.Nature
Reu.Mol. Cell Biol.7:225-230.
Purification of Cell Organelles
Battye,F. L., and K. Shortman. 1991. Flow cyrometryand cell-
separationprocedures.Curr. Opin. lmmunol. 3:238-241.
de Duve, C. 1,975. Exploring cellswith a centrifuge.Science
189:.1,86-1.94.
The Nobel Prize lectureof a pioneerin the study of
cellular organelles.
406 C H A P T E R9 | V t S U A L t Z t N GF, R A C T | O N A T | N G
A ,N D C U L T U R T NC
de Duve, C., and H. Beaufay.1981. A short history of tissue
fractionation.J. Cell Biol. 9tz293s-299s.
Howell, K. E., E. Devaney,and J. Gruenberg.1,989.Subcellular
fractionation of tissueculture cells.TrendsBiochem. Sci.t4:4448.
Ormerod, M. G., ed. 1990. FIow Cytometry:A Practical
Approach. IRL Press.
Rickwood, D. 1992. Preparatiue Centrifwgation: A Practical
Approach. IRL Press.
lsolation, Culture, and Differentiation of Metazoan Cells
Bissell,M. J., A. Rizki, and I. S. Mian. 2003. Tissuearchitec-
ture: the ultimate regulator of breastepithelialfunction. Curr. Opin.
Cell Biol. l5:753-762.
Davis, J. M., ed. 1.994.Basic Cell Cultwre:A Practical
Approach. IRL Press.
Edwards,B., et al. 2004. Flow cyrometryfor high-throughput,
high-contentscreening.Curr. Opin. Chem. Biol. 8:392-398.
Goding, I.W. 1996. Monoclonal Antibodies:Principlesand
Practice.Production and Application of Monoclonal Antibodies in
Cell Biology, Biochemistry, and Immunology, 3d ed. Academic
Press.
Griffith, L. G., and M. A. Swartz.2006. Capturing complex 3D
tissuephysiologyin vitro. Nature Reu.Mol. Cell. Biol.7z211J24.
Krutzik, P.,et al. 2004. Analysisof protein phosphorylationand
cellular signalingeventsby flow cytometry: techniquesand clinical
applications.Clin. Immwnol. 1L0z206- 221.
Paszek,M. J., and V. M. Weaver.2004.The tensronmounrs:
mechanicsmeetsmorphogenesisand malignancy.!. Mammary
Gland Biol. Neoplasia9:325-342.
Shaq A. J., ed. 1996. Epithelial Cell Cubure.IRL Press.
. Tyson, C. A., and J. A. Frazier, eds. L993. Methods in Toxicology.
Vol. I (Part A): In Vitro Biological Systems.Academic Press.Descri6es
methods for growing many types of primary cells in culture.
GE L L S
cLASSIC EXPERIMENT
ORGANELLES
SEPARATING
Beaufayet al., 1964,BiochemJ.92=191
In the 1950s and 1960sscientistsused
two techniquesto study cell organelles:
microscopy and fractionation. Christ-
ian de Duve was at the forefront of cell
fractionation. In the early 1950s he
used centrifugation to distinguish a
new organelle, the lysosome, from
previously characterizedfractions: the
nucleus, the mitochondrial-rich frac-
tion, and the microsomes.Soon there-
after he used equilibrium-density cen-
trifugation to uncover yet another
organelle.
Background
Eukaryotic cells are highly organized
and composed of cell structures
known as organelles that perform
specific functions. Although mi-
croscopyhas allowed biologiststo de-
scribethe location and appearanceof
various organelles,it is of limited use
in uncoveringan organelle'sfunction.
To do this, cell biologists have relied
on a technique known as cell fraction-
ation. Here, cells are broken open,
and the cellular componentsare sepa-
rated on the basis of size, mass, and
density using a variety of centrifuga-
tion techniques.Scientistscould then
isolate and analyze cell components
of different densities, called fractions.
Using this method, biologists had
divided the cell into four fractions:
nuclei, mitochondrial-rich fraction,
microsomes,and cell sap.
De Duve was a biochemist inter-
ested in the subcellular locations of
metabolic enzymes. He had already
completed a large body of work on the
fractionation of liver cells, in which he
had determined the subcellular loca-
tion of numerous enzymes.By locating
theseenzymesin specificcell fractions,
he could begin to elucidate the func-
tion of the organelle.He noted that his
work was guided by two hypotheses:
the "postulate of biochemical homo-
geneity" and "the postulate of single
location." In short, these hypotheses
proposethat the entire compositionof
a subcellular population will contain
the same enzymes and that each en-
zyme is located at a discretesite within
the cell. Armed with these hypotheses
and the powerful tool of centrifuga-
tion. de Duve further subdivided the
mitochondrial-rich fraction. First, he
identified the light mitochondrial frac-
tion, which is made up of hydrolytic
enzymesthat are now known to com-
posethe lysosome.Then, in a seriesof
experiments describedhere, he identi-
fied another discrete subcellular frac-
tion, which he called the peroxisome,
within the mitochondrial-rich fraction.
The Experiment
De Duve studied the distribution of
enzymesin rat liver cells. Highly ac-
tive in energy metabolism, the liver
contains a number of useful enzymes
to study. To look for the presenceof
various enzymesduring the fractiona-
tion, de Duve relied on known tests,
called enzyme assays,for enzYme
activity. To retain maximum enzyme
activity, he had to take precautions,
which included performing all frac-
tionation steps at OoC becauseheat
denaturesprotein, compromising en-
zyme actlvrty.
De Duve used rate-zonal centrifu-
gation to separatecellular components
by successivecentrifugation steps. He
removed the rat's liver and broke it
apart by homogenization. The crude
preparation of homogenizedcells was
then subjectedto relatively low-speed
centrifugation. This initial step sepa-
rated the cell nucleus,which collectsas
sediment at the bottom of the tube'
from the cytoplasmic extract' which
remains in the supernatant. Next, de
Duve further subdivided the cytoplas-
mic extract into heavy mitochondrial
fraction, Iight mitochondrial fraction,
and microsomal fraction. He accom-
plished separating the cytoplasm by
employing successive centrifugation
steps of increasingforce. At each step
he collected and stored the fractions
for subsequentenzyme analysis' Once
the fractionation was comPlete, de
Duve performed enzyme assaysto de-
termine the subcellular distribution of
eachenzyme.He then graphically plot-
ted the distribution of the enzyme
throughout the cell. As had been
shown previously, the activity of cy-
tochrome oxidase, an important en-
zyme in the electron transfer system'
was found primarily in the heavy mito-
chondrial fractions. The microsomal
fraction was shown to contain another
previously characterized enzYme,
glucose-5-phosphatase. The light mito-
chondrial fraction, which is made up
of the lysosome,showed the character-
istic acid phosphataseactivity. Unex-
pectedly, de Duve observed a fourth
pattern when he assayeduricase activ-
ity. Rather than following the pattern
of the referenceenzymes,uricaseactiv-
ity was sharply concentrated within
the light mitochondrial fraction. This
sharp concentration, in contrast to the
broad distribution, suggested to de
Duve that the uricase might be se-
cluded in another subcellular popula-
tion separate from the lYsosomal
enzymes.
To test this theorS de Duve em-
ployed a technique known as equilib-
rium density-gradient centrifugation,
which separates macromolecules on
the basis of density. Equilibrium
density-gradientcentrifugation can be
performed using a number of different
ORGANELLES
SEPARATING 407
 oxidase, segregatedinto the samefrac- its function. His careful work resulted
tions as uricase.Becauseeach of these in the uncovering of two organelles:
enzymes either produced or used hy- the Iysosomeand the peroxisome. His
drogen peroxide, de Duve proposed work also provided important clues to
Organelle
that this fraction represented an the organelles'function. The lysosome,
) fraction
organelle responsiblefor the peroxide where de Duve found so many poten-
1.09
Lysosomes metabolism and dubbed it the peroxi- tially destructiveenzymes.is now
A^
1.11 ( 1 . 1 2g / c m 3 )
ot some. known to be an important site for
M i t o c h o n d r i -a | degradation of biomolecules.The per-
( 1 . 1 8g / c m s ) oxisome has been shown to be the site
Discussion
of fatty acid and amino acid oxidation,
Peroxisomes.f De Duve's work on cellular fractiona- reactions that produce a large amount
( 1 . 2 3g / c m s ) |
tion provided an insight into the func- of hydrogen peroxide. In 1974, de
tion of cell structures as he sought to Duve received the Nobel Prize for
Before After map the location of known enzymes. Physiology and Medicine in recogni-
centrifugation centrifugation Examining the inventory
of enzymesin tion of his pioneering work.
A FIGURE 1 Schematic depictionof a given cell fraction gave him clues to
the separationof the lysosomes,
mitochondria, and peroxisomes by
equilibriumdensitycentrifugation. The
mitochondrial-richfractionfromrate-zonal
centrifugationwasseparated in a sucrose
gradient,andthe organelles wereseparated
on the basisof density[From Lodish etal,
MolecularCellBiology,3d
ed, W H Freeman and Cytochromeoxidase
Company, p 166]

gradients,includingsucroseand glyco-
gen. In addition, the gradient can be c"
made up in eitherwater or "heavy wa-
o4
ter," which contains the hydrogen iso-
tope deuterium in place of hydrogen. 6r
In his experiment de Duve separated Uricase
o2
the mitochondrial-rich fraction pre- o)
pared by rare-zonalcentrifugation in ol
0)
each of these different gradients (Fig-
ure 1). If uricasewere part of a sepa-
rate subcellularcompartment,it would
separate from the lysosomal enzymes
in each gradient tested.De Duve per-
formed the fractionations in this series
of gradients, then performed enzyme Acid phosphatase
assaysas before.In eachcase,he found
uricasein a separatepopulation than
the lysosomal enzyme acid phos-
phataseand the mitochondrial enzyme
20 40 60 80
cytochromeoxidase (Figure2).By re-
Percent
heightin tube
p e a t e d l yo b s e r v i n gu r i c a s ea c r i v i t yi n a
a FIGURE 2 Graphicalrepresentation of the enzymeanalysisof productsfrom a
distinct fraction from the acivity of
sucrosegradient.Themitochondrial-rich fraction
wasseparated asdepicted in Figure10.,1
,
the lysosomal and mitochondrial en-
andthenenzyme assayswereperformed Therelative concentrationof activeenzyme is
zymes,de Duve concluded that uricase
plottedon they axis;the heightin thetubeisplottedon the x axisThepeakactivities of
was part of a separateorganelle. The cytochrome (rop)andacidphosphatase
oxidase (bottom) areobserved nearthetop of the
experiment also showed that two other tube Thepeakactivityof uricase (middle)migratesto the bottomof the tube [Adaptedfrom
enzymes,catalaseand o-amino acid Beaufayet al , 1964,Biochem J 92:191l

408 C H A P T E R9 | v t S u A L t z t N G ,F R A C T | O N A T | N G
A ,N D C U L T U R T N C
GE L L S

Molecularmodelof a phospholipid
bilayersunoundedby water,as
determinedby molecular
dynamrcs @ NationalInstitutes
calculations
Inc
Researchers,
of Health/Photo
embranesparticipate in many aspectsof cell struc-
ture and function. The plasma membranedefinesthe
cell and separatesthe inside from the outside. In
eukaryotes, membranesalso define the intracellular organelles
such as the nucleus and lysosome.These biomembranesall
have the same basic architecture-a phospholipid bilayer-
but they are not static; their function is not to prevent all
exchange across a border. Each cellular membrane has its
own set of proteins that allow it to carry out its multitude of
specificfunctions (Figure 10-1).
Prokaryotes, the simplest and smallest cells, are about
t-2 pm in length and are surrounded by a single plasma
membrane;in most casesthey contain no internal membrane-
limited subcompartments(seeFigure 1-2a). However, this
single plasma membrane contains hundreds of different
types of proteins that are integral to the function of the
cell. Some of these proteins catalyze ATP synthesisand ini-
tiation of DNA replication, for instance. Others represent
the many types of membrane transport proteins that en-
able specific ions, sugars, amino acids, and vitamins to
cross the otherwise impermeable phospholipid bilayer to
enter the cell and that allow specific metabolic products
to exrt.
In eukaryotic cellsthe plasma membrane (Figure 10-2) is
not a site for ATP generation or DNA synthesis.Eukaryotic
plasma membranesare studded with a multitude of membrane
transpoft proteins that allow selectiveimport and export of
small molecules and ions. Receptorsin the plasma membrane
are proteins that allow the cell to recognizechemical signals-
CHAPTER
BIOMEMBRANE
STRUCTURE
many sent by neighboring cells-present in its environment
and adlust its metabolism or, especiallyduring development,
its pattern of gene expressionin response.Other specialized
plaima membrane proteins allow the cell to adhere to other
cells and to components in the surrounding fibrous extra-
cellular matrix. Many plasma membrane proteins bind
components of the cytoskeleton, the densenetutork of pro-
tein filaments that permeatesthe cytosol and mechanically
supports cellular membranes,interactions that are essential
for the cell to assumeits specific shape and for many types
of cell movements.
The plasma membrane bends, folds, and flexes in three
dimensions. Some segments bleb inward, incorporating
components from the extracellular medium into intracel-
lular vesicles(seeFigure 9-2). Virusessuch as HIV bud out-
ward from the cell-surface membrane, enveloping them-
selves with a bit of the plasma membrane that contains
virus-specificproteins (Figure 10-3).
OUTLIN E
10.1 B i o m e m b r a n e sL:i p i d C o m p o s i t i o n
4't1
and StructuralOrganization
10.2 Biomembranes:ProteinComponents
and BasicFunctions
10.3 s ,p h i n g o l i p i d sa,n d C h o l e s t e r o l :
P h o s p h o l i p i dS
Synthesisand IntracellularMovement 429
409
 Lipid-anchor
protein
Peripheral
memDrane
I protein

Hydrophilic
phospholipid
h e a dg r o u p

Phospholipid
bilayer

Hydrophilic
Cytosol fatty acyl
Lipid-anchored
protein P e r i p h e r a l s i d ec h a i n s
memorane
I n t e g r am
l embrane protein
protein

FIGURE 10-1 Fluidmosaicmodelof biomembranes. A bilayer anchored proteins

of phospholipids-3 nm thickprovides
the basicarchitecture
of all
cellular
membranes; membrane proteins
giveeachcellular
membrane membrane
itsuniquesetof f unctionsIntegral
(transmembrane)proteinsspan
the bilayer
andoftenformdimersandhigher-order oligomersLipid-
Each organellein a eukaryotic cell (Figures1-2, 10-2 and
10-4)containsa unique complementof protelns-some em-
beddedin its membrane(s),others containedin its aqueous
interior space,the lumen-that enable it to carry our lrs
characteristic cellular functions. For instance, the internal
pH of the lysosome, an organelle that contains many
P l a s m am e m b r a n e
aretetheredto oneleafletby a longcovalently
attachedhydrocarbon chainPeripheralproteinsassociate
withthe
primarily
by specific
noncovalent interactions
with
proteins
integral or membrane lipids.lAfterD Enqelman.
2005,
/Vature
438:578-80l
degradativeenzymes,is about 5, compared to pH 7.2 of the
cytosol, the aqueous part of the cytoplasm. The lysosome
membrane contains ATP-powered H* pumps that use the
energy of hydrolysis of ATP phosphoanhydride bonds to
pump protons from the cytosol into the lumen of the lyso-
some, lowering its pH.
We begin our examination of biomembranesby consid-
ering their lipid components. These not only affect mem-
brane shapeand function but also play important roles in
anchoring proteins to the membrane, modifying membrane
protein activities,and transducingsignalsto the cytoplasm.
'We
then consider the structure of membrane proteins.
Many, such as transport proteins and receptors, have large
segmentsthat are imbedded in the hydrocarbon core of the

HIV core Plasma

memDrane

Lysosome Multivesicular body

FIGURE 10-3 Eukaryotic cellmembranes are dynamic
A FIGURE 10-2 Cellularmembranes The plasma memorane structures.An electronmicrographof the plasmamembrane of
defines
theexterior
of thecellandcontrolsthemovement of molecules an HIV-infected
cell,showingHIVparticles buddingintotheculture
betweenthe cytosolandthe extracellular
mediumDifferent typesof medium. As theviruscorebudsfromthecell,it becomes enveloped
organelles
andsmaller vesicles
enclosedwithintheirown distinctive by a membrane derived fromthe cell'splasmamembrane that
membranescarryout special
functronssuchasgeneexpression, energy contains
specificviralproteins[From
W SundquistandU vonSchwedler.
production,
membrane synthesis,
andintracellular
transport. University
of Utahl

410 CHAPTER
1O I B I O M E M B R A NSET R U C T U R E

A FIGURE 10-4 Stackedmembranes of the Golgicomplex.
Notethe irregular
shapeandcurvature
of thesemembraneslFrom
C HopkinsandJ Burden,
lmperial London
College l ing properties.
phospholipid bilayer, and we will focus on the principal
classesof such membrane proteins. Finally, we consider
how phospholipids and cholesterol are synthesizedin cells
and distributedto the many membranesand organelles.Cho-
lesterol is an essentialcomponent of the plasma membrane
of all animal cells but is toxic to the organism if present in
CXCCSS.
Biomembranes: LipidComposition
and StructuralOrganization
In Chapter 2 we learnedthat phosphoglyceridesare the prin-
cipal building blocks of most biomembranes.Like the two
other principal classesof membrane lipids, sphingolipids
and cholesterol(Figure10-5),phosphoglycerides are amphi-
pathic molecules-they consist of two segmentswith very
different chemical properties. In phosphoglyceridesand
sphingolipids the hydrocarbon "tails" of the fatty acid side
chains are hydrophobic and partition away from water,
whereasthe "head groups" are strongly hydrophilic, or wa-
ter loving, and tend to interact with water molecules.
Steroids such as cholesterol, in contrast, are mostly hy-
drophobic except for one hydrophilic hydroxyl group. AII
three types of phospholipids have the necessaryqualities to
form membranesand play different roles in the function of
the cell.
s p o n t a n e o u s lFy o r mB i l a y e r s
P h o s p h o l i p i dS
The amphipathic nature of phospholipids,which governs
their interactions,is critical to the structureof biomembranes.
When a suspensionof phospholipids is mechanicallydis-
B I O M E M B R A N E LSI:P I DC O M P O S I T I OANN D S T R U C T U R AOLR G A N I Z A T I O N
persed in aqueous solution, the phospholipids aggregate
into one of three forms: sphericalmicellesand liposomes
and sheetlikephospholipid bilayers, which are two mole-
culesthick (Figure 10-6). The type of structureformed by a
pure phospholipid or a mixture of phospholipidsdepends
on several factors, including the length of the fatty acyl
chains, their degreeof saturation, and temperature.In all
three structures, the hydrophobic effect causesthe fatty
acyl chainsto aggregateand excludewater moleculesfrom
the "core." Micelles are rarely formed from natural phos-
phoglycerides, whose fatty acyl chains generally are too
bulky to fit into the interior of a micelle. However, micelles
are formed if one of the two fatty acyl chains is removed
from the phosphoglyceride by hydrolysis, forming a
lysophospholipid.In aqueoussolution, common detergents
and soapsform micellesthat behaveas tiny ball bearings,
thus giving soap solutions their slippery feel and lubricat-
Under suitable conditions, phospholipids of the com-
position present in cells spontaneously form symmetric
phospholipid bilayers.Each phospholipid layer in this
lameilar structureis called a leaflet.The hydrophobic fatty
acyl chains in each leaflet minimize contact with water by
aligning themselvestightly together in the center of the
bilayer, forming a hydrophobic core that is about 3-4 nm
thick (Figure 10-6b). The close packing of thesenonpolar
'Sfaals
tails is stabilized by van der interactions between
thesehydrocarbon chains. Ionic and hydrogen bonds sta-
bilize the interaction of the phospholipid polar head
groups with one another and with water. Electron mi-
croscopy of thin membrane sectionsof cells stained with
osmium tetroxide, which binds strongly to the polar head
groups of phospholipids,revealsthe bilayer structure (Fig-
ure 10-6a). A cross section of a single membrane stained
with osmium tetroxide looks like a railroad track: two
thin dark lines (the stained head group complexes)with a
uniform light space of about 2 nm between them (the
h y d r o p h o b i ct a i l s ) .
A phospholipid bilayer can be of almost unlimited size-
from micrometers (pm) to millimeters (mm) in length or
width-and can contain tens of millions of phospholipid
molecules. Becauseof their hydrophobic core, bilayers are
virtually impermeable to salts, sugars,and most other small
hydrophilic molecules. The phospholipid bilayer is the
basic structural unit of nearly all biological membranes;
although they contain other molecules (e.g., cholesterol,
glycolipids, proteins), biomembraneshave a hydrophobic
core that separatestwo aqueoussolutions and acts as a per-
meability barrier.
P h o s p h o l i p i dB i l a y e r sF o r ma S e a l e d
C o m p a r t m e nS t u r r o u n d i n ga n l n t e r n a l
A q u e o u sS p a c e
Phospholipid bilayers can be generatedin the laboratory by
simpleexperimentalmanipulations;theseutilize either chemi-
cally pure phospholipidsor lipid mixtures of the composition
41'l
 (a) Phosphoglycerides H e a dg r o u p
H

PE

gocH

o,r.--r. o..o1.o..^..r-i?tt'
-cH, PC
o
Hydrophobictait
A | .*
^,'\'- N
\JTH
I PS
o4o

o
Plasmalogenl3ir...

O-^-?^O-i-O-^
ro
o
o

(b)Sphingolipids
OH Il l 9H, ^,
1 .."' '3
-P-^ Nl
-cH,
6"
SM

cHg
(c) Sterols

HO HO
ch"olesterol ttntT:lit stisqrasterol

A FIGURE 10-5 Threeclasses of membranelipids.(a)Most Various fattyacylchainsareconnected to sphingosine by an amide

phosphoglycerides arederivatives of glycerol 3-phosphate (red), bond Thesphingomyelins (SM),whichcontaina phosphocholine
whichcontains two esterified fattyacylchains thatconstitute the headgroup,arephospholipids Othersphingolipids areglycolipids in
hydrophobic "tail" anda polar"headgroup"esterified to the whicha singlesugarresidue or branched oligosaccharide isattached
phosphate. Thefattyacidscanvaryin lengthandbe saturated (no to thesphingosine backbone. Forinstance, thesimpleglycolipid
doublebonds)or unsaturated (one,two, or threedoublebonds). glucosylcerebroside (GlcCer) hasa glucose headgroup.(c)The
In phosphatidylcholine (PC), the headgroupischolineAlsoshown majorsterols in animals (cholesterol), fungi(ergosterol), andplants
arethe molecules attachedto the phosphate groupin threeother (stigmasterol) differslightlyin structure, butallserveaskeycomponents
commonphosphoglycerides: phosphatidylethanolamine (PE), of cellular membranes Thebasicstructure of steroids isa four-ring
phosphatidylserine (PS), andphosphatidylinositol (Pl)Plasmalogens hydrocarbon (yellow). Likeothermembrane lipids, sterols are
containonefattyacylchainattached to glycerol by an esterlinkage amphipathic Thesinglehydroxyl groupisequivalent to the polar
andoneattached by an etherlinkage; thesecontainsimilar head headgroupin otherlipids; theconjugated ringandshorthydrocarbon
g r o u p s a s p h o s p h o g l y c e(rbi )dSepsh i n g o l i p i d s a r e d e r i v a t i v e s o f c h a i n f o r m t h e h y d r o p h o b i c[ S
t aei el Hs p r o n g e t,a2l0 0 1N, atureRev.
sphingosine (red),an aminoalcohol with a lonqhvdrocarbon chain Mot Celt Biot2:5041

412 CHAPTER
1O I B I O M E M B R A NSET R U C T U R E
 (at M e m b r a n eb i l a y e r that mediate the transport of specific molecules across this
otherwise impermeable bilayer. The secondproperty of the
bilayer is its stability. The bilayer structure is maintained by
hydrophobic and van der Waals interactions between the
lipid chains. Even though the exterior aqueousenvironment
can vary widely in ionic strength and pH, the bilayer has the
strength to retain its characteristic architecture. Third, all
phospholipid bilayers can spontaneouslyform sealedclosed
compartments where the aqueous space on the inside is

Phospholipid
bilayer

P o l a rh e a d
g roups 'l

g o l un, u u a /
groups

(c)

P h o s p h o l i p i disn s o l u t i o n

"1r..,
Disperse D i s s o l v ep h o s p h o l i p i d s
phospholipids i n s o l v e n ta n d a p p l y
in water t o s m a l lh o l e
in oartition

FIGURE 10-6 The bilayerstructureof biomembranes.

(a)Electron micrograph of a thin sectionthroughan erythrocyte
membrane stained withosmiumtetroxide Thecharacteristic "railroad
track"appearance of the membrane indicatesthe presenceof two
polarlayers, consistentwiththe bilayer structure for phospholipid
membranes (b)Schematic interpretation of the phospholipid bilayer
in whichpolargroupsfaceoutwardto shield the hydrophobic fatty
acyltailsfromwaterThehydrophobic effectandvanderWaals
interactions betweenthe fattyacyltailsdrivethe assembly of the
Liposome
bilayer(Chapter 2).(c)Cross-sectional viewsof two otherstructures
formedbydispersal of phospholipids in waterA spherical micelle has a EXPERIMENTAL FIGURE 10-7 Formationand studyof
a hydrophobic interior
composed entirely
of fattyacylchains; a pure phospholipidbilayers.(Iop)A preparation of biological
spherical liposome consists of a phospholipid bilayer surrounding an membranes istreatedwith an organic solvent,suchasa mixture of
aqueous centerlPart(a)courtesy of J D Robertson ] chloroform andmethanol (3:1),whichselectively the
solubilizes
phospholipids andcholesterol. Proteins andcarbohydrates remain
in an insolubleresidue.Thesolvent is removed by evaporation
(Bottom/eft)lf the lipidsaremechanically dispersed in water,they
found in cell membranes(Figure 10-7). Studieson these bi- spontaneously forma liposome, shownin crosssection, with an
layers have shown that they possessthree important proper- internalaqueous compartment. (Bottom right)A planarbilayer,also
ties. First, the hydrophobic core is an impermeable barrier shownin crosssection, canformovera smallholein a partition
that prevents the diffusion of water-soluble (hydrophilic) separatingtwo aqueous phases; suchbilayers canbe usedto study
solutesacrossthe membrane.Importantly, this simple barrier the movement of solutesfromonesolution to the otherthroughthe
function is modulated by the presenceof membrane proteins membrane

A N D S T R U C T U R AOL R G A N I Z A T I O N O 413
B I O M E M B R A N E LSI P I DC O M P O S I T I O N
 < FIGURE 10-8 Thefacesof cellular
Mitochondrion membranes. Theplasmamembrane, a
s i n g l eb i l a y emr embranen , c l o s et h
s ec e l l
I n t h i sh i g h l ys c h e m a tri ce p r e s e n t a t i o n ,
internal cytosol (greenstipple) andexternal
environment (purple) definethe cytosolic (red)
andexoplasmic (black) facesof the bilayer
Vesicles andsomeorganelles havea single
Matrix
Intermembranespace membrane andtheirinternal aoueous soace
(purple) istopologically equivalent to the
Exoplasmic outside of thecell.Threeorganelles-the
face nucleus, mitochondrion, andchloroplast
(whichis not shown)-areenclosed by two
membranes separated bya smallintermembrane
space. Theexoplasmic facesof the innerand
outermembranes aroundtheseorganelles
borderthe intermembrane spacebetween
Cytosolic them Forsimplicity, the hydrophobic membrane
face
interior isnotindicated in thisdiaqram
P l a s m am e m b r a n e

Exterior

IntermembranesDace

- Exoplasmic
separatedfrom that on the outside. An "edge" of a phos-
pholipid bilayer, as depicted in Figure 10-6b with the hydro-
carbon core of the bilayer exposedto an aqueoussolution, is
unstable; the exposed fatty acyl side chains would be in an
energeticallymuch more stable state if they were not adja-
cent to water molecules but surrounded by other fatty acyl
chains (hydrophobic effect; Chapter 2). Thus in aqueous
solution, sheetsof phospholipid bilayers sponraneouslyseal
their edges,forming a spherical bilayer that enclosesan
Cytosolic
aqueous central compartment. The liposome depicted in face
Figure 10-6 is an example of such a structure viewed in
cross sectton.
This physical chemical property of a phospholipid
bilayer has important implications for cellular membranes:
no membrane in a cell can have an "edge" with exposed A FIGURE 10-9 Facesof cellularmembranesare conserved
hydrocarbon fatty acyl chains. All membranes form closed during membranebuddingand fusion.Redmembrane surfaces
compartments, similar in basic architecture to liposomes. arecytosolicfaces;blackareexoplasmic faces.Duringendocytosisa
Becauseall cellular membranesenclosean entire cell or an segment of the plasmamembrane budsinwardtowardthecytosol
internal compartment, they have an internal face (the surface andeventually pinchesoff a separate
vesicle.
Duringthisprocess the
oriented toward the interior of the compartment) and an faceof the plasma
cytosolic membrane remains facingthe cytosol
andtheexoplasmic faceof the newvesicle membrane facesthe
externalface (the surfacepresentedto the environment).
lumenDuringexocytosis an intracellular
vesicle
fuseswith the
More commonl5 we designatethe two surfacesof a cellu-
plasmamembrane, andthe lumenof thevesicle (exoplasmicface)
Iar membrane as the cytosolic face and the exoplasmic face.
connectswith theextracellularmediumProteins thatspanthe
This nomenclature is useful in highlighting the topological membrane retaintheirasymmetric orientation
duringvesicle budding
equivalence of the faces in different membranes, as dia- andfusionevents; in particular
thesamesegment alwaysfacesthe
grammed in Figures 10-8 and 10-9. For example. the cyrosol

414 CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
exoplasmic face of the plasma membrane is directed away
from the cytosol, toward the extracellular spaceor external en-
vironment, and definesthe outer limit of the cell. The cytosolic
face of the plasma membrane faces the cytosol. Similarly for
organellesand vesiclessurroundedby a singlemembrane,the
cytosolic face facesthe c1'tosol.The exoplasmic face is always
directed away from the cytosol and in this caseis on the inside
of the organelle in contact with the internal aqueous space,or
lumen. That the inside, or lumen, of thesevesiclesis topologi-
cally equivalentto the extracellularspaceis most easilyunder-
stood for vesiclesthat ariseby invagination(endocytosis) of the
plasma membrane. This processresults in the external face of
the plasma membrane becoming the internal face of the vesicle
membrane, and in the vesiclethe cytosolic face of the plasma
membraneremainsfacing the cytosol (seeFigure 10-9).
Two distinct membranessurround three organelles-the
nucleus, mitochondrion, and chloroplast; the exoplasmic
surface of each membrane faces the spacebetween the two
membranes.This can perhaps best be understood by refer-
ence to the endosymbiont hypothesis,discussedin Chapter
1, which posits that mitochondria and chloroplasts arose
early in the evolution of eukaryotic cells by the engulfment spm
| I
of bacteria capable of oxidative phosphorylation or photo-
synthesis,respectively(seeFigure6-20). Thus the exoplasmic
face of the mitochondrial inner membrane, derived from the
exoplasmicface of the ancestralbacterial plasma membrane,
as well as the exoplasmic face of the outer mitochondrial
membrane,derived from the exoplasmicface of the ancestral
plasma membrane, both face the intermembranespace.
Natural membranes from different cell types exhibit a
variety of shapes,which complementa cell'sfunction (Figure
10-10; seeFigures10-3 and 10-4).The smooth,flexiblesur-
face of the erythrocyte plasma membrane allows the cell to
squeezethrough narrow blood capillaries.Somecells have a
long, slenderextensionof the plasma membrane,called a cil-
ium or flagellum, which beatsin a whiplike manner.This mo-
tion causesfluid to flow acrossthe surfaceof a sheetof cells FIGURE 10-10Variationin biomembranes in differentcell
or a sperm cell to swim toward an egg. Membranes are dy- types.(a)A smooth,flexible membrane coversthe surfaceof the
namic structures;Figure 10-3 shows an HIV virus budding discoid cell
erythrocyte as seen in thisscanningelectron micrograph.
from the surfaceof a human cell. In infectedcells,specificvi- (b)Tuftsof cilia(Ci)projectfromthe ependymal cellsthat linethe
ral proteins becomeinsertedinto the plasma membrane,and brainventricles lPart(a)Copyright@omiKron/Photo Inc.
Researchers,
segmentsof the plasma membrane envelop the viral core, or Part(b)fromR G Kessel andR H Kardon, andOrgans:
1979,Tissues A
nucleocapsid, that contains the viral RNA genome as the of Scanning
Text-Atlas Electron W
Microscopy, H Freeman and Company]
virus buds from the cell. The membrane-coatedvirus then
pinches off from the plasma membrane and is releasedinto sphingolipids, and steroids, which differ in their chemical
the surrounding medium. Internal cellular membranessuch structures,abundance,and functions in the membrane.
as the Golgi complex (seeFigure 10-4) are constantly bud- Phosphoglycerides, the most abundant class of lipids in
ding off membrane vesiclesinto the cytosol. Thesethen fuse most membranes, are derivativesof glycerol 3-phosphate(see
with other membranesto transport the luminal contentsfrom Figure 10-5a).A typical phosphoglyceride moleculeconsistsof
one organelleto another (Chapter 14). a hydrophobic tail composedof two fatty acyl chains esterified
to the fvvo hydroxyl groups in glycerol phosphate and a polar
head group attached to the phosphate group' The two fatty
s o n t a i nT h r e eP r i n c i p a l
B i o m e m b r a n eC
acyl chains may differ in the number of carbons that they con-
Classes of Lipids tain (commonly 1.6or 18) and their degreeof saturation(0, 1,
As noted above, a typical biomembrane is assembledfrom or 2 double bonds).A phosphoglycerideis classifiedaccording
three classes of amphipathic lipids: phosphoglycerides, to the nature of its head group. In phosphatidylcholines,

A N D S T R U C T U R AOL R G A N I Z A T I O N
B I O M E M B R A N E SL:I P I DC O M P O S I T I O N 415
 the most abundant phospholipids in the plasma membrane,
the head group consistsof choline, a positively chargedalco-
hol, esterifiedto the negarivelycharged phosphate.In other
phosphoglycerides,an OH-containing molecule such as
ethanolamine, serine, and the sugar derivative inositol is
linked to the phosphate group. The negatively charged
phosphate group and the positively charged groups or the
hydroxyl groups on the head group interacr strongly with
water. At neutral pH, some phosphoglycerides (e.g.,
phosphatidylcholineand phosphatidylethanolamine) carry no
net electriccharge,whereasothers (e.g.,phosphatidylinositol
and phosphatidylserine)carry a single net negative charge.
Nonetheless,the polar head groups in all phospholipidscan
pack togetherinto the characteristicbilayer structure.
The plasmalogensare a group of phosphoglyceridesthat
contain one fatty acyl chain attached to carbon 2 of glycerol
by an esterlinkage and one long hydrocarbon chain attached
to carbon 1 of glycerol by an ether (C-O-C) rather than
an ester linkage. The abundance of plasmalogensvaries
among tissuesand speciesbut is especiallyhigh in human
brain and heart tissue. The additional chemical stability of
the ether linkage in plasmalogens, compared to the ester
linkage, or the subtle differencesin their three-dimensional
structure compared with that of other phosphoglycerides
may have as yet unrecognizedphysiologic significance.
A secondclassof membranelipid is the sphingolipids.All
of thesecompounds are derived from sphingosine,an amino
alcohol with a long hydrocarbon chain, and contain a long-
chain faty acid attached in an amide linkage to the sphingosine
amino group. Like phosphoglycerides,sphingolipids have a
phosphate-based polar head.In sphingomyelin,the most abun-
dant sphingolipid, phosphocholine is attached to the terminal
hydroxyl group of sphingosine(seeFigure 10-5b).Thus sphin-
gomyelin is a phospholipid, and its overall structure is quite
similar to that of phosphatidylcholine. Sphingomyelinsare
similar in shape to phosphoglyceridesand can form mixed
bilayers with them. Other sphingolipids are amphipathic
glycolipids whose polar head groups are sugars that are not
linked via a phosphategroup. Glucosylcerebroside, the sim-
plest glycosphingolipid, contains a singleglucoseunit attached
to sphingosine. In the complex glycosphingolipids called
gangliosides,one or two branched sugar chains (oligosaccha-
rides) containing sialic acid groups are attached to sphingosine.
Glycolipids constitute 2-10 percent of the total lipid in plasma
membraneslthey are most abundant in nervoustissue.
Cholesterol and its analogs constiture the third impor-
tant class of membrane lipids, the steroids.The basic struc-
ture of steroidsis a four-ring hydrocarbon. The structuresof
the principal yeast sterol (ergosterol)and plant phytosterols
(e.g., stigmasterol) differ slightly from that of cholesterol,
the major animal sterol (seeFigure 10-5c).The small differ-
ences in the biosynthetic pathways of fungal and animal
sterols and in their structures are the basis of most antifun-
gal drugs currently in use. Cholesterol, Iike the rwo orher
sterols, has a hydroxyl substituent on one ring. Although
cholesterolis almost entirely hydrocarbon in composition, it
is amphipathic becauseits hydroxyl group can interact with
water. Cholesterol is especially abundant in the plasma
4'16 . c H A p r E Rt o l BToMEMBRAN
s rER U c r u R E
membranesof mammalian cells but is absent from most
prokaryotic and all plant cells.As much as 30-50 percent of
the lipids in plant plasma membranes consist of certain
steroids unique to plants. Between50 and 90 percent of the
cholesterolin most mammalian cells is presentin the plasma
membrane and associatedvesicles.Cholesterol and other
sterols are too hydrophobic to form a bilayer structure on
their own. Instead, at concentrationsfound in natural mem-
branes,these sterols must intercalate between phospholipid
moleculesto be incorporated into biomembranes.
In addition to its structural role in membranes,choles-
terol is the precursor for severalimportant bioactive mole-
cules.They include bile acids,which are made in the liver and
help emulsify dietary fats for digestionand absorption in the
intestinesl steroid hormones produced by endocrine cells
(e.g.,adrenalgland, ovar5 testes);and vitamin D produced in
the skin and kidneys. Another critical function of cholesterol
is its covalent addition to Hedgehogprotein, a key signaling
moleculein embryonic development(Chapter 16).
Most Lipidsand Many ProteinsAre Laterally
M o b i l ei n B i o m e m b r a n e s
In the two-dimensional plane of a bilayer, thermal motion
permits lipid molecules to rotate freely around their long
axes and to diffuse Iaterally within each leaflet. Because
such movements are lateral or rotational, the fatty acyl
chains remain in the hydrophobic interior of the bilayer. In
both natural and artificial membranes,a typical lipid molecule
Heat
Gel-likeconsistency Fluidlikeconsistency
si
FIGURE 10-11Geland fluid forms of the phospholipid
bilayer.(Iop)Depiction of gel-to-fluid
transitionPhospholipids
with longsaturated fattyacylchains tendto assemble intoa highly
ordered,gel-like bilayer in whichthereislittleoverlapof the
nonpolar tailsin thetwo leaflets. Heatdisorders
the nonpolar tails
andinduces a transition froma gelto a fluidwithina temperature
rangeof onlya few degrees. As thechainsbecome disordered, the
bilayeralsodecreases in thickness. (Bottom)
Molecular models of
phospholipid monolayers in gelandfluidstates,asdetermined by
molecular dynamrcs calculations. lBottombasedonH Helleretal, 1993.
J PhysChem97:8343 l
exchangesplaces with its neighbors in a leaflet about 107
times per second and diffuses several micrometers per sec-
ond at 37 "C. Thesediffusion ratesindicatethat the viscos-
ity of the bilayer is 100 times as great as that of water-
about the same as the viscosity of olive oil. Even though
lipids diffuse more slowly in the bilayer than in an aqueous
solvent, a membrane lipid could diffuse the length of a typ-
ical bacterialcell (1 pm) in only 1 secondand the length of
an animal cell in about 20 seconds.Vhen fluid artificial
pure phospholipid membranesare cooled below 37'C, the
lipids can undergo aphase transition from a liquidlike (fluid)
state to a gel-like (semisolid)state, analogousto the liquid-
solid transition when liquid water freezes(Figure 10-11).
Below the phase transition temperature, the rate of diffu-
sion of the lipids drops precipitously. At usual physiologic
temperatures,the hydrophobic interior of natural mem-
branes generally has a low viscosity and a fluidlike consis-
tency, in contrast to the gel-like consistency observed at
lower temperatures,
In pure membrane bilayers, phospholipids and sphin-
golipids rotate and move laterallg but they do not sponta-
neously migrate, or flip-flop, from one leaflet to the other'
The energetic barrier is too high; migration would require
moving the polar head group from its aqueous environ-
ment through the hydrocarbon core of the bilayer to the
aqueoussolution on the other side. Specialmembranepro-
teins discussedin Chapter 1.1 are required for membrane
lipids and other polar molecules to flip from one leaflet to
the other.
The lateral movements of specific plasma-membrane
proteins and lipids can be quantified by a technique called
fluorescencerecouery after photobleaching /FRAP/. Phos-
pholipids containing a fluorescent substituent are used to
monitor lipid movement. For proteins, a fragment of a mon-
oclonal antibody that is specificfor the exoplasmic domain
of the desired protein and that has only a single antigen-
binding site is tagged with a fluorescent dye. \fith this
method, described in Figure 10-1.2, the rate at which

(a)

M e m b r a n ep r o t e i n Fluorescentreagent Bleachedarea
I

Bleachwith
Label taser
+
E a

lhl

6
Fluorescence
before bleaching
ri
3 3000
6

! zooo
a)
o
3
o
rooo
o
o

f
L 100 150
T i m e( s )
A EXPERIMENTAL FIGURE 10-12Fluorescence recoveryafter labeled molecules thataremobilein the membrane. (b)Results
photobleaching(FRAP)experimentscan quantify the lateral of FRAP experiment with human hepatoma cellstreatedwith a
movementof proteinsand lipidswithin the plasma fluorescent antibody specificfor the asialoglycoprotein receptor
membrane.(a)Experimental protocolStep(E): Cellsarefirst proteinThefindingthat 50 percent of thefluorescence returned to
labeledwith a fluorescent
reagent thatbindsuniformly to a specific the bleached areaindicates that 50 percent of the receptor molecules
membrane lipidor proteinStep(E): A laserlightisthenfocused in the illuminated membrane patchweremobileand50 percent
on a smallareaof thesurface,irreversibly
bleaching the bound wereimmobile Because the rate of fluorescence recovery is
reagentandthusreducing thefluorescence in the illuminatedarea oroportional to the rateat whichlabeled molecules moveintothe
Step(B): Intime,thefluorescence of the bleached patchrncreases bleached region, the diffusioncoefficient of a proteinor lipidin the
asunbleached fluorescent
surface molecules diffuseintoit and membrane canbe calculated fromsuchdata.[See Y I Henisetal,
bleachedonesdiffuseoutwardTheextentof recovery of 1990,J CellBiol.111:14091
fluorescencein the bleachedpatchisproportronal to thefractionof

N N D S T R U C T U R AOL R G A N I Z A T I O N
B I O M E M B R A N E SL:I P I DC O M P O S I T I O A 417
 o/o)
(MOt
C0MP0$Tl0N
SOURCUI.()CATION PC PE+ PS SM CH()TESIEBOT

Plasma membrane (human eryrhrocytes) 2I 29 2t 26

Myelin membrane (human neurons) 16 37 13 34

Plasmamembrane (E. coli) 0 85 0 0

Endoplasmic reticulum membrane (rat) 54 26 5 7

Golgi membrane (rat) 45 20 t3 l-)

Inner mitochondrial membrane (rat) 45 45 2 7

Outer mitochondrial membrane (rat) 34 46 2 11.

Primary leaflet location Exoplasmic Cytosolic Exoplasmic Both

PC : phosphatidylcholine;
PE : phosphatidylethanolamine;
PS: phosphatidylserine;
SM - sphingomyelin.
souRcE:S7.DowhanandM.Bogdanov,2002,inD.E.VanceandJ.E Vance,eds.,Biochemistryof
Lipids,Lipoproteins,andMembranes,Elsevier.

membrane molecules move-the diffusion coefficient-can
be determined, as well as rhe proportion of the molecules
that are laterallymobile.
The results of FRAP studieswith fluorescence-labeled
phospholipidshave shown that in fibroblast plasma mem-
branes, all the phospholipids are freely mobile over dis-
tancesof about 0.5 pm, but most cannot diffuse over much
longer distances.These findings suggestthat protein-rich
regions of the plasma membrane about 1 pm in diameter
separatelipid-rich regionscontaining the bulk of the mem-
brane phospholipid. Phospholipids are free to diffuse
within such regions but not from one lipid-rich region to
an adjacent one. Furthermore,the rate of lateral diffusion
of lipids in the plasma membrane is nearly an order of
magnitude slower than in pure phospholipid bilayers:
diffusion consrantsof 10-8 cm2/sand I0-7 cm2lsare char-
acteristic of the plasma membrane and a lipid bilayer,
respectively.This difference suggeststhat lipids may be
tightly but not irreversibly bound to certain integral pro-
teins in some membranes, as indeed has recently been
d e m o n s t r a t e d( s e eF i g u r e 1 0 - 1 7 b e l o w ) .
L i p i dC o m p o s i t i o nI n f l u e n c e st h e p h y s i c a l
Propertiesof Membranes
A typical cell contains myriad types of membranes,each
with unique properties bestowed by its particular mix of
Iipids and proteins. The data in Table 10-1 illustrate the vari-
ation in lipid composition in different biomembranes.Several
phenomena contribute to these differences.For instance,
there are differencesin the relative abundancesof phospho-
glyceridesand sphingolipids between membranesin the en-
doplasmic reticulum (ER), where phospholipids are synthe-
sized, and the Golgi, where sphingolipids are synthesized.
The proportion of sphingomyelin as a percentageof total
membrane lipid phosphorus is about six times as high in
Golgi membranesas it is in ER membranes.In other cases,
the movement of membranesfrom one cellular compartment
to another can selectivelyenrich certain membranesin lipids
such as cholesterol.In responding to differing environments
throughout an organism, different types of cells generate
membranes with differing lipid compositions. In the cells
that line the intestinal tract, for example, the membranes
that face the harsh environment in which dietary nutrients
are digested have a sphingolipid-to-phosphoglyceride-to-
cholesterol ratio of 1:1,;1 rather than the 0.5:1.5:1 ratio
found in cells subject to less stress.The relatively high con-
centration of sphingolipid in this intestinal membrane may
increaseits stability becauseof extensivehydrogen bonding
by the free -OH group in the sphingosine moiety (see
F i g u r e1 0 - 5 ) .
The degreeof bilayer fluidity dependson the lipid com-
position, structure of the phospholipid hydrophobic tails,
'Waals
and temperature. As aheady noted, van der interac-
tions and the hydrophobic effect causethe nonpolar tails
of phospholipids to aggregate.Long, saturated fatty acyl
chains have the greatest tendency to aggregate,packing
tightly together into a gel-like state. Phospholipids with

418 CHAPTER
1O I B I O M E M B R A NSET R U C T U R E

SM and
cholesterol
short fatty acyl chains, which have less surface area and
therefore fewer van der'Waals interactions, form more fluid
bilayers. Likewise, the kinks in cis- unsaturated fatty acyl
chains (Chapter2) result in their forming lessstablevan der
Waals interactions with other lipids, and hence more fluid
bilayers,than do straight saturatedchains,which can pack
more tightly together.
Cholesterolis important in maintaining the appropriate
fluidity of natural membranes,a property that appearsto
be essentialfor normal cell growth and reproduction. Cho-
lesterol restricts the random movement of phospholipid
head groups at the outer surfacesof the leaflets, but its
effect on the movement of long phospholipid tails depends
on concentration.At cholesterolconcentrationspresentin
the plasma membrane, the interaction of the steroid ring
with the long hydrophobic tails of phospholipidstends to
immobilize these lipids and thus decreasebiomembrane
fluidity. At lower cholesterolconcentrations,however,the
steroid ring separatesand dispersesphospholipid tails,
causing the inner regions of the membrane to become
slightly more fluid.
The lipid composition of a bilayer also influencesits
thickness, which in turn may influence the distribution of
other membrane components,such as proteins, in a particu-
lar membrane.The resultsof biophysical studieson artificial
membranesdemonstratethat sphingomyelin associatesinto
B I O M E M B R A N E SL:I P I DC O M P O S I T I O N
< FIGURE 10-13Effectof lipid compositionon bilayer
thicknessand curvature.(a)A puresphingomyelin (SM)bilayer
isthickerthanone formed from a phosphoglyceridesuch as
(PC).Cholesterol
phosphatidylcholine effecton
hasa lipid-ordering
phosphoglyceridebilayersthatincreasestheirthicknessbut doesnot
affectthethicknessof the moreordered SMbilayer. (b)Phospholipids
suchasPChavea cylindrical shapeandformmoreor lessflat
monolayers, whereasthose with headgroupssuchas
smaller
phosphatidylethanolamine (PE)havea conical shape(c)A bilayer
enrichedwith PCin theexoplasmic leafletandwith PEin the
face,asin manyplasma
cytosolic membranes, wouldhavea natural
curvature lAdaptedfromH Sprongetal,2001,NatureRev Mol CellBiol
22504l
a more gel-like and thicker bilayer than phospholipids do
(Figure 10-13a). Cholesteroland other moleculesthat de-
creasemembrane fluidity also increasemembrane thickness.
Becausesphingomyelintails are akeady optimally stabilized,
the addition of cholesterolhas no effect on the thicknessof a
sphingomyelin bilayer.
Another property dependenton the lipid composition of
a bilayer is its curvature, which dependson the relative sizes
of the polar head groups and nonpolar tails of its con-
stituentphospholipids.Lipids with long tails and largehead
groups are cylindrical in shape; those with small head
groupsare cone shaped(Figure10-13b).As a result,bilayers
composed of cylindrical lipids are relatively flat, whereas
those containing large numbers of cone-shapedlipids form
curved bilayers(Figure 10-13c).This effect of lipid compo-
sition on bilayer curvature may play a role in the formation
of highly curved membranes,such as sites of viral budding
(seeFigure 10-3) and formation of internal vesiclesfrom
the plasma membrane (seeFigure 10-9) and in specialized
stable membrane structures such as microvilli. Severalpro-
teins bind to the surfaceof phospholipid bilayers and cause
the membrane to curve; such proteins are important in
formation of transport vesiclesthat bud from a donor mem-
brane (Chapter 14).
Lipid Compositionls Different in the Exoplasmic
and CytosolicLeaflets
A characteristicof all membranesis an asymmetryin lipid
composition across the bilayer. Although most phospho-
lipids are present in both membrane leaflets' they are
commonly more abundant in one or the other leaflet. For
instance,in plasma membranesfrom human erythrocytes
and certain canine kidney cells grown in culture' almost
all the sphingomyelin and phosphatidylcholine, both of
which form less fluid bilayers, are found in the exoplas-
mic leaflet. In contrast, phosphatidylethanolamine,phos-
phatidylserine, and phosphatidylinositol, which form
more fluid bilayers, are preferentially located in the cy-
tosolic leaflet. This segregationof lipids acrossthe bilayer
A N D S T R U C T U R AOLR G A N I Z A T I O N
419

o
_______>|
c:o
I
(cH,)
-n
| diacylglycerol.As we will see in Chapter 15, both water
cHg
A FIGURE 10-14 Specificityof phospholipases. Eachtypeof
p h o s p h o l i p acsl e a v eosn eo f t h e s u s c e p t i bbl e o n d ss h o w ni n r e d
T h eg l y c e r ocla r b o na t o m sa r ei n d i c a t ebdy s m a lnl u m b e r sI n
i n t a c ct e l l so, n l yp h o s p h o l i p iidnst h ee x o p l a s ml ieca f l eot f t h e
plasmm a e m b r a naer ec l e a v ebdy p h o s p h o l i p a si netsh e
s u r r o u n d i nmge d i u mP h o s p h o l i p aCs,e a c y t o s o le i cn z y m ec,l e a v e s briefly translocatedto the exoplasmicface, presumably by
c e r t a i np h o s p h o l i p iidnst h e c y t o s o l li e
c a f l eot f t h e p l a s m a
membrane
may influence membrane curvature (see Figure 10-13c).
Unlike particular phospholipids, cholesterol is relatively
evenly distributed in both leaflets of cellular membranes.
The relative abundanceof a particular phospholipid in the
t w o l e a f l e t so f a p l a s m a m e m b r a n e c a n b e d e t e r m i n e d
experimentally on the basis of the susceptibilityof phos-
pholipids to hydrolysis by phospholipases,enzymesthat
cleavevarious bonds in the hydrophilic ends of phospho-
lipids (Figure 10-14). \7hen added to the external
medium, phospholipasescannot cross the membrane, and
thus they cleave off the head groups of only those lipids
present in the exoplasmic face; phospholipids in the
cytosolic leaflet are resistant to hydrolysis becausethe
enzymescannot penetrateto the cytosolic face of the plasma
memDrane.
How the asymmetric distribution of phospholipids in
membrane leafletsarisesis still unclear.As noted, in pure bi-
layers phospholipids do not sponraneouslymigrate, or flip-
flop, from one leaflet to the other. To a first approximation,
the asymmetry in phospholipid distribution results from
synthesisof these lipids in the endoplasmic reticulum and
Golgi. Sphingomyelinis synthesizedon the luminal (exo-
plasmic) face of the Golgi, which becomesthe exoplasmic
face of the plasma membrane. In contrast, phosphoglyc-
erides are synthesizedon the cytosolic faceof the ER mem-
brane, which is topologically identical with the cytosolic face
of the plasma membrane(seeFigure 10-8). Clearly,this ex-
planation does not account for the preferential location of
CHAPTER
1O I B I O M E M B R A NSET R U C T U R E
phosphatidylcholinein the exoplasmic leaflet. Movement of
this phosphoglyceride,and perhaps others, from one leaflet
to the other in some natural membranesis catalyzedby ATP-
powered transport proteins called flippases,which are dis-
cussedin Chapter 11.
The preferential location of lipids on one face of the
bilayer is necessaryfor a variety of membrane-basedfunc-
tions. For example,the head groups of all phosphorylated
forms of phosphatidylinositol (see Figure 10-5; PI) face
the cytosol. Stimulation of many cell-surfacereceptorsby
their corresponding hormone results in activation of the
cytosolic enzyme phospholipase C, which can then hy-
drolyze the bond connecting the phosphoinositolsto the
soluble phosphoinositols and membrane-embeddeddia-
cylglycerol participate in intracellular signaling pathways
that affect many aspects of cellular metabolism. Phos-
phatidylserinealso is normally most abundant in the cy-
tosolic leaflet of the plasma membrane.In the initial stages
of platelet stimulation by serum, phosphatidylserine is
a flippase enzyme,where it activatesenzymesparticipar-
ing in blood clotting.
C h o l e s t e r oal n d S p h i n g o l i p i d C
s l u s t e rw i t h
S p e c i f i cP r o t e i n si n M e m b r a n eM i c r o d o m a i n s
Membrane lipids are not randomly distributed (evenly
mixed) in eachleaflet of a bilayer.One hint that lipids may
be organized within the leaflets was the discovery that the
lipids remaining after the extraction of plasma membranes
with nonionic detergents predominantly conrain two
species:cholesteroland sphingomyelin.Becausethesetwo
lipids are found in more ordered, less fluid bilayers, re-
searchers hypothesized that they form microdomains,
termed lipid rafts, surrounded by other, more fluid phos-
pholipids that are more readily extracted by detergents.
Some biochemical and microscopicevidencesupports the
existenceof lipid rafts, which in natural membranesare
typically 50 nm in diameter. Rafts can be disrupted by
methyl-B-cyclodextrin,which specificallyextracts choles-
terol out of membranes,or by antibiotics, such as filipin,
that sequestercholesterolinto aggregateswithin the mem-
brane. Such findings indicatethe importance of cholesterol
in maintaining the integrity of these rafts. As judged by
their presencein the complex of cholesterol and sphin-
gomyelin remaining after detergent extraction, lipid rafts
in plasma membranesare thought to be enrichedfor a sub-
set of plasma membraneproteins,including those that par-
ticipate in sensing extracellular signals and transmitting
them into the cytosol. Thus by bringing many key proteins
into close proximity these lipid-protein complexes may
facilitate signalingby cell-surfacereceptorsand the subse-
quent activation of cytosolic events. However, much re-
mains to be learned about the structure and bioloeical
function of lipid rafts.

Biomembranes:Lipid Composition
and Structural Organization
r The eukaryotic cell is demarcatedfrom the external en-
vironment by the plasma membrane and organized into
membrane-limited internal compartments (organellesand
vesicles).
r The phospholipid bilayer, the basic structural unit of all
biomembranes,is a two-dimensional lipid sheet with hy-
drophilic facesand a hydrophobic core, which is imperme-
able to water-solublemoleculesand ions (seeFigure 10-6).
r The primary lipid components of biomembranesare
phosphoglycerides,sphingolipids, and sterols such as cho-
lesterol(seeFigure 10-5).
r Most lipids and many proteins are laterally mobile
biomembranes.
r Membranes can undergo phasetransitions from the fluid
to gel-like states depending on the temperature and com-
position of the membrane.
r Different cellular membranesvary in lipid composition
(see Table 10-1). Phospholipids and sphingolipids are
asymmetricallydistributed in the two leafletsof the bilayer,
whereas cholesterol is fairlv evenlv distributed in both
leaflets.
r Natural biomembranesgenerally have a viscous con-
sistencywith fluidlike properties.In general,membrane
fluidity is decreasedby sphingolipidsand cholesteroland
increasedby phosphoglycerides.The lipid composition of a
membrane also influencesits thickness and curvature (see
F i g u r e1 0 - 1 3 ) .
r Lipid rafts are microdomains containing cholesterol,
sphingolipids,and certain membrane proteins that form in
the plane of the bilayer. These aggregatesmight facilitate
signaling by certain plasma-membranereceptors.
Biomembranes: Protein
and BasicFunctions
Components
Membrane proteins are defined by their location within or at
the surfaceof a phospholipid bilayer.Although every biolog-
ical membrane has the same basic bilayer structure, the pro-
teins associatedwith a particular membrane are responsible
for its distinctive activities. The kinds and amounts of pro-
teins associatedwith biomembranesvary depending on cell
type and subcellular location. For example, the inner mito-
chondrial membrane is 75 percent protein; the myelin mem-
brane that surrounds nerve axons, only 18 percent.The high
phospholipid content of myelin allows it to electricallyinsu-
late the nerve from its environment, as we discussin Chap-
ter 23. The importance of membrane proteins is suggested
from the finding that approximately a third of all yeastgenes
B I O M E M B R A N E SP:R O T E I NC O M P O N E N T A
encode a membrane protein. The relative abundance of
genesfor membrane proteins is greater in multicellular or-
ganisms in which membrane proteins have additional func-
tions in cell adhesion.
The lipid bilayer presentsa distinctive two-dimensional hy-
drophobic environment for membrane proteins. Someproteins
contain segmentsthat arc imbedded within the hydrophobic
core of the phospholipid bilayer; other proteins are associated
with the exoplasmic or cytosolic leaflet of the bilayer. Protein
domains on the extracellular surface of the plasma membrane
generally bind to extracellular molecules, including external
signalingproteins, ions, and small metabolites(e.g.,glucose,
fatty acids), as well as proteins on other cells or in the external
environment. Segmentsof the protein within the plasma mem-
brane have a variety of functions, including those that form
channelsand pores in transport proteins that move molecules
and ions into and out of cells.Domains lying along the cytoso-
lic face of the plasma membrane have a wide range of func-
tions, from anchoring cytoskeletalproteins to the membraneto
triggering intracellular signaling pathways.
In many cases,the function of a membrane protein and
the topology of its polypeptidechain in the membranecan be
predicted on the basis of its similarity with other well-
characterizedproteins. In this section' we examine the char-
acteristicstructural featuresof membraneproteins and some
'We
of their basic functions. will describe the structures of
severalproteins to help you get a feel for the way membrane
proteins interact with membranes.More completecharacter-
ization of the properties of various types of membrane proteins
is presentedin later chapters that focus on their structures
and activitiesin the context of their cellular functions.
ProteinsInteractwith Membranes
in Three DifferentWaYs
Membrane proteins can be classifiedinto three categories-
integral, lipid-anchored, and peripheral-on the basisof the
nature of the membrane-proteininteractions(seeFigure 10-1)'
Integral membrane proteins, also called transrnembrane
proteins, span a phospholipid bilayer and comprise three
segments.The cytosolic and exoplasmic domains have hy-
drophilic exterior surfaces that interact with the aqueous
solutions on the cytosolic and exoplasmic faces of the
membrane. These domains resemble segments of other
water-solubleproteins in their amino acid composition and
structure. In contrast, the membrane-spanning segments
usually contain many hydrophobic amino acidswhose side
chains protrude outward and interact with the hydropho-
bic hydrocarbon core of the phospholipid bilayer. In all
transmembrane proteins examined to date, the membrane-
spanning domains consist of one or more a helicesor of
multiple B strands. Becausethey must be inserted into
membranes, the ribosomal synthesisand posttranslational
processingof integral membrane proteins differs from that
of soluble cytosolic proteins and is discussedseparatelyin
Chapters 13 and 1'4.
S N D B A S I CF U N C T I O N S
421
 Lipid-anchored membraneproteins are bound covalently
to one or more lipid molecules.The hydrophobic segmentof
the attachedlipid is embeddedin one leaflet of the membrane
and anchors the protein to the membrane. The polypeptide
chain itself does not enter rhe phospholipid bilayer.
Peripheral membrane proteins do not directly contact the
hydrophobic core of the phospholipid bilayer. Instead they are
bound to the membraneeither indirectly by interactionswith in-
tegral or lipid-anchored membraneproteins or directly by inter-
actionswith lipid headgroups.Peripheralproteins can be bound
to either the cytosolic or the exoplasmicfaceof the plasmamem-
brane. In addition to theseproteins, which are closelyassociated
with the bilayer,cytoskeletalfilamentscan be more looselyasso-
ciated with the cytosolic face, usually through one or more pe-
ripheral (adapter) proteins. Such associationswith the cy-
toskeleton provide support for various cellular membranes,
helping to determine cell shapeand mechanicalproperties, and
play a role in the two-way communication betweenthe cell in-
terior and the exterior, as we learn in Chapter 17. Finally, pe-
ripheral proteins on the outer surfaceof the plasma membrane
and the exoplasmicdomainsof integralrn.rntr"n. proreinsare
often anached to components of the extracellular matrix or to
the cell wall surrounding bacterial and plant cells, providing a
crucial interface betlveenthe cell and its environmenr.
(a)
Extracellu
lar
domain
M e mb r an e - s p a
nning
helices
Cytosolic
domain
A FIGURE10-15 Structureof glycophorin A, a typical single-
passtransmembrane protein. (a)Diagramof dimericglycophorin
showingmajorsequencefeaturesand its relationto the membrane
The single23-residue membrane-spanning o helixin eachmonomeris
composedof amino acidswith hydrophobic (uncharged)
(redand greenspheres). By bindingnegatively chargedphospholipid
headgroups,the positively chargedarginlneand lysineresidues
spheres) nearthe cytosolicsideof the helixhelpanchorglycophorin
in the membraneBoththe extracellular and the cytosolicdomains
a r e r i c h i n c h a r g e dr e s i d u eas n d p o l a ru n c h a r q e dr e s i d u e st h
422 CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
Most Transmembrane ProteinsHave
M e m b r a n e - S p a n n i nagH e l i c e s
Solubleproteins exhibit hundreds of distinct localizedfolded
structures, or motifs (see Figure 3-9). In comparison, the
repertoireof folded structuresin the transmembranedomains
of integral membrane proteins is quite limited, with the hy-
drophobic ct helix predominating. Proteinscontaining mem-
brane-spanning a-helical domains are stably embedded in
membranes becauseof energeticallyfavorable hydrophobic
and van der rWaalsinteractionsof the hydrophobic sidechains
in the domain with specificlipids and probably also by ionic
interactionswith the polar head groups of the phospholipids.
A single cr-helical domain is sufficient to incorporate
an integral membrane protein into a membrane. However,
many proteins have more than one transmembranecr helix.
Typically, a membrane-embeddedcr helix is composed of a
continuous segment of 20-25 hydrophobic (uncharged)
amino acids (seeFigure 2-14).The predictedlength of such
an ct helix (3.75 nm) is just sufficient to span the hydrocar-
bon core of a phospholipid bilayer. In many membrane pro-
teins, these helices are perpendicular to the plane of the
membrane, whereas in others, the helicesrraversethe mem-
brane at an oblique angle.The hydrophobic side chains pro-
trude outward from the helix (Figure 10,15) and form van
(bI
extracellular
domainisheavily glycosylated,
withthecarbohydrate
sidechains(green diamonds) attached to specific
serine,
threonine,
andasparagine (b)Molecular
residues modelof thetransmemprane
domainof dimeric glycophorin correspondingto resrdues73-96.The
sidechains hydrophobic sidechainsof theo helixin onemonomer aresnownIn
pink;thosein theothermonomer, in greenResidues depictedas
(blue space-filling participate
structures in intermonomer vanderWaals
interactions
thatstabilize
thecoiled-coildimer[part(b)adapted
from
K R M a c K e n z i e e t a l ,1 9 9 7 ,S c i e n c e 2 T 6 : 1 3I1
:e
 (b) Back H a l fh e l i c e s

Exterior

Membrane

Cytosol

FIGURE 10-16Structuralmodelsof two multipassmembrane membrane-spanning

several o helicesthatareat obliqueangles, the
proteins.(a)Bacteriorhodopsin,a photoreceptor in certainbacteria thatpenetrate
two helices onlyhalfway through the membrane
Theseven hydrophobic in bacteriorhodopsin
crhelices traverse the (purple
withyellowarrows), andonelongmembrane-spanning helix
lipidbilayerroughly to the planeof the membrane
perpendicular A witha "break"or distortionin the middle(purple withyellowline)'
retinalmolecule(black) attached
covalently to onehelixabsorbs light Theglycerolmoleculein the hydrophilic "core"iscolored red The
Thelargeclass of G protein-coupledreceptors in eukaryotic cellsalso wasapproximately
structure positioned in the hydrocarbon coreof the
hassevenmembrane-spanning crhelices;theirthree-dimensional membrane byfindingthe most hydrophobic 3 mm slabof the protein
structureisthoughtto besrmilarto thatof bacteriorhodopsin. (b)Two to the membrane
perpendicular plane.lPart (a)AfterH Lueckeetal,
viewsof theglycerol channelGlpf,rotated180"with respect to each 1ggg,J Mol. Biol 291:899 Part(b) after J Bowie,20O5, Nature438:58'l-589,
otheralongan axisperpendicular to the planeof the membrane. Note and D Fu et al , 2000, Sclence290:481-486.1

der.il/aals interactions with the fatty acyl chains in the bi- detail is bacteriorhodopsin' a protein found -in the mem-
layer.In conrrast,the hydrophilic amide peptide bonds are in brane of certain photosynthetic bacteria; it illustrates the
the interior of the cr helix (seeFigure :-4;; e".h carbonyl generalstructureof all theseproteins (Figure-10-16a)'Ab-
(C:O) group forms a hydrogen bond with the amide hy- iorption of light by the retinal group covalently attached to
drogen the amino acid four residuestoward the C- this protein causesa conformational change in the protein
"-to--of helix. These polar groups are shieldedfrom that results in the pumping of protons from-the cytosol
terminus of the
the hydrophobic interior of tLe membrane. across the bacterial membrane to the extracellular space'
To help you get a better senseof the structuresof proteins The proton concentration gradient thus generatedacrossthe
with a-helical domains,we will briefly discussthree Jifferent membrane is used to synthesizeATP (Chapter 1'2)' In the
kinds of such proteins: glycophorin A, G protein-coupled high-resolution structure of bacteriorhodopsinthe positions
receptors,a^d aqrr"pori.i(*"i.riglycerol channels). of all the individual amino acids, retinal, and the surround-
blycophorin- A, the major p-rotein in the erythrocyte ing lipids are clearly defined. As might be expected,virtually
plasma membrane, is a ..pr.r.., itive single-passtiansmem- ali oi the amino acids on the exterior of the membrane-
braneprotein, which containsonly one membrane-spanning
cr helix (Figure10-15).The transmembranehelix of one gly-
cophorin A polypeptide associateswith the corresponding
transmembrane helix in a second glycophorin A to form a
coiled-coildimer (Figure10-15b).Suchinteractionsof mem-
brane-spanningct helicesare a common mechanismfor cre-
ating dimeric membraneproteins, and many membranepro-
teins form oligomers (two or more polypeptides bound
together noncovalently) by interactions between their mem-
b r a n e - s p a ni n g h e l i c e s .
A large and important group of integralproteinsis defined
by the presenceof sevenmembrane-spanning a helices;this in-
cludes the large family of G protein-coupled cell-surfacere-
ceptors discussedin Chapter 15. One such mwbipasstrans-
membrane protein whose structure is known in molecular

B I O M E M B R A N EP
SR C O M P O N E N TASN D B A S I CF U N C T I O N S
: OTEIN
423
 the middle, and more strikingly, there are two c helixes
that penetrate only halfway through rhe membrane. The
N-termini of these helicesface each other (yellow N's in
the figure), and together they span the membrane at an
oblique angle. Thus some membrane-embeddedhelices-
and other nonhelical structureswe will encounter later-
do not traversethe entire bilayer. As we will seein Chap-
ter 1.1,these short helixes in aquaporins form part of the
glycerol/water-selectivepore in the middle of each sub-
unit. This highlights the considerablediversity in the ways
membrane-spanningcr helices interact with the lipid bi-
layer and with other segmentsof the protein.
The specificity of phospholipid-protein interactions is
revealedin great detail in the structure of a different aqua-
porin, aquaporin 0 (Figure 10-17). Aquaporin 0 is the
most abundant protein in the plasma membrane of the
fiber cells that make up the bulk of the lens of the mam-
malian eye. Like orher aquaporins,it is a tetramer of iden-
tical subunits.The protein's surfaceis not coveredby a set
of uniform binding sites for phospholipid molecules.In-
stead, fatty acyl side chains pack tightly against the
Podcast:
AnnularPhospholipiO,
$|
Membrane
FIGURE 10-17Annularphospholipids. Sideviewof the
three-dimensional structure of onesubunitof the lens-specific
aquaporin 0 homotetramer, crystallized
in the presence of the
phospholipid
dimyristoylphosphatidylcholine, a phospholipid with
14 carbon-saturated
fattyacylchainsNotethe lipidmorecures
forminga bilayershellaroundthe proteinTheproteinisshownas
a surfaceplot(thelighterbackground molecule).
Thelipid
molecules areshownin space-fill format;the polarlipidhead
groups(greyandred)andthe lipidfattyacylchains(blackand
grey)forma bilayer with almostuniformthrckness aroundthe
protein.
Presumably, in the membrane, lipidfattyacylchains will
coverthewholeof the hydrophobic surface of the protein;only
the mostordered of the lipidmolecules will be resolvedin the
crystallographic
structure[AfterA Lee,2005,Nature 438:569_570.
and T. Gonen et al , 2005, Nature 439:633-688 l
CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
irregular hydrophobic outer surface of the proteinl some
of the fatty acyl chains are straight, in the all-trans confor-
mation (Chapter 2), whereas others are kinked in order to
interact with bulky hydrophilic side chains on rhe surface
of the protein. Some of the lipid head groups are parallel
to the surface of the membrane, as is the casein purified
phospholipid bilayers. Others, however, are oriented al-
most at right anglesto the plane of the membrane. Thus
there can be specific interactions between phospholipids
and membrane-spanning proteins, and the function of
many membrane proteins can be affected by the specific
types of phospholipid presentin the bilayer.
M u l t i p l e p S t r a n d si n P o r i n sF o r m
M e m b r a n e - S p a n n i n" gB a r r e l s "
The porins are a class of transmembrane proteins whose
structurediffersradicallyfrom that of other integralproreins
basedon a-helical transmembranedomains. Severaltypes of
porins are found in the outer membrane of gram-negative
bacteria such as E. coli and in the outer membranesof mito-
chondria and chloroplasts.The outer membrane prorecrsan
intestinal bacterium from harmful agents (e.g., antibiotics,
bile salts,and proteases)but permits the uptake and disposal
of small hydrophilic molecules, including nutrients and
waste products. Different types of porins in the outer mem-
brane of an E. coli cell provide channelsfor the passageof
specific types of disaccharidesor other small molecules as
well as of ions such as phosphate.The amino acid sequences
of porins contain none of the long hydrophobic segments
typical of integral proteins with o-helical membrane-span-
ning domains. X-ray crystallography has revealed that
porins are trimers of identical subunits. In each subunit,
16 B strands form a sheet that twists into a barrel-shaped
structurewith a pore in rhe center (Figure 10-18). Unlike a
typical water-solubleglobular protein, a porin has a hy-
drophilic interior and a hydrophobic exrerior; in this sense.
porins are inside out. In a porin monomer, the outward-
facing side groups on each of the B strands are hydrophobic
and form a nonpolar ribbonlike band that encirclesthe out-
side of the barrel. This hydrophobic band interacts with the
fatty acyl groups of the membrane lipids or with other porin
monomers. The side groups facing the inside of a iorin
monomer are predominantly hydrophilic; they line the pore
through which small water-solublemoleculescrossthe mem-
brane. (Note that the aquaporins discussedabove, despite
their name, are not porins and contain multiple trurrrr.ri--
braneo. helices.)
CovalentlyAttached HydrocarbonChains
A n c h o rS o m eP r o t e i n st o M e m b r a n e s
In eukaryotic cells,severaltypes ofcovalently attachedlipids
anchor some otherwise typically water soluble proteins to
one or the other leaflet of the plasma membraneor other cel-
lular membranes.In theselipid-anchored proteins, the lipid
 group is bound through a thioether bond to the -SH group
of a b-terminal cysteineresidue.In somecases'a secondger-
anylgeranylgroup or a fatty acyl palmitate group is linked to
a nearby cysteine residue. The additional hydrocarbon an-
chor is thought to reinforce the attachment of the protein to
the membrane. For example, Ras' a GTPase superfamily
protein that functions in intracellular signaling (Chapter 16),
is recruited to the cytosolic face of the plasma membrane by
such a double anchor.Rab proteins' which also belong to the
GTPase superfamily, are similarly bound to the cytosolic
surface of intracellular vesicles by prenyl anchors; these
proteins are required for the fusion of vesicleswith their
target membranes(ChaPtet 1"4).
Some cell-surface proteins and specialized proteins
with distinctivecovalently attachedpolysaccharidescalled

Periplasm

A FIGURE 10-18Structuralmodelof one subunitof OmpX'

a porin found in the outer membraneof E. coli' All porinsare Exterior
t r i m e r itcr a n s m e m b r apnreo t e i n sE a c hs u b u n iitsb a r r esl h a p e d ,
w i t h B s t r a n dfso r m i n gt h ew a l la n da t r a n s m e m b r apnoer ei n t h e
c e n t e rA b a n do f a l i p h a t i(ch y d r o p h o bainc dn o n c y c l isci)d ec h a r n s
(yellow) anda borderof aromatic (ring-containing) sidechains(red)
positionthe oroteinin the bilaver. lAfterG E Schulz,20O0, Curr. Opin
StrucBiol 10:443|

Cytosol

hydrocarbon chains are embedded in the bilayer' but the

protein itself does not enter the bilayer. The anchors used to NHst
insert proteins at the cytosolic face are not used for the exo- (b)Prenylation
(a)Acylation
plasmicface and vice versa.
One group of cytosolicproteins are anchoredto the cy- A FIGURE 10-19Anchoringof plasma-membrane proteins
linked hydrocarbon groups'
tosolic face of a membrane by a fatty acyl group (e.g., to the bilayer by covalently
(a)Cytosolicproteinssuchasv-Srcareassociatedwith the plasma
myristate or palmitate) covalently attachedto an N-terminal
membrane to the
througha singlefattyacylchainattached
glycine residue(Figure 10-t9a). Retention of such proteins
at the membrane by the N-terminal acyl anchor, called
acylation, may play an important role in a membrane-
associatedfunction. For example,v-Src, a mutant form of
a cellular tyrosine kinase, induces abnormal cellular
growth that can lead to cancer but only when it has a bothof whichareunsaturated.
groups, (c)Thelipidanchoron the
myristylatedN-terminus. exoplasmic of
surface the plasmamembrane isglycosylphos-
A second group of cytosolic proteins are anchored to (GPl).
phatidylinositol Thephosphatidylinositol part(red)of this
membranes by a hydrocarbon chain attached to a cysteine anchoicontainstwo fattyacylchains thatextendintothe bilayer.The
residueat or near the C-terminus(Figure 10-19b). Someof phosphoethanolamine unit(purple)
in the anchorlinksit to the
these chains arc prenyl anchors, built from S-carbon Thetwo greenhexagons
protein. represent sugarunits,whichvaryin
isoprene units, which, as detailed in the following section, number,nature,andarrangement GPIanchorsThe
in different
are also used in the synthesisof cholesterol. In these pro- complete of a yeastGPIanchorisshownin Figure13-14
structure
M o l C e l lB i o l 2 : 5 0 4 1
teins, a 1S-carbon farnesyl or 20-carbon geranylgeranyl l A d a p t e d f r o m H S p r o n g e t a l, 2 0 0 1 , N a t u r e R e v

B I O M E M B R A N EP
SR C O M P O N E N TASN D B A S I CF U N C T I O N S
: OTEIN
425
 proteoglycans(Chapter 19) are bound to the exoplasmic
face of the plasma membrane by a third type of anchor
group, glycosylphosphatidylinositol (GpI). The exact
structures of GPI anchors vary gready in different cell
(Figure 10-19c). Therefore GpI anchors are glycolipids.
The GPI anchor is both necessaryand sufficient for bind-
All Transmembrane proteinsand Glycolipids
Are AsymmetricallyOrientedin the Bilayer
faces.The orientation of different types of transmembrane
proteinsis establishedduring their synthesis,as we describe
in Chapter 13. Membrane proteins have never been ob-
served to flip-flop across a membrane; such movemenr, re-
quiring a transient movement of hydrophilic amino acid
residues through the hydrophobic inteiior of rhe mem-
brane, would be energeticallyunfavorable. Accordingly, the
asymmetric topology of a transmembraneprotein, which is
established during its biosynthetic insertion lnro a mem_
brane, is maintained throughout the protein's lifetime. As
drial membrane,chloroplastlamellae,and severalother in_
tracellular membranes.Becausethe carbohydratechains of
glycoproteins and glycolipids in the plasma membrane
426 CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
L i p i do r
protein A antigen
L i p i do r
p rotein O antigen
L i p i do r
protein B antigen
FIGURE 10-20 HumanABOblood group antigens.These
antigensareoligosaccharidechainscovalently
attached to glycolipids
or
glycoproteins
in the plasmamembrane. Theterminaloligosaccharide
sugarsdistinguishthethreeantigens.
Thepresence or absence of the
glycosyltransferases
thataddgalactose(Gal)or N-acetylgalactosamine
(GalNAc)to O antigendetermine a person's
bloodtype
extend into the extracellular space,they are available to in-
teract with components of the extracellular matrix as well
as lectins (proteins that bind specific sugars), growth fac-
tors. and antibodies.
One important consequenceof such interactions is il-
lustrated by the A, B, and O blood group antigens.These
three structurally related oligosaccharidecomponents of
certain glycoproteinsand glycolipids are expressedon the
surfaces of human red blood cells and many other cell
types (Figure 10-20). AII humans have the enzymes for
synthesizingO antigen. Personswith type A blood also
have a glycosyltransferaseenzyme that adds an extra
modified monosaccharide called N-acetylgalactosamine
to O antigen to form A antigen. Those with type B blood
have a different transferasethat adds an extra galactoseto
O antigen to form B antigen. People with both trans-
ferasesproduce both A and B antigen (AB blood type);
those who lack thesetransferasesproduce O antigen only
(O blood type).
Peoplewhose erythrocyteslack the A antigen, the B anti-
gen, or both on their surface normally have antibodies
against the missing antigen(s)in their serum. Thus if a type
A or O person receivesa transfusion of type B blood, anti-
bodies against the B antigen will bind to the introduced red
cells and trigger their destruction. To prevent such harmful
reactions, blood group typing and appropriate matching of
blood donors and recipients are required in all transfusi,ons
( T a b l e1 0 - 2 ) .
 ANTIBODIES
SERUM CAN TYPES
BTtlOl)
RECEIVE
GRI)UP
BI()OD ONRBCS-
ANTIGTNS

Anti-B A and O
A A

Anti-A B and O
ts, B

None All
AB A and B

Anti-A and anti-B O

O O

"See Figure 10-20 for antigen structures.

Lipid-BindingM o t i f s H e l pT a r g e tP e r i p h e r a l
Proteinsto the Membrane
Many water-soluble enzymesuse membrane phospholipids
as their substratesand thus must bind to membranesurfaces'
As exemplified by the phospholipases,many such enzymes
initially bind to the polar head groups of membrane phos-
pholipids to carry out their catalytic functions. As noted ear-
iier, phospholipaseshydrolyze various bonds in the head
groups of phospholipids (seeFigure 1.0-1'4).These enzymes
have an important role in the degradation of damaged or
aged cell membranes and also are active components in
many snake venoms. The mechanismof action of phospho-
lipase 42 illustrates how such water-solubleenzymescan re-
versibly interact with membranes and catalyzereactions at
the interface of an aqueoussolution and lipid surface (inter- Active
facial chemistry).'Whenthis enzymeis in aqueoussolution, site
its Ca2*-containing active site is buried in a channel lined

(b)

FIGURE10-21 Interfacial binding surface and mechanism

the catalyticsite (Figure10-21b).

P r o t e i n sC a n B e R e m o v e df r o m M e m b r a n e s
b y D e t e r g e n t so r H i g h - S a l S
t olutions

acidsthat
change,openinga channellinedwith hydrophobicamino
As a phospholipid moves
leadsfrom the bilayerto the catalyticsite
Ca2* ion (green)binds to the
into the channel,an enzyme-bound
positioning the esterbond to be cleaved(red)next to
headgroup,
Curr'Opin
the catalyticsite. lPart(a)adaptedfrom M H Gelbet al ' 1999'
hydrophobic part of a detergentmolecule is attracted to StrucBiol 9:428 Parl(b),seeD Blow,1991,Nature351:4441

C O M P O N E N TASN D B A S I CF U N C T I O N S 427
B I O M E M B R A N EP
SR: OTEIN
 I O N I CD E T E R G E N T S

-t
H"C o
H C - C H 2 - C H z- C O O - N a +
ll
H 3 C - ( C H 2 ) 1 1- O - S - O - N a +

o
Sodium deoxycholate Sodium dodecylsulfate (SDSI

N O N I O N I CD E T E R G E N T S

HsQ o-(cH2)7-CH3
9Hs
1l
H3c-c-cH2-?1
/o_rcnz_cH2_o)e5_H
(Average)
HsC CHs

(potyoxyet Octylglucoside
nyrrr
lirlS
i, Il lll- pheno|) (octyl-p-D-glucopyranoside)
""r'
A FIGURE 10-22 Structuresof four commondetergents.The naturalproduct;
theothersaresynthetic.Althoughionicdetergents
hydrophobic
partof eachmolecule isshownin yellow;the commonly causedenaturation of proteins,
nonionicdetergents
do
hydrophilic
part,in blue Thebilesaltsodiumdeoxvcholate isa not andarethususefulin solubilizing
integral
membrane proteins

water (Figure 10-22).Ionic detergents,such as sodium de_

soluble in water. The membrane-spanningdomains, how_
oxycholate and sodium dodecylsulfate(SDS), contain a
ever, are rich in hydrophobic and uncharged residues (see
charged group; nonionic detergents,such as Triton X_100
Figure 10-15). When separated from membranes, these
and octylglucoside,lack a charged group. At very low con_
exposed hydrophobic segmenrstend to interact with one
centrations, detergentsdissolve in pure water as isolated
molecules.As the concentration incriases,the moleculesbe_
gin to form micelles-small. spherical aggregatesin which
hydrophilic parts of the molecule, f".e Lut*ard and the
hydrophobic parts clusrerin rhe center (seeFigure 10_6(c)).
The critical micelle concentration (CMC) at which micelles
form is characteristicof each detergentand is a function
of proteins can rhen be purified by affinity chromatography
the structuresof its hydrophobic and hydrophilic parts.
and other techniques used in purifying water-soluble pro_
Ionic detergents bind to the exposed hydrofhobi. ,._
teins (Chapter3).
gions of membrane proteins as well as to the hvdrophobic
As discussedpreviously, most peripheral proteins are
cores of water-solubleproteins. Becauseof rheir .iurg., .
bound to specific transmembraneproieins or membrane
these detergentsalso disrupt ionic and hydrogen borrds.Ai
phospholipids by ionic or other weak interactions. Gener_
high concenrrations,for example, sodium Jodecylsulfate
ally, peripheral proteins can be removed from the membrane
completely denatures proteins by binding to every side
by solutions of high ionic strength (high salt concentrations),
chain, a properry that is exploited in SDSgel electrophoresis
which disrupt ionic bonds, or by chemicalsthat bind divaleni
(seeFigure 3-35). Nonionic detergenlsgenerally do
not de_ cations such as Mgt*. Unlike integral proteins, most periph_
nature proteins and are thus useful in extracting proteins
eral proteins are soluble in aqueoussolution ,r..J rroi be
from membranesbefore the proteins are purified.-Tirese "rrd
de_ solubilized by nonionic detergents.
tergentsact in different ways at different concentrations.
At
high concentrations (above the CMC), they solubilize
bio_
logical membranesby forming mixed micelies of detergent,
phospholipid, and integral membrane proteins (Figure
t0_ Biomembranes:Protein Components
23). At low concentrarions (below the CMC). these
deter_
gents bind to the hydrophobic regions of most integral and BasicFunctions
mem_
brane proteins, making them soluble in aqueoussolution. r Biological membranes usually contain both integral
Treatmenr of cultured cells with a buffered salt solution (transmembrane)proteins and peripheral membrarr. p.o_
containing a nonionic detergentsuch as Triton X_100 extracts teins,which do not enter the hydrophobic core of the bilaver
water-solubleproteins as well as integral membraneprotelns. ( s e eF i g u r el 0 - I ) .
As noted earlier, the exoplasmic and cytosolic domains
of r Most integral membrane proteins contain one or more
integral membrane proteins g.rr.."ily hydrophilic and membrane-spanninghydrophobic a helices bracketed by
"r.
428 . cHAprE1 R0 | B T o M E M B R A s rNREu c r u R E
 < FIGURE 10-23Solubilization of integral
membraneproteinsbY nonionic
detergents.At a concentration higherthan
Concentration concentration
micelle
itscritical (CMC),a
above CMC detergent lipidsandintegral
solubilizes
membrane formingmixedmicelles
proteins,
containing detergent,protern,andlipid
molecules At concentrationsbelowthe CMC,
nonionic detergents(eg., octylglucoside,
TritonX-100)candissolve membrane proteins
withoutformingmicelles by coating the
membrane-spanning regions

Dissolved
but not
forming
micelles

hydrophilic domains that extend into the aqueous solu- One focus will be on fatty acids, the precursors of the phos-
tions surrounding the cytosolic and exoplasmicfacesof the
membrane(seeFigures10-15 and 1,0-16).
r Fatty acyl side chains as well as the polar headsof mem-
brane lipids groups pack tightly and irregularly around the
hydrophobic segmentsof integral membrane proteins.
r All transmembraneproteins and glycolipids are asym-
metrically oriented in the bilayer; invariably carbohy-
drate chains are attachedto the exoplasmicsurfaceof the
proteln. helps controls calcium metabolism; and other biologically
r The porins, unlike other integral proteins, contain activelipids.
membrane-spanningB sheetsthat form a barrel-likechan-
nel through the bilayer (seeFigure 10-18).
o
r Long-chain lipids attachedto certain amino acids anchor
H 3 C- ( C H 2 ) nc- - o - c H 2
some proteins to one or the other membrane leaflet (see ol
F i g u r e1 0 - 1 9 ) .
H3C-(CH2)"-c-o-cH
r The binding of a water-solubleenzyme (e.g.,a phospho-
lipase, kinase, or phosphatase)to a membrane surface ?l
brings the enzyme close to its substrateand in some cases H3C-(CH2),-c-o-cH2
activatesit. Such interfacial binding is often due to the at- Triacylglycerol
traction between positive chargeson basic residuesin the
protein and negativechargeson phospholipid head groups
in the bilayer. Sfe focus our discussion of lipid biosynthesis and
movement on the major lipids found in cellular mem-
r Transmembraneproteins are selectivelysolubilized and
branes and their precursors.In lipid biosynthesis'water-
purified with the use of nonionic detergents.
soluble precursors are assembled into membrane-
intermediates that are then converted into
"sroci"tei lipid products. The movement of lipids, espe-
membrane
cially membtuni .o-ponents' between different or-
SPhingoliPids,
Phospholipids, g"n.il., is critical for maintaining the proper composition
Synthesis
and Cholesterol: i.rd p.op..ties of membranes and overall cell structure,
b.rt ou, understandingof such intracellular lipid transport
Movement
and Intracellular is still rudimentarY'
In this section,we considersome of the specialchallenges A fundamental principle of membrane biosynthesis is
that a cell faces in synthesizingand transporting lipids, that cells synthesizenew membranes only by the expansion
which are poorly soluble in the aqueousinterior of cells. of existing membranes. Although some early steps in the

MOVEMENT 429
SS
PHOSPHOLIPID P,H I N G O L I P I DASN,D C H O L E S T E R OSLY: N T H E S IASN D I N T R A C E L L U L A R
 synthesisof membrane lipids take place in the cytoplasm,
the final steps are catalyzedby enzymesbound to preexist-
ing cellular membranes,and the products are incoiporated
into the membranesas they are generated.Evidencefor this
phenomenon is seen when cells are briefly exposed to ra-
dioactiveprecursors(e.g.,phosphateor fatty all the
"iidr;,
phospholipids and sphingolipids incorporating these pre-
cursor substancesare associatedwith intracellular mem_

membranes. FIGURE 10-24 Bindingof a fatty acidto the hydrophobic

pocketof a fatty-acid-binding protein(FABP). Thecrystal
structure of adipocyte FABp(ribbondiagram) reveals that the
Fatty Acids Synthesists Mediated hydrophobic bindingpocketisgenerated fromtwo B sheets that
by Severallmportant Enzymes a r en e a r l a
y t r i g h ta n g l e tso e a c ho t h e l f o r m i n ga c l a m - s h e l l - l i k e
structureA fattyacid(carbons yellow;oxygens red)interacts
noncovalently with hydrophobic aminoacidresidues withinthis
pocket. [SeeA Reese-Wagoner
et al , j999, Biochim Biophys Acta
23:144 1(2-3). 106-1 16 l

urated and unsaturatedchains.

Fatty acids are synthesizedfrom the two-carbon building
..
block acetate,CH3COO-. In cells,both acetateand the in_
termediates in fatty acid biosynthesis are esterified to the
large water-solublemoleculecoenzymeA (CoA), as exempli-
fied by the srructure of acetyl CoA:
SmallCytosolicProteinsFacilitate
Movement of Fatty Acids
In order to be transported through the cell cytoplasm free,
or unesterified,fatty acids (those unlinked to a CoA). com_
monly are bound by fatty-acid-binding proteins (FABps),

CoenzymeA (CoA)

Acetyl CoA is an important intermediatein the metabo- which belong to a group of small cytosolic proteinsthat
lism of glucose,fatty acids,and many amino acids,as detailed facilitate the intracellular movement of many Iipids.These
in Chapter 12. It also contributes acetyl groups in many proteins contain a hydrophobic pocket lined by
biosynthetic pathways. Saturated fatty' aC-ids B sheets
lno double (Figure 10-24). A long-chain fatty acid can fit into this
bonds) containing 14 or 16 carbon atoms are made from pocket and interact noncovalently with the surrounding
acetyl CoA by two enzymes,acetyl-CoA carboxylase and protetn.
fatty
acid synthase.In animal cells,theseenzymesare found in the
plants, they are found in chloroplasts.palmitoyl
7t9sof;.in
CoA (16 carbon fatty acylgroup linked to C-oe)can be elon_
gated to 18-24 carbons by the sequential addition of two_
carbon units in the endoplasmicreticulum (ER) or sometimes
in the mitochondrion. Desaturaseenzymes,also locatedin the
ER, introduce double bonds at specificpositionsin somefatty
acids, yielding unsaturated fatty acidi. Oleyl CoA (oleat!
linked to CoA, see Table 2-4), for example,is formed by re_
moval of two H atoms from stearyl CoA. In contrast to free
fatty.acids, fatty acyl CoA derivativesare soluble in aqueous
solutions becauseof the hydrophilicity of the CoA sesment.

430 . cHAprER
to I BToMEMBRA
s rNREU c r u R E
 I
con
\, ?
- *6-5-CoA
F a t t Y a c YG
l oA CMP
CDP-choline
+
c-@@choline c--@
H'"tCl --C H , Glycerol phosphate

OH OH Phosphatidic z;' Diacyl-

glycerol
Choline
acid
2CoA< OH .;
Cytosol
2.)
>!Y

ER
membrane
E
@

o-g
Lumen GPAT LPAAT x;:
(acvltransferases)
Choline

A FIGURE 10-25Phospholipid synthesis. Becausephospholipids

areamphipathic molecules, the laststages synthesis
of theirmultistep
takeplaceat the interface
between a membrane andthe cytosoland
arecatalyzedby membrane-associated enzymesStep([): Twofatty
acidsfromfattyacylCoAareesterified to the phosphorylated
glycerolbackbone, formingphosphatidic acld,whosetwo long leaflet.
formedto theexoPlasmic
hydrocarbon chains anchorthe molecule to the membrane. Step(Z):

Incorporationof Fatty Acids into Membrane through vesicle-mediatedmechanismssimilar to those dis-

L i p i d sT a k e sP l a c eo n O r g a n e l l eM e m b r a n e s
Fatty acids are not directly incorporated into phospholipids;
rather, in eukaryotic cells they are first converted into CoA
esters.The subsequentsynthesisof many diacyl glycetophos-
pholipids from fatty acyl CoAs, glycerol 3-phosphate,and
polar head-groupprecursorsis carried out by enzymesassoci-
ated with the cytosolic face of the ER membrane,usually the
smooth ER, in animal cells(Figure10-25);the ER is described
in detail in Chapters9 and L3. Mitochondria synthesizesome
of their own membranelipids and import others.
Sphingolipidsare derivativesof sphingosine,an amino alco-
hol that containsa long, unsaturatedhydrocarbonchain (Fig-
ure 10-5). Sphingosineis made in the ER, beginningwith the
coupling of a palmitoyl group from palmitoyl CoA to serine;
the subsequentaddition of a second fatty acyl group to form
N-acyl sphingosine(ceramide)also takesplace in the ER. The
later addition of a polar head group to ceramidein the Golgi
yieldssphingomyelin, whose head group is phosphorylcholine,
and various glycosphingolipids, in which the head group may
be a monosaccharideor a more complex oligosaccharide(see
Figure 10-5b). Somesphingolipidsynthesiscan also take place
in mitochondria. In addition to serving as the backbone for
sphingolipids, ceramide and its metabolic products are
important signaling molecules that can influence cell growth'
proliferation,endocytosis, resistance to stress,and apoptosis.
After their synthesisis completed in the Golgi, sphin-
golipids are transported to other cellular compartments
.orr.J in Chapter 14. In contrast,phospholipids,as well as
cholesterol,can move betweenorganellesby different mech-
anisms,describedbelow.
s o v e P h o s p h o l i p i dfsr o m O n e
F l i p p a s eM
MembraneLeafletto the OppositeLeaflet
Even though phospholipids are initially incorporated into
the cytosolic leaflei of the ER membrane' various phospho-
lipids are asymmetrically distributed in the two leaflets of
tlie ER membrane and of other cellular membranes' As
noted above, phospholipids spontaneouslyflip-flop from
one leaflet to the other only very slowly' For the ER mem-
brane to expand by growth of both leaflets and have asym-
metrically distributed phospholipids, its phospholipid com-
ponents must be able to rapidly and selectivelyflip-flop from
tne membraneleaflet to the other. Although the mechanisms
leafletto the other (seeFigure 1'1'-1'6).
The usual asymmetric distribution of phospholipids in
membrane leaflets is broken down as cells (e'g', red blood
cells) become senescentor undergo apoptosis' For instance,
phosphatidylserineand phosphatidylethanolamineare prefer-
..rtl"ity locatedin the cytosolic leaflet of cellular membranes'

O 431
, D C H O L E S T E R OSLY
P H O S P H O L I P I DSSP, H I N G O L I P I DASN SN D I N T R A C E L L U L AMRO V E M E N T
: N T H E S IA

Cholesterolls Synthesizedby Enzymes
i n t h e C y t o s o la n d E RM e m b r a n e
Next we focus on cholesterol,the principal sterol in animal
cells.The first stepsof cholesterolsynthesis(Figure 10-26)-
protein, even though both its subsrrateand its product are
water soluble.The water-solublecatalyticdomain of HMG_
are high, binding of cholesterolto this domain causesthe
protein to bind to two other integral ER membrane pro-
teins, Insig-1 and Insig-2. This in turn induces ubiquitina-
t i o n ( s e e F i g u r e 3 - 2 9 ) o f H M G - C o A r e d u c t a s ea n d i t s
degradationby the proteosomepathway, reducingthe pro-
duction of mevalonate,the key intermediatein cholesterol
biosynthesis.
Atherosclerosis,frequently calledcholesterol-dependent
clogging of the arteries, is characterized by the pro-
gressivedeposition of cholesteroland other lipids. cells. and
e x t r a c e l l u l am
r a r r i x m a t e r i a li n t h e i n n e rl a y e i o f r h e w a l l o f
an artery. The resulting distortion of the artery's wall can
Iead, either alone or in combination with a blood clot, ro
major blockage of blood flow. Atherosclerosisaccounts for
75 percent of deaths due to cardiovascular diseasein the
United States.
Cholesterolis synthesizedmainly in the liver. perhaps
.
the most successfulanti-atherosclerosismedications aie
the statins. Thesedrugs bind to HMG-CoA reductaseand
directly inhibit its activity, thereby lowering cholesterol
biosynthesis.As a consequence, the amount of low-density
lipoproteins (see Figure 14-27)-the small, membrane-
envelopedparticles containing cholesterol esterifiedto fatty

Acetyl CoA * acetoacetylCoA

S-CoA
II
J
-o/'-AAs-coa HMG-CoA
,HMG-CoA
reductase
J
Mevalonate
J
J
OPP lsoPentenyladenosine
lsopentenylpyrophosphat"'z
(lPP) ' M a n y o t h e ri s o p r e n o i d s

Squalene

V i t a m i nD
*
- B i l ea c i d s
CholesterolI Steroid hormones
'
HO Cholesterolesters
M o d i f i e dp r o t e i n s( H e d g e h o g )
FIGURE 10-26Cholesterol biosyntheticpathway.The which hasthe basicfive-carbonisoprenoidstructurelpp can be
regulated
rate-controlling
stepin cholesterol
biosynthesis
isthe convertedinto cholesteroland into manyother lipids,often through
conversion
of B-hydroxy-B-methylglutaryl
CoA(HMG_CoA) into the polyisoprenoid intermedlates
shown here.Someof the numerous
mevalonic
acidby HMG-CoA reductase,
an ER_membrane protein. compoundsderivedfrom isoprenoidintermediates and cholesterol
Mevalonateisthenconvertedintoisopentenyl
pyrophosphate
(lpp), itselfare indicated.

432 CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
acids that often and rightly are called "bad cholestslol"-
drops in the blood, reducing the formation of atheroscle-
rotic plaques.I
Mevalonate,the 6-carbon product formed by HMG-CoA
reductase,is convertedin severalstepsinto the 5-carbon iso-
prenoid compound isopentenylpyrophosphate (IPP) and its
stereoisomer,dimethylallyl pyrophosphate (DMPP) (see
Figure 70-26). Thesereactionsare catalyzedby cytosolic en-
zymes, as are the subsequentreactionsin the cholesterol
synthesispathway, in which six IPP units condenseto yield
squalene,a branched-chain30-carbon intermediate.Enzymes
bound to the ER membrane catalyzethe multiple reactions
that convert squaleneinto cholesterol in mammals or into
related sterols in other species.One of the intermediatesin
this pathway, farnesylpyrophosphate,is the precursor of the
prenyl lipid that anchors Ras and related proteins to the
cytosolicsurfaceof the plasmamembrane(seeFigure10-19)
as well as other important biomolecules(seeFigure 10-26\.
C h o l e s t e r oal n d P h o s p h o l i p i dAs r e T r a n s p o r t e d
B e t w e e nO r g a n e l l e sb y S e v e r a M l echanisms
As already noted, the final stepsin the synthesisof choles-
terol and phospholipids take place primarily in the ER,
although some of thesemembranelipids (plasmalogens) are
produced in mitochondria and peroxisomes. Thus the
plasma membraneand the membranesbounding other or-
ganellesmust obtain theselipids by meansof one or more in-
tracellulartransportprocesses. Membranelipids accompany
both soluble and membrane proteins during the secretory
pathway describedin Chapter 14; membranevesiclesbud
from the ER and fuse with membranesin the Golgi complex,
and other membrane vesiclesbud from the Golgi complex
and fusewith the plasmamembrane(Figure10-27a).How-
ever,severallines of evidencesuggestthat there is substantial
interorganelle movement of cholesterol and phospholipids
through other mechanisms. For example, chemical in-
hibitors of the classicsecretorypathway and mutationsthat
impede vesiculartraffic in this pathway do not prevent cho-
lesterolor phospholipidtransport betweenmembranes.
A second mechanism entails direct protein-mediated
contact of ER or ER-derivedmembraneswith membranesof
other organelles(Figure 10-27b). In the third mechanism,
small lipid-transfer proteins facilitate the exchangeof phos-
pholipids or cholesterol between different membranes (Fig-
r:.teIO-27c). Although such transfer proteins have beeniden-
tified in assaysin vitro, their role in intracellular movements
of most phospholipids is not well defined.For instance,mice
with a knockout mutation in the gene encoding the phos-
phatidylcholine-transferprotein appear to be normal in
most respects,indicating that this protein is not essentialfor
cellularphospholipidmetabolism.
As noted earlier, the lipid compositions of different or-
ganellemembranesvary considerably(seeTable 10-1).Some
of th.r. differencesare due to different sitesof synthesis.For
example,a phospholipid calledcardiolipin, which is localized
to the mitochondrial membrane, is made only in mitochon-
dria and little is transferredto other organelles.Differential
transport of lipids also plays a role in determining the lipid
compositions of different cellular membranes.For instance,
even though cholesterol is made in the ER, the cholesterol
molar ratio) is
concentration (cholesterol-to-phospholipid
-1.5-13-fold higher in the plasma membranethan in other
organelles(ER, Golgi, mitochondrion, lysosome)' Although
the mechanismsresponsiblefor establishingand maintaining
thesedifferencesare not well understood' we have seenthat
the distinctivelipid composition of eachmembranehas a ma-
ior influenceon its physical and biological properties'
and Cholesterol:
Phospholipids, Sphingolipids,
Movement
Synthesisand Intracellular
r Saturatedand unsaturatedfatty acidsof variouschain
Iengthsare components sphingolipids'
of phospholipids,
and triglycerides.

(b) (c)
{a)

Binding
proteln
m
,z\y\

Vesicle
\,/
Hypothetical Binding
proteins protein

Cytosol Cytosol Cytosol

FIGURE 10-27Proposedmechanisms of transportof betweenmemDranes thatis mediated by membrane-embedded

phospholipids between membranes. In proteinsIn mechanism (c),transfer by small,soluble
is mediated
and
cholesterol
(a),vesicles ln proteins
lipid-transfer lAdaptedfrom F R andD Wustnel
Maxfield 2002'
mechanism lipidsbetweenmembranes
transfer
mechanism(b),lipidtransfer
isa consequenceof directcontact I Clin lnvest 110:891 l

e 433
: N T H E S IASN D I N T R A C E L L U L AMRO V E M E N T
P H O S P H O L I P I DSSP,H I N G O L I P I DASN,D C H O L E S T E R OSLY
 Fatty acids are synthesizedfrom acetyl CoA by water-
luble enzymesand modified by elongation and desatura-
tion in the endoplasmicreticulum (ER).
r The final stepsin the synthesisof glycerophospholipids,
plasmalogens, and sphingolipids are catalyzed by mem-
brane-associatedenzymesprimarily on the cytosolic face of
the ER (seeFigure 10-25).
r Each type of lipid is initially incorporated the pre-
existing membraneson which it is made.
r Most membranephospholipids are preferentially distrib-
uted in either the exoplasmic or the cytosolic leaflet. This
a.symmetryresults in part from the action of phospholipid
flippases.
The initial stepsin cholesterolbiosynthesistake place in
e cytosol, whereasthe last stepsare catalyzedby enzymes
sociatedwith the ER membrane.
r The rate-controlling step in cholesterol biosynthesisis
catalyzedby HMG-CoA reductase,whose rransmembrane
segmentsare embeddedin the ER membrane and contain a
sterol-sensingdomain.
r Considerableevidenceindicates that Golgi-independent
vesicular transport, direct protein-mediated contacts
between different membranes,soluble protein carriers, or
all three may account for some inter-organelletransport of
cholesteroland phospholipids(seeFigure 10-27).
for transporting cholesterol and phospholipids between or-
ganelle membranes remain poorly characterized.In particu-
lar, we lack a detailed understandingof how various trans-
port proteins move lipids from one membrane leaflet to
another (flippaseactivity) and into and out of cells.Such un-
derstanding will undoubtedly require a determination of
many high-resolutionstructuresof thesemolecules,their cap-
ture in various stagesof the transport process,and careful ki-
netic and other biophysicalanalysesof their function, similar
to the approachesdiscussedin Chapter 11 for elucidatingthe
operation of ion channelsand ATP-poweredpumps.
Recent advancesin solubilizing and crystallizing integral
membrane proteins have led to the delineation of the molec-
ular structuresof many important types of proteins, such as
ion channels,ATP-powered ion pumps, and aquaporins, as
we will seein Chapter 11. However, many important classes
of membraneproteins have proven recalcitrant to even these
new approaches.For example, we lack the structure of any
protein that transports glucoseinto a eukaryotic cell. As we
will learn in Chapters 15 and 16, many classesof receptors
span the plasma membrane with one or more o helixes.per-
haps surprisingl5 we lack the molecular structure of the
transmembrane segment of any eukaryotic cell-surfacere-
ceptor, and so many aspectsof the function of theseproteins
are still mysterious. Elucidating the molecular structures of
these and many other types of membrane proteins will clar-
ify many aspectsof molecular cell biology.

KeyTerms

acylation 425 internalface414

One fundamental question in lipid biology concernsthe gen-
eration, maintenance,and function of the asymmetricdistri- brle acld 416 Ieaflet411
bution of lipids within the leafletsof one membrane and the cholesterol 41.5 lipid raft 420
critical micelle liposome411
concentration(CMC) 428 lumen4L0
cytosolic face414 micelle411
detergent 427 peripheralmembrane
exoplasmic face414 protein422
external face414 phosphoglyceide 415
flippase 420 phospholipidbllayer 4 11
fluorescencerecovery after plasmologen 415
photobleaching(FRAP)41 7 porin 423
Major controversiessurround the existenceof lipid rafts glycolipid 416
in biological membranesand their function in cell signaling.
prenyl anchor425
GPI anchor 426 receptorproteins409
Many biochemicalstudiesusing model membranesshow that
HMG-CoA reductase432 sphingolipid 416
hydrophobic41 1 statrn432
hydrophilic 411 sterol-sensing domain432
integral membrane
protein 421

structure and are too small to be resolved by fluorescence
microscopy.Proving their existencein cellswill require devel_
opment of new biophysicaland microscopictools.
Despiteconsiderableprogressin our understandingof the
cellular metabolism and movement of lipids, the mechanisms
Review the Concepts
l. \flhen viewed by electron microscopy the lipid bilayer is
often describedas looking like a railroad track. Explain how
the structure of the bilayer createsthis image.

434 o cHAprE1
Ro I BToMEMBRA
s rNREU c r u R E

2. Biomembranes contain many different types of lipid
molecules.What are the three main types of lipid molecules
found in biomembranes?How are the three types similar,
and how are they different?
3. Proteinsmay be bound to the exoplasmicor cytosolic
face of the plasma membrane by way of covalently
attached lipids. -What are the three types of lipid anchors
responsiblefor tethering proteins to the plasma membrane
bilayer, and which type is used by cell-surfaceproteins
that face the external medium and by glycosylated
proteoglycans?
4. Lipid bilayers are consideredto be two-dimensional
fluids; what does this mean?'Whatdrivesthe movementof
lipid molecules and proteins within the bilayer? How can
such movement be measured?What factors affect the degree
of membrane fluidity?
5. Phospholipid biosynthesisat the interface between the
endoplasmicreticulum (ER) and the cytosol presentsa num-
ber of challengesthat must be solved by the cell. Explain
how each of the following is handled.
a. The substratesfor phospholipid biosynthesisare all
water soluble, yet the end products are not.
b. The immediate site of incorporation of all newly synthe-
sized phospholipids is the cytosolic leaflet of the ER mem-
brane, yet phospholipids must be incorporated into both
leaflets.
c. Many membrane systemsin the cell, for example' the
plasma membrane, are unable to synthesizetheir own phos-
pholipids, yet these membranesmust also expand if the cell
is to grow and divide.
6. Fatty acidsmust associatewith lipid chaperonesin order
to move within the cell. \7hy are these chaperonesneeded,
and what is the name given to a group of proteins that are
responsible for this intracellular trafficking of fatty acids?
'What
is the key distinguishing feature of theseproteins that
allows fatty acids to move within the cell?
7. What are the common fatty acid chains in glycerophos-
pholipids, and why do these fatty acid chains differ in their
number of carbon atoms by multiples of 2?
8. The biosynthesisof cholesterol is a highly regulated
'What
process. is the key regulated enzyme in cholesterol
biosynthesis?This enzyme is subject to feedback inhibition.
What is feedback inhibition? How does this enzyme sense
cholesterollevelsin a cell?
9. It is evident that one function of cholesterolis structural
becauseit is the most common single lipid molecule in the
plasma membrane of mammals. Yet cholesterol may also
'Strhat
have other functions. aspectsof cholesterol and its
metabolism lead to the conclusion that cholesterolis a mul-
tifunctional membrane lipid?
10. Phospholipidsand cholesterolmust be transportedfrom
their site of synthesisto various membrane systemswithin
cells. One way of doing this is through vesicular transport'
as is the case for many proteins in the secretory pathway.
However, most phospholipid and cholesterolmembrane-to-
membrane transport in cells is not by Golgi-mediatedvesic-
'S7hat
ular transport. is the evidence for this statement?
\fhat appear to be the maior mechanismsfor phospholipid
and cholesteroltransport?
11. Explain the following statement: The structure of all
biomembranesdependson the chemical properties of phos-
pholipids, whereas the function of each specific biomem-
brane dependson the specific proteins associatedwith that
membrane.
12. Name the three groups into which membrane-associated
proteins may be classified.Explain the mechanism by which
each group associateswith a biomembrane.
13. Although both facesof a biomembraneare composed
of the same general types of macromolecules,principally
lipids and proteins, the two faces of the bilayer are not
iJentical. What accounts for the asymmetry between the
two faces?
Analyze the Data
The behavior of receptor X (XR)' a transmembraneproteln
present in the plasma membrane of mammalian cells, is be-
ing investigated.The protein has been engineeredas a fusion
protein containing the green fluorescentprotein (GFP) at its
N-terminus. GFP-XR is a functional protein and can replace
XR in cells.
a. Cells expressingGFP-XR or artificial lipid vesicles
(liposomes)containing GFP-XR are subjectedto fluores-
cence recovery after photobleaching (FRAP). The intensity
of the fluorescenceof a small spot on the surfaceof the cells
(solid line) or on the surfaceof the liposomes(dashedline) is
measured prior to and following laser bleaching (arrow)'
The data are shown below
5000
o
q)
c
q)
o
c)
o
0)
L
f
1000
25 50 75 100
T i m e( s )
\What explanation could account for the differing behavior
of GFP-XR in liposomesversusin the plasma membrane of
a cell?
b. Tiny gold particles can be attached to individual
moleculesand their movement then followed in a light mi-
croscopeby single-particletracking. This method allows one
to observe the behavior of individual proteins in a mem-
brane. The tracks generatedduring a 5-secondobservational
period by a gold particle attached to XR present in a cell
ANALYZETHE DATA 435
 (left) or in a liposome (middle) or to XR adheredto a micro- Simons,K., and \7. L. C. Vaz. Model sysrems,lipid rafts, and
scopeslide (right) are shown below. cell membranes.2004.Annw. Reu.Bio\hys. Biomolic. Strwct.
33:269-295.
Thmm, L. K., V. K. Kiessling,and M. L. Wagner.2001.Membrane
€ dynamics.Encyclopediaof Life Sciences.Nature Publishing Group.
Vance,D. E., and J. E. Vance.2002. Biochemistryof Lipids,
Lipoproteins, and Membranes, 4th ed. Elsevier.
XR present XR present XR presenton a
in a cell in a liposome m i c r o s c o p es l i d e Van Meer, G.2006 Cellular lipidomics.EMBO J.
24:31.59-31.65.
\fhat additional information do these data provide beyond Yeager,P.L.2001,.Lipids. Encyclopediaof Life Sciences. Narure
what can be determinedfrom rhe FRAp data? PublishingGroup.

c. Fluorescenceresonanceenergy transfer (FRET) is a .. Zimmerberg,J., and M. M. Kozlov. 2006. How proteinsproduce

technique by which a fluorescent molecule, following its
excitation with the appropriatewavelengthof light, can trans-
fer its emissionenergy ro and excite a nearby different fluo-
rescent molecule (seeFigure 15-1,4).Cyan fluorescenr pro-
tein (CFP) and yellow fluorescentprotein (yFp) are relared
to GFP but fluorescear cyan and yellow wavelengthsrather
than at green. If CFP is excited with the appropnare wave,
length of light and a YFP molecule is very near, rhen energy
can be transferred from CFP emission and used to excite
YFP, as indicated by a loss of emission of cyan fluorescence
and an increasein emissionof yellow fluorescence.CFP-XR
and YFP-XR are expressedrogetherin a cell line or are both
incorporated into liposomes. The number of molecules of
YFP-XR and CFP-XR per cm2 of membrane is equivalent in
the cells and the liposomes.The cells and liposomesare then
irradiated with a wavelengthof light that causesCFp but not
YFP to fluoresce. The amount of cyan (CFp) and yellow
(YFP) fluorescenceemitted by the cells (solid line) or lipo-
somes(dashedline) is then monitored, as shown below.
cellufarmembranecurvature.Nature Reu.Mol. Cetl Biol.7:9-1,9.
10.2 Biomembranes: Protein Components
and Basic Functions
Bowie,J. Solvingrhe membraneprotein folding problem. 2005.
Nature 438:581-589.
Cullen,P.J.,G.E. Cozier,G. Banting,and H. Mellor. 2001.
Modular phosphoinositide-bindingdomains:their role in signalling
and membranetrafficking. Curr. Biol. 11:R882-R893.
Engelman,D. Membranesare more mosaicthan fluid. 2005.
Nature 438:578-580.
Lanyi, J. K., and H. Luecke.2001. Bacteriorhod,oosin. Curr.
Opin. Struc.Biol. 1l:415-519.
Lee, A. G. A greasygrip. 2005. Nature 438:569-570.
MacKenzie,K. R., J. H. Prestegard,and D. M. Engel,man.1997.
A transmembranehelix dimer: structureand implications.Science
276:131-133.
Mclntosh, T. J., and S. A. Simon. 2006. Rolesof bilayer mate-
rial propertiesin function and distribution of membraneproreins
Ann. Reu.Biophys. B iomolec.Struct. 3 5 :177-198.
^ Schulz,G- E. 2000. B-Barrelmembraneproteins. Curr. Opin.
Struc. Biol. 10:443447.

10.3 Phospholipids, Sphingolipids, and Cholesterol:

20 Synthesis and Intracellular Movement
o Bloch, K. 1965. The biological synthesisof cholesterol.Science
c
o Yellow light 150:1.9-28.
c
0) Daleke,D. L., and J. V. Lyles.2000. Identification and purifica-
c tion of aminophospholipidflippases.Biochim. Biophys. Acia
o 1' "n
o t486:1.08-127.
o
q)
Futerman,A., and H. Riezman.2005. The inns and outs of
sphingolipidsynrhesis.TrendsCell Biol. lS:312-318.
L
Hajri, T., and N. A. Abumrad. 2002. Fatty acid rransport across
membranes:relevanceto nutrition and metabolic pathology.Ann.
Reu.Nutr. 22:383-41,5.
475 500 525 Henneberry,A. L., M. M. \7right, and C. R. McMaster. 2002.
(nm)of emittedfluorescent The major sitesof cellular phospholipid synthesisand molecular
Wavelength light determinanrs oI fatty acid and lipid head group specificity. Mol.
$7hat can be deducedabout XR from thesedata? Biol. Cell 13:3148-3161.
. Holthuis, J. C. M., and T. P. Levine.2005. Lipid traffic: floppy
References drivesand a superhighwayNature Reu.Molec. Cblt Biot. 6JO9:r20.
Ioannou, Y. A. 2001. Multidrug permeasesand subcellular
10.1 Biomembranes: Lipid Composition cholesteroltransporr.Nature Reu.Mol. Cetl Biol. 2:657-66g.
and Structural Organization Kent, C. 1995. Eukaryotic phospholipid biosynthesis.Ann. Reu.
Biochem.64:315-343.
. McMahon, H., and J. L. Gallop. 2005. Membrane curvature Maxfield, F. R., and I. Tabas.2005. Role of cholesteroland
ii{ 1^e1hir^rigms of dynamic cell mimbrane remodeling.Nature lipid organizationin disease.Nature 438:61,2-621,.
438:590-596.
Stahl,A., R. E. Gimeno, L. A. Tartaglia,and H. F. Lodish. 2001.
Mukherjee, S., and F. R. Maxfield. 2004. Membrane domains.
Annu. Reu.Cell Deu.Biol.2O:839-866. F"y.".1d transport proteins:a current view of a growing family.
TrendsEndocrinol. Metab. 12(6\:266-273.
_ Simons,K., and D. Toomre.2000. Lipid rafts and signaltrans_ van Meer, G., and H. Sprong.2004. Membrane lipids and
duction. Nature Reu.Mol. Cetl Biol. t:Zi-4t. vesiculartraffic. Curr. Opin. Cell Biol. 16:373-378.

436 CHAPTER
10 | B T o M E M B R A NSET R U C T U R E
 CHA PTER

TRANSMEMBRANE
TRANSPORTOF
IONSAND SMALL
MOLECULES
A studyof mutantzebrafish with palestripesledto the
identificationof a sodium/calcium
transporter that regulates
the
darkness of humanskin @ ChristinaMicek

water. Plasma and other membranes serve as both barriers

I n all cells, the plasma membrane forms the permeability
I barrier that separatesthe cytoplasm from the exterior en- and conduits.
I vironment, thus topologically and biochemicallydefining a Integral membrane proteins called transport proteins,
cell and distinguishingit from its surroundings.As a perme- embedded in the plasma membrane and other membranes
ability barrier, the plasma membrane allows import of essen- within cells by multiple transmembranedomains, permit the
tial nutrients, ensuresthat metabolic intermediatesremain in controlled and selective transport of molecules and ions
the cell where they belong, and enableswaste products to acrossthe membrane.In some cases,this involves molecules
leave the cell. By preventing the unimpeded movement of moving from a higher to a lower concentration, a thermody-
moleculesinto and out of cells, the plasma membrane main- namically spontaneousprocessthat does not require the in-
tains essentialdifferencesbetweenthe composition of the ex- put of energy.Examples include the movement of water or
tracellular fluid and that of the cytoplasm; for example, the glucosefrom the blood into body cells.
concentration of NaCl in the blood and extracellular fluids In other cases.moleculesmust be moved "uphill" across
of animals is generallyabove 150 mM, similar to that of the a membrane-against a concentration gradient-a thermo-
seawaterin which all cells are thought to have evolved' dynamically unfavorable processthat can only occur when
whereas the Na+ concentration in the cytoplasm is tenfold an external source of energy is available. Examples include
lower. In contrast, the potassium ion concentration is higher
in the cytoplasm than outside.
OUTLINE
The plasma membrane, like all cellular membranes, is
basedon a bilayer of phospholipids into which proteins and Overviewof MembraneTransport 438
11.1
other lipids are embedded.If the plasma membrane were a
pure phospholipid bilayer, it would be an excellentchemical 11.2 Uniport Transportof Glucoseand Water 441
barrier, impermeable to virtually all ions, amino acids' sug-
ars, and other water-solublemoleculesthat must selectively 1 1 . 3 A T P - P o w e r ePdu m p sa n d t h e I n t r a c e l l u l alro n i c
enter or leave a cell. In fact, only a few gasesand uncharged Environment
small moleculescan readily diffuse across a pure phospho-
1 1 . 4 N o n g a t e dl o n C h a n n e l sa n d t h e R e s t i n g
lipid membrane(Figure11-1).However,plasmamembranes M e m b r a n eP o t e n t i a l 458
must not only serve as barriers but also must play another'
almost contradictory role. They must permit the selective andAntiporters
by Symporters
11.5 Cotransport 465
transport of material and information between the cell's
Transport
11.6 Transepithelial 47O
interior and exterior spaces,which often involves regulated
entrv or exit of many diverse small biomolecules,ions, and

437
 Gases
K+
co2, N2,02 Lysine
Exterior
, Ti- +++
Small
uncharged E t h an o l Plasma
polar membrane
molecules o
li H,o Cytosol
il-
NH2-C-NH2 Water 2 Na*
Slightly ADP + P;
Urea permeable
Large K* ATP
uncharged
polar Na*/K* K+ Na+/lysine
1,"1""ut"" Glucose,fructose pump channel symporter

FIGURE 11-2 Multipletransportproteinsfunctiontogether

lons in the plasmamembraneof metazoancells.Graotenrs are
K t , M g z * , C a 2 + ,C l - , indicatedby triangleswith thetip pointing towardlower
HCO3-, HPO42- concentration TheNa+/K+ATPase in the plasma membrane uses
energyreleased byATPhydrolysis to pumpNa+out of the celland
Charqed K* inward;thiscreates a concentration gradient of Na+that is
polar- Amino acids, ATP, greater out thanin andoneof K+thatisgreater
glucose 6-phosphate,
in thanout
molecules
proteins, nucleic acids Movement of positively
charged K* ionsout of thecellthrough
membrane K+ channelproteins creates an electricpotentialacross
the plasma membrane-the cytosolic faceisnegative with respect
to
A FIGURE 11-1 Relativepermeabilityof a purephospholipid theextracellular face.A sodium-lysine transporter, a typical
bilayerto variousmolecules. ispermeable
A bilayer to small sodium-aminoacidtransporter,movesNa+ionstogetherwith
hydrophobic molecules
andsmalluncharged polarmolecules,
slightly lysine fromtheextracellular mediumintothecell."Uphill"movement
permeable to waterandurea,andessentrallyimpermeable
to ions of theaminoacidispowered by "downhill"movement of Na+ions,
andto largepolarmolecules powered bothbytheout-greater-than-in Na+concentration gradient
andbythe negative potential on the inside of thecellmembrane,
concentraring protons within lysosomesto generate a low whichattracts the positively
charged Na* ionsTheultimate source
of
pH in the lumen. Often this energy is provided by mechanis- theenergy to poweraminoaciduptakecomesfromtheATp
hydrolyzed bythe Na*/K+ATPase, sincethispumpcreates boththe
tically coupling the energy-releasinghydrolysis of the termi-
Na- ionconcentration gradient and,viathe K+ channels, the
nal phosphoanhydride bond in ATp; such transporrers are
membrane potential,whichtogether powerinfluxof Na+ions
called ATP-powered pumps. When such pumps rransport
ions, suchas Na* and K+, they generateacrossthe membrane
properties that enable different cell types to function appro-
an electricalgradient, or potential, as well as a chemicalcon-
priately. We also explain how subcellularmembranesencom-
centration gradient. The energy stored in such gradientscan
pass specificcombinations of transport proteins that enable
subsequently be used to do work or convey information.
cells to carry out essenrialphysiological processes,including
Other transport proteins couple the movement of one mole-
the maintenanceof cytosolicpH, the accumulationof sucrose
cule or ion againstits concentration gradient with the move-
and salts in plant cell vacuoles,and the directed flow of wa-
ment of another down its gradient, using the energyreleased
ter in both plants and animals. Epithelial cells, such as rhose
by the downhill movement of one moleculeor ion to thermo-
lining the small intestine, transport ions, sugars, and other
dynamically drive the uphill movement of another.
small moleculesand water from one side ro the other.'Wewill
Frequently, several different types of transport proteins
see how this understanding has led to the development of
work in concert to achievea physiological function. An ex-
sports drinks and also therapiesfor cholera.
ample is seenin Figure 11-2,where an ATp-powered rrans-
porter pumps Na* our of the cell and K+ ions inward; this
pump establishesthe opposing concenrration gradients of Overviewof MembraneTransport
Na* and K+ ions acrossthe plasmamembrane.The human
Many different proteins contribute to the transport of ions
genome encodeshundreds of different types of transport
and small moleculesacross membranes.As we learn about
proteins that use the energystored in the Na+ concentration
different transport processesin this chapter,we will seehow
different kinds of membrane-embeddedproteins accomplish
the task of moving moleculesin different ways.

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s s
quent sections,we describe the structure and ooeration of M e m b r a n e sb y S i m p l eD i f f u s i o n
specific examples of each class and show how members of As we saw above,only gases,suchas 02 and CO2, and small
families of homologous transport proteins have different uncharged polar molecules, such as urea and ethanol. can

438 ' CHAPTER

11 T R A N S M E M B R A NTER A N s p o R To F r o N s A N D S M A L LM o L E c u L E S
|
readily move by simple diffusion across an artificial mem-
brane composed of pure phospholipid or of phospholipid
and cholesterol(seeFigure 11-1). Such moleculesalso can
diffuse acrosscellular membraneswithout the aid of trans-
port proteins. No metabolic energy is expended because
movement is from a high to a low concentration of the mol-
ecule,down its chemical concentration gradient. As noted in
Chapter2, suchtransportreactionsare spontaneousbecause
they have a positive AS value (increasein entropy) and thus
a negativeAG (decreasein free energy).
The relative diffusion rate of any substanceacrossa pure
phospholipid bilayer is proportional to its concentration
gradient across the bilayer and to its hydrophobicity and
size; the movement of charged moleculesis also affected by
any electric potential across the membrane. tWhen a phos-
pholipid bilayer separatestwo aqueous compartments,
membrane permeability can be easily determined by adding
a small amount of radioactive material to one compartment
and measuring its rate of appearancein the other compart-
ment. The greater the concentration gradient of the sub-
stance,the faster its rate of movement acrossa bilayer.
The hydrophobicity of a substanceis measuredby its par-
tition coefficientK, the equilibrium constant for its partition
betweenoil and water. The higher a substance'spartition co-
efficient,the more lipid solubleit is. The first and rate-limiting
step in transport by simple diffusion is movement of a mole-
cule from the aqueoussolution into the hydrophobic interior
of the phospholipid bilayer,which resemblesoil in its chemi-
cal properties.This is the reasonthat the more hydrophobic a
moleculeis, the fasterit diffusesacrossa pure phospholipid bi-
layer. For example, diethylurea, with an ethyl group
(CH3CH2-) attachedto eachnitrogen atom of urea, has a K
of 0.01,whereasureahasa K of 0.0002 (seeFigure11-1).Di-
ethylurea,which is 50 times (0.01/0.0002)more hydrophobic
than urea, will thereforediffuse through phospholipid bilayer
membranesabout 50 times faster than urea. Similarly, fatty
E a
ATP-powered pumps lon channels
(100-103ions/s) (1o7-108 ions/s)
Closed
ATP ADP+P,
Open
1'l-3 Overviewof membranetransportproteins'
FIGURE
Gradientsareindicated with thetip pointing
bytriangles toward
or both.E Pumps
potential,
electrical utilize
lowerconcentration,
released by ATP to
hydrolysis power movement of specific
the energy
ions(redcircles)
or smallmoleculesagainsttherrelectrochemical
permitmovement ions(orwater)
of specrfic
gradientE Channels
downtheirelectrochemical gradient S whichfallinto
Transporters,
threegroups,facilitatemovement of small
specific molecules or ions
acids with longer hydrocarbon chains are more hydrophobic
than those with shorter chains and will diffuse more rapidly
acrossa pure phospholipid bilayer at all concentrations.
If a transported substancecarries a net charge,its move-
ment is influencedby both its concentrationgradient and the
membrane potential, the electric potential (voltage) across
the membrane. The combination of thesetwo forces, called
the electrochemicalgradient, determinesthe energetically fa-
vorable direction of transport of a chargedmoleculeacrossa
membrane.The electric potential that exists acrossmost cel-
lular membranesresults from a small imbalance in the con-
centration of positively and negatively charged ions on the
two sides of the membrane. \7e discusshow this ionic im-
balance,and resulting potential' arise and are maintained in
Sections17.4 and 1I.5.
MembraneProteinsMediate Transportof Most
M o l e c u l e sa n d A l l l o n s A c r o s sB i o m e m b r a n e s
As is evident from Figure 1'1'-L,very few molecules and no
ions can cross a pure phospholipid bilayer at appreciable
rates by simple diffusion. Thus transport of most molecules
into and out of cells requires the assistanceof specialized
membrane proteins. Even transport of moleculeswith rela-
tively large partition coefficients(e'g., urea and certain gases
such as CO2) is frequently acceleratedby specific proteins
becausetheir transport by simple diffusion usually is not suf-
ficiently rapid to meet cellular needs.
All transport proteins are transmembraneproteins con-
taining multiple membrane-spanningsegmentsthat gener-
ally are o.helices.By forming a protein-lined pathway across
the membrane, transport proteins are thought to allow
movement of hydrophilic substanceswithout their coming
into contact with the hydrophobic interior of the membrane'
Here we introduce the various types of transport proteins
coveredin this chapter(Figure11-3).
E
Transporters
(02-104 molecules/s)
aa
Uniporter Symporter Antiporter
tr tr q
Uniporterstransporta singletypeof moleculedownitsconcentratton
gradientEE.Cotransport proteins(symporters,EE, andantiporters,
EE) catalyzethe movement of onemolecule againstitsconcentration
gradient(blackcircles),
drivenby movement of oneor moreions
do*n .n electrochemicalgradient(red Differences
circles). in the
mechanisms by thesethreemajorclasses
of transport of proteins
accountfor theirvaryingratesof solutemovement
O V E R V I E WO F M E M B R A N ET R A N S P O R T O 439
 ATP-poweredpumps (or simply pumps) are ATpasesthat
use the energy of AIP hydrolysis to move ions or small mole-
culesacrossa membraneagainsta chemicalconcentrationgra-
dient, an electricpotential,or both. This process,referredto as
activetransport, is an exampleof a coupledchemicalreaction
(Chapter2). In this case,transport of ions or small molecules
"uphill" against an electrochemicalgradient, which requires
energy,is coupled to the hydrolysis of ATp, which releasesen-
ergy. The overall reaction-ATP hydrolysis and the ,,uphill',
movement of ions or small molecules-is energeticallyfavor-
able. The Na*/K* pump shown in our overview figure (see
Figure 11-2) is an exampleof an Alp-powered pump.
Channel proteins transport water, specific ions, or hy-
drophilic small moleculesdown their concentrationor electric
potential gradients via facilitated transport (or facilitated
diffusion), the protein-assistedmovement of a substance
In contrast,antiporters and symporterscouple the movement
of one type of ion or moleculeagainst its concentrationgra,
dient with the movement of one or more different ions doun
its concentrationgradient, in the same (symporter) or differ-
ent (antiporter) directions.Theseproteins ofren are calledco-
transporters, referring to their ability to transporr rwo or
more different solutes simultaneously.In Figure 11-2, lysine
is moved into the cell via a Na+/lysine symporter.
Like AIP pumps, cotransportersmediate coupled reactions
in which an energeticallyunfavorable reaction (i.e., uphill
movement of one type of molecule) is coupled to an energet-
ically favorable reaction, the downhill movement of another.
Note, however, rhat the nature of the energy-supplyingre-
action driving active transport by these two classesof pro-
teins differs. ATP pumps use energy from hydrolysis of ATp,
whereas cotransportersuse the energy stored in an electro-
chemical gradient. This latter processsometimesis referred
to as secondary activetransport.
Table 11-1 summarizesthe four mechanisms by which
small molecules and ions are transported across cellular
membranes.Conformational changesare essentialto the
function of all transport proteins. MP-powered pumps and
transporters undergo a cycle of conformational change ex-
posing a binding site (or sites)to one side of the membrane
in one conformation and to the other side in a secondcon-
formation. Becauseeach such cycle results in movement of
only one (or a few) substrate molecules,these proteins are
charactertzedby relatively slow rates of transport ranging
from 100to 10aions or moleculesper secondlseefigure t i-:;.
Ion channels shuttle between a closed state and an oDen
state, but many ions can passthrough an open channel with-
out any further conformational change. For this reason,
channelsare characterizedby very fast rates of transport, up

PROPEBTY SIMPLE
DIFFUSION FACITITATED
TflANSPOBI ACTIVE
TRANSP()RT COTRANSP()RT-
Requires specific
proteln
+
Solute transported
against its gradient
+
Coupled to ATp
hydrolysis

Driven by movement
of a cotransported ion
down its gradient
+
Examples of 02, CO2, steroid Glucose and Ions,small Glucose and amino
molecules transported hormones,many amino acids hydrophilic acids (symporters);
drugs (uniporters);ions molecules,lipids various ions and
and water (channels) (ATP-powered pumps) sucrose (antiporters)
Also called secondary dctiue transport

440 CHAPTER
11 I T R A N S M E M B R A NT E
R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s
to 10Eions per second.'Wewill begin with the simplesttrans- Consequently,there is a maximum transport rate V^"* that
port proteins, the moleculesresponsiblefor the transport of is achievedwhen the concentration gradient acrossthe
glucose and water, before moving on to progressivelymore membrane is very large and each uniporter is working at its
complex transport molecules. maximal rate.

4. Transport is specific.Each uniporter transports only a

single speciesof molecule or a singlegroup of closely re-
Overview of Membrane Transport lated molecules.A measureof the affinity of a transporter
for its substrateis K-, which is the concentration of sub-
r The plasma membrane regulatesthe traffic of molecules
strate at which transport is half maximal.
into and out of the cell.
r \With the exception of gases(e.g.,Oz and COz) and small Theseproperties also apply to transport mediated by the
hydrophobic molecules,most molecules cannot diffuse other classesof proteins depicted in Figure 11-3.
across a pure phospholipid bilayer at rates sufficient to One of the best-understood uniporters is the glucose
meet cellularneeds.
r Three classesof transmembraneproteins mediate trans-
port of ions, sugars, amino acids, and other metabolites
across cell membranes:ATP-powered pumps, channels'
and transporters(seeFigure 11-3).
r In active transport, a transport protein couples move-
ment of a substrate against its concentration gradient to
ATP hydrolysis.
r In facilitated diffusion, a transport protein assistsin the
movement of a specificsubstrate(moleculeor ion) down its
concentration gradient.
r In secondaryactive transport, or cotransport, a transport
protein couples movement of a substrate against its con-
centration gradient to the movement of a secondsubstrate 500
down its concentrationgradient(seeTable 11-1).
ot

UniportTransportof Glucose o
o
and Water 3 zso
GLUT2 (liver cellsl
Most animal cells utilize glucose as a source for ATP pro-
duction; they employ a glucoseuniporter to take up glucose 0)

from the blood or other extracellular fluid down its concen-

tration gradient. Many cellsutilize membranetransport pro-
teinscalledaquaporinsto increasethe rate of water movement
acrosstheir surface membranes.Here, we discussthe struc- 2 3 4 5 6 7 8 9 1011121314
ture and function of theseand other uniport proteins. Externalconcentrationof glucose(mM)
Km

SeveralFeaturesDistinguishUniport Transport A EXPERIMENTAL FIGURE 11-4 Cellularuptakeof glucose

mediated by GLUT proteins exhibitssimpleenzymekinetics
from SimpleDiffusion
and greatly exceedsthe calculatedrate of glucoseentry solely
The protein-mediated transport of glucose and other small by simplediffusion.Theinitialrateof glucose uptake(measured as
hydrophilic moleculesacrossa membrane, a processknown micromoles per
of cells hour)
permilliliter in the firstfew seconds is
as uniport, exhibits the following distinguishingproperties: plottedagainst increasing glucose concentration in the extracellular
medium. In thisexperiment, the concentration
initial of glucose in
l. The rate of facilitated diffusion by uniporters is far higher the cellsisalways zero BothGLUT1, expressed by erythrocytes, and
than simple diffusion through a pure phospholipid bilayer. GLUT2, expressed by livercells,greatlyincrease the rateof glucose
uptake (burgundy andtancurves) compared with thatassociated
2. Becausethe transported moleculesnever enter the hy- with simple diffusion (bluecurve) at all externalconcentrations Like
drophobic core of the phospholipid bilayer, the partition enzyme-catalyzed reactions,GLUT-facilitated uptakeof glucose
exhibitsa maximum rate(V'"^).TheK' istheconcentration at which
coefficient K is irrelevant.
the rateof glucose uptakeishalfmaximal GLUT2, with a K' of
3. Transport occurs via a limited number of uniporter about20 mM, has a much lower affinityfor glucose than GLUT1,
moleculesrather than throughout the phospholipid bilayer. with a K- of about1 5 mM

AND WATER
T F GLUCOSE . 441
U N I P O R TT R A N S P O R O
 Exterior GLUTl I Glucose
Iu c o s e 1l
ll
Outward-facing
Cytosol
conformation
A FIGURE 11-5 Modelof uniporttransportby GLUT1.In one
conformation, theglucose-bindingsitefacesoutward;in theother,
the bindingsitefacesinwardBinding of glucoseto theoutward-
facingsite(stepn) triggers
a conformationalchangein thetransporter
suchthatthe bindingsitenowfacesinwardtowardthecytosol
(stepZ) Glucose thenisreleasedto the insideof the cell(stepB).
enzyme-catalyzedreaction involving a single substrate.The
kinetics of transport reactions mediated by other types of
proteins are more complicatedthan for uniporters. Nonethe-
less,all protein-assistedtransport reactionsoccur faster than
allowed by simple diffusion, are subsrrare-specific as re-
flected in lower K- values for some substratesthan others,
and exhibit a maximal rate (V*"*).
GLUT1UniporterTransportsGlucoseinto Most
M a m m a l i a nC e l l s
Most mammalian cells useblood glucoseas the malor source
of cellular energy and express GLUT1. Since the glucose
concentration usually is higher in the extracellular medium
(blood, in the caseof erythrocytes)than in the cell, GLUT1
generally catalyzesthe net import of glucose from the extra-
cellular medium into the cell. Under this condition, V_.* is
achievedat high external glucoseconcentrations.
Like other uniporters, GLUT1 alternates between two
conformational states: in one, a glucose,binding site faces
the outside of the membranel in the other, a glucose-binding
site faces the inside. Figure 11-5 depicts the sequenceo?
The kinetics of the unidirectional transport of glucose
from the outside of a cell inward via GLUT1 can be de-
scribed by the same rype of equation used ro describe a
simple enzyme-catalyzedchemical reaction. For simplicitS
let's assumethat the substrateglucose, S, is present i"itiatiy
only on the outside of the membrane.In this case.we can
wnte
K- V-u*
So,t+GLUT1 : So,t- GLUT1 .- Si, + GL1;rI1
442 C H A P T E R1 1 | T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E S
Outward-facing
conformation
Finally,
thetransporter undergoes the reverseconformationalchange,
regeneratingthe outward-facingbindingsite(stepE). lf thecon-
of glucose
centration ishigherinsidethecellthanoutside, thecycle
willworkin reverse(step4 -+ step[), resultingin netmovement
of glucose
frominsideto out Theactualconformational chanqesare
probablysmallerthanthosedepicted here.
where So,, - GLUT1 representsGLUT1 in the outward-
facing conformation with a bound glucose.This equation is
similar to the one describing the path of a simple enzyme-
catalyzed reaction where the protein binds a single substrate
and then transforms it into a different molecule. Here, how-
ever,no chemical modification occurs to the GLUT1-bound
sugar; rather, it is moved across a cellular membrane.
Nonetheless,the kinetics of this transport reaction are simi-
lar to those of simple enzyme-catalyzedreactions, and we
can use the same derivation as that of the Michaelis-Menten
equation in Chaprer 3 to derive the following expressionfor
u, the initial transport rate for S into the cell catalyzed by
GLUT1:
V-"*
v: __ u (11_t)
^_
1*?
where C is the concentration of So,t (initiallg the concentra-
tion of Si" : 0). V-"*, the rate of transport when all mole-
cules of GLUT1 contain a bound S, occurs at an infinitely
high So",concentration.The lower the value of K-, the more
tightly the substratebinds to the transporter and the greater
the transport rate at a fixed concentration of substrate.
Equation 11-1 describesthe curve for glucoseuptake by ery-
'1.'J.-4
throcytes shown in Figure as well as similar curves for
other uniporters.
For GLUT1 in the erythrocyte membrane, the K- for
glucosetransport is 1.5 mM; at this concentration, roughly
half the transporters with outward-facing binding iites
would have a bound glucose and transport would occur at
50 percent of the maximal rate. Since blood glucoseis nor-
mally 5 mM, the erythrocyte glucose transporter usually is
functioning at 77 percentof the maximal rate, as can be seen
from Equation 11-1. GLUT1 and the very similar GLUT3
are expressedby erythrocytes and other cells that need to
take up glucosefrom the blood continuously at high rates;
the rate of glucose uptake by such cells will remain high
regardlessof small changesin the concentration of blood
glucose.
In addition to glucose, the isomeric sugars D-mannose
and n-galactose,which differ from l-glucose in the configu-
ration at only one carbon atom, are transported by GLUT1
at measurablerates. However, the K- for glucose (1.5 mM)
is much lower than the K- for D-mannose (20 mM) or o-
galactose(30 mM). Thus GLUT1 is quite specific,having a
much higher affinity (indicated by a lower K-) for the nor-
mal substrateo-glucosethan for other substrates.
GLUT1 accounts for 2 percent of the protein in the
plasma membrane of erythrocytes. After glucose is trans-
ported into the erythrocyte, it is rapidly phosphorylated,
forming glucose 6-phosphate,which cannot leave the cell.
Becausethis reaction, the first step in the metabolism of glu-
cose (seeFigure 12-3), is rapid and occurs at a constant rate,
the intracellular concentration of glucose is kept low even
when glucose is imported from the medium. Consequently
the concentration gradient of glucose outside greater than
inside the cell is maintained at a sufficiently high ratio to
support import of additional glucose molecules and main-
tain a constant rate of slucosemetabolism.
T h e H u m a nG e n o m eE n c o d e sa F a m i l yo f S u g a r -
TransportingGLUTProteins
The human genome encodesat least 12 highly homologous
GLUT proteins, GLUT1-GLUTI2, that are all thought to
contain 12 membrane-spanning ct helices,suggestingthat they
evolved from a single ancestraltransport protein. Although
no three-dimensionalstructure of GLUT1 is available, de-
tailed biochemical studies have shown that the amino acid
residuesin the transmembranect helicesare predominantly
hydrophobic; several helices, however, bear amino acid
residues(e.g., serine, threonine, asparagine,and glutamine)
whose side chains can form hydrogen bonds with the hy-
droxyl groups on glucose. These residues are thought to
form the inward-facing and outward-facing glucose-binding
sitesin the interior of the protein (seeFigure 11-5).
The structuresof all GLUT isoforms are thought to be
quite similar, and all transport sugars.Nonetheless,their
differential expressionin various cell types and isoform-
specificfunctional propertiesenabledifferent body cellsto
regulate glucose metabolism independently and at the
sametime maintain a constant concentrationof glucosein
the blood. For instance,GLUT3 is found in neuronal cells
of the brain. Neurons depend on a constant influx of glu-
cose for metabolism, and the low K- of GLUT3 for glu-
cose,like that of GLUT1, ensuresthat thesecells incorpo-
rate glucosefrom brain extracellular fluids at a high and
constant rate.
GLUT2, expressedin liver and the insulin-secreting B
cellsof the pancreas,has a K- of :20 mM, about 13 times
higher than the K^ of GLUT1. As a result, when blood glu-
coserisesfrom its basal level of 5 mM to 10 mM or so after
a meal, the rate of glucose influx will almost double in
GlUT2-expressing cells, whereas it will increase only
slightly in GLUT1-expressingcells (seeFigure 11-4).In liver,
the "excess" glucose brought into the cell is stored as the
polymer glycogen.In islet B cells, the rise in glucosetriggers
secretionof the hormone insulin, which in turn lowers blood
glucose by increasingglucose uptake and metabolism in
muscle and by inhibiting glucose production in liver (see
Figure 1,5-32).
Another GLUT isoform, GLUT4, is expressedonly in
fat and musclecells,the cellsthat respond to insulin by in-
creasingtheir uptake of glucose,thereby removing glucose
from the blood. In the absenceof insulin, GLUT4 is found
in intracellular membranes,not on the plasma membrane,
and is unable to facilitate glucoseuptake. By a processde-
tailed in Chapter 15, insulin causesthese GLUT4-rich in-
ternal membranesto fuse with the plasma membrane, in-
creasing the number of GLUT4 molecules on the cell
surface and thus the rate of glucose uptake. A defect in
this process, one principal mechanism by which insulin
lowers blood glucose,is one of the causesof adult onset,
or type II, diabetes,a diseasemarked by continuously high
blood glucose.
TransportProteinsCan Be EnrichedWithin
Artificial Membranesand Cells
Although transport proteins can be isolated from mem-
branes and purified, the functional properties of these pro-
teins can be studied only when they are associatedwith a
membrane.Most cellular membranescontain many different
types of transport proteins but a relatively low concentration
of any particular one, making functional studies of a single
protein difficult. To facilitate such studies, researchersuse
two approachesfor enriching a transport protein of interest
so that it predominatesin the membrane.
In one common approach, a specifictransport protein is
extracted and purified; the purified protein then is reincor-
porated into pure phospholipid bilayer membranes,such as
liposomes (seeFigure 10-6). For example, all of the integral
proteins of the erythrocyte membrane can be solubilized by
a nonionic detergent, such as octylglucoside. The glucose
uniporter GLUT1 can be purified by antibody affinity chroma-
tography (Chapter 3) on a column containing a specificmon-
oclonal antibody and then incorporated into liposomes
made of pure phospholipids.
Alternatively, the geneencoding a specifictransport pro-
tein can be expressedat high levels in a cell type that nor-
mally does not expressit. The difference in transport of a
substanceby the transfectedand by control nontransfected
cells will be due to the expressedtransport protein. In these
systems,the functional properties of the various membrane
proteins can be examined without ambiguity. As an exam-
ple, overexpressingGLUT1 in lines of cultured fibroblasts
increasesseveralfoldtheir rate of uptake of glucose,and ex-
pressionof mutant GLUT1 proteins with specificamino acid
alterations can identify residues important for substrate
binding.
AND WATER
T F GLUCOSE
U N I P O R TT R A N S P O R O 443
OsmoticPressureCausesWater to Move Across In higher plants, water and minerals are absorbed
Membranes from the soil by the roots and move up the plant
through conducting tubes (the xylem); water loss from the
Movement of water in and out of cells is an important fea-
plant, mainly by evaporation from the leaves,drives these
ture of the life of both plants and animals. Aquaporins are a
movements of water. Unlike animal cells, plant, algal, fun-
family of membrane proteins that allow water and a few
gal, and bacterial cells are surrounded by rigid cell walls,
other small uncharged molecules,such as glycerol, to cross
which resistthe expansionof the volume of the cell when the
biomembranes. But before discussingthese transport pro-
intracellular osmotic pressureincreases.Sfithout such a
teins, we need to review osmosis,the force that powers the
wall, animal cells expand when internal osmotic pressurein-
movement of water.
'Water creases-if that pressurerises too much, the cells will burst
tends to move acrossa semioermeablemembrane
like overextended balloons. Becauseof the cell wall in
from a solution of low solute concentiation to one of high
plants, the osmotic influx of water that occurs when such
concentration, a processtermed osmosis,or osmotic flow. In
cells are placed in a hypotonic solution (even pure water)
other words, since solutions with a high concentration of
leads to an increasein intracellular pressurebut not in cell
dissolvedsolute have a lower concentration of water, water
volume.In plant cells,the concentrationof solutes(e.g.,sug-
will spontaneouslymove from a solution of high water con-
ars and salts)usually is higher in the vacuole (seeFigure 9-7)
centration to one of lower. In effect, osmosisis equivalentto
"diffusion" of water. Osmotic pressureis defined as the hy- than in the cytosol, which in turn has a higher solute con-
centration than the extracellular space. The osmotic pres-
drostatic pressure required ro stop the net flow of water
sure, called turgor pressure,generatedfrom the entry of wa-
acrossa membrane separatingsolutions of different compo-
ter into the cytosol and then into the vacuole pushes the
sitions (Figure t1-6).In this context, the "membrane" may
cytosol and the plasma membrane against the resistant cell
be a layer of cellsor a plasmamembranethat is permeableto
wall. Plant cellscan harnessthis pressureto help them grow.
water but not to the solutes.The osmotic pressureis directly
Cell elongation during growth occurs by a hormone-induced
proportional to the difference in the concentration of the
localized loosening of a defined region of the cell wall, fol-
total number of solute molecules on each side of the mem-
Iowed by influx of water into the vacuole, increasingits size
brane.For example,a 0.5 M NaCl solutionis actually0.5 M
and thus the size of the cell. I
Na* ions and 0.5 M Cl- ions and has the same osmouc
pressureas a 1 M solution of glucoseor sucrose.
Although most protozoans (like animal cells) do not
The movement of water across the plasma membrane
have a rigid cell wall, many contain a contractile vacuole
also determinesthe volume of individuaf cells,which must
that permits them to avoid osmotic lysis. A contractile
be regulatedto avoid damageto the cell. Osmotic pressureis
vacuole takes up water from the cytosol and, unlike a
the force powering the movement of water in biological
plant vacuole, periodically discharges its contents
systems.
through fusion with the plasma membrane. Thus even
though water continuously enters the protozoan cell by
osmotic floq the contractile vacuole prevents too much
water from accumulating in the cell and swelling it to the
bursting point.
H y d r o s t a t i pc r e s s u r e
Water-permeable
r e q u r r e dt o p r e v e n l
membrane
n e t w a t e rf l o w
I
Aquaporinslncreasethe Water Permeabilityof
* C e l lM e m b r a n e s
Small changesin extracellular osmotic strength causemost
S o l u t i o nA Solution B
animal cells to swell or shrink rapidly. N7henplaced in a hy-
CA UB
potonic solution (i.e., one in which the concentration of
solutes is lower than in the cytosol), animal cells swell ow-
ing to the osmotic flow of water inward. Converselg when
placed in a hypertonic solution (i.e., one in which the con-
centration of solutes is higher than in the cytosol), animal
cells shrink as cytosolic water leaves the cell by osmotic
A FIGURE flow. Consequently,cultured animal cells must be main-
11-6 Osmoticpressure. SolutionsA andB are
separatedby a membrane thatispermeable tained in an isotonic medium, which has a solute concen-
to waterbut
impermeable to allsolutes.lf Cs(thetotalconcentrationof solutes tration and thus osmotic strength identical with that of the
in
solutionB)isgreater thanC4,waterwilltendto flow across the cell cytosol.
membrane fromsolution A to solutionB Theosmoticpressure n In contrast, frog oocytesand eggsdo not swell when placed
between thesolutions isthe hydrostaticpressurethatwouldhaveto in pond water of very low osmotic strength, even though their
be appliedto solutionB to prevent thiswaterflow.Fromthevan,t internal salt (mainly KCI) concentrationis comparableto that
Hoffequation,osmoticpressure isgivenby n = ,q16',- Co),whereR of other cells (:159 mM KCI). Theseobservationswere what
rsthe gasconstant andf istheabsolute temperature first led investigatorsto suspectthat the plasma membranesof

444 CHAPTER
11 I T R A N S M E M B R A NT E
R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E S
ZN
AquaporinBurstsin HypotonicSolution
Video:Frog Oocyte Expressing
0 . 5m i n 1 . 5m i n
A EXPERIMENTAL FIGURE 11-7 Expression of aquaporinby
frog oocytesincreasestheir permeabilityto water. Frogoocytes,
whichnormally areimpermeable to wateranddo notexpress an
aquaporinprotein,weremicroinjectedwith mRNAencoding
aquaporin Thesephotographs showcontroloocytes (bottomcellin
eachpanel)andmicroinjected oocytes(topcellin eachpanel)at the
indicated
timesaftertransferfroman isotonic
saltsolution(0 1 mM)
to a hvpotonic (0 035 M) Thevolumeof the control
saltsolution
erythrocytesand other cell types,but not frog oocytes,contain
water-channel proteins that acceleratethe osmotic flow of
water. The experimentalresultsshown in Figure 1L-7 demon-
stratethat an aquaporin in the erythrocyteplasma membrane
functionsas a water channel.
In its functional form, aquaporin is a tetramerof identi-
cal 28-kDa subunits (Figure 11-8a). Each subunit contains
six membrane-spanningct helices that form a central pore
through which water moves (Figure11-8b, c). At its center,
the :2-nm-long water-selective gate, or pore, is only 0.28
nm in diameter-only slightly larger than the diameter of a
water molecule. The molecular sieving properties of the
constriction are determined by several conserved hy-
drophilic amino acid residueswhose side-chainand car-
bonyl groups extend into the middle of the channel.Several
water moleculesmove simultaneouslythrough the channel,
each of which sequentially forms specific hydrogen bonds
and displacesanother water molecule downstream. The for-
mation of hydrogen bonds between the oxygen atom of wa-
ter and the amino groups of two amino acid side chains en-
sures that only water passesthrough the channel; even
protons cannot passthrough, allowing ionic gradients to be
maintained across membranes even when water is flowing
across.
Mammals express a family of aquaporins; 11 are
known in humans.Aquaporin 1 is expressedin abun-
dance in erythrocytes,and the homologous aquaporin 2 is
2 . 5m i n 3 . 5m i n
oocytesremained unchanged becausetheyarepoorlypermeable to
the
water In contrast, oocytes
microinjected expressing aquaporin
swelledandthenburstbecause of an osmoticinfluxof water,
indicatingthataquaporin protein[Courtesy
isa water-channel of
GregoryM PrestonandPeter Hopkins
Agre,Johns School
University of
Medicine SeeL 5 King, D Kozono,and P Agre ,2004, Nat Rev.Mol Cell
Biol 5:687-981
found in the kidney epithelial cells that resorb water from
the urine, thus controlling the amount of water in the body.
The activity of aquaporin2 is regulatedby vasopressin,also
called antiduretic hormone. The regulation of the activity of
aquaporin 2 in resting kidney cells resembles that of
GLUT4 in fat and muscle in that when its activity is not re-
quired, when the cells are in their resting state and water is
excreted to form urine, aquaporin 2 is localized to intracel-
lular vesiclemembranes,not to the plasma membrane' and
'$7hen
so is unable to catalyze water import into the cell. the
polypeptide hormone vasopressinbinds to the cell-surface
vasopressinreceptor, it activates a signaling pathway (de-
tailed in Chapter 15) that causesthese aquaporin 2-con-
taining vesiclesto fuse with the plasma membrane' increas-
ing the rate of water uptake and its return into the
circulation instead of the urine. Inactivating mutations in ei-
ther the vasopressinor the aquaporin 2 gene causediabetes
insipidus, a diseasemarked by excretion of large volumes of
dilute urine. This finding establishesthe etiology of the dis-
easeand demonstratesthat the level of aquaporin 2 is rate
limiting for water resorption from urine being formed by
the kidney. I
Other members of the aquaporin family transport hy-
droxyl-containing molecules such as glycerol rather than
water. Human aquaporin 3, for instance,transports glycerol
and is similar in amino acid sequenceand structure to the
Escherichiacoli glyceroltransport protein G/pF.
AND WATER
T F GLUCOSE
U N I P O R TT R A N S P O R O 445
 Extracellular
vestibule

Exterior

Cytosol

NHs* coo-
FIGURE 11-8 Structureof the water-channelprotein form partof the water-selective gate (c)Sideviewof the porein a
aquaporin.(a)Structural modelof thetetrameric proteincomprising singleaquaporin subunitin whichseveral watermolecules (red
four identical subunitsEachsubunitformsa waterchannel, asseen oxygens andwhitehydrogens) areseenwithinthe 2-nm-long water-
in thisviewlookingdownon the proteinfromtheexoplasmic side gatethat separates
selective the waterfilledcytosolicand
Oneof the monomers isshownwith a molecular surface in which extracellular
vestibules Thegatecontains highlyconserved arginine
the poreentrance canbe seen(b)Schematic diagram of the andhistidine residues,
aswellasthetwo asparagine residues(blue)
topology of a singleaquaporinsubunitin relationto the membrane. whosesidecharns formhydrogen bondswith transported waters.
Threepairsof homologous transmembrane o helices (A andA,,B (Keygateresidues arehighlighted in blue) Transportedwatersalso
andB',andC andC')areoriented in the opposite direction with formhydrogen bondsto the main-chain carbonyl groupof a cysteine
respect to the membrane andareconnected bytwo hydrophilic residueThearrangement of thesehydrogen bondsandthe narrow
loopscontaining shortnon-membrane-spanning helices and porediameter of 0.28nm prevent passage of protons(i.e.,HrO+)or
conserved asparagine (N)residues.
Theloopsbendintothecavity otherions lAfterH.Suietal, 2OO1, Nature414.8]2SeealsoT.Zeuthen,
formedbythe sixtransmembrane helices, meetinqin the middleto 2001,TrendsBiochem Sci.26:77,andK Murata etal, 2000,Nature4O7:599|

r Most biological membranesare semipermeable,more

Uniport Transport of Glucoseand Water
r Protein-catalyzedtransport of a soluteacrossa membraneoc-
curs much faster than passivediffusion, exhibits a V^"* when
the limited number of transporter moleculesare saturatedwith
substrate,and is highly specificfor substrare(seeFigure 11-4).
r Uniport proteins, such as the glucosetransporters (GLUTs),
are thought to shuttle between two conformational states,
one in which the substrate-bindingsite faces outward and
one in which the binding site facesinward (seeFigure 11-5).
r All membersof the GLUT protein family transporr sugars
and have similar structures.Differencesin their K- values,
expressionin different cell types, and substratespecificities
are important for proper sugar metabolism in the body.
r Two common experimental systems for studying the
functions of transport proteins are liposomes containing a
purified transport protein and cells transfected with the
gene encoding a particular transport protein.
permeableto water than to ions or most other solutes.
'Water
moves by osmosis across membranes from a solu-
tion of lower solute concentration to one of higher solute
concentration.
r The rigid cell wall surrounding plant cells prevents their
swelling and leads to generation of turgor pressurein re-
sponseto the osmotic influx of water.
r In responseto the entry of wate! protozoans maintain
their normal cell volume by extruding water from contrac-
tile vacuoles.
r Aquaporins are water-channel proteins that specifically
increasethe permeability of biomembranesfor water (see
F i g u r e1 1 - 8 ) .
r Aquaporin 2 in the plasma membrane of certain kidney
cells is essentialfor resorption of water from urine being
formed; the absenceof aquaporin 2 leads to the medical
condition diabetesinsipidus.

446 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E S

ATP-Powered Pumpsand the
lonicEnvironment
lntracellular
In previous sections,we focused on transport proteins that
move molecules down their concentrations gradients. Here
we focus our attention on a major class of proteins-the
ATP-powered pumps-that use the energy releasedby hy-
drolysis of the terminal phosphoanhydride bond of ATP to
transport ions and various small molecules across mem-
branes against their concentration g.adietttt. All ATP-
powered pumps are transmembrane proteins with one or
more binding sites for ATP located on subunits or segments
of the protein that face the cytosol. Although theseproteins
commonly are called ATPases, they normally do not hy-
drolyze ATP into ADP and P1unlessions or other molecules
are simultaneouslytransported.Becauseof this tight cou-
pling between ATP hydrolysis and transport, the energy
stored in the phosphoanhydridebond is not dissipatedbut
rather used to move ions or other molecules uphill against
an electrochemicalgradient. As Figure 11-2 illustrates,
these concentration gradients are used by other transport
proteins to power uphill movement of yet other types of
molecules.
Different Classesof PumpsExhibit
Structuraland Functional
Characteristic
Properties
The general structures of the four classesof ATP-powered
pumps are depictedin Figure 11-9, with specificexamplesin
each classlisted below the figure. Note that the membersof
three of the classes(R R and V) only transport ions' whereas
members of the ABC superfamily primarily transport small
moleculessuch as amino acids and sugars.
All P-classion pumps possesstwo identical catalytic o.
subunits, each of which contains an MP-binding site. Most
also have two smaller B subunits that usually have regula-
tory functions. During transport, at least one of the a

Exoplasmic
face

vo

Cytosolic 2Ht
face

ATP ADP + P; ATP

ADP + P;
P-classpumps V-class proton pumps F-classproton pumps ABC superfamily
P l a s m am e m b r a n eo f p l a n t s f, u n g i , V a c u o l a rm e m b r a n e si n Bacterialplasma Bacterialplasma
bacteria(H+pump) plants,yeast,other fungi membrane m e m b r a n e s( a m i n oa c i d ,
sugar,and peptide
P l a s m am e m b r a n eo f h i g h e r E n d o s o m aal n d l y s o s m a l l n n e rm i t o c h o n d r i a l transporters)
e u k a r y o t e (sN a + / Kp+u m p ) m e m b r a n e si n a n i m a l memDrane
cells M a m m a l i a np l a s m a
A p i c a lp l a s m am e m b r a n eo f T h y l a k o i dm e m b r a n e membranes (transporters
m a m m a l i a ns t o m a c h( H r / K rp u m p ) P l a s m am e m b r a n eo f of chloroplast o f p h o s p h o l i p i d ss,m a l l
plasma membraneof all eukarvotic osteoclastsand some l i p o p h i l i cd r u g s ,c h o l e s t e r o l ,
tr ta )- K l O n e YI U D U I eC e l l s o t h e rs m a l l m o l e c u l e s )
ceils (La- pump,
S a r c o p l a s m i cr e t i c u l u m m e m b r a n e
i n m u s c l e c e l l s ( C a 2 *p u m p )

A FIGURE 11-9 The four classesof ATP-poweredtransport the reverse

directronto utilizeenergyin a protonconcentratlonor
proteins.Thelocationof specificpumpsareindicated beloweach gradient
electrochemical to synthesizeATP All of
members the large
pumpsarecomposed crsubunits,
of two catalytic which ABCsuperfamily of proteins containtwo transmembrane (T)domains
classP-class
becomephosphorylated aspartof thetransport cycleTwoB andtwo cytosolicATP-binding (A)domains, whichcoupleATP
subunits, in someof thesepumps,mayregulate
present transport to solutemovement.
hydrolysis Thesecoredomains arepresent as
Onlyoneo andB subunitaredepicted. V-class
andF-classpumps separatesubunitsin some ABC (depicted
proteins here)
but are fused
do not formphosphoprotein intermediates andtransportonly intoa singlepolypeptidein otherABCproteins[See andM
T.Nishi
protonsTheirstructuresaresimilar andcontainsimilarproteins,
but Forgac,2Oo2,Nature Rev.Mol CellBiol 3:94; C Toyoshimaet al , 2000,
noneof therrsubunitsarerelated pumpsV-class
to thoseof P-class Nature405.64-/,D Mclntosh,2000, Nature Struc Biol 7:532; and T Elston,
pumpscoupleATPhydrolysis to transportof protons a
against H W a n g ,a n d G O s t e r ,1 9 9 8 ,N a t u r e3 9 1 : 5 1 0 l
concentrationgradient,
whereas F-classpumpsnormally operatein

O N I CE N V I R O N M E N T O
DU M P SA N D T H E I N T R A C E L L U L AI R
A T P - P O W E R EP 447
 subunitsis phosphorylated(hencethe name "P" class),and ATP-Powered lon PumpsGenerateand Maintain
the transported ions move through the phosphorylated sub- l o n i cG r a d i e n t sA c r o s sC e l l u l a rM e m b r a n e s
unit. The amino acid sequencearound the phosphorylated
residue is homologous in different pumps. This class in- The specificionic composition of the cytosol usually differs
cludes the Na+/K+ ATPasein the plasma membrane, which greatly from that of the surrounding extracellular fluid. In
generatesthe low cytosolic Na* and high cytosolic K* con- virtually all cells-including microbial, plant, and animal
centrationstypical of animal cells (SeeFigure 11-2). Certain cells-the cytosolic pH is kept near 7.2 regardlessof the ex-
Ca'- ATPasespump Ca'* ions out of the cytosol into the ex- tracellular pH. In the most extreme casethere is a million-
ternal medium; others pump Ca2* from the cytosol into the fold difference in H+ concentration between the pH of the
endoplasmicreticulum or into the specializedER called the cytosol of the epithelial cells lining the stomach and the pH
sarcoplasmicreticulum, which is found in muscle cells. An- of the stomach lumen. Also, the cytosolic concentration of
other member of the P class,found in acid-secretingcells of K* is much higher than that of Na*. In both invertebrates
the mammalian stomach,transportsprotons (H+ ions) out and vertebratesthe concentration of K+ is 20-40 times
of and K* ions into the cell. higher in cells than in the blood, while the concenrrationof
The structures of V-classand F-classion pumps are sim- Na* is 8-12 times lower in cells than in the blood (see
ilar to one another but unrelatedto, and more complicated Figure 11-2 and Table 11-2). Some Caz* in the cytosol is
than, P-classpumps. V- and F-classpumps contain several bound to the negatively charged groups in ATP and other
different transmembraneand cytosolicsubunits.All known
V and F pumps transport only protons and do so in a
processthat does not involve a phosphoproteinintermedi-
ate. V-classpumps generallyfunction to maintain the low
pH of plant vacuoles and of lysosomesand other acidic
vesiclesin animal cells by pumping protons from the cy-
tosolic to the exoplasmicface of the membrane against a
lON (mM)
CELL (mM)
Bt00D
proton electrochemicalgradient.The H* pump that gener-
ates and maintains the plasma membrane electric potential
SQUID AXON
in plant, fungal, and bacterialcellsalso belongsto this class. (INVERTEBRATE)-
F-classpumps are found in bacterial plasma membranes
and in mitochondria and chloroplasts.In contrasrto V-class K* 400 20
pumps, they generally function as a kind of reverseproron
pump, in which the energy releasedby the movement of Na* 50 440
protons from the exoplasmic to the cytosolic face of the
membrane down the proton electrochemicalgradient is cl 40-150 560
used to power the synthesisof ATP from ADp and pi.
Becauseof their importance in ATP synthesisin chloro- caz* o.ooo3 10
plastsand mitochondria, F-classproton pumps, commonly
calledATP synthases,are treatedseparatelyin Chapter 12. a t 3oo-4oo 5-10
The final class of ATP-powered pumps is a large family
of multiple membersthat are more diversein function than MAMMALIAN CELL
those of the other classes.Referred to as the ABC (ATp- (VERTEBRATE)
binding cassette)superfamily, this class includes several
hundred different transporr proteins found in organisms K* r39 4
ranging from bacteria to humans. As detailed below, some
of these transporr proteins were first identified as mul- Na- 12 I45
tidrug-resistance proteins that, when overexpressed in can-
c e r c e l l s ,e x p o r t a n t i c a n c e d
r r u g s a n d r e n d e ir u m o r s r e s i s t - cl 4 1.16
ant to their action. Each ABC protein is specificfor a single
substrateor group of relatedsubsrrates, which may be ions, HCO3 1.2 29
sugars,amino acids, phospholipids,cholesterol,peptides,
polysaccharides,or even proteins. All ABC rransporr pro- x- 138 9
teins share a structural organization consisting of four
"core" domains:two transmembrane(T) domains,forming Mgt* o.g 1.5
the passagewaythrough which transported moleculescross
the membrane, and two cyrosolic ATp-binding (A) do- ca2* < o.ooo2 1.g
mains. In some ABC proteins,mostly those in bacteria,the -The
core domains are present in four separarepolypeptides; in large nerve axon of the squid has been widely used in studies of the
mechanism of conduction of electric impulses.
others,the core domains are fusedinto one or two multido- rX representsproteins, which have
a net negative charge at the neutral
main polypeptides. pH of blood and cells.

448 CHAPTER
11 I T R A N S M E M B R A NT E
R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s
molecules,but it is the concentration of free, unbound Ca"*
that is critical to its functions in signaling pathways and
muscle contraction. The concentration of free Ca2* in the
cytosol is generallylessthan 0.2 micromolar (2 x 10-7 M),
a thousand or more times lower than that in the blood. Plant
cells and many microorganisms maintain similarly high cy-
tosolic concentrations of K* and low concentrations of
Caz* andNa* evenif the cells are cultured in very dilute salt
solutions.
The ion pumps discussedin this section are largely re-
sponsible for establishing and maintaining the usual ionic
gradients acrossthe plasma and intracellular membranes.In
carrying out this task, cells expend considerableenergy.For
example, up to 25 percent of the ATP produced by nerve and
kidney cells is used for ion transport, and human erythro-
cytes consume up to 50 percent of their available ATP for
this purpose; in both cases, most of this ATP is used to
power the Na+/K+ pump. The resultant Na+ and K*
gradients in nerve cells are essentialfor their ability to con-
duct electric signals rapidly and efficiently, as we detail in
Chapter 23. Certain enzymesrequired for protein synthesis
in all cells require a high K+ concentration and are inhibited
by high concentrations of Na*; these would ceaseto func-
tion without the operation of the Na*/K* pump. In cells
treated with poisonsthat inhibit the production of ATP (e.g.,
2,4-dinitrophenol in aerobic cells), the pumping stops and
the ion concentrations inside the cell gradually approach
those of the exterior environment as ions spontaneously
move through channelsin the plasma membrane down their
electrochemicalgradients.Eventually treated cells die: partly
becauseprotein synthesisrequires a high concentration of
K* ions and partly becausein the absenceof a Na* gradient
acrossthe cell membrane, a cell cannot import certain nutri-
ents such as amino acids. Studieson the effectsof such poi-
sons provided early evidencefor the existenceand significance
of ion pumps.
MuscteRelaxationDependson Ca2+ATPases
That PumpCa2*from the Cytosolinto the
S a r c o p l a s m iRce t i c u l u m
In skeletal muscle cells, Ca2* ions are concentratedand
stored in the sarcoplasmicreticulum (SR); releaseof stored
Caz* ions from the SR lumen via ion channelsinto the cy-
tosol causescontraction, as discussedin Chapter 17. A P-
class Ca2* AIPase located in the SR membrane of skeletal
muscle pumps Ca2* from the cytosol back into the lumen of
the SR, thereby inducing muscle relaxation. Becausethis
muscle calcium pump constitutesmore than 80 percent of the
integral protein in SR membranes,it is easily purified from
other membrane proteins and has been studied extensively.
Determination of the three-dimensionalstructure of this pro-
tein in severalconformational statesthat representdifferent
steps in the pumping process has revealed much about its
mechanismof action.
In the cytosol of muscle cells, the free Ca2* concentra-
7 6
tion rangesfrom 10 M (restingcells)to more than 10 M
A T P - P O W E R EP
(contracting cells), whereas the total Ca2* concentration in
the SR lumen can be as high as 10-2 M. TYo soluble pro-
teins in the lumen of the SR vesiclesbind Ca"* and serveas
a reservoir for intracellular Caz*, thereby reducing the con-
centration of free Ca2* ions in the SR vesiclesand conse-
quently the energy needed to pump Ca2* ions into the SR
fro- tit. cytosol.-Theactivity of the muscle Ca2+ AIPase in-
creasesas the free Ca2* concentration in the cytosol rises.In
skeletal muscle cells the calcium pump in the SR membrane
works in concert with a similar Ca2* pump located in the
plasma membraneto ensurethat the cytosolic concentration
of free Ca2* in resting muscle remains below 1 pM.
The current model for the mechanism of action of the
Ca2* AIPase in the SR membrane involves multiple confor-
mational states. For simplicity' we group these into E1
states,in which the two binding sitesfor Caz* ,located in the
center of the membrane-spanningdomain, face the cytosol'
andE2 states,in which thesebinding sitesface the exoplas-
mic face of the membrane,pointing into the lumen of the SR.
Coupling of ATP hydrolysis with ion pumping involves sev-
eral conformational changesin the protein that must occur
in a defined order, as shown in Figure 11-10.'When the pro-
tein is in the E1 conformation, two Ca2* ions bind to two
hieh-affinitv bindine sitesaccessiblefrom the cytosolic side;
.r,".n thouei, the Ca)* concentration is very low (seeTable
11-2). calc]um ions still fill thesesites.Next, an ATP binds to
a site on the cytosolic surface(step 1). The bound ATP is hy-
drolyzed to ADP in a reaction that requires Mgz* , and the
liberated phosphate is transferred to a specific aspartate
residue in the protein, forming the high-energy acyl phos-
phate bond denoted by E1 - P (step2).The protein then un-
dergoesa conformational changethat generatesE2' in which
the affinity of the two Ca2*-binding sitesis reduced (Figure
11.-1.1a\and in which these sites are now accessibleto the SR
lumen (step 3). The free energy of hydrolysis of the aspartyl-
phosphate bond in E1 - P is greater than that in E2-P, and
this reduction in free energy of the aspartyl-phosphatebond
can be said to power the E1 -+ E2 conformational change.
The Ca2* ions spontaneouslydissociatefrom the low-affinity
sites to enter thi SR lumen, because even though the Caz*
concentration there is higher than in the cytosol, it is lower
than the K6 for Ca2* binding in the low-affinity state (step
into the SR lumen.
Much structural and biophysical evidencesupports the
model depicted in Figure 11-10' For instance, the muscle
calcium pump has been isolated with phosphate linked to
the key aspartate residue, and spectroscopic studies have
detected slight alterations in protein conformation during
the E1 -+ E2 conversion. The two phosphorylated states
can also be distinguishedbiochemically; addition of ADP to
phosphorylated E1 results in synthesisof AIP, the reverseof
it.p f i" Figure 11-10, whereas addition of ADP to phos-
phorylated E2 does not. Each principal conformational
O N I CE N V I R O N M E N T
DU M P SA N D T H E I N T R A C E L L U L AI R
449
 Ca2t-bindrng
SR lumen
E1 E1
c?'*. \
"rl
',/ I
,\, I
)
Cytosol
\
Phosphorylat Y
aspartate
I
Conformational
ATP-
change
binding
stte Calcium
rerease
<+-

E2 E2 E2
A FIGURE 11-10 Operationalmodel of the Ca2+ATpasein the membrane. In the figure,-P indicates
a high-energyaspartyl
the SRmembraneof skeletalmusclecells.Onlyoneof the two phosphate bond;-P indicates a low-energybond.Because the
catalytic
o subunits
of thisP-class pumpis depictedE1and E2are affinityof Ca2*for the cytosolic-facing
bindingsitesin E1is a thou-
alternative
conformations of the proteinin whichthe Ca2+-binding sandfold greater thanthe affinityof Ca2*for the exoplasmic-facing
sitesareaccessible
to the cytosolicand exoplasmicfaces,respecrrvery sitesin E2,thispumptransports Ca2+unidirectionally
fromthe cytosol
An orderedsequence of stepslE), as diagrammed here,is essential to the 5RlumenSeethe textand Figure11-11for moredetails.
for coupling
ATPhydrolysis andthe transport of Ca2*ionsacross [SeeC Toyoshima andG Inesi,2004, Ann Rev.Biochem73:269-92]

state ofthe reaction cycle can also be characterizedby a dif-
ferent susceptibility to various proteolytic enzymessuch as
trypsln.
As seen in the three-dimensionalstructure of the Ca2*
pump in the E1 state,the 10 membrane-spanningct helicesin
the catalytic subunit form the passagewaythrough which
Ca"- ions move, and amino acidsin four of thesehelicesform
the two high-affinity E1 Ca2+-binding sites (Figure 11-11a,
left). One site is formed out of negatively charged oxygen
atoms from the carboxyl groups (COO-) of glutamate and
aspartate side chains, as well as from water molecules.The
other site is formed from side- and main-chain oxygen
atoms. Thus most of the water moleculesthat normally sur-
round a Ca2* ion in aqueous solution are replaced by oxy-
gen atoms attachedto the protein. In contrast, in the E2 state
'1.1-a,
(Figure right), severalof thesebindine side chains have
moved fractions of a nanometer and are Jnable to interact
with Ca2* ions, accounting for the low affinity of the E2
state for Ca2* ions.
The cytoplasmic part of the Ca2* pump consists of
three domains that are well separatedfrom each other in
the E1 state (Figure 11-11b). Each of thesedomains is
connected to the membrane-spanning helices by short seg-
ments of amino acids, and movements of these cytosolic
domains will causecorrespondingmovementsof attached
membrane-spanning o' helices. The phosphorylated
residue, Asp 351, is located on the p domain, and the
adenosine moiety of ATp binds to the N domain. Follow-
ing ATP and Ca2* binding, the N domain moves until rhe
1 phosphateof the bound ATP becomesadiacentro the as-
partate on the P domain that is to receivethe phosphate.
Although the details of these conformational changesare
not yet clear, this movement is thought to be transmitted
by scissorlike motions of the connecting segments to
movements of several membrane-spanning a helices.
These changesare especiallyapparent in the four helices
that contain the two Ca2*-binding sites:the changespre-
vent the bound Ca2+ ions from dissociatingback rntothe
cytosol but enable them to dissociateinto the exoplasmic
medium. These changes also weaken the binding of the
two Ca'- ions, as can be seenby comparing the structures
in Figure 11-11a. This weakening enablesthe bound ions
to dissociate into the exoplasmic space, here, the SR
lumen. After the Caz* ions dissociate and the asoartvl
phosphate is hydrolyzed, the protein reverts back io the
E1 conformation.
All P-classion pumps, regardlessof which ion they trans-
port, are phosphorylated on a highly conserved aspartate
residue during the transport process.Thus the operational
model in Figure 11-11 is generally applicable to all of these
ATP-powered ion pumps. In addition, the catalytic a sub-
units of all the P pumps examined to date have a similar
molecular weight and, as deduced from their amino acid
sequencesderived from cDNA clones. have a similar
arrangement of transmembrane o. helices and cytosol-
f a c i n g A , P , a n d N d o m a i n s ( s e eF i g u r e 1 1 - 1 0 ) . T h e s e

450 ' cHAPTER

11 | T R A N s M E M B R ATNREA N s p o RoTF r o N sA N D s M A L LM o L E c u L E s
 (a) El state Ca2*-bound state Ca2*-free

'-{ \,t
\- I

(b)
SR lumen

Membrane

Ca2*
Cytosol
ee

Actuator
d o m a i n( A )

b i n d i n gd o m a i n( N )

A FIGURE 11-11Structureof the catalyticotsubunitof the segment formsthreedomains: the nucleotide-binding domain
muscleca2* ATPase. (a)Ca2*-binding
sitesin the low-affinity
E2state N (blue),the phosphorylation domain P (green),and the actuator
withoutboundions,andin the E1state(/eff),with two bound
(flEtht), domainA (beige) thatconnects two of the membrane-spanning
calcium ions Sidechainsof keyamrnoacidsarewhite,andtheoxygen helices(c)Modelsof the pumpin the E1state(left)andE2state
atomson the glutamate andaspartatesidechainsarered.Inthe high- (right).Notethe differences between the E1andE2statesin the
affinityE1conformation, Ca2*ionsbindat two sitesbetweenhelices 4, conformations of the nucleotide-binding andactuator domains; these
5, 6, and8 insidethe membrane. Onesiteisformedout of negatively movements power the conformational changes of the membrane-
charged oxygenatomsfromglutamate andaspartate sidechainsandof spanning crhelices(purple)thatconstitute the Ca'*-binding sites,
watermolecules (notshown),andthe otherrsformedout of side-and converting themfromonein whichthe Ca2"-binding sitesare
main-chain oxygenatomsSeven oxygenatomssurround the Ca2*ion accessibleto the cytosolicface(E1state)to one in whichthe now
in bothsites(b)Three-dimensional modelof the proteinin the E1 loosely boundcalcium areaccessible
ions to the exoplasmic face(E2
statebasedon the structure determined by x-raycrystallography state)[Adapted from C ToyoshimaandH Nomura,2002, Nature,
Thereare10transmembrane fourof which(purple)
o helices, 418:605-1 1;C Toyoshima 2004,Ann Rev.
andG lnesi, Biochem 73'269-92:
containresidues thatparticipate
in Ca2 bindingThecytosolic andE Gouaux andR MacKinnon, 2005, Science 310:1461 l

findings strongly suggestthat all such proteins evolved
from a common precursor although they now transport
different ions.
C a l m o d u l i nR e g u l a t e st h e P l a s m aM e m b r a n e
Ca2*PumpsThat Control CytosolicCa2+
Concentrations
As we explain in Chapter 15, small increasesin the concen-
tration of free Ca2* ions in the cytosol trigger a variety of
cellular responses.In order for Caz* to function in intracel-
lular signaling,the concentration of Ca2* ions free in the cy-
tosol usually must be kept below 0.1'-0.2 pM. Animal,
yeast, and probably plant cells^expressplasma membrane
bat* ATParesthat transport Ca2* out of the cell against its
electrochemical gradient. The catalytic c subunit of these
P-classpumps is similar in structure and sequenceto the c
subunit of the muscle SR Ca2* PumP.
The activity of plasma membrane Ca"- ATPasesis reg-
ulated by calmodulin, a cytosolic Ca'*-binding protein

O N I CE N V I R O N M E N T O
A T P - P O W E R E DP U M P SA N D T H E I N T R A C E L L U L AI R 451
(seeFigure 3-31). A rise in cytosolic Ca2* inducesthe bind-
ing of Ca2* ions to calmodulin, which triggers allosteric
activation of the Ca2* ATPase.As a result, the export of
Ca2* ions from the cell accelerates,quickly restoring the
low concentration of free cytosolic Ca2* characteristicof
the resting cell.
N a * / K + A T P a s eM a i n t a i n st h e I n t r a c e l l u t aN
a n d K + C o n c e n t r a t i o nisn A n i m a l C e l l s
A secondimportant P-classion pump present in the plasma
membraneof all animal cellsis the Na+/K+ ATpase.This ion
pump is a tetramer of subunit composition c2B2. (ClassicEx-
periment 11.1 describesthe discovery of this enzyme.)The
small, glycosylatedB polypeptide helps newly synthesizedct
subunits to fold properly in the endoplasmicreticulum but
apparentlyis not involved directly in ion pumping. The amino
acid sequenceand predicted tertiary structure of the catalytic
cr subunit are very similar to those of the muscle SR Ca2+
MPase (seeFigure 11-11). In particular,the Na+/K+ ATpase
has segmentson the cytosolic face that link the ATp-binding
N domain, the phosphorylated aspartate on the p domain,
rj
A FIGURE 11-12Operationalmodelof the Na+/K*ATpase
in the plasmamembrane.Onlyoneof thetwo catalytic
subunits of thisP-class pumpisdepictedlt isnot knownwhether
justoneor bothsubunits in a srngle ATpase molecule
ions.lonpumpingbythe Na+/K+ATpase involves
dephosphorylation, andconformational chanqes
i n t h em u s c lC
e a 2 *A T P a s( se e eF i q u r 1
e 1 - 1 0 )I .nt h i sc a s e .
452 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
and the A domain to the membrane-embedded segment.The
overall transport process moves three Na+ ions out of and
two K- ions into the cell per ATP molecule hydrolyzed.
The mechanism of action of the Na*/l(+ ATpase, out-
'1,1,-12,
lined in Figure is similar to that of the musclecalcium
pump, except that ions are pumped in both directions across
the membrane, with each ion moving against its concentra-
r a+ tion gradient. In its E1 conformation, the Na*/K* ATpase
has three high-affinity Na*-binding sites and two low-
affinity K*-binding sitesaccessiblero the cytosolic surfaceof
the protein. The K- for binding of Na+ to these cytosolic
sitesis 0.5 mM, a value considerablylower than the intracel-
lular Na* concentration of :12 mM; as a result. Na* ions
normally will fully occupy these sites. Conversely,the affinity
of the cytosolic K+-binding sitesis low enough that K+ ions,
transported inward through the protein, dissociatefrom E1
into the cytosol despitethe high intracellular K+ concentra-
tion. During the E1 -+ E2 transition, the three bound Na+
ions become accessibleto the exoplasmic face, and simulta-
neouslythe affinity of the three Na*-binding sitesdrops. The
three Na+ ions, transported oufward through the protein
and now bound to the low-affinity Na+ sitesexposedto the
OverviewAnimation:BiologicalEnergyInterconversions
ffi
I
2K*
E2f
Dephosphorylation
and conformational
change
E2
hydrolysis
of the E2-Pintermediatepowersthe E2-+ E1
o conformationalchangeandconcomitant transportof two K*
ionsinward.Na+ionsareindicated by redcircles;
K+ ions,by
transport purplesquares;high-energy
acylphosphate bond,by -p; low-
phosphorylation, energyphosphoesterbond,by -P [SeeK Sweadner andC Donnet,
similar to those 2001,BiochemI 356:6875,
fordetails
of thestructure
of theo subunit
I
T F I O N SA N D S M A L L M O L E C U L E S
exoplasmicface, dissociateone at a time into the extracellu- primary sphericallysosomewith a volume of 4.18 x 10-15 ml
lar medium despitethe high extracellularNa* concentration. (diameterof 0.2 pm) will contain just 252 protons. At pH 7, the
Transition to the E2 conformation also generatestwo high- sameorganellewould have only an averageof only 0.2 protons
affinity Kt sites accessibleto the exoplasmic face. Because in its lumen, and thus pumping of only approximately 250 pro-
the K- for K* binding to thesesites (0.2 mM) is lower than teins is necessaryfor lysosomeacidification.
the extracellularK* concentration(4 mM), thesesiteswill fill By themselves,ATP-powered proton pumps cannot
with K+ ions as the Na* ions dissociate.Similarly,during the acidify the lumen of an organelle (or the extracellular space)
E2 -+ E1 transition, the two bound K* ions are transported becausethese pumps are electrogenic; that is, a net move-
inward and then releasedinto the cytosol. ment of electric charge occurs during transport. Pumping of
Certain drugs (e.g.,ouabain and digoxin) bind to the ex- just a few protons causesa buildup of positively charged
oplasmic domain of the plasma membrane Na-/l(- ATPase H* ions on the exoplasmic (inside) face of the organelle
and specificallyinhibit its ATPaseactivity. The resulting dis- membrane. For each H+ pumped across' a negative ion
ruption in the Na*/I(* balanceof cellsis strong evidencefor (e.g., OH- or Cl-) will be "left behind" on the cytosolic
the critical role of this ion pump in maintaining the normal face, causing a buildup of negatively charged ions there.
K* and Na* ion concentration gradients. These oppositely charged ions attract each other on oppo-
site faces of the membrane, generating a charge separation,
or electric potential, across the membrane. As more and
V-ClassH* ATPasesMaintain the Acidity
more protons are pumped, the excessof positive chargeson
of Lysosomesand Vacuoles the exoplasmic face repels other H* ions, soon preventing
All V-classATPasestransport only H* ions. These proton pumping of additional protons long before a significant
pumps, presentin the membranesof lysosomes,endosomes, iransmembrane H* concentration gradient is established
and plant vacuoles, function to acidify the lumen of these (Figure 11-13). In fact, this is the way that P-classH+
organelles.The pH of the lysosomallumen can be measured
preciselyin living cells by use of particles labeledwith a pH-
sensitivefluorescent dye. ufhen these particles are added to
the extracellular fluid, the cells engulf and internalize them
(phagocytosis;seeChapter 17), ultimately transporting them (a) ATP ADP + P1
into lysosomes.The lysosomal pH can be calculated from
the spectrum of the fluorescenceemitted. Maintenance of
the 10O-fold or more proton gradient betweenthe lysosomal
Cytosol
lumen (pH :4.5-5.0) and the cytosol (pH :7.0) dependson
a V-classATPaseand thus ATP production by the cell. The
low lysosomal pH is necessaryfor optimal function of the @
NeutralpH
many proteases,nucleases,and other hydrolytic enzymesin
the lumen; on the other hand, a cytosolic pH of 5 would dis-
rupt the functions of many proteins optimized to act at pH7
and lead to death of the cell.
The ATP-powered proton pumps in lysosomal and vac-
(b) ATP ADP + Pr
uolar membraneshave been isolated, purified, and incorpo-
rated into liposomes.As shown in Figure11-9 (center),these
V-class proton pumps contain two discrete domains: a cy-
tosolic hydrophilic domain (V1) and a transmembrane do-
main (Ve) with multiple subunits forming each domain.
Binding and hydrolysis of ATP by the B subunits in V1 pro- H+ cl- Cl
H+ No electric
vide the energy for pumping of H* ions through the proton- potential
Acidic pH H*
conducting channel formed by the c and a subunits in Ve.
Unlike P-class ion pumps, V-class proton pumps are not
phosphorylated and dephosphorylatedduring proton trans-
port. The structurally similar F-classproton pumps, which
FIGURE 11-13 Effectof V-class H* pumpson H-
we describein Chapter 12, normally operatein the "reverse"
concentrationgradientsand electricpotential gradientsacross
direction to generateATP rather than pump protons; their cellularmembranes. (a)lf an intracellular
organellecontains only
mechanismof action is understood in great detail. V-classpumps,protonpumpinggenerates potential
an electric across
Pumping of relatively few protons is required to acidify an the membrane, facing
cytosol- side and
negative lumenal-side positive
intracellular vesicle.To understandwhy, recall that a solution of pH.(b)lf the organelle
changein the intraluminal
but no significant
pH 4 has an H* ion concentration of 10-a moles per liter, or membrane alsocontainsCl- channels,anionspassivelyfollowthe
10 7 molesof H* ions per milliliter. Sincethere are6.02 x 1023 pumpedprotons, in an accumulation
resulting of H+ andCl- ions
atoms of H per mole (Avogadro'snumber), then a milliliter of a rnthe lumen(lowluminalpH)but no electricpotential the
across
pH 4 solution contains6.02 x 1016H* ions. Thus at pH 4, a membrane.

O N I CE N V I R O N M E N T
DU M P SA N D T H E I N T R A C E L L U L AI R
A T P - P O W E R EP 453
pumps generate a cytosol-negative potential across plant membrane-spanningcr helices, form the pathway through
and yeastplasma membranes. which the transported substance (substrate)crossesthe
In order for an organellelumen or an extracellular space membrane and determine the substrate specificity of each
(e.g.,the lumen of the stomach) to becomeacidic, movement ABC protein. The sequencesof the A domains are approxi-
of protons must be accompaniedeither by (1) movementof mately 30-40 percent homologous in all members of this
an equal number of anions (e.g.,Cl-) in the samedirection superfamily,indicating a common evolutionary origin. Some
or by (2) movement of equal numbers of a different carion in ABC proteins also contain an additional exoplasmic sub-
the opposite direction. The first processoccurs in lysosomes strate-bindingsubunit or regulatory subunit.
and plant vacuoles, whose membranes contain V-class H+ The plasma membrane of many bacteria contains nu-
ATPasesand anion channels through which accompanying merous permeases that belong to the ABC superfamily.
Cl- ions move (Figure 11-13b). The secondprocessoccurs Theseproteins use the energy releasedby hydrolysis of ATP
in the lining of the stomach,which contains a P-classH*/K* to transport specificamino acids, sugars,vitamins, and even
ATPasethat is not electrogenicand pumps one H+ outward peptides into the cell. Since bacteria frequently grow in soil
and one K* inward. Operation of this pump is discussed or pond water where the concentration of nutrients is low,
later in the chapter. these ABC transport proteins enable the cells to import nu-
trients against substantialconcentration gradients.Bacterial
BacterialPermeases Are ABC proteinsThat permeasesgenerally are inducible; that is, the quantity of a
lmport a Varietyof Nutrientsfrom the transport protein in the cell membrane is regulated by both
Environment the concentration of the nutrient in the medium and the
metabolic needsof the cell.
As noted earlier, all members of the very large and diverse
In the E. coli vitamin B12permease,a typical bacterial
ABC superfamily of transport proteins contain two trans-
ABC protein whose structure is known in molecular detail
membrane (T) domains and two cytosolic ATP-binding (A) (seeFigure 11-18), the two transmembranedomains and
domains (Figure 1.1-14).The T domains, each built of 10
two cytosolic ATP-binding domains are formed by four
separatesubunits.Gram-negativebacteria,such as E. coli,
contain, besidesthe plasma membrane, an outer mem-
brane also built of a phospholipid bilayer (seeFigure 1-2).
This outer membrane contains porin proteins (seeFigure
10-18) that render it highly permeableto most small mol-
Endoplasm ecules, including amino acids and vitamins. Thus these
moleculescan enter into the periplasmic spacein between
the plasma and outer membranes(seeFigure 1-2). A solu-
ble vitaminBl2-binding protein, located in the periplasmic
Membrane
space, binds vitamin B12 tightly and directs it to the T
subunits of the permease, through which the vitamin
Cytoplasm crossesthe plasma membranepowered by ATP hydrolysis.
Precisely how transport occurs is not known, but it is
thought that B12 binding is signaledto the nucleotide hy-
drolysis sites where the affinity for ATP increases,a pre-
requisite for a productive transport cycle. The two ATP-
binding domains then carry out a highly cooperarive
ATP-binding and hydrolysis reaction that is concurrent
with a substantial conformational change in the mem-
brane-spanning segments;this change somehow allows
the bound substrateto pass between the two membrane-
spanning domains into the cell cytoplasm. Then the trans-
porter returns to the resting state through the dissociation
A FfGURE11-14 Structureof the Escherichia coli BtuCD
protein,an ABCtransportermediatingvitamin B12uptake.The of ADP and inorganic phosphate.
complete transporter isassembled fromfoursubunits. Mutant E. coli cells that are defectivein any of the 812
two identical
membrane-spanning subunrts (green),
andtwo ATp-binding permease subunits or the soluble periplasmic-binding
subunits
(blue).Molecules of cyclotetravanadate,
an analogof the phosphate protein are unable to transport B12 into the cell but are
groupsin ATP, arelocated in theATp-binding sitesandaredepicted able to transport other molecules such as amino acids
in ball-and-stick
Approxtmate boundariesof the membrane bilayer whose uptake is facilitated by other transport proteins.
areindicated bythe grayshaded area,withtheexternal surface at Such genetic analysesprovide srrong evidencethat these
the top andthe cytoplasm at the bottom [AfterK Locheret al, 2002. permeasesfunction to transport specific solutes into bac-
Sclence295:1091-98 l terial cells.

454 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E s
T h e A p p r o x i m a t e l y5 0 M a m m a l i a nA B C
TransportersPlay Diverseand lmportant Roles
i n C e l la n d O r g a n P h y s i o l o g y
Discovery of the first eukaryotic ABC protein to be recog-
nized came from studies on tumor cells and cultured cells
that exhibited resistanceto several drugs with unrelated
chemical structures.Such cells eventually were shown to
express elevated levels of a multidrwg-resistance(MDR)
transport protein known as MDR1. This protein usesthe
energy derived from ATP hydrolysis to export a large vari-
ety of drugs from the cytosol to the extracellularmedium.
The Mdrl gene is frequently amplified in multidrug-resist-
ant cells,resulting in a large overproduction of the MDRl
pfoteln.
Most drugs transported by MDRl are small hydropho-
bic molecules that diffuse from the medium across the
plasma membrane, unaided by transport proteins, into the
cell cytosol, where they block various cellular functions.
Two such drugs are colchicine and vinblastine, which block
assemblyof microtubules (Chapter 18). ATP-poweredex-
port of such drugs by MDR1 reducestheir concentration in
the cytosol. As a result, a much higher extracellular drug
concentration is required to kill cells that express MDR1
than those that do not. That MDR1 is an AlP-powered
small-moleculepump has beendemonstratedwith liposomes
containing the purified protein. The AIPase activity of these
liposomesis enhancedby different drugs in a dose-dependent
manner corresponding to their ability to be transported by
MDR1.
About 50 different mammalian ABC transport proteins
are now recognized(Table 11-3)' Severalare expressedin
abundance in the liver, intestines, and kidney-sites where
natural toxic and waste products are removed from the
body. Substratesfor these ABC proteins include sugars'
amino acids, cholesterol' bile acids, phospholipids, pep-
tides, proteins, toxins, and foreign substances.The normal
function of MDR1 most likely is to transport various natu-
ral and metabolic toxins into the bile or intestinal lumen or
to the urine being formed in the kidney. During the course
of its evolution, MDRl appearsto have acquired the ability
to transport drugs whose structures are similar to those of
these endogenous toxins. Tumors derived from MDR-
expressingcell types,such as hepatomas(liver cancers),fre-
quently are resistant to virtually all chemotherapeutic
agents and are thus difficult to treat' presumably because
the tumors exhibit increasedexpressionof MDR1 or the re-
lated MDR2.
Severalhuman geneticdiseasesare associatedwith de-
fectiveABC proteins.The best studied is cystic fibrosis
(CF), causedby a mutation in the geneencodingthe cysticfi-
brosistransmembraneregulator (CFTR)' This CI- transport
protein is expressedin the apical plasma membranesof ep-
ithelial cellsin the lung, sweatglands' pancreas'and other tis-
sues.For instance,CFTR protein is important for resorption
of Cl- into cells of sweat glands' and babies with cystic fi-
brosis, if licked, often taste "salty." An increase in cyclic
AMP (cAMP), a small intracellular signalingmolecule,causes

PR()TEIN EXPRESSION
TISSUE IUNCTION CAUSEO
DISEASE PROTTIN
BYDETECTIVE

ABCB1(MDR1) Adrenal,kidney,brain Exportslipophilicdrugs

ABCB4 (MDR2) Liver Exportsphosphatidylcholine

into bile

ABCB11 Ltver Exportsbile saltsinto bile

CFTR Exocrinetissue TransportsCl ions Cysticfibrosis

ABCDr inperoxisoma' (ADL)

Adrenoreukodvstrophv
H:ffi;'"'# ::*ffn^1':':irr::ffitT;n.fl"-
fatty acids

ABCG5/8 Liver,instestine Exportscholesteroland other sterols p-Sitosterolemia

ABCA1 Ubiquitous Exporrscholesteroland phospholipid Tangier'sdisease

for uptakeinto high-densitY
lipoprotein(HDL)

O N I CE N V I R O N M E N T
D U M P SA N D T H E I N T R A C E L L U L AI R
A T P - P O W E R EP
455
 phosphorylation of CFTR and stimulates Cl- transport by
such cellsfrom normal individuals but not from CF individu-
als, who have a defectiveCFTR protein. (The role of cAMp
in numerous signaling pathways is covered in Chapter 15.)
The sequenceand predicted structure of the CFTR protein,
basedon analysisof the cloned gene,are very similar to those
of the MDR1 protein exceptfor the presenceof an additional
domain, the regulatory (R) domain, on rhe cytosolic face.
Moreover, the Cl--transporr acrivity of CFTR protein is en-
hanced by the binding of ATP, but it is uncertain whether or
not CFTR is actually an ATP-powered Cl- pump.l

C e r t a i nA B CP r o t e i n s" F l i p " p h o s p h o l i p i d as n d
O t h e r L i p i d - S o l u b lS
e u b s t r a t e fsr o m O n e
MembraneLeafletto the OppositeLeaflet
The substratesof mammalian MDR1 are primarily planar, ADP+ P,
lipid-solublemoleculeswith one or more positive charges; A FIGURE 11-15Flippase modelof transportby MDRl and
they all compete with one another for transport by MDR1, similarABCproteins.Stepll: Thehydrophobic portion(black)of a
suggestingthat they bind to the samesite or siteson the Dro- substrate molecule movesspontaneously fromthecytosol intothe
tein. In contrast to bacterial ABC proteins, all four domains cytosolic-facingleaflet
of the lipidbilayer,
whilethecharged end(red)
of mammalian MDRl are fused into a single 170,000-M\fl remains in thecytosolStepE: Thesubstrate diffuses
laterallyuntilit
protein. The recently determined three-dimensional struc- encounters andbindsto a siteon the MDRlproteinwithinthe bilayer.
ture of a homologous E. coli lipid-transport protein reveals StepS: Theproteinthen"flips"thecharged substratemolecule into
that the molecule is V shaped, with the apex in the mem- theexoplasmic leaflet,
an energetically unfavorable
reaction involving
brane and the arms containing the ATP-binding sites pro- conformational changes in the membrane-spanning domains thatare
powered bythecoupled hydrolysis of ATpbythecytosolicdomains
truding into the cytosol (Figure11-15).
Steps4 andE: Oncein theexoplasmic face,thesubstrate againcan
Although the mechanism of transporr by MDR1 and
diffuselaterally
in the membrane andultimately movesintothe
similar ABC proteins has not been definitively demon- phaseon theoutside
aqueous fromp Borst,
of thecell [Adapted N
strated, a likely candidate is the flippase model depicted in Zelcer,and A van Helvoort,2000, Biochim Biophys Acta 14g5.12g,1
Figure 11-15. According to rhis model, MDR1 ,,flips,' an
amphiphilic subsrraremolecule from the cytosolic to the ex-
oplasmic leaflet, an energericallyunfavorable reaction pow-
cellsmovesnormally through the secretorypathway to the cell
ered by the coupled ATPaseactivity of the protein.
surface(Chapter 1,4).At higher nonpermissiveremperarures,
Support for the flippase model of transport by MDRl however,the secprotein is nonfunctionaland secretoryvesicles
comes from ABCB4 (originally called MDR2), a homolo-
cannot fusewith the plasmamembrane,as they do in wild-type
gous protein present in the region of the liver-cell plasma
cells; so vesiclescontaining ABCB4 and other yeasrprorerns
membrane that faces the bile duct. ABCB4 movei ohos-
accumulatein the cells.After purifying thesesecretoryvesicles,
phatidylcholinefrom the cytosolicto the exoplasmicleaflet
investigatorslabeled them in vitro with a fluorescentphos-
of the plasma membrane for subsequentreleaseinto the
phatidylcholine derivative. The fluorescence-quenchingassay
bile in combination with cholesreroland bile acids, which
outlined in Figure 11-16 was usedto demonstrarethat the vesi-
themselvesare transported by other ABC family members.
cles containing ABCB4 exhibited an AlP-dependent flippase
Severalother ABC superfamilymembersparticipatein the
activity, whereasthose without ABCB4 did not.
c e l l u l a re x p o r ro f v a r i o u sl i p i d s( s e ef a U t e t l - : t .
ABCB4 was first suspectedof having phospholipidflippase
activity becausemice with homozygous loss-of-function muta-
tions in the ABCB4 geneexhibited defectsin rhe secretionof
ATP-PoweredPumps and the Intracellular lonic
phosphatidylcholineinto bile. To determinedirectly if ABCB4
Environment
was in fact a flippase, researchersperformed experiments on a
homogeneouspopulation of purified vesicleswith ABCB4 in r classesof transmembraneproteinscouple the energy-
the membrane and with the cytosolic face directed outward. ing hydrolysis of AIP with the energy-requiringtrans-
These vesicleswere obtained by introducing cDNA encoding port of substancesagainst their concentrationgradient: p-,
mammalian ABCB4 into a temperature-sensitiveyeasrsec mv- V-, and F-classpumps and ABC proteins (seeFigure 11-9).
tant. At lower, permissivetemperatureswhere the secproteinis r The combined action of P-classNa*/K* ATpasesin the
functional, the ABCB4 protein expressedby the transfected plasma membrane and homologous Ca2* Mpases in the

456 CHAPTER
11 I T R A N S M E M B R A NTER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s
 Externallabeledlipids:
u nprotected,

u nprotected,
Trans- a l l l a b e l e dl i p i d s
ATPaSe membrane
ABC84 quenched
(flippase) domain

ADP ADP
Add
ATP

o
q)
c
c)
o
0)
th
o)

T i m e( m i n )

a EXPERIMENTAL FIGURE 11-16ln vitro fluorescence- abilityto fluoresce (gray). Inthe presence of the quencher, only
quenchingassaycan detect phospholipidflippaseactivity of labeled phospholipid in the protected environment on the Inner
ABCB. A homogeneous population of secretoryvesiclescontaining leafletwillfluoresce Subsequent to the additionof the quenching
ABC84proteinwaspurified f roma yeastsecmutanttransfected with agent,thetotalfluorescence decreases with timeuntilit plateaus at
gene phospholipidscontaininga the pointat whichallexternal fluorescence isquenched and only the
IheABCB4 Step [: Synthetic
modifiedheadgroup(blue)wereincorporated primarily internal phospholipid fluorescence canbe detected. Theobservatton
fluorescently
StepZ: lf of greater fluorescence (lessquenching) in the presence of ATPthan
intotheouter,cytosolicleafletsof the purified
vesicles
ABC84actedasa flippase, thenon addition of ATPto the outsideof in itsabsence indicates thatABC84hasflippedsomeof the labeled
labeled phospholipid to the insideStep4: Addltionof detergent to the
a smallfractionof the outward-facing
thevesrcles,
StepB: Flipping vesicles generates micelles and makes all fluorescentlipids accessible
phospholipidswouldbe flippedto the insideleaflet.
quenching to the quenching agent and lowers the fluorescence to baseline
wasdetected by addinga membrane-impermeable
calleddithioniteto the mediumsurrounding thevesicles valueslAdapted fromS Ruetz andP Gros,1994, Cell771071]
compound
reacts
Dithionite with thefluorescent headgroup,destroying its

plasma membraneor sarcoplasmicreticulum createsthe usual
ion milieu of animal cells: high K*, low Ca2*, and low
Na* in the cytosol; low K+, high Ca2*, and high Na* in
the extracellular fluid.
r In P-classpumps, phosphorylation of the a (catalytic)
subunit and a changein conformational statesare essential
for coupling ATP hydrolysis to transport of H+, Na*, K*,
or Ca2* ions (seeFigures 11-10, L1.-1.L,and 1'1-12).
r V- and F-classATPases,which transport protons exclu-
sively, are large, multisubunit complexes with a Proton-
coniucting channel in the transmembrane domain and
ATP-binding sitesin the cytosolic domain.
ss H* pumps in animal lysosomal and endosomal
anes and plant vacuole membranesare responsible
intaining a lower pH inside the organellesthan in
the surroundingcytosol (seeFigure 11-13b).

O N I CE N V I R O N M E N T
DU M P SA N D T H E I N T R A C E L L U L AI R
A T P - P O W E R EP
457
 r AII members of the large and diverse ABC superfamily
of transport proteins contain four core domains: two
transmembrane domains, which form a pathway for
solute movement and determinesubstratespecificity,and
two cytosolic ATP-binding domains (see Figures 11-14
and 11-15).
r The ABC superfamily includes bacterial amino acid and
sugar permeasesand about 50 mammalian proteins (e.g.,
MDR1, ABCA1) that transport a wide array of substrates,
including toxins, drugs, phospholipids, peptides, and pro-
teins, into or out of the cell.
r According to the flippasemodel of MDR activity,a sub-
strate molecule diffuses into the cytosolic leaflet of the
plasma membrane, then is flipped to the exoplasmic
leaflet in an ATP-powered process,and finally diffuses
from the membrane into the extracellularspace(seeFig-
ure11-15).
r Biochemical experiments directly demonstrate that
ABCB4 (MDR2) possessesphospholipid flippase activity
(seeFigure 11-16).
Nongatedlon Channels
andthe
RestingMembranePotential
In addition to ATP-powered ion pumps, which transport
ions againsl their concentration gradients, the plasma
membranecontains channelproteins that allow the princi-
pal cellular ions (Na+, K+, Ca2+, and Cl-) to move
through them at different rates down their concenrrarron
gradients.Ion concentrationgradientsgeneratedby pumps
and selectivemovementsof ions through channelsconsti-
tute the principal mechanism by which a difference in volt-
age, or electric potential, is generatedacross the plasma
membrane.In other words, ATp-powered ion pumps gen-
erate differencesin ion concentrationsacross the plasma
membrane, and ion channels utilize these concentration
gradients to generatean electric potential across the mem-
b r a n e ( s e eF i g u r e I 1 - 2 ) .
In all cells the magnitude of this electric potential gener-
ally is -70 millivolts (mV) with the inside of the cell mem-
brane always negatiuewith respectto the outside.This value
does not seemlike much until we consider rhat the thickness
The ionic gradients and electric potential across the
plasma membrane play crucial roles in many biological
processes.As noted previously,a rise in the cytosolic Ca2+
concentration is an important regulatory signal, initiating
contraction in musclecells and triggering protein secretion
458 . cHAprE1
R1 | T R A N S M E M B RTARNAEN s p o R
o FT t o N sA N D S M A L LM O L E C U L E S
by many cells, such as that of digestive enzymes in ex-
ocrine pancreatic cells. In many animal cells, the com-
bined force of the Na* concentration gradient and mem-
brane electric potential drives the uptake of amino acids
and other molecules against their concentration gradient
by ion-linked symport and antiport proteins (seeSection
11.5). Furthermore,the conduction of action potentials by
nerve cells depends on the opening and closing of ion
channelsin responseto changesin the membranepotential
( C h a p t e r2 3 ) .
Here, we discussthe origin of the membrane electric po-
tential in resting cells, often called the cell's "resting poten-
tial"; how ion channels mediate the selectivemovement of
ions acrossa membrane;and useful experimentaltechniques
for characterizing the functional properties of channel
protelns.
SelectiveMovement of lons Createsa
Transmembrane ElectricPotentialDifference
To help explain how an electric potential across the
plasma membrane can arise, we first consider a set of
simplified experimental systems in which a membrane
separatesa 150 mM NaCl/15 mM KCI solution (similar
to the extracellular medium surrounding metazoan cells)
on the right from a 15 mM NaCl/150 mM KCI solution
(similar to that of the cytosol) on the left. A potentiome-
ter (voltmeter) is connectedto both solutions to measure
any difference in electric potential across the membrane.
If the membrane is impermeable to all ions, no ions will
flow across it. There will be no difference in voltage, or
electric potential gradient acrossthe membrane,as shown
i n F i g u r e1 1 - 1 7 a .
Now supposethat the membrane contains Na*-channel
proteins that accommodateNa+ ions but exclude K+ and
Cl- ions (Figure 11-17b). Na* ions then tend to move
down their concentration gradient from the right side to the
left, leaving an excessof negative Cl- ions compared with
Na- ions on the right side and generating an excessof pos-
itive Na- ions compared with Cl- ions on the left side. The
excessNa* on the left and Cl on the right remain near the
respectivesurfacesof the membrane becausethe excesspos-
itive chargeson one side of the membrane are attracted to
the excessnegative chargeson the other side. The resulting
separation of charge across the membrane constitutes an
electric potential, or voltage, with the left (cytosolic) side of
the membrane having excesspositive charge with respectto
the right.
As more and more Na* ions move through channels
across the membrane, the magnitude of this charge differ-
point at which the two opposing factors that determine the
( a ) M e m b r a n ei m p e r m e a b l et o N a + ,K + ,a n d C l - < E X P E R I M E N TFA
IGL U R1E1 - 1 7G e n e r a t i oonf a
transmembrane electric potential(voltage)dependson the
0 selectivemovementof ionsacrossa semipermeable
membrane.Inthisexperimental system, a membrane separates a
Potentiometer- (/eft)
from a
'1 mM NaCl/1
50 5
15 mM NaCl/150 mM KCIsolution
mM KCIsolution (flqhf);theseionconcentrations aresimilar to those
andblood,respectively
in cytosol lf the membrane separating the
two solutions is impermeable to allions(a),no ionscanmoveacross
the membrane andno difference in electflc potential isregistered on
Cell cytosol Extracellular the potentiometer connecting the two solutions lf the membrane is
medium permeable
selectively only to Na+ (b)or to K+ (c), then diffusion of
ionsthroughtheirrespective channels leadsto a separation of charge
1 5m M 1 5 0m M At equilibrium, the membrane potential
acrossthe membrane
Na'Cl- Na*Cl-
caused bythechargeseparation becomes equalto the Nernst
1 5 0m M 1 5m M on the potentiometer Seethetextfor
K*Cl-
Ex"or E6regtstered
potential
K+Cl-
Cytosolic Exoplasmic furtherexplanation
face face

( b ) M e m b r a n ep e r m e a b l eo n l Y t o N a * movement of Na* ions-the membrane electric potential

0 and the ion concentration gradient-balance each other out'
Membrane electric Potential =
At equilibrium, no net movement of Na- ions occurs across
+59 mV, cytosolic face of the
+60 membrane positive with the membrane. Thus this semipermeablemembrane, like all
respectto the exoplasmicface

store positive chargeson one side and negative chargeson

the other.

Na* channel

chemistry:
Chargeseparationacrossmembrane
RT. [Na,] (1.1,-2)
EN': l"
zF tNurl
( c ) M e m b r a n ep e r m e a b l eo n l Yt o K +

0
Membrane electric Potential =
-59 mV, cytosolic face of the
membrane negative with
respectto the exoplasmicface.

equation is written with the concentrationof ion in the ex-

tracellular solution (here the right side of the membrane)
K+channel placed in the numerator and that of the cytosol in the de-
nomrnator.
At 20'C, Equation 11-2 reducesto

INu'] (11-3)
E r . r:, 0 . 0 5 t9o s ' o ; 5 u , l
C h a r g es e p a r a t i o na c r o s sm e m b r a n e

M E M B R A N EP O T E N T I A L O 459
AND THE RESTING
N O N G A T E DI O N C H A N N E L S
 If [Na,]/[Na1] : 10, a tenfold ratio of concenrrarionsas
in Figure 11-17b, rhen El.l. : +0.059 V (or +59 mV). with
the left, cytosolic side positive with respectto the exoplasmic
right side.
If the membraneis permeableonly to K* ions and not to
Na* or Cl- ions, then a similar equationdescribesthe potas-
sium equilibrium potential E11:
:0.059loSroffi
EN"
The magnitude of the membrane electric potential is the
same (59 mV for a tenfold difference in i,on concenrra-
tions), except that the left, cytosolic side is now negatrue can be measured with a microelectrode inserted inside the
with respect to the right (Figure 11-17b), opposite io the
polarity obtained acrossa membraneselectiveiypermeable
to Na* ions.
T h e M e m b r a n ep o t e n t i a li n A n i m a l C e l l s
DependsLargelyon potassiumlon Movements
T h r o u g hO p e n R e s t i n gK + C h a n n e l s
Although restingK* channelsplay the dominant role in
ton pumps (seeFigure 11-1,3a).In aerobic bacterial cells
the inside negarive potential is generated by outward
pumping of protons during electron transporr, a process
( 1 1 - 4 ) similar to proton
pumping in mitochondrial inner mem-
branes that will be discussedin detail in Chapter 12 (see
Figure 12-16).
The potential acrossthe plasma membrane of large cells
cell and a reference electrode placed in the extracellular
fluid. The two are connectedto a potentiometer capable of
measuringsmall potential differences(Figure 11-1g). The
potential acrossthe surface membrane of most animal cells
generally does not vary with time. In contrast, neurons and
muscle cells-the principal types of electricallyactive cells-
undergo controlled changesin their membrane potential that
we discussin Chapter 23.

The plasma membranesof animal cellscontain many open

K+ channelsbut few open Na*, Cl , or Ca2* channels.es

Potentiometer
ions through thesechannels,called resting K+ channels,is
the major determinant of the inside-negativemembrane
p o t e n t i a l . L i k e a l l c h a n n e l s ,t h e s e a l r e r n a t eb e t w e e n a n

Microelectrode Referenceelectrode
f i l l e dw i t h in contactwith
conducting b a t h i n gm e d i u m
s a l ts o l u t i o n

Bathing medium
Quantitatively,the usual resting membranepotential of
-70 mY is closero +**++++ +++++++
the potassiumequilibrium potential,cal_ +
culated from the Nernst equation and the K+ concentrations
in cellsand surroundingmedia depictedin Table 11_2.Usu_
Cytosol
ally the potential is lower than ihat calculated from the P l a s m am e m b r a n e

EXPERIMENTAL FIGURE 11-18The electricpotentialacross

the plasmamembraneof living cellscan be measured.A
microelectrode, constructed by fillinga glasstubeof extremely small
d i a m e t ewr i t ha c o n d u c t i nf lgu i ds u c ha sa K C Is o l u t i o ni s, i n s e r t e d
gradient that drives the flow of ions through resting K+ intoa cellin sucha waythatthe surface membrane sealsitself
channelsis generatedby the Na+/K* ATpase describeJore_ aroundthe tip of the electrode. A reference electrode ls placedin
viously (seeFigures 11-2 and 11-12). In the absenc. of thi, the bathingmedium. A potentiometer connectinq thetwo
pump, or when it is inhibited, the K+ concenrrationgradient electrodes registers the potential, in thiscase-60 mV A potential
difference is registered onlywhenthe microelectrode is inserted into
cannot be maintained, and eventually the magnitude of the
the cell;no potential is registered if the microelectrode is in the
membrane potential falls to zero.
b a t h i n fgl u i d

460 C H A P T E R1 1 | T R A N S M E M B R A NTER A N S P O R O
T F | O N SA N D S M A L L M O L E C U L E S
( a ) S i n g l es u b u n i t ( b )T e t r a m e r i c h a n n e l
filter

Exterior

Membrane

Cytosol

a helices; theyconsist of a nonhelical"turret,"whichlinesthe upper

FIGURE 11-19Structureof restingK* channelfrom the
lividans.All K* channelproteinsare partof the pore;a shorto helix; and an extended loopthat protrudes
bacteriumStreptomyces
two intothe narrowest part of the poreand forms the filter.
ion-selectivity
tetramerscomprisingfouridentrcalsubunits,eachcontaining
conserved membrane-spanning calledby convention
cthelices, 55 ThisfilterallowsK* (purple spheres)but nototherionsto pass'Below
anda shorterB or poresegment (pink)(a)Oneof thef ilteristhecentralcavityor vestibule linedbythe inner,or 56 ct,
and56 (yellow),
viewedfromtheside,with keystructural features helixesThesubunits in gated K* channels,whichopenandclosein
thesubunits,
(b)Thecomplete viewedfromtheside response to specificstimuli,contain transmembrane
additional helices
indicated. tetramericchannel
not shown these
here; are in
discussed Chapter 23 Y
[See Zhou etal,
end(nqht)ThePsegments
(/eft)andthetop,or extracellular, are
located neartheexoplasmic surfaceandconnect the 55 and56 2001,Nature 414:43
l

l o n C h a n n e l sC o n t a i na S e l e c t i v i t yF i l t e r Severalrelated piecesof evidencesupport the role of P

segmentsin ion selection.First, the amino acid sequenceof
Formedfrom ConservedTransmembrane
thI P segmentis highly homologousin all known K* chan-
Segments .r.1, different from that in other ion channels' Second'
"nJ
All ion channels exhibit specificity for particular ions: K-
channelsallow K* but not closely related Na* ions to enter'
whereasNa* channelsadmit Na* but not K+. Determina-
tion of the three-dimensionalstructure of a bacterial K*
channel first revealed how this exquisite ion selectivity is
achieved.Comparisonsof the sequences of other K+, Na+,
and Ca2* channelsestablishedthat all suchproteinssharea
common structure and probably evolved from a single type other ions.
of channelprotein. Na* ions are smaller than K* ions. How, then' can a
Like alf other K+ channels,bacterial K+ channelsare channel protein exclude Na* ions, yet allow passageof
built of four identical subunits symmetrically arranged K*? The ability of the ion-selectivityfilter in K* channels
around a central pore (Figure 1'1'-1'9)'Each subunit contains to selectK* over Na* is due mainly to backbonecarbonyl
two membrane-spanning ct helices(55 and 56) and a short P oxygens on residues located in a Gly-Tyr-Gly sequence
(pore domain) segmentthat partly penetratesthe membrane thaiis found in an analogousposition in the P segmentin
bilayer. In the tetrameric K* channel, the eight transmem-
brane a helices(two from each subunit) form an "inverted
tepee," generating a water-filled cavity called the uestibule
in the central portion of the channel that extends halfway
through the membrane.Four extendedloops that are p^rt
of the four P segmentsform the actual ion-selectiuityfilter
in the narrow part of the pore near the exoplasmicsurface,
above the vestibule.

M E M B R A N EP O T E N T I A I
SN D T H E R E S T I N G
461
N O N G A T E DI O N C H A N N E L A
 ( a ) K * a n d N a +i o n s i n t h e p o r e o f a K +c h a n n e l( t o o v i e w )
< FIGURE 11-20Mechanism of ion selectivityand transportin
restingK+ channels. (a)Schematic diagrams of K+ andNa+ions
K+in water Na+in water
hydrated in solution andin the poreof a K+channelAs K+ ions
passthroughthe selectivity filter,theylosetheirboundwater
molecules andbecome coordinated instead to eightbackbone
carbonyl oxygens, fourof whichareshown,thatarepartof the
conserved aminoacidsin thechannel-lining loopof eachp segment
Thesmaller Na* ionswith theirtightershellof watermolecules
cannotperfectly coordinatewith thechannel oxygenatomsand
therefore passthroughthechannel onlyrarely(b)High-resolution
K* in K pore Naiin K pore electron density mapobtained fromx-raycrystallography showing
K+ ions(purple spheres) passing throughtheselectivity filter.Only
two of thediagonally opposed channel subunits areshown.Within
theselectivityfiltereachunhydrated K+ ionrnteracts with eight
carbonyl oxygenatoms(redsticks) liningthe channel, two from
eachof thefoursubunits, asif to mimictheeightwatersof
hydration(c)Interpretation of the electron density mapshowing
thetwo alternating statesbywhichK+ ionsmovethroughthe
( b ) K * i o n s i n t h e p o r e o f a K +c h a n n e l( s i d ev i e w )
channelIn state1, movingfromtheexoplasmic sideof thechannel
oo Inward, oneseesa hydrated
molecules,
K* ionwith itseightboundwater

o
K- ionsat positions 1 and3 withintheselectivity filter,
Exoplasmic
Tace
E anda fullyhydrated K+ ionwithinthevestibule DuringK+
ot , t movement eachionin state1 movesonestepinward,formingstate

| ..^'
o 2 Thusin state2 the K+ ionon theexoplasmic
haslostfourof itseightwaters,
sideof thechannel
the ionat position1 in statet has

- o movedto posltion
to position
2, andthe tonat position 3 in statet hasmoved
4 In goingfromstate2 to state1,the K+ at position
Carbonyl
oxygens
o 4 movesintothevestibule
anotherhydrated
andpicksup eightwatermolecules,
K* ionmovesintothechannel openingandthe
while

t
'4j
o otherK' ionsmovedownonestep.lpart(a)adapted
1998, Science280:56
Nature414.43l
parts(b)and(c)adapted
fromC.Armstrong,
fromy Zhouetal,2001.

Vestibule-.-
a-
K+ through the channel.A dehydratedNa* ion is too small to
bind to all eight carbonyl oxygens that line the selectivity
filter with the samegeometry as a Na+ ion surrounded by
( c ) l o n m o v e m e n tt h r o u g hs e l e c t i v i t fyi l t e r its normal eight warer molecules.As a result, Na* ions
would "prefer" to remain in water rather than enter the
oooo

cooo
o
oooo

lnreracr properly with the O atoms in the selectivityfilter.

Also, more energyis required to strip the waters of hydra_
tion from Ca2* than from K+.
Recent x-ray crystallographicstudies reveal that both
when open and closed, the channel contains K+ ions within
the selectivityfilter; without theseions the channel probablv
COO would collapse.The K+ ions are thought to be presenteither
at positions 1 and 3 or at2 and 4, each surrounded by eight
c carbonyl oxygen atoms (Figure 1I-20b and c). Severalk*
State 1 State 2 ions move simultaneouslythrough the channel such that
when the ion on the exoplasmic face that has been partiallv
stripped of its water of hydration moves into position 1, th!
ion at position 2 jumps to position 3 and the one at position
4 exits the channel (Figure l1-20c).
Although the amino acid sequencesof the p segmentin
Nat and K* channels differ somewhat, they are similar

462 C H A P T E R1 1 | TRANSMEMBRAN
TER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E S
> EXPERIMENTAL FIGURE 11-21Currentflow through
individualion channelscan be measuredby patch-clamping Deviceto maintainconstant
voltage acrossmembraneand
technique.(a)Basic experimental arrangement for measuring
to measurecurrentflow across
currentflowthroughindividual ionchannels in the plasma membraneat tip of Patch
membrane of a livingcell.Thepatchelectrode, filledwith a current- electrode
conducting salinesolution, isapplied,with a slightsuction, to the
plasma membrane. The0 5pm-diameter tip covers a regionthat
contains onlyoneor a few ionchannels. Thesecond electrode is
inserted throughthe membrane intothe cytosolA recording device Patchelectrodefilled
in the patchof with conductingsalt
measures currentflow onlythroughthechannels
solution
plasma membrane(b)Photomicrograph of the cellbodyof a
cultured neuronandthetip of a patchpipettetouching thecell
membrane, (c)Differentpatch-clamping configurations. lsolated,
detached patches arethe bestconfigurations for studying theeffects
on channels of ion
different concentrations and solutessuch as
extracellular hormones andintracellularsecond messengers (e g ,
cAMP). IPart(b)fromB Sakmann, 1992,Neuron 8:613 (Nobel also
lecture);
publishedinE Neher andB Sakmann,1992, SciAm 266(3):44 Parl(c\
adaptedfrom B Hille, 1992, lon Channelsof ExcitableMembranes,2d ed ,
r s s o c i a t e sp, 8 9 l
S i n a u eA

enough to suggestthat the general structure of the ion-

selectivity filters are comparable in both types of channels.
Presumablythe diameter of the filter in Na- channelsis
small enough that it permits dehydratedNa* ions to bind to
the backbone carbonyl oxygens but excludesthe larger K+ (c) Tip of micropiPette
ions from entering,but as yet no three-dimensionalstructure
of a Na* channelis available. lon channel
---/

PatchClampsPermit Measurementof lon

M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s
The technique of patch clamping enablesworkers to inves-
tigate the opening,closing,regulation,and ion conductance
of a single ion channel. In this technique, the inward or out-
ward movement of ions across a patch of membrane is
quantified from the amount of electric current needed to
maintain the membranepotential at a particular "clamped"
value (Figure 11'-21,aand b). To preserveelectroneutrality
and to keep the membranepotential constant' the entry of
each positive ion (e.g.,a Na* ion) into the cell through a
channel in the patch of membraneis balancedby the addi-
tion of an electron into the cytosol through a microelec-
trode inserted into the cytosol; an electronic device meas-
ures the numbers of electrons (current) required to
counterbalancethe inflow of ions through the membrane
channels. Conversely,the exit of each positive ion from the
cell (e.g., a K+ ion) is balancedby the withdrawal of an
electron from the cytosol. The patch-clamping technique
can be employed on whole cells or isolated membrane
patches to measure the effects of different substancesand
ion concentrations on ion flow (Figure 11-21c).
The patch-clamp tracings in Figure 1'1-22 rllustratethe use
of this techniqueto study the propertiesof voltage-gatedNa-
channelsin the plasma membraneof musclecells.As we dis-
cussin Chapter 23, thesechannelsnormally are closedin rest-
ing muscle cells and open following nervous stimulation.
On-cellpatch measuresindirecteffectof extracellularsolutes
on channelswithin membranepatch on intactcell
Cytosolic Exoplasmic
face face :.:
Inside-outdetachedpatch Outside-outdetachedpatch
measureseffectsof intra- measureseffectsof extra-
snchannels
c e l l u l asro l u t e o c e l l u l asro l u t e o
s nchannels
withinisolatedpatch withinisolatedpatch
Patchesof muscle membrane, each containing one Na* chan-
nel, were clamped at a voltage slightly less than the resting
membrane potential. Under these circumstances'translent
pulsesof .ot..nt crossthe membraneas individual Na- chan-
.r.ls op.tt and then close. Each channel is either fully open or
completelyclosed' From such tracings' it is possibleto deter-
mine the iime that a channel is open and the ion flux through
it. For the channelsmeasuredin Figure 11-22,the flux is about
10 million Na* ions per channelper second'a typical value for
ion channels. Replacement of the NaCl within the patch
pipette (corresponding to the outside of the cell) with KCI or
.ttttin. chloride abolishescurrent through the channels,con-
firming that they conduct only Na+ ions, not K* or other ions'

M E M B R A N EP O T E N T I A L O 463
AND THE RESTING
N O N G A T E DI O N C H A N N E L S
 Open Closed 1 0m s
T M i c r o i n i e cm
t RNA
5.0 oA l encodingchannel
I
I protein of interest
Plasma
membrane

Twoinside-out patches of muscle plasma membrane wereclamped

at a potential of slightly lessthanthatof the restinqmembrane Newly
potential. Thepatchelectrode contained NaCl.Thetransient lncubate24-48hfor
pulses synthesized
of electric svnthesisand
currentin picoamperes (pA),recorded aslargedownward p channel
m o v e m e n to f c h a n n e
deviations (bluearrows), indicate theopeningof a Na* channel protein
and proteinto plasma
movement of Na* ionsinwardacross the membrane Thesmaller memorane
deviations in currentrepresent background noiseTheaverage
c u r r e n t t h r o ua gnho p e nc h a n n ei sl 1 . 6p A ,o r 1 . 6x 1 0 1 2a m p e r e s .
'1
Since ampere: 1 coulomb(C)of chargepersecond, thiscurrentis
equivalent to the movement of about9900Na+ionsperchannel oer
d. :6 x 1 0 ' 2 C / s ) ( 1 0 -s3/ m s ) (x6 1 0 2 3
m i l l i s e c o n( 1 m o l e c u l e s / m+o l ) M e a s u r ec h a n n e l - Patchelectrode
96,500C/mol [SeeF.J Sigworth andE Neher, 1980,Narure 287:44]l
E 3:?:f,',-:,:flJil""
t e c hn i o u e

N o v e ll o n C h a n n e l sC a n B e C h a r a c t e r i z e d
A EXPERIMENTAL FIGURE 11-23Oocyteexpression assayis
by a Combinationof Oocyte Expressionand
usefulin comparingthe functionof normaland mutantforms
P a t c hC l a m p i n g of a channelprotein.A follicular
frogoocyteisfirsttreatedwith
collagenase
to removethesurrounding folliclecells,leaving a
denuded oocyte,whichis microinjectedwith mRNAencoding the
channelproteinunderstudy.lAdaptedfromT.p Smith, 1988,Irends
Neurosci
11:250l

At the concentrationsof Na6 and Nao,, shown in Figure 11-24,

which are typical for many mammalian cells, AG., the
change in free energy due to the concentration gradient, is
- 1.45 kcal for transporr of 1 mol
of Na+ ions from outside
to inside the cell, assumingthere is no membrane electric po-
tential. Note the free energy is negative, indicating ,porrt"-
neous movement of Na+ into the cell.
The free-energychange generated from the membrane
electric potenrial is given by

N a + E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e
Changein FreeEnergy(AG)
As mentioned earlier, two forces govern the movement of
rons across selectivelypermeablemembranes:the voltage
and the ion concentration gradient across the membrane.
The sum of theseforces,which may act in the samedirection
or rn opposite directions,constitutesthe electrochemicalgra_
dient. To calculatethe free-energychangeAG corresponJng
to the transport of any ion across a membrane, we need to
consider the independentcontributions from each of the
forces to the electrochemicalgradient.
For example, when Na* moves from outside to inside
the cell, the free-energychangegeneratedfrom the Na+ con-
centration gradient is given by
AG-: Pg (r1-6)
where F is the Faraday constant and E is the membrane electric
potential. lf E = -70 mV, then AG-, the free-energychangedue
to the membranepotential,is - 1.61 kcal for transpoft of 1 mol
of Na* ions from outside to inside the cell, assumingthere is no
Na+ concentration gradient. Sinceboth forces do in fact act on
Na- ions, the total AG is the sum of the two partial values:
AG : AG. + AG- : (-1.45) + (-1.61): -3.0d kcaVmole
In this example, the Na+ concentration gradient and the

AG.:RThm ( 11 - s )

464 . cHAprER 11 T R A N S M E M B R A NTER A N S e o R To F t o N s A N D S M A L LM O L E C U L E S

|
 EnergyInterconversions
@ Ou"ruiewAnimation:Biological
> FIGURE 11-24Transmembrane forcesactingon Na* ions'As lon concentration M e m b r a n ee l e c t r i c
of Na* ionsacrossthe plasma gradient potential
with all ions,the movement
membrane is governedby the sumof two separate forces-the ion
gradient potentialAt the lnside Outside Inside Outside
concentration andthe membrane electric
cells, +
internal andexternal Na+concentrations typicalof mammalian 1 2m M N a * 1 4 5m M N a + +
theseforcesusually actin thesamedirection, makingthe inward
movement of Na* ionsenerqeticallvfavorable. Na+ -70 mV

LG" = -1.45 kcal/mol acm = kcal/mol

or out of animal cells. The rapid, energetically favorable Free-energychangeduring transport

movement of Na+ ions through gated Na* channelsalso is of Na* from outsideto inside
critical in generating action potentials in nerve and muscle
cells, as we discussin Chapter 23. Inside Outside

Na+

-70 mV
Nongated lon Channelsand the Resting Membrane
Potential
r An inside-negativeelectric potential (voltage) of 50-70 AG = AGc+ AG. = -3.66 kcal/mol
mV exists acrossthe plasma membrane of all cells.
r In animal cells, the membrane potential is generatedpri-
marily by movement of cytosolic K* ions through resting
K* channelsto the external medium. Unlike the more com-
mon gated ion channels,which open only in responseto and
various signals, these nongated K+ channels are usually
CotransPortbY SYmPorters
open. Antiporters
r In plants and fungi, the membrane potential is main- In previous sectionswe saw how ATP-powered pumps gener-
tained by the ATP-driven pumping of protons from the cy- ate ion concentration gradients across cell membranesand
tosol to the exterior of the cell. how ion channel proteins use these gradients to establish an
r K* channelsare assembledfrom four identicalsubunits, electric potential acrossthesemembranes.In this section we
each of which has at least tlvo conservedmembrane-span- seehow cotransporters usethe energy stored in the electric po-
ning cr helices and a nonhelical P segment that lines the tential and conientration gradients of Na+ or H* ions to
ion pore and forms the selectivity filter (seeFigure 11-19). power the uphill moYement of another substance,which may
te a small organic moleculesuch as glucoseor an amino acid
r The ion specificity of K* channel proteins is due mainly
or a different ion (seeFigure 1'1,-2)-Fot instance' the ener-
to coordination of the selectedion with the carbonyl oxy-
gen atoms of specific amino acids in the P segments,thus
lowering the activation energy for passageof the selected
K* compared with other ions (seeFigure 1'1'-20).
r Patch-clamping techniques,which permit measurement
of ion movementsthrough single channels,are used to de-
termine the ion conductivity of a channel and the effect of
various signalson its activity (seeFigure 11-21,).
r Recombinant DNA techniquesand patch clamping allow Cotransporters share common features with uniporters
the expressionand functional characterization of channel such as the GLUT proteins. The two types of transporters
proteins in frog oocytes (seeFigure 11-23). exhibit certain structural similarities' operate at equiva-

r The electrochemical gradient across a semipermeable

membrane determines the direction of ion movement
through channel proteins. The two forces constituting the
electrochemicalgradient, the membrane electric potential
and the ion concentration gradient, may act in the same or
opposite directions (seeFigure 11-24).
tration gradient.

C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S 465

 I7hen the transported molecule and cotransported ion
move in the same direction, the process is called symport;
when they move in opposite directions, the processis called
antiport (seeFigure 11-3). Some cotransporrersrransporr
only positive ions (cations), while others transport only
negativeions (anions).An important example of a cation
plasma membrane.Yet other cotransportersmediatemove-
ment of both cations and anions together. Cotransporters
are presentin all organisms,including bacteria,plants, and
animals, and in this section we describe the operation and
function of several physiologically importanr symporrers
and antiporters.
Na--LinkedSymporterslmport Amino Acids
a n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h
ConcentrationGradients
Most body cells import glucose from the blood down the
concentration gradient of glucose, utilizing GLUT proteins
to facilitate this transport. However, certain cells, such as
those lining the small intestine and the kidney tubules, need
to import glucosefrom the intestinal lumen or forming urine
againsta very large concentrationgradient. Such cells utilize
a two-Na+ /one-glucosesymporter,;protein that couplesim-
port of one glucosemoleculeto the import of two Na* ions:
2 Na+o,. * glucoseout s 2 Na+;, * glucose1,
QuantitativelS the free-energychange for the symporr rrans-
port of two Na* ions and one glucosemoleculecan be written
Oveview Animation: BiologicalEnergyInterconversions
2Na+o OGlucose
Exrenol a
FIGURE 11-25 Operationalmodel for the two-Na+/one_
glucosesymporter.Simultaneous bindingof Na+andglucose
to the conformation
with outward-facing
bindingsites(stepIl)
causes a conformational
changein the proteinsuchthatthe
boundsubstratesaretransiently
occluded, unableto dissociate
intoeithermedium(stepZ) In stepEt the proteinassumes a
466 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E 5
lslucose,.I f Na,Il
AG: RTln.fr + 2RTlnffi + zF E ( 11_7)
lgtucoseo,,l [NaJ",]
Thus the AG for the overall reaction is the sum of the free-en-
ergy changesgenerated by the glucoseconcentration gradient
(1 molecule transported),the Na* concentrationgradient (2
Na+ ions transported),and the membrane potential (2 Na+
ions transported). As illustrated in Figure '1.'J.-24,
the free en-
ergy released by movement of Na+ into mammalian cells
down its electrochemical gradient has a free-energy change
AG of about -3 kcal per mole of Na* transported.Thus the
AG for transport of two moles of Na* inward is about -6
kcal. This negative free energy of sodium import is coupled to
the uphill transport of glucose, a processwith a positive AG.
'We
can calculate the glucose concentration gradient, inside
greater than outside, againstwhich glucosecan be transported
by realizing that at equilibrium for sodium-coupled glucose
import, AG : 0. By substituting the values for sodium import
into Equation 11-7 and sening AG : 0, we seethat
o : ^r,n_!4!!o'.,"L_ 6kcal
Lglucoseourl
and we can calculatethat at equilibrium, the ratio of gluco-
se;,/ glucoseo,,: :30,000. Thus the inward flow of two
moles of Na* can generatean intracellular glucoseconcen-
tration that is -30,000 times greater than the exterior con-
centration. If only one Na* ion were imported (AG of -3
kcal/mol) per glucose molecule, then the available energy
could generate a glucose concentration gradient (inside >
outside) of only about 1,70-fold.Thus by coupling the trans-
port of two Na+ ions to the transport of one glucose, the
two-Na*/one-glucose symporter permits cells to accumulate
a very high concentration of glucoserelative to the external
++
Inward-facing Outward-facing
conformation conformation
thirdconformation with inward-facingsitesDissociationof the
boundNa* andglucose intothecytosol(stepB) allowsthe protern
to revertto itsoriginaloutward-facingconformation (step[), ready
to transportadditional substrate[SeeE Wrightet.at,2OO4,physiology
19:370 for detailson the structureand function of thrs and related
transportersl
concentration. This means that glucosepresent even at very
low concentrations in the lumen of the intestine or in the
forming urine can be efficiently transported into the lining
cells and not lost from the body.
The two-Na+/one-glucose symporter is thought to con-
tain L4 transmembrane a helices with both its N- and C-
termini extending into the cytosol. A truncated recombi-
nant protein consisting of only the five C-terminal
transmembrane o helices can transport glucose independ-
ently of Na* across the plasma membrane, down its con-
centration gradient. This portion of the molecule thus func-
tions as a glucose uniporter. The N-terminal portion of the
protein, including helices 1-9, is required to couple Na+
binding and influx to the transport of glucoseagainst a con-
centration gradient.
Figure 11-25 depicts the current model of transport by
Na*/glucose symporters.This model entails conformational
changesin the protein analogousto those that occur in uni-
port transporters, such as GLUT1, which do not require a
fT
!l Symporter
eod.ust:The Two-Na+/one-Leucine
11-26 Three-dimensionalstructureof the two-Na--
A FIGURE
/one-feucinesymporterfrom the bacteriumAquifex aeolicus'
(a)TheboundL-leucine,
two sodiumions,anda chloride ionare
shownasCPKmodelsin yellow,purpleandgreen,respectively The
thatbindthe Na*or leuctne
cthelices are
threemembrane-spanning
coloredbrown,blue,andorange(b)Binding of thetwo sodiumions
cotransportedion (seeFigure 11-5). Binding of all substrates
to their sites on the extracellular domain is required before
the protein undergoesthe conformational change that tran-
sitions the substrate-bindingsites from outward to inward
facing; this ensures that inward transport of glucose and
Na* ions are coupled.
BacterialSymporterStructureRevealsthe
Mechanismof SubstrateBinding
No three-dimensional structure of a mammalian sodium
symporter has been determined' but the structuresof several
homologous bacterial sodium-amino acid transporters
have provided considerable information about symport
function. The bacterial two-Na*/one-leucine symporter
shown in Figure 1'1'-26aconsistsof 12 membrane-spanning
a helices.Two of the helices (numbers 1, and 6l have non-
helical segmentsin the middle of the membrane that form
part of the leucine-bindingsite.
to carbonyl main-chain oxygen atomsor carboxyl oxygens
side-chain
(red)that arepartof helices1 (brown),6 (blue),or 8 (orange)lt is
important thatoneof thesodiumions(top)isalsoboundto the
carboxyl groupof the transportedleucine(yellow). [FromA Yamashita
et al, Nature
2005, 437:215:seealsoD Yernool et al, 2004, 431:811
Nature
onthestructure
fordetails andfunctionof thisandrelatedtransporters
l
C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S 467
 Amino acid residues involved in binding the leucine
and the two sodium ions are located in the middle of the
membrane-spanning segment (as depicted for the two-
Na*/one-glucosesymporter in Figure 11-25) and are close
together in three-dimensional space. This demonstrates
that the coupling of substrate and ion transport in these
transporters is the consequenceof direct or nearly direct
physical interactions of the substrates.Indeed, one of the
sodium ions (number 1 in Figure 11-26b) is bound to the
carboxyl group of the transported leucine,indicating how
binding of sodium and leucine are coupled. Each of the
two sodium ions is bound to six oxyge., ,to-r. Sodium 1,
for example, is also bound to carbonyl oxygens of several
transporter amino acids as well as to carbonyl oxygens
and the hydroxyl oxygen of one threonine. Equally impor-
tantly, there are no water moleculessurrounding either of
the bound sodium atoms, as is the case for K+ ions in
potassium channels (see Figure 11-20). Thus as the
sodium ions lose their water of hydration in binding to the
transporter,they bind to six oxygen atoms with a similar
geometry. This reducesthe activation energy for binding
of sodium ions and preventsother ions, such as porassium,
from binding in place of sodium.
One striking feature of the structure depicted in Fig-
u r e 1 1 - 2 6 i s t h a t t h e b o u n d s o d i u m i o n s a n d l e u c i n ea r e
occluded-that is, they cannot diffuse out of the protein
to either the surrounding extracellular or cytoplasmic
media. Apparently the process of crystallization of this
protein with its bound substrateshas "trapped" it into an
intermediate transport step (see Figure 11-25) in which
the protein appears to be changing from a conformation
with an exoplasmic- to one with a cytosolic-facing bind-
ing site.
Na+-LinkedCa2+Antiporter ExportsCa2*from
C a r d i a cM u s c l eC e l l s
In cardiac muscle cells a tbree-Na+/one-Ca2* anti\orter.
rather than the plasma membrane Ca2* Alpase dis.ussej
earlier, plays the principal role in maintaining a low
concentration of Ca2* in the cytosol. The transporr reacrron
mediated by this cation antiporter can be written
3 Na+or, * Ca2*;, ; - 3 Na+1. * C"'*o,.
Note that the movement of three Na+ ions is required to
power the export of_oneCa2' ion from the .ytoto1, with a
lcal*J of :2 x 10-7 M, ro the extracellularmedium, with a
[C"t*] of 2 x 10-3 M, a gradientof some 10,000-fold.In all
muscle cells, a rise in the cytosolic Ca2* concentration in
cardiac muscle triggers conrraction; by lowering cytosolic
Ca'-, operation of the Na+/Ca2+ 2lliporter reducesthe
strengthof heart musclecontracrion.
The Na*/K+ ATPasein the plasma membrane of car-
diac muscle cells, as in other body cells, createsthe
N a * concentration gradient necessaryfor export of Caz*
468 . c H A p r E R1 1 | T R A N S M E M B R ATNREA N s p o RoTF t o N s A N D S M A L LM O L E C U L E S
by the Na*-linked Ca'* antiporter. As mentioned earlier,
inhibition of the Na*/K* AIPase by the drugs ouabain and
digoxin lowers the cytosolic K* concentration and, more
relevant here, simultaneouslyincreasescytosolic Na*. The
resulting reduced Na* electrochemicalgradient across the
membranecausesthe Na+-linked Ca2* antiDorterto func-
tion less efficiently. As a result, fewer Ca21 ions are ex-
ported and the cytosolic Ca2* concentration increases,
causing the muscle to contract more strongly. Becauseof
their ability to increasethe force of heart muscle contrac-
tions, drugs such as ouabain and digoxin that inhibit the
Na-/K- ATPase are widely used in the treatment of con-
gestiveheart failure. I
SeveralCotransportersRegulateCytosolicpH
The anaerobic metabolism of glucoseyields lactic acid, and
aerobic metabolism yields CO2, which adds water to form
carbonic acid (H2CO3).Theseweak acids dissociate,yield-
ing H- ions (protons); if these excessprotons were not re-
moved from cells, the cytosolic pH would drop precipi-
tously, endangering cellular functions. Two types of
cotransport proteins help remove some of the "excess" pro-
tons generatedduring metabolism in animal cells. One is a
Na- HCO j- /Cl antiporter, which imports one Na+ ion to-
gether with one HCO3-, in exchangefor export of one Cl
ion. The cytosolic enzymecarbonic anhydrasecatalyzesdis-
sociation of the imported HCO3- ions into CO2 and an
OH- (hydroxyl)ion:
HCO.- ;- CO, + OH
The OH- ions combine with intracellular protons, forming
water, and the CO2 diffuses out of the cell. Thus the overall
action of this transporter is to consume cytosolic H+ ions,
thereby raising the cytosolic pH. Also importanr in raising
cytosolic pH is a Na* /H* antiporter, which couplesentry of
one Na* ion into the cell down its concenrrationgradient to
the export of one H+ ron.
Under certain circumstances,the cytosolic pH can rise
beyond the normal range of 7 .2-7.5. To cope with the excess
OH- ions associatedwith elevated pH, many animal cells
utilize an anion antiporter that catalyzesthe one-for-one ex-
changeof HCO3- and Cl acrossthe plasma membrane.At
high pH, this C/ /HCO3- antiporter exports HCO3-
(which can be viewed as a "complex" of OH and CO2) in
exchangefor import of Cl-, thus lowering the cytosolic pH.
The import of CI- down its concentration gradient
(Cl -.air- ) Cl .r,oror)powers the transport.
The activity of all three of these anriport proteins depends
on pH, providing cells with a finely tuned mechanismfor con-
trolling the cytosolic pH. The two antiporters that operateto in-
creasecltosolic pH are activated when the pH of the cytosol
falls. Similarly, a rise in pH above 7.2 stimulates the
CI-iHCO3- antiporter, leading to a more rapid export of
HCO3- and decreasein the cytosolic pH. In this manner,the cy-
tosolic pH of growing cells is maintained very closeto pH7.4.
A PutativeCation ExchangeProteinPlaysa Key
R o l ei n E v o l u t i o no f H u m a nS k i nP i g m e n t a t i o n
Sequencingof the human, mouse, and rat genomesindi-
cates the presenceof hundreds of putative transport pro-
teins, but the functions of most of these are as yet un-
known. A particularly interesting human transporter
called SLC24A5 emerged from a study of zebrafish that
had abnormal skin color; in fish homozygous for the
golden mutation, the eponymous black horizontal stripes
were very pale (Figure 11-27a and b). Microscopy showed
that the mutant fish had a much lower amount of the
black pigment called melanin, and melanin vesicles,called
melanosomes,were much smaller and paler than normal
(Figure 11-27 c and d). Positional cloning of the golden
gene demonstrated that it encodesa putative cation ex-
change protein termed SLC24A5. Immunofluorescence
studies showed that the protein is found in intracellular
A FfGURE 11-27 Zebratishmutationsin the gene encodingthe
cation exchangerSLC24A5 causethe golden skin pigment
phenotype.Lateralviewsof adultwild-type(a)andgolden(b)
zebrafish Insets showmelanophores (arrowheads)
(inset, 0 5 mm) Goldenmutantshavemelanophores
average smaller, paler, andmoretransparent thannormal
electron micrographs of skinmelanophores
Transmission
type(c) andgolden(d)larvaeshowthat goldenskinmelanophores
(arrowheads showedges) arethinnerandcontarn
m e l a n o s o m e s t hnaonr m a S e a r si n ( c ) a n d( d ) ,1 0 0 0n m l F r o m
l c a lb
R L Lamason etal, 2005, Scrence310:1 782,1
membranes,likely in the membrane of the melanosomeor
its precursor,but the ions transported by SLC24A5 are not
yet known. However, the amino acid sequence of the
SLAC24A5 protein is closestto that of severalsodium/cal-
cium antiporters, so the protein is likely a sodium/calcium
antlporter.
Most strikingly, investigators showed that the human
version of SLC24A5 is highly similar in sequenceto the ze-
brafish protein; when the human protein is expressedin
mutant golden zebrafish,it complementsthe mutant pheno-
type and the fish have normal black stripes. The most evo-
lutionarily conservedform of the gene, or allele, most simi-
lar to the wild-type zebrafish gene predominates in
dark-skinned African and East Asian human populations.
In contrast. a version, or variant allele, of the SLC24A5
gene with a single amino acid change that is thought to
encode a less active protein is found in virtually all people
of European origin. Studies of the allele frequenciesin ad-
mixed populations indicate that different forms, or poly-
morphisms, in iust this cation transporter play a key role in
determining the darkness of human skin color' Clearly
much needsto be learned about the role of this transporter
in cell physiology and how a single point mutation in this
gene accountsfor the large differencesin skin pigmentation
characteristic of individuals of European, African, and
Asian origin.
t r o t e i n sE n a b l eP l a n t
N u m e r o u sT r a n s p o r P
Vacuolesto AccumulateMetabolitesand lons
The lumen of plant vacuolesis much more acidic (pH 3-6)
than is the cytosol (pH 7.5). The acidity of vacuolesis main-
tained by a V-classAlP-powered proton pump (seeFigure
1,1,-9)and by a pyrophosphate-poweredpump that is unique
to plants. Both of thesepumps, located in the vacuolar mem-
brane, import H* ions into the vacuolar lumen against a
concentration gradient. The vacuolar membrane also con-
tains Cl- and NO: channels that transport these anions
from the cytosol into the vacuole. Entry of these anions
against their concentration gradients is driven by the inside-
positive potential generated by the H* pumps. The com-
tined operation of these proton pumps and anion channels
producesan inside-positiveelectricpotential of about 20 mV
across the vacuolar membrane and also a substantial pH
gradient (Figure 11-28).
The proton electrochemical gradient across the plant
vacuole membrane is usedin much the sameway as the Na*
electrochemicalgradient acrossthe animal-cell plasma mem-
brane: to power the selectiveuptake or extrusion of ions and
small molecules by various antiporters. In the leaf, for ex-
ample, excesssucrosegeneratedduring photosynthesisin the
Scale bars,5 mm
day is stored in the vacuole; during the night' the stored su-
thatareon
crose moves into the cytoplasm and is metabolized to CO2
fromwild- and H2O with concomitant generation of ATP from ADP
and P;. A proton/sucrose antiporter in the vacuolar mem-
fewer brane operatesto accumulatesucrosein plant vacuoles.The
inward movement of sucrose is powered by the outward
movement of H*, which is favored by its concentration
C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S 469
 H*-pumping proteins
ADP+ P;
Cotransport by Symporters and Antiporters
PPi
r Cotransporters use the energy releasedby movement of
an ion (usually Ht or Na*) down its electrochemicalgra-
dient to power the import or export of a small molecule or
2H- H' different ion against its concenrrationgradient.
lon-channel + r The cells lining the small intestine and kidney tubules
P l a n tv a c u o l el u m e n
( p H= 3 - 6 ) + express symport proteins that couple the energetically
favorable entry of Na+ to the import of glucose and
Nat Ca2* Sucrose amino acids against their concentration gradients (see
Figure 11-25).
r The molecular structure of a bacterial Na+-amino acid
symporter revealshow binding of Na* and leucineare cou-
Cytosol
pled and provides a snapshot of an occluded transport in-
H* H+ ( p H= 7 . 5 )
Protonantiportproteins termediate in which the bound substratescannot diffuse
out of the protein.
A FIGURE 11-28 Concentration of ionsand sucroseby the
plant vacuole.Thevacuolar membrane contains two typesof r In cardiac musclecells,the export of Ca2* is coupled to
protonpumps(orange): a V-class H* ATpase (/eft)anda and powered by the import of Na* by a cation antiporter,
pyrophosphate-hydrolyzing protonpump(flght)thatdiffersfromall which transports 3 Na* ions inward for each Ca2* ion
otheriontransport proteins andprobably isuniqueto plantsThese exported.
pumpsgenerate a low luminalpHaswellasan inside-positive r As judged by mutations in zebrafish and polymor,
electric potential across thevacuolar membrane owingto the inward phisms in humans, the presumed sodium/calcium co-
pumpingof H* ions Theinside-positive potential powersthe
transporter SLC24A5 plays a major role in forming
movement of Cl- andNOr- fromthecytosol throughseparate
channel proteins (purple)Protonantiporters melanin granules and in regulating the darkness of
(green), powered by the
H + g r a d i e nat ,c c u m u l aNt ea + ,C a 2 +a, n ds u c r o si n human skin pigmentation.
e s i dteh ev a c u o l e .
[ A f t e r P R e aa n d D S a n d e r s1, 9 8 7 ,p h y s i o t p l a n t 7 1 . t 3 1 ; J M M a a t h u i sa n d r Two cotransportersthat are activated at low pH help
D S a n d e r s1, 9 9 2 ,C u r r O p i n C e l lB i o l . 4 . 6 6 1 ;a n d p A R e ae t a l , 1 9 9 2 . maintain the cytosolic pH in animal cells very close to
TrendsBiochem Sci. 17:348 l 7.4 despitemetabolic production of carbonic and lactic
acids.One, a Na*/H* antiporter,exports excessprotons.
The other, a Na*HCO3-lCl- cotransporrer, imports
HCO3 , which dissociatesin the cytosol to yield pH-
gradient (lumen > cytosol) and by the cytosolic-negative raising OH- ions.
potendal acro-ssthe vacuolar membrane (seeFigure 11-28). r A CI-/HCO3 antiporter that is activated at high pH
Uptake of Ca2* and Na+ into the vacuole from the cytosol functions to export HCO3 when the cytosolic pH rises
against their concentration gradients is similarly mediated above normal and causesa decreasein pH.
by proton antiporters.
r Uptake of sucrose,Na*, Ca2*, and other substances into
plant vacuoles is carried out by proton antiporters in the
Understandingof the transport proteins in plant vacuo-
vacuolar membrane.Ion channelsand proton pumps in the
lar membraneshas the potential for increasingagricul-
production in high-salt (NaCl) soils, which are found membrane are critical in generatinga large enough proton
throughout the world. Becausemost agriculturally useful concentration gradient to power accumulation of ions and
crops cannot grow in such saline soils, agricultural scientists metabolites in vacuoles by these proton antiporters (see
have long sought to develop salt-tolerant plants by tradi- Figure 11-28).
tional breedingmerhods.\With the availability of the cloned
gene encoding the vacuolar Na*/H* antiporter, researchers
can now produce transgenicplants that overexpressthis
transport protein, leading to increasedsequestrationof Na*
in the vacuole. For insrance,rransgenlctomato plants that Transepithel
iaI Transport
overexpressthe vacuolar Na*/H* antiporter can groq Previous sections illustrated how several types of trans-
flower, and produce fruit in the presenceof soil NaCl con- porters function together to carry out important cell func-
centrations that kill wild-type plants. Interestingly although tions (seeFigure 11,-2).Here,we exrend this concept by fo-
the leavesof thesetransgenictomato plants accumulatelarge cusing on the transport of several types of molecules and
amounts of salt, the fruit has a very low salt content. I ions acrossthe sheetlikelayers of epithelial cells that cover

470 CHAPTER
11 I T R A N S M E M B R A NTER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E 5
most external and internal surfacesof body organs. Like all
epithelial cells, an intestinal epithelial cell is said to be polar-
ized becauseits plasma membrane is organized into at least
two discreteregions.Typically, the surfacethat facesthe out-
side of the organism, here the lumen of the intestine,is called
the apical, or top, surface, and the surface that faces the
inside of the organism is called the basolateral surface (see
Figure 19-9).
Specializedregions of the epithelial-cellplasma mem-
brane, called cell junctions, connect the cells and provide
strength and rigidity to the cell sheet (seeFigure 1,9-9for
details). One of these types of cell junctions-the tight
junction-is of particular interesthere sincetight junctions
prevent many water-solublesubstanceson one side of an
epithelium from moving acrossto the other side through
the extracellular space betweencells. For this reason, ab-
sorption of nutrients from the intestinal lumen into the
blood occurs by the two-stage process called transcellwlar
transport: import of moleculesthrough the plasma mem-
brane on the apical side of intestinal epithelial cells and
their export through the plasma membrane on the blood-
facing (basolateral,or serosal) side (Figure 11-29). The
apical portion of the plasmamembrane,which facesthe in-
testinal lumen, is specialized for absorption of sugars,
amino acids, and other moleculesthat are produced from
food by various digestive enzymes.Numerous fingerlike
projections (100 nm in diameter) called microvilli greatly
increasethe area of the apical surfaceand so the number of
2 Na+/glucose
GLUTz symporter
Glucose Glucose Glucose
FI 2 Na'
t Na 2Na
Na+76+
ATPase
Apical
membrane
T i g h tj u n c t i o n
Cytosol
Low Na-
H i g hK *
A FIGURE 11-29Transcellular transportof glucosefrom the
intestinallumeninto the blood.TheNa*/K*ATPase in the
basolateralsurface membrane generatesNat andK* concentration
gradients(step1) Theoutwardmovement of K+ ionsthrough
nongated K* channels (notshown)generates an inside-negative
membrane potentialBoththe Na* concentration gradientand
the membrane areusedto drivethe uptakeof glucose
potential from
the intestinallumenbythetwo-Na'/one-glucose symporter located
in the apicalsurfacemembrane (step2) Glucose leavesthecellvia
facilitated
diffusioncatalyzed
by GLUT2, a glucoseuniporter located
in the basolateralmembrane (step3)
transport proteins it can contain, enhancingthe cell's ab-
sorptive capacitY.
Multiple TransportProteinsAre Neededto
Move Glucoseand Amino AcidsAcrossEpithelia
Figure 11-29 depicts the proteins that mediate absorption of
glucose from the intestinal lumen into the blood and illus-
trates the important concept that different types of proteins
are localizedto the apical and basolateralmembranesof ep-
ithelial cells.In the first stageof this process'a two-Na*/one-
glucosesymporter located in microvillar membranesimports
glucose, against its concentration gradient' from the intes-
tinal lumen acrossthe apical surfaceof the epithelial cells.As
noted above, this symporter couples the energeticallyunfa-
vorable inward movement of one glucosemoleculeto the en-
port, are pumped out across the basolateralmembrane,
which facesthe underlying tissue.Thus the low intracellular
Na* concentration is maintained. The Na*/K* ATPasethat
accomplishesthis is found exclusivelyin the basolateral
membraneof intestinalepithelialcells' The coordinatedoper-
ation of thesetwo transport proteins allows uphill movement
of glucoseand amino acids from the intestine into the cell.
This first stage in transcellular transport ultimately is pow-
ered by ATP hydrolysis by the Na*/l(- ATPase.
In the secondstage,glucoseand amino acidsconcentrated
inside intestinal cells by symportersare exported down their
concentrationgradientsinto the blood via uniport proteins in
the basolateralmembrane.In the caseof glucose,this move-
ment is mediatedby GLUT2 (seeFigure 11-29).As noted ear-
lier, this GLUT isoform has a relatively low affinity for glu-
cosebut increasesits rate of transport substantiallywhen the
glucosegradient acrossthe membranerises(seeFigure 11-4).
The net result of this two-stage processis movement of
Na* ions, glucose,and amino acidsfrom the intestinallumen
acrossthe intestinalepithelium into the extracellularmedium
that surrounds the basolateralsurfaceof intestinal epithelial
cells.Tight junctions betweenthe epithelialcellspreventthese
moleculesfrom diffusing back into the intestinal lumen, and
eventually they move into the blood. The increasedosmotic
pressurecreated by transcellular transport of salt' glucose,
and amino acids acrossthe intestinal epithelium draws water
from the intestinal lumen into the extracellular medium that
surrounds the basolateralsurface.In a sense,salts, glucose,
and amino acids "carry" the water along with them.
S i m p l eR e h y d r a t i o nT h e r a p yD e p e n d so n t h e
OsmoticGradientCreatedby Absorption of
G l u c o s ea n d N a -
An understandingof osmosisand the intestinal absorp-
tion of salt and glucose forms the basis for a simple
TRANSEPITHELIT
ARL ANSPORT 471
 therapy that savesmillions of lives each year, particularly in
Cl /HCO3
Iess-developedcountries. In these countries, cholera and antiporter Cl- channel
other intestinal pathogens are major causesof death of
at- CI
young children. A toxin released by the bacteria acrivates
chloride secretion by the intestinal epithelial cells into the HC03- HCO3- K+ channel
lumen; water follows osmotically, and the resultant massive
K+
K"
loss of water causesdiarrhea, dehydration,and ultimately J.",oon'"
anhvdrase
v+

death. A cure demands not only killing the bacteria with I

ATP
H*/K*
antibiotics but also rehydration-replacement of the water
that is lost from the blood and other tissues. coz
I
+ OH <-H2O
ADP+P1
----+ H+
ATPase
2
Simply drinking water does nor help, becauseit is ex-
cretedfrom the gastrointestinal tract almost as soon as it en- Basolateral Apical
ters. Howeve! as we have just learned. the coordinated memorane memDrane
Tight junction
transport of glucoseand Na* acrossthe inrestinalepithe-
Cytosol
lium creates a transepirhelial osmotic gradient, forcing pH7 2
movement of water from the intestinal lumen acrossthe cell
layer and ultimately into the blood. Thus giving affected FIGURE 11-30Acidificationof the stomachlumenby
children a solution of sugarand salt to drink (but not sugar parietalcellsin the gastriclining.Theapicalmembrane of parietal
or salt alone)causesthe osmoticflow of water into the blood cellscontainsan H*/K* ATPase (a P-class
pump)aswellasCl and
from the intestinallumen and leadsto rehydration.Similar K* channel proteinsNotethecyclicK+transport across
theapical
sugar-saltsolu ns are the basisof popular drinks used by membrane: K+ ionsarepumpedinwardbythe H+/K+ATpase and
athletesto get gar as well as water into the body quickly exitviaa K* channel.Thebasolateral membranecontainsan anion
antiporterthatexchanges HCO, andCl ions Thecombined
and efficiently.
operationof thesefourdrfferent proteins
transport andcarbonic
anhydrase acidifies
thestomach lumenwhilemaintaininq
the neutral
pHandelectroneutralityof thecytosol
P a r i e t aC
l e l l sA c i d i f yt h e S t o m a c hC o n t e n t s
W h i l e M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H
The mammalian stomach conrainsa 0.1 M solution of hy-
combineswith CO2 that diffusesin from the blood, forming
drochloric acid (HCl). This strongly acidic medium kills
HCO3-. Catalyzedby the basolateralanion antiporter, this
many ingested pathogens and denatures many ingested
bicarbonateion is exported acrossthe basolateralmembrane
proteins before they are degraded by proteolytic enzymes
(and ultimately into the blood) in exchangefor a Cl- ion. The
(e.g.,pepsin)that function at acidic pH. Hydrochloric acid
Cl- ions then exit through Cl- channelsin the apical mem-
is secretedinto the stomach by specializedepithelial cells
brane, entering the stomach lumen. To preserveelectroneu-
calledparietal cells(alsoknown as oxyntic cells)in the gas-
trality, eachCl- ion that movesinto the stomachlumen across
tric lining. These cells contain a H* /K+ ATpase in their
the apical membraneis accompaniedby a K* ion that moves
ounvard through a separateK* channel.In this way, the ex-
cessK* ions pumped inward by the H*lK* AIPase are re-
turned to the stomachlumen, thus maintaining the normal in-
tracellular K+ concentration. The net result is secretion of
equal amounts of H+ and Cl- ions (i.e.,HCI) into the stom-
ach lumen, while the pH of the cytosol remains neutral and
chondria in parietal cellsproduce abundant ATp for use by
the excessOH ions, as HCO3 , are transported into the
the H+/K* ATpase.
blood.
If parietal cells simply exported H+ ions in exchange
for K* ions, the loss of protons would lead to a rise in the
concentrationof OH ions in the cytosol and thus a
m a r k e d i n c r e a s ei n c y t o s o l i c p H . ( R e c a l l t h a t [ H + ] x
TransepithelialTransport
t O H - ] a l w a y s i s a c o n s t a n t , 1 , 0 - 7 4M 2 . ) p a r i e t a l c e l l s
avoid this rise in cytosolic pH in conjunction with acidifi- r The apical and basolateralplasma membrane domains of
cation of the stomach lumen by using CI-/HCO3- an- epithelial cells contain different transport proteins and
tiporters in the basolateral membrane to export the carry out quite different transport processes.
"excess" OH ions from the cytosol to the blood. As r In the intestinal epithelial cell, the coordinated opera-
noted earlieq this anion antiporter is activated at high cy- tion of Na+-linked symporters in the apical membrane
tosolic pH. with Nat/K* ATPasesand uniporters in the basolateral
The overall process by which parietal cells acidify the membrane mediates transcellular transport of amino
stomachlumen is illustratedin Figure 11-30. In a reacrroncat- acids and glucosefrom the intestinal lumen to the blood
alyzed by carbonic anhydrase,the ,,excess',cytosolic OH (seeFigure 17-29).

472 CHAPTER
11 I T R A N S M E M B R A NTER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s
 r The combined action of carbonic anhydraseand four dif-
ferent transport proteins permits parietal cells in the stom-
ach lining to secreteHCI into the lumen while maintaining
their cytosolicpH near neutrality (seeFigure 11-30).
In this chapter, we have explained the action of specific
membranetransport proteins and their impact on certain as-
pectsof human physiology; such a molecular physiology ap-
proach has many medical applications. Even todaS specific
inhibitors or activators of channels,pumps, and transporters
constitute the largest single class of drugs. For instance, an
inhibitor of the gastric H+/K+ ATPase that acidifies the
stomach is the most widely used drug for treating stomach
ulcers and gastric reflux syndrome. Inhibitors of channel
proteins in the kidney are widely used to control hyperten-
sion (high blood pressure);by blocking resorption of water
from forming urine into the blood, thesedrugs reduce blood
volume and thus blood pressure.Calcium-channelblockers
are widely employed to control the intensity of contraction
of the heart. Drugs that inhibit a particular potassium chan-
nel in B islet cells enhancesecretionof insulin (seeFigure 15-
32) and are widely used to treat adult-onset (type II) dia-
betes.
\7ith the completion of the human genome project, we
are positioned to learn the sequencesof all human mem-
brane transport proteins. Already we know that mutations
in many of them causedisease-cystic fibrosis, due to mu-
tations in CFTR, is one example. This exploding basic
knowledge will enable researchersto identify new types of
compounds that inhibit or activate just one of these mem-
brane transport proteins and not its homologs.An important
challenge,however,is to understandthe role of an individual
transport protein in each of the severaltissuesin which it is
expressed.
Another major challenge is to understand how each
channel, transporter, and pump is regulated to meet the
needsof the cell. Like other cellular proteins, many of these
proteins undergo reversiblephosphorylation, ubiquitina-
tion, and other covalent modifications that affect their activ-
ity, but in the vast majority of cases,we do not understand
how this regulation affectscellular function. Many channels,
transporters,and pumps normally reside on intracellular
membranes,not on the plasma membrane, and move to the
plasma membrane only when a particular hormone is pres-
ent. The addition of insulin to muscle, for instance,causes
the GLUT4 glucose transporter to move from intracellular
membranesto the plasma membrane, increasingthe rate of
glucose uptake. We noted earlier that the addition of vaso-
pressin to certain kidney cells similarly causesan aquaporin
to traffic to the plasma membrane,increasingthe rate of wa-
ter transport. But despitemuch research,the underlying cel-
lular mechanismsby which hormones stimulate the move-
ment of transport proteins to and from the plasma
membrane remain obscure.
KeyTerms
ABC superfamlly 448 isotonrc444
active transport 440 membranepotential439
antiport 466 microvrlli471
aquapoins 444 Na*/K* ATPase452
corransport 465 patchclamping463
electrochemical P-classpumps448
gradient 439 restingKt channels460
facilitated transport 440 simple diffusion439
F-classpumps 448 symport466
flippasemodel456 tight junctions47L
gated channel 440 transcellularluansport471
GLUT proteins 443 uniport 44L
hypertonic 444 V-classpumps448
hypotonic 444
Reviewthe Concepts
1. The basic structural unit of a biomembrane is the phos-
pholipid bilayer.Acetic acid and ethanol are each composed
of two carbons, hydrogen and oxygen, and both enter cells
by passivediffusion. At pH 7' one is much more membrane
permeable than the other' I7hich is more permeable and
why? Predict how the permeability of each is altered when
the pH is reducedto 1.0, a value typical of the stomach.
2. Uniporters and ion channels support facilitated diffu-
sion across biomembranes.Although both are examples of
facilitated diffusion, the rates of ion movement via an ion
channel are roughly 10a- to 1Os-foldfaster than that of mol-
eculesvia a uniporter. \7hat key mechanistic difference re-
sults in this large differencein transport rate?
3. Name the three classesof transporters.Explain which of
theseclassesis able to move glucoseor bicarbonate(HCO3 ),
for example, againstan electrochemicalgradient. In the case
of bicarbonate, but not glucose, the AG of the transport
process has two terms. What are these two terms' and why
doesthe secondnot apply to glucose?'Whyare cotransporters
often referredto as examplesof secondaryactivetransport?
4. GLUT1, found in the plasma membraneof erythrocytes,
is a classic example of a uniporter. Design a set of experi-
ments to prove that GLUT1 is indeed a glucose-specificuni-
porter rather than a galactose-or mannose-specificuni-
port.r. Glucose is a 6-carbon sugar while ribose is a
s-carbon sugar.Despite this smaller size, ribose is not effi-
ciently transported by GLUT1. How can this be explained?
5. Name the four classesof ATP-powered pumps that pro-
duce active transport of ions and molecules.Indicate which
of theseclassestransport ions only and which transport pri-
marily small molecules.The initial discovery of one classof
these ATP-powered pumps came from studying not the
transport of a natural substratebut rather artificial substrates
used as cancer chemotherapy drugs. \fhat do investigators
now think are common examplesof the natural substratesof
this particular classof ATP-powered pumps?
R E V I E WT H E C O N C E P T S . 473
 6. Genome sequencingprojects continue, and the complete Why are tight junctions essentialfor the process?Rehydra-
genome sequencesfor an increasing number of organisms tion supplementssuch as sport drinks include a sugar and a
are known. How does this information allow us to state the salt. Why are both important to rehydration?
total number of transporters or pumps of a given type in ei-
ther mice or humans?Many of the sequence-identified trans-
porters or pumps are "orphan" proteins, in the sensethat Analyze the Data
their natural substrate or physiological role is not known.
How can this be, and how might one establishthe physio- Imagine that you are investigating the transepithelial trans-
logical role of an orphan protein? port of radioactive glucose. Intestinal epithelial cells are
grown in culture to form a complete sheet so that the fluid
7. As we saw in the section Perspectivesfor the Future, spe-
bathing the apical domain of the cells (the apical medium) is
cific inhibitors or activators of channels,pumps, and trans-
completely separatedfrom the fluid bathing the basolateral
porters constitute the largest single classof drugs produced by
domain of the cells (the basolateral medium). Radioactive
the pharmaceutical industry. Skeletal muscle contraction is
114c-labeled;glucoseis addedto the apicalmedium, and the
causedby elevation ofCa2* concentrarionin the cytosol. \fhat
appearanceof radioactivity in the basolateralmedium is
is the expectedeffect on musclecontraction of selectivedrug in-
monitored in terms of counts per milliliter (cpm/ml), a meas-
hibition of sarcoplasmicreticulum (SR)P-classCa2* Alpase?
ure of radioactivity per unit volume.
8. The membrane porential in animal cells, but not in
plants, depends largely on resring K* channels.How do Treatment 1: The apical and basolateralmedia each
thesechannelscontribute to the restingpotential?Why are contain 150 mM Na+ (curve1).
these channelsconsideredto be nongated channels?How Treatment 2: The apical medium contains 1. mM Na+,
do thesechannelsachieveselectivityfor K+ versusNa*? and the basolateralmedium contains 150 mM Na+ (curve2).
9. Patch clamping can be used to measurethe conductance Treatment3: The apical medium contains 150 mM Na+,
properties of individual ion channels. Describe how patch and the basolateralmedium contains L mM Na+ (curve 3).
clamping can be used to determine whether or not the gene
coding for a putative K* channel actually codesfor a K+ or Radioactivity in BasolateralMedium
Na- channel.
E 200 Treatments1, 3
10. Plants use the proton electrochemicalgradient across
the vacuole membrane to power the accumulation of salts a 150
and sugarsin the organelle.This createsa hypertonic situ- E ,oo
ation. Why does thii not resuk in the planr iell bursting?
How does the plasma membrane Na*/K* ATpase allow Hrn
i5 ""
animal cells to avoid osmotic lysis even under isotonic
conditions? fo
11. In the caseof the bacterialsodiumJeucinetransporter,what T i m e( m i n )i n 1 a C - g l u c o s e
is the key distinguishing feature about the bound sodium ions
that ensuresthat other ions, particularlyK+, do not bind? De- a. What is a likely explanation for the different results ob-
scribethe symporr processby which cells lining the small intes- tained in treatments 1 and 3 versus treatment 2?
tine import glucose. !7hat ion is responsiblefor the transport, In additional studies, the drug ouabain, which inhibits
and what two particular featuresfacilitate the energetically Na*/K* ATPases,is included as noted.
favored movement of this ion acrossthe plasma membrane? Treatment 4: The apical and basolateralmedia con-
12. Sequencingseveral genomes, including that of the ze- tain 150 mM Na+ and the apical media contains ouabain
brafish, has revealeda number of receptorsand transporters. ( c u r v e4 )
In one case, positional cloning of the zebrafish golden gene Treatment 5: The apical and basolateralmedia contain
identified a putative cation exchangeprotein calledSLC24A5. 150 mM Na- and the basolateral media contains ouabain
When the golden gene is mutated, the normally black hori- (curve5)
zontal stripes are pale or golden in appearancebecausethe
amount of black pigment or melanin is greatly reduced.What Radioactivity in BasolateralMedium
is the name of the melanin-containingvesiclespresentin fish
and humans?Design an experimentto identify where the pro- t zoo
F
tein encodedby the golden geneis expressedand localizedin a 150
the zebrafish.What is the term usedwhen a mutant geneand
its phenotype,for insrancegolden in zebrafish,is rescuedby E ,oo
expressingan orthologousgenefrom another animal? c50
o
13. Movement of glucosefrom one side to the other side of
on
the intestinal epithelium is a major example of transcellular cx
2468
transport. How doesthe Na*/I(* Alpase power the process? T i m e ( m i n )i n 1 a C - g l u c o s e

474 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E S
b. Vhat is a likely explanation for the different resulrs ob,
tained in treatment 4 versustreatment 5?
c. A population of epithelialcellsusedin the abovestudieshas
beenengineeredto expressGLUT1 rather than GLUT2 in their
basolateralmembrane. These engineeredcells appear to be
much lessrobust than the parental cellsand do not survive long
in culture. \fhat is a reasonableexplanation for this findine?
References
Uniport Transport of Glucose and Water
Engel,A., Y. Fujiyoshi, and P. Agre. 2000. The importance of
aquaporin water channel protein structures.EMBO /. 19:800-806.
Hedfalk, K., et al. 2005. Aquaporin gating. Cwrr. Opinion
Structural Biology 16:1-1 0.
Hruz, P. W., and M. M. Mueckler.2001. Structuralanalysisof
the GLUT1 facilitativeglucosetransporter (review).Mol. Memb.
Biol. 18:183-193.
King, L. S., D. Kozono, and P. Agre.2004. From structureto
disease:the evolvingtale of aquaporin biology. Nat. Reu.Mol. Cell
Biol. 5:687-598.
Maurel, C. 1997. Aquaporins and water permeabilityof plant
membranes.Ann. Reu.Plant Physiol. Plant Mol. Biol. 48:399430.
Mueckler,M. 1.994.Facilitativeglucosetransporters.Eur. l.
Biocbem. 219:713-725.
Schafer,J. A. 2004. Renal water reabsorption:a physiologicret-
rospectivein a molecular era.Kidney Int. Swppl.9lS20-27.
Schultz,S. G. 2001. Epithelialwater absorption:osmosisor co-
transport?Proc. Nat'l. Acad. Sci.USA 98:3628-3630.
'Wang,
Y., K. Schulten,and E. Tajkhorshid.2005.'What makes
an aquaporin a glycerol channel?A comparativestudy of AqpZ and
GlpF structure. Structure13 11107-1118.
Verkman, A. S. 2005. Novel roles of aquaporinsrevealedby
phenotypicanalysisof knockout mice. Rey.Physiol. Biochem.Phar-
macol. 155:31.-55.
ATP-Powered Pumps and the Intracellular lonic
Environment
Borst, P.,N. Zelcer,and A. van Helvoort. 2000. ABC trans-
porters in lipid transport. Biochim. Biophys. Acta 1486:1,28-744.
Guerini, D., L. Coletto, and E. Carafoli. 2005. Exporting cal-
cium from cells. Cell Calcium 38:281,-289.
Davidson,A. L., and J. Chen. 2004. ATP-bindingcassettetrans-
porters in bacteria.Ann. Reu.Biochem. T3:241-268.
Davies,J., F, Chen, and Y. Ioannou. 2000. Transmembranemolecu-
lar pump activity of Niemann-Pick C1 protein. Science290.2295J298.
Gottesman,M. M. 2002. Mechanismsof cancerdrug resistance.
Ann. Reu.Med. 53:615-627.
Gottesman,M.M., and V. Ling. 2006.The molecularbasisof
multidrug resistancein cancer:the early yearsof P-glycoproteinre-
search.FEBS Lett. 580:998-1009.
Inoue, T., S. Wilkens, and M. Forgac.2003. Subunit structure,
function, and arrangementin the yeastand coatedvesicleV-
ATPases.J. Bioenerg.Biomembr. 35:29'1,299.
Jencks,!7. P. 1995. The mechanismof coupling chemicaland
physicalreactionsby the calcium ATPaseof sarcoplasmicreticulum
and other coupled vectorial systems.Biosci. Rept. 15:283-287.
Kiihlbrandt, \7. 2004. Biology,structureand mechanismof
p-type ATPases.Nature Reu.Mol. Cell Biol. 5:282-295.
K. P. Locher,A. Lee, and D. C. Rees.2002. The E. coli BtuCD
structure:a framework for ABC transDorterarchitectureand
mechanism.Science296 109l.
K. Obara, et al. 2005. Structuralrole of countertransportre-
vealedin Ca'* pump crystal structurein the absenceof Ca'- PNAS.
IO2:1.4489-74495.
Ostedgaard,L. S., O. Baldursson,and M. J. I0elsh. 2001. Regu-
lation of the cysticfibrosis transmembraneconductanceregulator
Cl channelby its R domain.l. Biol. Chem.276:7689-7692.
Raggers,R. J., et al. 2000. Lipid traffic: the ABC of transbilayer
movement.Traffic l:226-234.
Rea, P.A., et al. 1,992.Vacuolar H*-translocating pyrophos-
phatases:a new categoryof ion translocase.TrendsBiochem. Sci.
17:348-353.
Riordan, J. 2005. Assemblyof functional CFTR chloride chan-
nels.Ann. Reu.Physiol. 67:701.-71.8.
Tovoshima.C.. and G. Inesi.2004. Structuralbasisof ion
pumping by Ca2-ATPase of the sarcoplasmicreticulum. Ann. Reu.
Biochem.T3:269.
Verkman, A. S., G. L. Lukacs, and L..|. Galietta.2005. CFTR
chloride channeldrug discovery-inhibitors as antidiarrhealsand
activatorsfor therapy of cystic fibrosis. Curr. Pharm. Des. 12:2235-
11 14
Nongated lon Channels and the Resting Membrane
Potential
Clapham, D. L999. Unlocking family secrets:K* channeltrans-
membranedomains. Cell 97 :547-S 5 0.
Cooper,E. C., and L. Y. Jan. 1999.lon channelgenesand hu-
man neurologicaldisease:recentprogress,prospects,and challenges.
Proc. Nat'(. Acad. Sci. USA96:47594766.
Dutzler, R., et al. 2002. X-ray structureof a CIC chloride chan-
nel at 3.0 A revealsthe molecularbasisof anion selectivity.Nature
415:287-294.
Gouaux, 8., and R. Mackinnon. 2005. Principlesof selectiveion
transport in channelsand pumps. Science310:146t-1.465.
Hille, B. 2001. Ion Channelsof Excitable Membranes,3d ed.
SinauerAssociates.
Jentsch,T., M. Podt,J. Fuhrmann, and A. Zdebik 2005.
Physiologicalfunctions of CLC Cl- channelsgleanedfrom
human geneticdiseaseand mousemodels.Ann. Reu.Physiol.
67:779-807.
K*
Jiang, Y., et al. 2003. X-ray structureof a voltage-dependent
channel.N ature 423 :33-41..
MacKinnon, R. 2004. Potassiumchannelsand the atomic basis
of selectiveion conduction.Nobel Lecturereprinted rn Biosci. Rep.
24:7 5-1.00.
Montell, C., L. Birnbaumer,and V. Flockerzi.2002. The TRP
channels,a remarkably functional famrly.Cell 108:595-598.
Neher,E. 1,992.lon channelsfor communicationbetweenand
within cells.Nobel Lecturereprinted in Neuron 8:605-612 and Sci-
ence256498-502.
Neher,E., and B. Sakmann.1992. The patch clamp technique.
Sci.Am. 266(3):28-35.
Roux, B. 2005. Ion conduction and selectivityin K* channels.
2005. Ann. Reu.Biophys. Biomol. Struct. 34:153-t71.
Zhou,Y., J. Morais-Cabral,A. Kaufman, and R' MacKin-
non. 2001. Chemistryof ion coordination and hydration re-
vealed by a K* channel-Fab complex at 2 A resolution. Nature
4t4:43-48.
Cotransport by Symporters and Antiporters
Alper, S. L., M. N. Chernova,and A. K. Stewart.2001. Regula-
tion of Na+-independentCl-iHCO3- exchangersby pH. J. Pan-
creas2:1,7I-175 .
Barkla, B., and O. Pantoja. 1996. Physiologyof ion transport
acrossthe tonoplast of higher plants. Ann. Reu. Plant Physiol. Plant
Mol. Biol. 47:1.59-184.
REFERENCES 475
 Orlowski, J., and S. Grinstein.2004. Diversity of the mam-
malian sodium/proton exchanger SLC9 gene family. Pflugers Arch.
4472549-565.
Ifakabayashi, S., M. Shigekawa,and J. Pouyssegur.1997. Mole-
cular physiology of vertebrate Nat/lI* exchangers.Physiol. Reu.
77:51-74.
'Wright,
E. M. 2001. Renal Na(*)-glucosecotransporters.Am. J.
Physiol. Renal Physiol. 280:F10-F18.
\7right, E. M., and D. D. Loo. 2000. Coupling befweenNa*,
sugar,and water transport acrossthe intestine.Ann. NY Acad. Sci.
9L5:54-66.
A. Yamashita,et al. 2005. Crystal structureof a bacterialhomo-
logue of Na+/Cl--dependentneurorransmirtertransporters.Nature
437:215.
476 C H A P T E R1 1 I T R A N S M E M B R A NTER A N S P O R O
T F I O N SA N D S M A L L M O L E C U L E S
Zhang, H-X., and E. Blumwald. 2001. Transgenicsalt-tolerant
tomato plants accumulatesalt in foliage but not in fruit. Nature
Biotech. l9:765-769.
Transepithelial
Transport
Furuse,M., and S. Tsukita. 2006. Claudins in occludingjunc-
tions of humans and flies. TrendsCell Biol. l6:I8t-188.
Goodenough,D. A. 1999. Pluggingthe leaks.Proc. Nat'(. Acad.
Sci.USA 96:3t9.
Mitic, L., and J. Anderson. 1,998.Molecular architectureof
tight junctions.Ann. Reu.Physiol. 60:t21-1,42.
Rao, M. 2004. OraI rehydration therapy: new explanations for
an old remedy.Ann. Reu.Physiol. 66':385417.
cLASSIC EXPERIMENT
STUMBLING
UPONACTIVETRANSPORT
J. 5kou, 1957, Biochem. Biophys. Acta 23:394
In the mid-1950s Jens Skou was a
young physician researchingthe effects
of local anestheticson isolated lipid bi-
layers. He needed an easily assayed
membrane-associated enzymeto use as
a marker in his studies. lfhat he dis-
covered was an enzyme critical to the
maintenanceof membrane potential,
the Na+/K+ ATPase, a molecular
pump that catalyzesactive transport.
Background
During the 1950s many researchers
around the world were actively investi-
gating the physiology of the cell mem-
brane, which plays a role in a number
of biological processes.It was well
known that the concentration of many
ions differs inside and outside the cell.
For example, the cell maintains a
lower intracellularsodium (Na*) con-
centration and higher intracellular
potassium(K*) concentrationthan is
found outside the cell. Somehow the
membrane can regulate intracellular
salt concentrations. Additionally,
movement of ions across cell mem-
branes had been observed, suggesting
that somesort of transport is systemis
present. To maintain normal intracel-
lular Na* and K* concentrations,the
transport systemcould not rely on pas-
sive diffusion becauseboth ions must
move across the membrane against
their concentration gradients. This
energy-requiringprocesswas termed
actrvetransport.
At the time of Skou's experiments,
the mechanismof active transport was
still unclear.Surprisingly Skou had no
intention of helping to clarify the field.
He found the Na*/K* ATPase com-
pletely by accident in his search for an
abundant, easily measuredenzyme ac-
tivity associatedwith lipid membranes.
A recent study had shown that mem-
11
branes derived from squid axons con-
tained a membrane-associatedenzyme
that could hydrolyze ATP. Thinking that
this would be an ideal enzyme for his
purposes,Skou setout to isolatesuchan
ATPasefrom a more readily available
source,crab leg neurons.It was during
his characterization of this enzyme that
he discoveredthe protein's function.
The Experiment
Sincethe original goal of his study was
to characterize the ATPase for use in
subsequentstudies, Skou wanted to
know under what experimental condi-
tion its activity was both robust and
reproducible. As often is the casewith
the characterization of a new eflzyme,
this requires careful titration of the
various components of the reaction.
Before this can be done, one must be
sure the system is free from outside
sourcesof contamination.
In order to study the influence of
various cations, including three that
are critical for the reaction-No-, K-,
and Mg2*-Skou had to make sure
that no contaminating ions were
brought into the reaction from another
source. Therefore all buffers used in
the purification of the enzyme were
prepared from salts that did not con-
tain these cations. An additional
s o u r c eo f c o n t a m i n a t i n gc a t i o n s w a s
the ATP substrate, which contains
three phosphate groups, giving it an
overall negative charge. Becausestock
solutions of ATP often included a
cation to balance the charge, Skou
converted the ATP used in his reac-
tions to the acid form so that balancing
cations would not affect the experi-
ments. Once he had a well-controlled
environment, he could characterizethe
enzyme activity. These precautions
were fundamental to his discovery.
S T U M B L I N GU P O N A C T I V ET R A N S P O R T
Skou first showed that his enzyme
could indeed catalyzethe cleavageof
ATP into ADP and inorganic phos-
phate. He then moved on to look for
the optimal conditions for this activity
by varying the pH of the reaction, and
the concentrations of salts and other
cofactors, which bring cations into the
reaction. He could easily determine a
pH optimum as well as an optimal
concentration of Mg'-, but optimizing
Na* and K* proved to be more diffi-
cult. Regardlessof the amount of K-
added to the reaction, the enzymewas
inactive without Na-. Similarly, with-
out K+, Skou observed only a low-
level ATPase activity that did not
increase with increasing amounts of
Na-.
Theseresults suggestedthat the en-
zyme required both Na* and K* for
optimal activity. To demonstrate that
this was the case,Skou performed a se-
ries of experiments in which he meas-
ured the enzyme activity as he varied
both the Na+ and K* concentratrons
in the reaction (Figure 1). Although
both cations clearly were required for
significant activity, something interest-
ing occurred at high concentrationsof
each cation. At the optimal concentra-
tion of Na* and K-, the ATPaseactiv-
ity reacheda peak. Once at that peak,
further increasing the concentration
did not affectthe ATPaseactivity.Na'
thus behavedlike a classicenzymesub-
strate,with increasinginput leading to
increased activity until a saturating
concentration was achieved, at which
t h e a c t i v i t y p l a t e a u e d .K * , o n t h e
other hand, behaveddifferently.'Sfhen
the K+ concentration was increased
beyond the optimum, ATPase activity
declined.Thus while K* was required
for optimal activity, at high concentra-
tions it inhibited the enzyme. Skou
reasoned that the enzyme must have
477
 (b) 40
Mg6mM/l
K120 mM/l

NaCl40 mMll

K3mM/l
NaCl20 mMll
q, 20
a

N a C l1 0 m M , / l
N a C l3 m M l l

N a C l0 m M l l

0 20 40 60 80 100 120 0 50 100 150 200

KCImM/l NaClmM,/l
A FIGURE 1 Demonstration of the dependence of the Na*/K* enzymeactivity up to a peakandthenlevels
increases out.Thisgraph
ATPase activityon the concentrationof eachion. Thegraphon alsodemonstrates
the dependence on low levels
of theactivity of
the leftshowsthatincreasing
K- leadsto an inhibition
of theATPase K+ [ndaptedfromJSkou,1957,Biochem BiophysActa23:3941
activityThegraphon the rightshowsthatwith increasing Na-, the

separate binding sites for Na* and
K*. For optimal ATPaseactivity, both
must be filled. However, at high con-
centrations K- could comDetefor the
'
N a - b i n d i n g s i t e . l e a d i n gr o e n z y m e
inhibition. He hypothesized that this
enzyme was involved in active trans-
port, that is, the pumping of Na+ out
of the cell, coupled ro the import of K*
into the cell. Later studieswould prove
that this enzymewas indeed the pump
that catalyzed active transport. This
finding was so exciting that Skou de-
voted his subsequentresearchto study-
i n g t h e e n z y m e .n e v e r u s i n g i t a s a
marker. as he initiallv intended.
Discussion
Skou's finding that a membrane AT-
Pase used both Na* and K* as sub-
strateswas the first step in understand-
ing active transport on a molecular
level. How did Skou know to test both
Nao and K*? In his Nobel lecture in
1.997,he explained that in his first at-
tempts at characterizing the ATPase,
he took no precautions to avoid the
use of buffers and ATP stock solutions
that containedNa* and K*. Pondering
the puzzling and unreproducible re-
sults that he obtained led to the real-
ization that contaminating salts might
be influencing the reaction. When he
repeated the experiments, this time
avoiding contamination by Na and K
at all stages,he obtained clear-cut, re-
producibleresults.
The discovery of the Na*/K*
ATPasehad an enormous impacr on
membrane biology, leading to a better
understandingof the membranepoten-
tial. The generation and disruption of
membrane potential forms the basis of
many biological processes,including
neurotransmissionand the coupling of
chemical and electricalenergy.For this
fundamental discovery, Skou was
awarded the Nobel Prize for Chem-
istry rn 1997.

478 CHAPTER
11 | T R A N S M E M B R A NT E
R A N S P O ROTF | O N SA N D S M A L LM O L E C U L E S
 CHAPTER

CELLULAR
ENERGETICS
lmmunofluorescence micrograph showingthe intertwinednetworko{
mitochondria (red)in a cellfrom the ovaryof the Himalayan
Tahr
mountaingoat Theunusualtwin nucleiin thiscellarestainedblue
lcourtesyof M Davidson]

rom the growth and division of a cell to the beating of a
heart to the electrical activity of a neuron that underlies
thinking, life requires energy.Cells are complex systems
in which a multitude of chemical reactions and transport
processesare coordinately regulatedin time and space.Cells
cannot generateand maintain their highly organized struc-
tures and conduct extensivemetabolism (e.g., carbohydrate
synthesis)without material and energy from their environ-
ments. This chapter describesthe molecular mechanismsby
which cells use sunlight or chemical nutrients as sourcesof
energy,with a specialfocus on how cellsconvert theseexter-
nal sourcesof energy into a biologically universal, intracel-
lular, chemical energy carrier, adenosine triphosphate, or
ATP (Figure 1.2-1.).ATP,found in all types of organismsand
presumably present in the earliest life-forms, is generated
from ADP and inorganic phosphate (HPO42-, often abbre-
viated as P1).Cells use the energy releasedduring hydrolysis
of the terminal high-energyphosphoanhydridebond in ATP
(seeFigure 2-31.)to power many otherwise energeticallyun-
favorable processes.Examples include the synthesisof pro-
teins from amino acids and of nucleic acids from nucleotides
(Chapter 4), transport of moleculesagainst a concentration
gradient by ATP-powered pumps (Chapter 11), contraction
of muscle (Chapter 1.7),and beating of cilia (Chapter 18).
The energy to drive ATP synthesisfrom ADP (AG :
7.3 kcallmol) is produced primarily by two processes:aero-
bic oxidation, which occurs in mitochondria in nearly all eu-
karyotic cells (Figure 1.2-1.,top), and photosynthesis,which
occursin chloroplastsonly in leaf cellsof plants (Figure 12-1,
bottoml and certain single-celled organisms, such as
cyanobacteria.Two additional processes,glycolysis and the
citric acid cycle, are also important direct or indirect sources
of ATP in both animal and plant cells.
In aerobic oxidation, breakdown products of sugars
(carbohydrates) and fatty acids (hydrocarbons)-both de-
rived in animals from the digestion of food-are converted
by oxidation with 02 to carbon dioxide and water. The en-
ergy releasedfrom this overall reaction is transformed into
the chemical energy of phosphoanhydride bonds in ATP.
This is analogous to burning wood (carbohydrates)or oil
(hydrocarbons)to generateheat in furnacesor motion in au-
tomobile engines: both consume 02 and generate carbon
dioxide and water. The key difference is that cells break the
overall reaction down into many intermediate steps. This
permits the amount of energy releasedin any given step to
OUTLINE
12.1 FirstStepsof Glucoseand FattyAcid Catabolism:
and the CitricAcid Cycle
Glycolysis
12"2 The ElectronTransportChainand Generation
of the Proton-MotiveForce
12.3 Harnessingthe Proton-MotiveForcefor
Energy-RequiringProcesses 503
12.4 and Light-AbsorbingPigments 511
Photosynthesis
12.5 MolecularAnalysisof Photosystems 517
12.6 CO2MetabolismDuring Photosynthesis 524

479
 Energy
sou rce
Stage lll
Chemical Electron -----) Proton--J ATP
bond oxidation FADH2 transport motive
(citricacid cycle) (electron force
Slucose \ \
carriers) Cj, H2O (H+gradient)
\pyruvate)
ATP NADH

ATP

Stage 3
Photons Energyabsorption + Electron * Proton----+ ATP
( s un l i gh t ) by pigmentsand
direct transfer
to electrons

FIGURE 12-1 Overviewof aerobicoxidationand pigments (stage1);the absorbed energyis usedto bothoxidize
photosynthesis. Eukaryotic cellsusetwo fundamental mechanisms waterto 02 andestablish conditions (stage2) necessary for the
to convertexternal sources of energyintoATP(Top)ln aerobic generation of ATP(stage3) andcarbohydrates fromC02(carbon
o x i d a t i o n" ,f u e l "m o l e c u l e( p
s r i m a r isl yu g a ras n df a t t ya c i d s ) fixation,stage4) Bothmechanisms involve the production of
undergo p r e l i m i n apr yr o c e s s i ni ngt h e c y t o s oel , g , b r e a k d o wonf reduced high-energy electron carriers (NADH,NADPH, FADHr) and
glucose to pyruvate (stagel), and arethentransferred into movement of electrons downan electrical potential in an electron
m i t o c h o n d r iwah, e r et h e ya r ec o n v e r t ebdy o x i d a t i ow ni t h0 2 t o t r a n s p ocr th a i nt h r o u g hs p e c i a l i zm
e de m b r a n eEsn e r gfyr o mt h e s e
c a r b o nd i o x i d e a n dw a t e r( s t a g e sl l a n dl l l ) a n dA T Pi sg e n e r a t e d electrons is released andcaptured asa proton-motive force(proton
(stagelV).(Bottom)In photosynthesis, whichoccursin electrochemical gradient) that isthenusedto driveATPsynthesis
c h l o r o p l a stths e, r a d i a net n e r g yo f l i g h ti sa b s o r b ebdy s p e c i a l i z e d Bacteria utilizecomparable processes

match closely the amount of energyrequired for the next in-
termediate stage of the process. If there were not a close
match, excessreleasedenergywould be lost as heat (which
would be very inefficient) or not enough energy would be
releasedto drive the next step in the process (which would
be ineffective).
In photosynthesis,the radiant energyof light is absorbed
by pigments such as chlorophyll and used to make ATP and
carbohydrates(primarily sucroseand starch).Unlike aerobic
oxidation, which uses carbohydratesand 02 to generate
CO2, photosynthesisusesCO2 as a substrateand generates
O2 and carbohydratesas products.
This reciprocal relationship betweenaerobic oxidation
in mitochondria and photosynthesisin chloroplastsunder-
lies a profound symbiotic relationship between photosyn-
thetic and nonphotosyntheticorganismsand is responsible
for much of the life on earth. The oxygen generatedduring
photosynthesisis the source of virtually all the oxygen in
the air, and the carbohydrates produced are the ultimate
sourceof energyfor virtually all nonphotosyntheticorgan-
isms. (An exceptionis bacterialiving in deep oceanvents-
and the organismsthat feed on them-that obtain energy
for converting COz into carbohydrates by oxidation of geo-
Iogically generatedreduced inorganic compounds released
by the vents.)
At first glance,it would seemthat the molecular mecha-
nisms underlying the reciprocal processesof photosynthesis
and aerobicoxidation have little in common. However. a rev-
olutionary discoveryin cell biology establishedthat bacteria,
mitochondria, and chloroplastsall use the same mechanism,
known as chemiosmosis,to generateAIP from ADP and P1.In
chemiosmosis(alsoknown as chemiosmoticcoupling), a pro-
ton electrochemicalgradient is generatedacrossa membrane,
driven by energyreleasedas electronstravel through an elec-
tron transport chain. The energystoredin this gradient,called
the proton-motive force, is useddirectly to power the synthe-
sis of AIP and other energy-requiringprocesses(Figure12-2).
In this chapter,we explore the molecular mechanismsof the
tv'/oprocesses that sharethis central mechanism,focusingfirst
on aerobicoxidation and then on photosynthesis.
FirstStepsof Glucose
and FattyAcid Catabolism:Glycolysis
and the CitricAcid Cycle
In an automobile engine, hydrocarbon fuel is oxidatively
and explosively converted in an essentiallyone step process
to mechanicalwork (i.e., driving a piston). The processis

480 c H A P T E R1 2 | CELLULAR
ENERGETTCS
 Low pH r Sincetotal energystored in the bonds of the initial fuel
Radiant H+ H+ H+ moleculesis substantiallygreaterthan that required to drive
energy
(light) the synthesisof a singleATP molecule (-7.3 kcal/mole),
Positive
electric many ATP moleculesare produced.
{ potential
1,4 f

In our discussionof aerobic oxidation, we will be tracing

the fate of the two main energy-producingdigestiveproducts
of food: sugars (principally glucose) and fatty acids. Under
(chlorophyll) |
certain conditions amino acids also feed into thesemetabolic
NADH
FADH2 pathways.
^ ADP+P; ATP The complete aerobic oxidation of each molecule of glu-
cose yields 6 molecules of CO2 and the energy releasedis
coupled to the synthesisof as many as 30 moleculesof ATP.
The overall reaction is
Membrane
Chemical impermeable
b o n d si n Exoplasmicface
carbohydrates H'
to H* c6Hpo5 + 6 02 + 30 Pi2- + 30 ADP3- + 30 H+ --+
andlipids Synthesis 6 CO2 + 30 ATP4- + 36 H2O, AG : 586 kcal/mol
of ATP

A FIGURE 12-2 Proton-motive force.Transmembrane proton Glucose oxidation in eukaryotes takes place in four
c o n c e n t r a t iaon de l e c t r i c(avl o l t a g eg)r a d i e n tcso, l l e c t i v eclayl l e d stages(seeFigure 12-1):
theproton-motive force,aregenerated duringaerobicoxidation
andphotosynthesis in eukaryotes and prokaryotes (bacteria) I. Conversion in the cytosol of one 6-carbon glucosemol-
H i g h - e n e r eg lye c t r o ngse n e r a t ebdy l i g h ta b s o r p t i obny p i g m e n t s ecule to two 3-carbon pyruvate molecules(glycolysis)
( e g , c h l o r o p h yol lr)h e l di n t h e r e d u c efdo r mo f e l e c t r ocna r r i e r s
( e g , N A D HF, A D H 2m) a d ed u r i n gt h e c a t a b o l i somf s u g a ras n d II. Pyruvate oxidation to CO2 in the mitochondrion via a
l i p i d sp a s sd o w na n e l e c t r otnr a n s p o cr th a i n( b l u ea r r o w s r) e , leasing 2-carbon acetyl CoA intermediate (citric acid cycle)
energythroughout the processTheenergyis recovered by
c o u p l i n igt sr e l e a steo p u m p i n g p r o t o nas c r o stsh e m e m b r a n(er e d III. Electron transport to generatea proton-motive force
arrows), generating the proton-motive force In chemiosmotic
coupling, the energyreleased whenprotonsflow downthe IV. ATP synthesisin the mitochondrion (oxidative phos-
gradientthroughATPsynthase drivesthe synthesis of ATPThe phorylation)
proton-motive forcecanalsopowertransport of metabolites across
t h e m e m b r a naeg a i n st th e i rc o n c e n t r a t igorna d i e natn dr o t a t i o o nf In this section,we discussstagesI and II: the biochemical
b a c t e r i fal la g e l l a . pathways that break down glucose and fatty acids to CO2,
generating some ATP and high-energy electrons in the
process; the fate of the releasedelectrons (stage III) is de-
relatively inefficient in that both substantial amounts of the scribedin the next section.
chemical energy stored in the fuel are wasted as they are
convertedto unusedheat and substantialamounts of fuel are During Glycolysis(Stagel), CytosolicEnzymes
only partially oxidized and releasedas carbonaceous,some-
ConvertGlucoseto Pyruvate
times toxic, exhaust.In the competition to survive,organisms
cannot afford to squander their sometimes limited energy Glycolysis occurs in the cytosol in both eukaryotes and
sources on an equivalently inefficient process. Cells have prokaryotes and does not require molecular oxygen; thus
evolved incredibly efficient mechanisms for hydrocarbon it is called anaerobic glucose catabolism (biological break-
(fatty acid) and carbohydrate (sugar)combustion coupled to down of complex to simpler substances).A set of 10 water-
ATP synthesis.That mechanism is aerobic oxidation. Each soluble cytosolic enzymescatalyzethe reactionsconstituting
stage of fuel conversion to energy comprisesmultiple steps the glycolytic pathway (glyco, "sweet"; /ysis, "split"), in
that are catalyzedor mediatedby specificproteins.This strat- which one molecule of glucose is converted to two
egy providesthe following advantages: moleculesof pyruvate (Figure 12-3). All the reaction inter-
mediates produced by these enzymes are water soluble'
r By dividing the processinto multiple stepsthat generate phosphorylated compounds called metabolic intermedi-
severalenergy-carryingintermediates,bond energy is effi- ates.ln addition to chemicallyconvertingone glucosemol-
ciently channeledinto the synthesisof ATP and energy lost eculeinto theseintermediatesand the two pyruvates,these
as heat is reduced. enzymatic reactions generatefour ATP molecules by phos-
phorylation of four ADPs (reactions7 and 10), a process
r Different fuels are reducedto common intermediatesthat called substrate-levelphosphorylation (to distinguish it
can then sharesubsequentpathwaysfor combustionand from the oxidative phosphorylation that generatesATP in
AIP synthesis. the third stageof aerobic oxidation). Unlike later stagesof

F T R SS : L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
A N D F A T T YA C I D C A T A B O L I S MG
T T E P SO F G L U C O S E 481
> FIGURE 12-3 The glycolyticpathway.Glucose isdegraded to
pyruvate, Tworeactions consume ATf formingADPand
phosphorylated sugars (red),two generate ATPfromADPby
substrate-level phosphorylation (9reen), andoneyieldsNADHby
reduction of NAD*(yellow)Notethatallthe intermediates between
glucose andpyruvate arephosphorylated compounds Reactions
1,
3, and 10,with singlearrows, areessentially irreversible (large
n e g a t i vAeGv a l u e su)n d e or r d i n a rcyo n d i t i o ni nsc e l l s

I
E ,r ArP
'' - t J r^u^3^ 2- OH
l\ noP

k
V - u
^6^
ru3
2

Fructose1,6-bisphosphate
F
E
OHH
HO
ill
OH
Dihydroxyacetone
tr Glyceraldehyde
OH
I
H

HC- C -c-H
HO3PO-C C-C-H 3-phosphate
I phosphate
(2 moleculesl I I
HH
H O oPo3'
1L,roo.*r,
tr , l l n-,' r o o , * , r .
tv OHH
1,3-Bisphosphoglycerate iltl
ATP formation in mitochondria and chloroplasrs,a proron- (2 molecules) U.TU-U-U-L
"l
motive force is not involved in substrate-levelphosphory- 4l
''' HOO2-
lation. However, substrate-levelphosphorylation requires ll lrz^DP
the addition (in reactions1 and 3) of two phosphatesfrom 2ArP
lL> OHH
two ATPs. Thesecan be rhought of as "pump priming" re- 3-Phosphoglycerate - o - ci_l tc1_ c _ H
actions,which introduce a little energyup front in order to {2 molecules)
I
effectivelyrecover more energy downstream. Thus glycol- 4l HO OPO32-
ysis yields a net of only two ATP molecules per glucose
E'',"""',],]',,i,]l!
ll
molecule. t OHH
ill
2-Phosphoglycerate
The balanced chemical equation for the conversion of {2 molecules)
glucose to pyruvate shows that four hydrogen atoms (four '-o.Po
1l oH
protons and four electrons)are also released: tr ini)ilell

ll'>z u,o
tv- o H
Phosphoenolpyruvate - o _ c _ c : c _ HI
oo (2 molecules)
' o.Po I
C6H12O6-zCH.-J J Or* 4H++4e- I
r ' " ,, , : 1 1 2 A D P
Glucose Pvruvate t0 ^
"'''" [ 2ATP
v ooH
(For convenience,we show pyruvate here in its un-ionized Pyruvate - o - c t_t cl _ c _ H
form, pyruvic acid, although at physiologicalpH it would be (2 molecules)
I
H
largely dissociated.)All four electrons and two of the four
protons are transferred (Figure 12-3, reaction 6) to two The overall chemical ecuation for this first stage of glu-
moleculesof the oxidized form of nicotinamide adeninedin- cosemetabolism is
ucleotide (NAD*) to produce the reducedform, NADH (see
F i g u r e2 - 3 3 ) : C 6 H 1 2 O 6+ 2 N A D + + 2 A D p 3 - + 2 p i 2 - - )
2C3H4Or+2NADH+2ATP4-
2H* + 4 e- -r 2 NAD+ --+2 NADH
After glycolysis, only a fraction of the energy available in
Later we will seethat the energycarried by the electronsin glucose has been extracted and converted to ATP and
NADH and an analogous carrier FADH2, the reduced NADH. The rest remains in the covalent bonds of the two
form of flavin adeninedinucleotide (FAD), can be used to pyruvate molecules.The ability to efficiently convert the en-
make additional ATPs via the electron transDort chain. ergy in pyruvate to ATP dependson molecular oxygen. As

482 CHAPTER
12 I CELLULAE
RN E R G E T I C S
we will see,in the presenceof oxygen (aerobic conditions),
energyconversionis highly efficient.In its absence(anaerobic
conditions), the processis much lessefficient.
The Rateof Glycolysisls Adjustedto Meet
the Cell'sNeedfor ATP
Enzyme-catalyzed reactions and metabolic pathways are
regulated by cells so as to produce the needed amounts of
metabolites but not an excess.The primary function of the
oxidation of glucoseto CO2 via the glycolytic pathway is to
produce NADH and FADH2, whose oxidation in mitochon-
dria generates ATP. Operation of the glycolytic pathway
(stageI), as well as the citric acid cycle (stageII), is continu-
ously regulated,primarily by allostericmechanisms,to meet
the cell's need for AIP (seeChapter 3 for generalprinciples
of allostericcontrol).
Three allosterically controlled glycolytic enzymesplay a
key role in regulating the entire glycolytic pathway (Figure
12-3). Hexokinase (step 0 ) is inhibited by its reaction prod-
uct, glucose6-phosphate.Pyruuatekinase (step IO) is inhib-
ited by ATP, so glycolysis slows down if too much ATP is
present.The third enzyme,phosphofructokinase-l (step B),
is the principal rate-limiting enzyme of the glycolytic path-
way. Emblematic of its critical role in regulating the rate of
glycolysis,this enzyme is allosterically controlled by several
molecules(Figure12-4).
For example, phosphofructokinase-1is allosterically
inhibited by ATP and allosterically actiuated by AMP. As a
result, the rate of glycolysisis very sensitiveto the cell's en-
ergy charge, reflected in the ATP:AMP ratio. The allosteric
inhibition of phosphofructokinase-1by ATP may seem
unusual, sinceATP is also a substrateof this enzyme.But the
affinity of the substrate-bindingsite for ATP is much higher
(has a lower K-) than that of the allosteric site. Thus at low
High [ATP]
High [AMP]
FruCtOSe
2,6-bisphosphate
A FIGURE 12-4 Allostericregulationof glucosemetabolism.
Thekeyregulatoryenzyme in glycolysis,
phosphofructokinase-1,
is
activated
allosterically byAMpandfructose 2,6-bisphosphate,
which
areelevatedwhenthecell'senergystoresarelow.Theenzyme is
by ATp(whenenergystores
inhibited arehigh)andcitrate,bothof
whichareelevatedwhenthe cellisactivelv qlucose
oxidizinq to COr
Laterwe will seethat citrateis generatedduringstagell of glucose
oxidation.Phosphofructokinase-2 (PFK2)is a bifunctionalenzyme:its
A N D F A T T YA C I D C A T A B O L I S MG
T T E P SO F G L U C O S E
F T R SS
concentrations,ATP binds to the catalytic but not to the in-
hibitory allosteric site, and enzymatic catalysis proceedsat
near maximal rates. At high concentrations,ATP also binds
to the allosteric site, inducing a conformational changethat
reduces the affinity of the enzyme for the other substrate,
fructose 6-phosphate,and thus reducesthe rate of this reac-
tion and the overall rate of glycolysis.
Another important allosteric activator of phosphofruc-
tokinase-1 is frwctose 2,5-bisphospbate.This metabolite is
formed from fructose 6-phosphate by an enzyme called
pbosphofructokinase-2.Fructose 6-phosphate accelerates
the formation of fructose 2,6-bisphosphate,which in turn
activates phosphofructokinase-1.This type of control is
known as feed-forward actiuation, in which the abun-
dance of a metabolite (here,fructose 5-phosphate)induces
an acceleration in its subsequent metabolism. Fructose
2,6-bisphosphateallostericallyactivatesphosphofructoki-
nase-1in liver cellsby decreasingthe inhibitory effect of high
ATP and by increasingthe affinity of phosphofructokinase-1
for one of its substrates,fructose 6-phosphate.
The three glycolytic enzymesthat are regulated by allostery
catalyzereactions with large negative AGo' values-reactions
that are essentially irreversible under ordinary conditions.
Theseenzymesthus are particularly suitablefor regulatingthe
entire glycolytic pathway. Additional control is exerted by
glyceraldehyde 3-phosphate dehydrogenase,which catalyzes
the reduction of NAD+ to NADH (seeFigure 12-3, step 6).
If cytosolic NADH builds up owing to a slowdown in mito-
chondrial oxidation, this reaction becomesthermodynami-
cally lessfavorable.
Glucosemetabolismis controlled differently in various
mammalian tissues to meet the metabolic needs of the
organism as a whole. During periods of carbohydratestar-
vation, for instance,it is necessaryfor the liver to release
glucoseinto the bloodstream.To do this, the liver converts
gh [citrate]
Phospho{ructo- FTUCtOSe
Fructose
kinase formsfructose
activity 2,6-bisphosphatefromfructose
6-phosphate,anditsphosphatase catalyzes
activity the reverse
whichrsreleased
Insulin,
reaction. bythe pancreas whenblood
glucoselevelsarehigh,promotesPFK2 kinase andthus
activity
At low bloodglucose,
glycolysis
stimulates glucagon isreleased by
the pancreasandpromotes PFK2 phosphatase in the liver,
activity
indirectlyslowingdown glycolysis
: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
483
 (a) (b)
ANAEROBIC (FERMENTATION)
METABOLISM AEROBIC IV]ETABOLISIV]

Yeast Muscle
CYTOSOL CYTOSOt CYTOSOL

c6H1206
c6H1206 c 6 H1 2 0 6
Glucose
Glucosc Glucose

2ADP+2NAD++2P, 2 A D P + 2 N A D + + 2P ; 2ADP+2NAD++2P;
Glycolysis Glycolysis Glycolysis

2ATP+2NADH+2P; 2ATP+2NADH+2P;
+2HrO +2H2O +2HrO
oo oo oo
iltl iltl tl
cH3-c-c-oH cH3-c-c-oH cH3-c c-oH
Pyruyic acid hFuvic actld Pyruvic acid

Pyruvate I
I
l,-- NADH+ H+
decarboxylase ,
LactateI
f. " dehydrogenase N
l>co,
v | \> NAD*
v
o oHo MITOCHONDRION
tl ttl
x2 cH3-cH cH3-cH-C-OH
Acetaldchyde Lastlc acid

NADH + H+

NAD' coz
CoA-SH
Pyruvate
x2 d e h y dr o g e n a s e
NAD+

NADH

Overallreactionsof anaerobicmetabolism:
G l u c o s+e 2 A D P+ 2 P , ----> 2 ethanol+ 2 CO2+2 ATP+ 2 H2O
2 , ---> 2 lactate + 2 ATP+ 2 H1O
G l u c o s e + 2 A D P +P
C i t r i ca c i d
NADH
cycle

Oxidative
NAD+ phosphorylation

-28 ADP + -28 Pi

-28 AfP + -28 H2O

A FIGURE 12-5 Anaerobicversusaerobicmetabolismof glucose.The 2CO2

ultimatefateof pyruvate formedduringglycolysis depends on the presenceor
absence of oxygen. In theformation of pyruvate fromglucose, onemolecule Overallreactionof aerobicmetabolism:
of NAD+isreduced (byaddition of two electrons) to NADHfor eachmolecule Glucose+ 6 02 + -30 ADP + -30 Pi ---->
of pyruvate formed(seeFigure12-3, reaction6) (a)In the absence of oxygen,
6 CO2+ 36 H2O+ -30 ATP
two electrons aretransferred from eachNADHmolecule to an acceptor
molecule to regenerate NAD+,whichis required for continued glycolysis.
In
yeasts(/eft),acetaldehyde isthe electronacceptorandethanolisthe product.
Thisprocess iscalledalcoholic fermentationwhen oxygenis limitingin muscle
cells(nght),NADHreduces pyruvate to formlacticacid,regenerating NAD+.
(b) In the presence of oxygen,pyruvateistransported into mitochondriaFirstit
isconverted by pyruvate dehydrogenase intoonemolecule of CO2andone
of aceticacid,the latterlinkedto coenzyme A (CoA-SH) to form acetylCoA,
concomitant with reduction of onemolecule of NAD+to NADH.Further
metabolism of acetylcoA andNADHgenerates approximately an additional
28 molecules of ATPperglucose molecule oxidized.

484 C H A P T E R1 2 I CELLULAR
ENERGETICS
the polymer glycogen,a storageform of glucose(Chapter 2),
directly to glucose 6-phosphate (without involvement of
hexokinase,step [). Under theseconditions, there is a re-
duction in fructose 2,6-bisphosphatelevels and decreased
phosphofructokinase-1activity (Figure 12-4). As a result,
glucose6-phosphatederived from glycogenis not metabo-
lized to pyruvate; rather, it is converted to glucose by a
phosphataseand releasedinto the blood to nourish the
brain and red blood cells,which dependprimarily on glu-
cose as an energy fuel. In all cases,the activity of these reg-
ulated enzymesis controlled by the level of small-molecule
metabolites, generally by allosteric interactions, or by
hormone-mediated phosphorylation and dephosphoryla-
tion reactions (Chapter 15 gives a more detailed discus-
sion of hormonal control of glucose metabolism in liver
and muscle).
G l u c o s el s F e r m e n t e dU n d e r
A n a e r o b i cC o n d i t i o n s
Many eukaryotesare obligate aerobes:they grow only in the
presenceof molecular oxygen and metabolizeglucose(or re-
lated sugars)completely to CO2, with the concomitant pro-
duction of a large amount of ATP. Most eukaryotes, how-
ever, can generate some ATP by anaerobic metabolism. A
few eukaryotesare facubatiue anaerobes:they grow in either
the presenceor the absenceof oxygen. For example, an-
nelids,mollusks,and someyeastscan live and grow for days
without oxygen.
In the absenceof oxygen, yeasts convert the pyruvate
produced by glycolysisto one molecule each of ethanol and
CO2; in these reactions two NADH molecules are oxidized
to NAD* for each two pyruvates converted to ethanol,
thereby regeneratingthe supply of NAD- (Figure 12-5a,
left). This anaerobic degradation of glucose, called fermen-
tation, is the basis of beer and wine production.
Oxygen deprivation can also affect glucosemetabolism
in animals. During prolonged contraction of mammalian
skeletal muscle cells-for example, during exercise-oxy-
gen within the muscle tissue can becomelimited and glu-
cosecatabolism is limited to glycolysis(stageI). As a con-
sequence, muscle cells convert the pyruvate from
glycolysis to two molecules of lactic acid by a reduction
reaction that also oxidizes two NADHs to NAD*s (Figure
L2-5a, right). Although the lactic acid is releasedfrom the
muscle into the blood, if the contractions are sufficiently
rapid and strong, the lactic acid can transiently accumu-
late in that tissue and contribute to muscle and joint pain
during exercise.Once it is secretedinto the blood, some of
the lactic acid passesinto the liver, where it is reoxidized
to pyruvate and either further metabolizedto C02 aerobi-
cally or converted back to glucose.Much lactate is metab-
olized to CO2 by the heart, which is highly perfused by
blood and can continue aerobic metabolism at times when
exercising, oxygen-poor skeletal muscles secretelactate.
Lactic acid bacteria (the organisms that spoil milk) and
other prokaryotes also generateATP by the fermentation
of glucoseto lactate.
F T R SS
T T E P SO F G L U C O S E
A N D F A T T YA C I D C A T A B O L I S MG
U n d e rA e r o b i cC o n d i t i o n sM
, itochondria
EfficientlyOxidizePyruvateand Generate
ATP(Stagesll-lv)
In the presenceof oxygen, pyruvate formed by glycolysis
is transported into mitochondria, where it is oxidized by
02 to CO2 and H2O via a seriesof oxidation reactions. The
overall process by which cells use 02 and produce COz is
collectively termed cellular respiration (Figure 12-5b). Re-
actions in the mitochondria (stagesII-IV) generatean esti-
mated 28 additional ATP molecules per original glucose
molecule, far outstripping the ATP yield from anaerobic
glucosemetabolism.
Oxygen-producing photosynthetic cyanobacteria ap-
peared about 2.7 billion years ago' The subsequentbuildup
in the earth's atmosphere of sufficient oxygen during the
next approximately billion years opened the way for organ-
isms to evolve the very efficient aerobic oxidation pathway,
which in turn permitted the evolution, especially during
what is called the Cambrian explosion' of large and complex
body forms and associatedmetabolic activities. In effect,
mitochondria are ATP-generating factories, taking full
advantageof this plentiful oxygen. We first describe their
structure and then the reactions they employ to degrade
pyruvate.
M i t o c h o n d r i aA r e D y n a m i cO r g a n e l l e s
with Two Structurallyand Functionally
D i s t i n c tM e m b r a n e s
Mitochondria (Figure 12-6) are among the larger organelles
in the cell. A mitochondrion is about the size of an E. coli
bacterium, which is not surprising, becausebacteria are
thought to be the evolutionary precursorsof mitochondria (see
Chapter 6 and the discussionof endosymbiont hypothesis,
below). Most eukaryotic cells contain many mitochondria,
collectively occupying as much as 25 percent of the volume
of the cytoplasm. The numbers of mitochondria in a cell,
hundreds to thousandsin mammalian cells, are regulated to
match the cell's requirements for ATP (e.g., stomach cells'
which use a lot of ATP for acid secretion,have many mito-
chondria). Analysis of fluorescentlylabeled mitochondria in
living cellshas shown that mitochondria are highly dynamic.
They undergo frequent fusions and fissions that generate
tubular, sometimesbranched networks (Figure 1'2-7),which
may account for the wide variety of mitochondrial mor-
phologiesseenin different types ofcells. Fusionsand fissions
apparently play a functional role as well becausegeneticdis-
ruptions in GTPasesuperfamily genesrequired for thesedy-
namic processescan disrupt function, such as maintenance
of proper inner membrane electrical potential, and cause
human disease,such as the neuromuscular diseaseCharcot-
Marie-Tooth subtype 2A.
The details of mitochondrial structure can be observed
with electron microscopy (seeFigure 9-8). Mitochondria
have two distinct kinds of concentricallyrelated membranes.
The outer membrane definesthe smooth outer perimeter of
the mitochondrion. The inner membrane has numerous
: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
 Video: Mitochondrion Reconstructedby ElectronTomography
(b)
F6F1complexes

Intermembrane
space

C r i s t a ej u n c t i o n s

FIGURE 12-6 Internalstructureof a mitochondrion. bluespheres), andgranules (largeyellowspheres) (b)Computer-

( a )S c h e m a tdi ica g r a m
s h o w i ntgh ep r i n c i p m
a le m b r a n a
ensd generated modelof a section of a mitochondrionfromchicken brain.
compartments Thecristae formsheets andtubesby invagination Thismodelisbased on a three-dimensionalelectron microscopicimage
o f t h ei n n e rm e m b r a naen dc o n n e ct o
t t h ei n n e m r embrane calculatedfroma seriesof two-dimensionalelectron micrographs
throughrelatively smalluniformtubularstructures calledcnsta recorded at regularintervalsThistechnique isanalogous to a three-
junctionsTheintermembrane spaceappears continuous with the dimensional x-raytomogram or CATscanusedin medical imaging
lumenof eachcristaTheFeF,complexes (smallredspheres), Notethetightlypacked cristae(yellow-green),
the innermembrane
whichsynthesize ATP, areintramembrane particles thatprotrude (lightblue),andtheoutermembrane (darkblue)[part(a)courtesyof
fromthecristae andinnermembrane intothe matrixThematrix T. Frey;part (b) from T. Freyand C Mannella,2000, TrendsBiochem Sci
contains the mitochondrial DNA(bluestrand), ribosomes (small 25:319l

invaginationscalled cristae (seeFigure 12-6). These mem-

Video: MitochondrialFusionand Fission (d branes topologically define two submitochondrial compart-
ments: the intermembrane space, between the outer and
inner membranes,and the matrix, or central compartment,
which forms the lumen within the inner membrane.'When
individual mitochondria fuse, each of their distinct comparr-
ments intermixes (e.g.,matrix with matrix, inner membrane
with inner membrane). Fractionation and purification of
these membranes and compartments have made it possible
to determine their protein, DNA, and phospholipid compo-
sitions and to localize each enzyme-catalyzedreaction to a
specific membrane or comparrment. About 1000 polypep-
tides are required to make and maintain mitochondria and
E X P E R I M E N TFA
IGL U RtE2 - 7 M i t o c h o n d r iuan d e r g o permit them to function. Only a small number of these-13
rapidfusionand fissionin living cells.Mitochondria labeled in humans-are encoded by mitochondrial DNA genes,
with a fluorescent proteinin a livingnormalmurineemoryontc while the remaining proteins are encoded by nuclear genes
fibroblastwereobserved usingtime-lapse fluorescence ( C h a p t e r6 ) .
microscopy Several mitochondria undergoing fusion(top)or The most abundant protein in the outer membrane is
fission(bottom)areartificially highllghted jn blueandwith mitochondrial porin, a transmembrane channel protein
arrowslModified fromD C Chan, 2006,Cell125(])j241-12521 similar in structure to bacterial porins (seeFigure 10-1S).
Ions and most small molecules(up to about 5000 Da) can

486 c H A P T E R1 2 | CELLULAE
RN E R G E T T C S
readily pass through these channel proteins when they are
open. Although there may be metabolic regulation of the
opening of mitochondrial porins and thus the flow of
metabolites across the outer membrane, the inner mem-
brane with its cristae are the major permeability barriers
between the cytosol and the mitochondrial matrix, limiting
the rate of mitochondrial oxidation.
Protein constitutes 76 percent of the total weight of the
inner membrane-a higher fraction than in any other cel-
lular membrane. Many of these proteins are key partici-
pants in cellular respiration. They include ATP synthase,
proteins responsiblefor electron transport, and a wide va-
riety of transport proteins that permit the movement of
metabolites between the cytosol and the mitochondrial
matrix. The human genomeencodes48 membersof a fam-
ily of mitochondrial transport proteins. One of these is
called the ADP/ATP carrier, an antiporter that moves
newly synthesizedATP out of the matrix and into the in-
ner membrane space(and subsequentlythe cytosol) in ex-
'!flithout
changefor ADP originating from the cytosol. this
essential antiporter, the energy trapped in the chemical
bonds in mitochondrial ATP would not be availableto the
rest of the cell.
The invaginating cristae greatly expand the surface area
of the inner mitochondrial membrane (see Figure 12-6),
enhancing its capacity to generateATP. In typical liver mi-
tochondria, for example,the area of the inner membrane,
including cristae, is about five times that of the outer mem-
brane. In fact, the total area of all inner mitochondrial mem-
branes in liver cells is about 1,7 times that of the plasma
membrane. The mitochondria in heart and skeletal muscles
contain three times as many cristae as are found in typical
liver mitochondria-presumably reflecting the greater de-
mand for AIP by muscle cells.
Note that plants have mitochondria and perform cellular
respiration as well. In plants, stored carbohydrates,mostly
in the form of starch, are hydrolyzed to glucose.Glycolysis
then produces pyruvate, which is transported into mito-
chondria, as in animal cells. Mitochondrial oxidation of
pyruvate and concomitant formation of ATP occur in pho-
tosynthetic cells during dark periods when photosynthesisis
not possible and in roots and other nonphotosynthetic tis-
suesat all times.
The mitochondrial inner membrane, cristae, and matrix
are the sites of most reactions involving the oxidation of
pyruvate and fatty acids to CO2 and H2O and the coupled
synthesisof ATP from ADP and P1, with each reaction
occurring in a discretemembrane or spacein the mitochon-
drion (Figure12-8).
The last three of the four stagesof glucoseoxidation are
r StageII. Conversionof pyruvate to acetyl CoA, followed
by oxidation to CO2 in the citric acid cycle.Theseoxidations
are coupled to reduction of NAD* to NADH and of FAD
to FADH2. (Fatty acid oxidation follows a similar route,
with conversion of fatty acyl CoA to acetyl CoA.) Most
of the reactionsoccur in or on the membrane facing the
matrIX.
T T E p SO F G L U C o 5 EA N D F A T T YA C I D C A T A B O L I S MG
F T R SS
r StageIII. Electron transfer from NADH and FADH2 to 02
via an electron transport chain within the inner membrane,
which generatesa proton-motive force acrossthat membrane.
r StageIV. Harnessingthe energy of the proton-motive
force for ATP synthesisin the mitochondrial inner mem-
brane. StagesIII and IV are together called oxidative
phosphorylation.
In Stagell, Pyruvatels Oxidizedto CO2
and High-EnergyElectronsStored
in ReducedCoenzymes
Pyruvate formed during glycolysis in stage I in the cytosol
is transportedinto the mitochondrial matrix (Figure 12-8).
StageII metabolism accomplishesthree things: (1) it con-
verts the 3-carbon pyruvate to three moleculesof CO1' Q)
it generates high-energy electron carriers (NADH and
FADH2) that will be used for electrontransport (stageIII);
and (3) it generatesa GTP molecule, which is then con-
verted to ATP:
GTP + ADP i- GDP + AIP
StageII can be subdivided into two distinct parts: (1) the
generationof acetyl CoA plus one molecule of COz and one
NADH and (2\ the conversion of acetyl CoA to two mole-
cules of CO2 and the high-energy intermediatesNADH (3
molecules),FADH2, and GTP.
Generation of Acetyl CoA \il/ithin the mitochondrial matrix,
pyruvate reactswith coenzymeA, forming CO2 and acetyl CoA
and NADH (Figure 12-8). This reaction, catalyzedby pyruuate
- 8.0 kcaUmol)and
dehydrogena.sais highly exergonic(AG"' :
essentiallyirreversible.
Acetyl CoA (Figure 1'2-9) plays a central role in the oxi-
dation of fatty acids and amino acids.In addition, it is an in-
termediate in numerous biosynthetic reactions' including
transfer of an acetyl group to histone proteins and many
mammalian proteins, and synthesisof lipids such as choles-
terol. In respiring mitochondria, however' the acetyl group
of acetyl CoA is almost always oxidized to CO2 via the cit-
ric acid cycle.
Citric Acid Cycle Nine sequentialreactions operate in a cy-
cle to oxidize acetyl CoA to CO2. The cycle is referred to by
severalnames:the citric acid cycle, the tricarboxylic acid (or
TCA) cycle, and the Krebs cycle. The net result is that for
each acetyl group entering the cycle as acetyl CoA, two mol-
eculesof CO2, three of NADH' and one each of FADH2 and
GTP are produced.
As shown in Figure 12-1'0,the cycle begins with con-
densation of the two-carbon acetyl group from acetyl CoA
with the four-carbon molecule oxaloacetate to yield the
six-carboncitric acid (hencethe name citric acid cycle).In
both reactions 4 and 5, a CO2 molecule is releasedand
NAD+ is reduced to NADH. Reduction of NAD- to
NADH also occurs during reaction 9; thus three NADHs
: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
487
 Outer mitochondrial membrane {permeable to metabolites)

coz Intermembrane space

StageI
Glucose
2 NAD*-J
I
2 NADH.f" z efP
J
2 Pyruvate - Pyruvate Acetyl CoA -----?2COt
C i t r i ca c i d
cycre

Mitochondrlal matrix

Succinate
2 e- + 2H* + !Or---+ Hrg
Qz Hzo
Fumarate

FoF,complex

A FIGURE 12-8 Summaryof aerobicoxidationof glucoseand FADH2, GTBandCO2.Stagelll: Electron transport reduces oxygen to
fatty acids.Stagel: Inthecytosol, glucoseisconverted to pyruvate waterandgenerates a proton-motive force.Electrons (blue)from
(glycolysis)
andfattyacidto fattyarylCoA hTruvate andfattyacylCoA reduced coenzymes aretransferred viaelectron-transport complexes
thenmoveintothemitochondrion Mitochondrialporins maketheouter (blueboxes) to 02 concomitant withtransport of H* ions(red)fromthe
membrane permeable to thesemetabolites, butspecificrranspon matrixto theintermembrane space, generating theproton-motive force
proteins(coloredovals)in theinnermembrane arerequired to import ElectronsfromNADHflowdirectly fromcomplex I to complex lll,
pyruvate (yellow)
andfattyacids(blue)intothematrixFattyacyl bypassing complex ll ElectronsfromFADH, flowdirectly fromcomplex ll
groupsaretransferred fromfattyarylCoAto an intermediate carriel to complex lll,bypassing complex I StagelV:ATPsynthase, the FoFl
transported across
the innermembrane (blueoval),andthenreattached complex (orange), harnesses the proton-motive forceto synthesize ATp
to CoAon thematrixsideStagell: Inthemitochondrial matrix, rnthematrix,Antiporter proteins(purpleandgreenovals) transport ADp
pyruvateandfattyacylCoAareconverted to acetylCoAandthen andP;intothematrixandexporlhydroxyl groupsandATpNADH
oxidized,releasing
CO2Pyruvate isconverted to acetylCoAwiththe generated in thecytosol isnottransported directly to thematnxoecause
formation of NADHandCOr;two carbons fromfattyacylCoAare theinnermembrane rsimpermeable to NAD+andNADH;instead, a
convefted to acetylCoAwiththeformation of FADH,andNADH shuttlesystem (red)transportselectrons fromcytosolic NADHto NAD+in
Oxidation of acetylCoAin thecitricacidcycleqenerates NADHand the matrix02 diffuses intothematrix,andCOrdiffuses our

are generatedper turn of the cycle.In reaction 7, two elec-
trons and two protons are transferredto FAD, yielding the
reducedform of this coenzyme,FADH2. Reaction 7 is dis-
tinctive becauseit not only is an intrinsic part of the citric
acid cycle (stageII), but also it is caralyzedty a membrane-
attached enzyme that is an intrinsic part of the electron
transport chain (stageIII). In reaction 6, hydrolysis of the
high-energythioester bond in succinyl CoA is coupled to
synthesisof one GTP by subsrrate-levelphosphorylation.
(BecauseGTP and ATP are interconvertible,this can be

A (CoA)
Coenzyme
a F I G U R E l 2 ' 9T h e s t r u c t u r e o fa c e t y lC o A . T h j s c o m p o u n d i s a c i d s , a n d m a n y a m i n o a c il d
t asl.s o c o n t r i b u t e s a c eg tr yolu p s i n
an importantintermediatein the aerobicoxidationof pyruvate,fatty many biosynthetic pathways

488 c H A P T E R1 2 | CELLULAE
RN E R G E T T C S
 N A D H+ H -
HrO coo
I
nu
t-
HO-C-H c-coo
I tl
HC
QH, I
t-
V iaa
vvv
coo-
/
Malate crs-Aconitate

I
Hro- coo- coo-
I
.1.,, CH" t
\tl
UN
coo- t-
H-C-COO
HC I I
I coo- nu
HO-C-H
coo- I t- E /l
Fumarate Z CH, coo-
QH,
l- I'
C H "= c:o lsocitrate
// FA t-' I
,r
Y coo coo-
Succinate 4-Keto-
FADH, GDP + Pi glutarate C O r + N A D H+ H '
+ H2O

GTP + HSCoA + N A D H+ H -

a FIGURE 12-10The citricacidcycle.AcetylCoAis metabolized of NADH,onemolecule of FADH2, andonemolecule of GTP

to C02andthe high-energy electron
carriers NADHandFADH2. In Thetwo carbonatomsthatenterthe cyclewith acetylCoAare
reaction 1, a two-carbon acetylresiduefromacetylCoAcondenses y lo A I n s u c c t n aat en d
h i g h l i g h t ei ndb l u et h r o u g hs u c c i n C
with the four-carbon molecule oxaloacetate to formthe six-carbon fumarate, whicharesymmetric molecules, theycanno longer
citrate.In the remaining reactions(2-9)eachmolecule of citrate be specifically denoted. lsotope-labeling studies haveshownthat
iseventually converted backto oxaloacetate, losingtwo C02 thesecarbonatomsarenot lostin theturn of the cyclein which
molecules In eachturn of the cycle,four pairsof
in the process. theyenter;on average, onewill be lostasC02duringthe next
electrons areremoved fromcarbonatoms,forminqthreemolecules turn of the cycle and the otherin subsequent turns.

consideredan ATP-generatingstep.) Reaction 9 regener-
ates oxaloacetate,so the cycle can begin again. Note that
molecular 02 does not participatein the citric acid cycle.
Most enzymesand small moleculesinvolved in the cit-
ric acid cycle are soluble in the aqueousmitochondrial ma-
trix. Theseinclude CoA, acetylCoA, succinylCoA, NAD*,
and NADH, as well as most of the eight cycle enzymes.
Succinate dehydrogenase(reaction 7), however, is a com-
ponent of an integral membraneprotein in the inner mem-
brane,with its active site facing the matrix. When mitochondria
are disrupted by gentle ultrasonic vibration or osmotic ly-
sis, non-membrane-boundenzymesin the citric acid cycle
are releasedas very large multiprotein complexes.Within
such complexes the reaction product of one enzyme is
thought to pass directly to the next enzymewithout diffus-
ing through the solution. However,much work is neededto
determine the structuresof these large enzyme complexes
as they exist in the cell.
Since glycolysis of one glucose molecule generatestwo
acetyl CoA molecules, the reactions in the glycolytic path-
way and citric acid cycle produce six CO2 molecules, 10
NADH molecules, and two FADH2 molecules per glucose
molecule(Table 12-1). Although thesereactionsalso gener-
ate four high-energyphosphoanhydridebonds in the form of
two ATP and two GTP molecules' this represents only a
small fraction of the available energy releasedin the com-
plete aerobic oxidation of glucose.The remaining energy is
itored as high-energy electrons in the reduced coenzymes
NADH and FADH2. The goal of stagesIII and IV is to re-
cover this energy in the form of ATP.
T r a n s p o r t e risn t h e I n n e r M i t o c h o n d r i a l
MembraneHelp Maintain AppropriateCytosolic
and Matrix Concentrationsof NAD* and NADH
In the cytosol NAD+ is required for step 6 of glycolysis (see
Figure 12-3), and in the mitochondrial matrix NAD+ is re-
qrri..d for conversion of pyruvate to acetyl CoA and for
th... tt.pt in the citric acid cycle (4, 5, and 9 in Figure 12-
FADH2 to FAD as it reduces02 to water and converts the
energy stored in the high-energy electrons in the reduced

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
A N D F A T T YA C I D C A T A B O L I S MG
F I R S Ts T E P SO F G L U C O S E
489
 c02M0ltcljlts NAD+
Ml)LECULES FADM{]TECULES
fltAcTt0N PBODUCED REOUCED
TONADH REDUCED
T0tADH2 (OR
ATP GTP)
L glucose molecule to 2 pyruvate molecules 0 2 0 z

2 pyruvates to 2 acetyl CoA molecules a

L z 0 0
2 acetyl CoA to 4 CO2 molecules 4 t) z 2
Total 5 10 2 +
,

forms of these molecules into a proton-motive force. Even simple. However, the inner mitochondrial membrane is im-
though 02 is not involved in any reaction of the citric acid permeableto NADH. To bypassthis problem and permit the
cycle, in the absenceof 02, this cycle soon stops operating as electrons from cytosolic NADH to be transfer red indirectly to
the intramitochondrial suppliesof NAD* and FAD dwindle 02 via the electron transport chain, cells use severalelectron
due to the inability of the electron transport chain to oxidize shuttles to transfer electrons from cytosolic NADH to NAD+
NADH and FADH2. These observations raise the question in the matrix. Operation of the most widespreadshuttle-the
of how a supply of NAD+ in the cytosol is regenerated. malate-aspartateshwttle-is depicted in Figure 12-11. For
If the NADH from the cytosol could move into the mito- every complete "turn" of the cycle,there is no overall change
chondrial matrix and be oxidized by the electron transport in the numbers of NADH and NAD* moleculesor the inter-
chain and if the NAD+ product could be transported back mediatesaspartateor malate used by the shuttle.However, in
into the cytosol, regenerationof cytosolic NAD+ would be the cytosol, NADH is oxidized to NAD+, which can be used

Cytosol NADHcytosot NAD*cytosol

Aspartate oxaroacetate \E/ , t",.,"

( \ - K e t olgu t a r a t eG l u t a m a t e
Gluta

Mitochondrial
inner membrane

Glutamate o - K e t o gI u t ar a t e
_->-_)

r r - K e t o g l u t ar a t e G l u t a m a t e
I \q/ ,,"nuY,1ll';,"."',
Aspartate Oxaloacetate €----------- Malare
/E\
Matrix NADHmatrix NAD*m"tri"

FIGURE 12-11The malateshuttle.Thiscyclical series of reactions directly

crossthe innermembrane, isconverted to aspartate
transfers electrons fromNADHin thecytosol (intermembrane space) by additionof an aminogroupfromglutamateInthistransaminase-
a c r o stsh e i n n e rm i t o c h o n d r m i ael m b r a n w e ,h i c hi s i m p e r m e a b l e catalyzedreaction in the matrix,glutamate is converted to
to NADHitself,to NAD* in the matrixThenet resultisthe cr-ketoglutarateStep[: A secondantrporter (redoval)exports
replacement of cytosolic NADHwith NAD+and matrixNAD+ aspartateto the cytosolin exchange for glutamateStep@: A
with NADHStepll: Cytosolic malatedehydrogenase transfers cytosoltc
transamrnase converts aspartate to oxaloacetate and
electrons fromcytosolic NADHto oxaloacetate, formingmalate. ct-ketoglutarate
to glutamate, completing the cycleTheblue
S t e pf , l : A n a n t i p o r t e( b
r l u eo v a l )i n t h e i n n e rm i t o c h o n d r i a l arrowsreflectthe movement of the cr-ketoglutarate,the red
m e m b r a nter a n s p o r m t sa l a t ei n t ot h e m a t r i xi n e x c h a n gf eor arrowsthe movement of glutamate,andthe blackarrowsthat
cr-ketogIutarate. StepS: MitochondriaI malatedehydrogenase of aspartate/malate lt is noteworthy that,asaspartate and
converts malatebackto oxaloacetate, reducing NAD+in the matrix malatecycleclockwise, glutamate andcr-ketoglutarate cyclein
to NADHin the processStepE: Oxaloacetate. whichcannot the oppositedirection

490 CHAPTER
12 I CELLULAR
ENERGETICS
for glycolysis,and in the matrix, NAD* is reducedto NADH,
which can be usedto generateATP via stagesIII and IV
NADH.yrorot + NAD;".. * --+NAD.*y,oro1
* NADH-.1'1"
MitochondrialOxidation of Fatty Acids
GeneratesATP
Up to now, we have focusedmainly on the oxidation of car-
bohydrates,namely glucose,for ATP generation.Fatty acids
are another important source of cellular energy. Cells can
take up either glucose or fatty acids from the extracellular
spacewith the help of specifictransporter proteins (Chapter
11). Should a cell not needto immediatelyburn thesemole-
cules,it can store them as a polymer of glucosecalled glyco-
gen (especiallyin muscleor liver) or as a trimer of fatty acids
covalently linked to glycerol, called a triacylglycerol or
triglyceride. In some cells, excessglucose is converted into
fatty acids and then triacylglycerols for storage. However,
unlike microorganisms, animals are unable to convert fatty
acids to glucose.lWhen the cells need to burn these energy
storesto make ATP (e.g.,when a resting muscle beginsto do
work), enzymesbreak down glycogen to glucose or hy-
drolyze triacylglycerols to fatty acids, which are then oxi-
dized to generateATP:
o of four reactionsin which all the carbon atoms are converted
cH3-(CH2)"-C- O-CH2
^l
YI
cH3-(cH2),-c-o-cH * 3 Hro +
?l
cH3- (cH2)"-c-o-cH2
Triacylglycerol
O HO-CH
3 CH3-(CH2)"-C-OH + HO-CH2
Fatty acid
Fatty acidsare the major energysourcefor many tissues,
particularly adult heart muscle.In humans,in fact, the oxi-
dation of fats is quantitatively more important than the ox-
idation of glucose as a source of ATP. The oxidation of 1 g
of triacylglyceride to CO2 generatesabout six times as
much AIP as does the oxidation of 1 g of hydrated glyco-
gen. Thus triglycerides are more efficient than carbohy-
drates for storage of energy,in part becausethey are stored
in anhydrous form and can yield more energy when oxi-
dized and also becausethey are intrinsically more reduced
(have more hydrogens)than carbohydrates.In mammals,
the primary site of storageof triacylglyceridesis fat (adi-
pose)tissue,whereasthe primary sitesfor glycogenstorage
are muscle and the liver.
Just as there are four stagesin the oxidation of glucose,
there are four stagesin the oxidation of fatty acids.To opti-
mize the efficiency of ATP generation,part of stageII (citric
acid cycle oxidation of acetyl CoA) and all of stagesIII and
IV of fatty acid oxidation are identical to those of glucose
F I R S TS T E P SO F G L U C O s EA N D F A T T YA C I D C A T A B O L I S MG
oxidation. The differenceslie in the cytosolic stageI and the
first part of the mitochondrial stageII. In stageI, fatty acids
are convertedto a fatty acyl CoA in the cytosol in a reaction
coupled to the hydrolysis of ATP to AMP and PPl (inorganic
pyrophosphate)(seeFigure 12-8):
o
R-C O- + HSCoA + ATP ------->
Fatty acid
o
R-C-SCOA+AMP+PPI
Fatty acyl CoA
Subsequenthydrolysis of PP1to two molecules of P;
drives this reaction to completion. To transfer the fatty
acyl group into the mitochondrial matrix, it is covalently
transferred to a molecule called carnitine, moved across
the inner mitochondrial membrane by an acylcarnitine
transporter protein (seeFigure 12-8' blue oval), and then
on th; matrix side, the fatty acyl group is released from
carnitine and reattached to another CoA molecule' The
activity of the acyl carnitine transporter is regulated to
prevent oxidation of fatty acids when cells have adequate
energy (ATP) supPlies.
In the first part of stage II' each molecule of a fatty acyl
CoA in the mitochondrion is oxidized in a cyclical sequence
two at a time to acetyl CoA with generation of FADH2 and
NADH (Figure 1,2-1'2a).For example, mitochondrial oxida-
tion of each molecule of the 18-carbon stearic acid'
HO-CH2
l
GlYcerol
section, the reduced NADH and FADH2 with their high-
energyelectronsfrom stageII will be used in stageIII to gen-
erct; a proton-motive force that in turn is used in stageIV to
power ATP synthesis.
PeroxisomalOxidation of Fatty Acids
GeneratesNo ATP
Mitochondrial oxidation of fatty acids is the major sourceof
ATP in mammalian liver cells, and biochemistsat one time
believedthis was true in all cell types. However, rats treated
with clofibrate, a drug that affectsmany featuresof lipid me-
tabolism, were found to exhibit an increasedrate of fatty acid
oxidation and a large increasein the number of peroxisomes
in their liver cells.This finding suggestedthat peroxisomes'as
well as mitochondri a, can oxidize fatty acids' These small or-
carbonsin the fatty acyl chain, or (Cs)' medium-(C3-Crz),
: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E
491
 > FIGURE12-12 Oxidation of fatty acids in ( a ) M I T O C H O N D R I AOLX t D A T | O N (b)PEROXTSOMAL
OXtDATION
mitochondria and peroxisomes.In both
mitochondrial oxidation(a)and peroxisomal
oxidation(b),fatty acidsare convertedto
o
acetylCoA by a seriesof four enzyme-catalyzed R- CH2-CH2-CHr-C-SCoA
reactions(showndown the centerof the fioure) Fatty acyl CoA
A fatty acylCoA moleculeis convertedto u..tyl
CoA and a fatty acylCoA shortenedby two
carbonatoms Concomitantly, one FADmolecule
is reducedto FADH2and one NAD* molecule
is reducedto NADH The cycleis repeatedon o H r O+ ' t / z O ,
il
the shortenedacylCoA untilfatty acidswith R- CH2-CH: CH-C-SCoA
an evennumberof carbonatomsare completelv
converted to acetylCoA In mitochondrra, HrO
electrons
from FADH2and NADHenterthe electrontransport
chainand ultimately are usedto generateATp;the
acetylCoA generatedisoxidizedrn the citricacid
cycle,resultingin release of CO2and ultimately the
synthesis of additionalATP Becauseperoxisomes NADH
lackthe electrontransportcomplexescomposlng exportedfor
the electrontransportchainand the enzymesof reoxidation
the citricacidcycle,oxidationof fatty acidsin these
organellesyieldsno ATPlAdapted fromD L Netson
andl\,4M Cox,Lehninger principlesof
Biochemistry,3d
ed , 2000,WorthPublishersl

R-CHr-C-SCoA
I
o
Acyl CoA shortened
by two carbon atoms
+
o
Citricacid l
+- H3C-C-SCoA Acetyl CoA
cycre exported
Acetyl CoA

stead it is transported into the cytosol for use in the synthe-

sis of cholesterol(Chapter10) and other metabolites.

oxidation of fatty acids, which is coupled to generation of
ATP, peroxisomal oxidation of fatty acids is not linked to
ATP formation, and energy is releasedas heat.
The reaction pathway by which fatty acids are degraded
to acetyl CoA in peroxisomesis similar to that used in mito_
dases, peroxisomes contain abundant catalase, which
quickly decomposesthe H2O2, a highly cytotoxic metabo_
lite. NADH produced during oxidation of fatty acids is ex-
ported and reoxidized in the cytosol; there is no need for a
malate/aspartateshuttle here. peroxisomesalso lack the cit_
ric acid cycle, so acetyl CoA generatedduring peroxisomal
degradation of fatty acids cannot be oxidized further: in-
First Steps of Glucoseand Fatty Acid Catabolism:
Glycolysisand the Citric Acid Cycle
r In a processknown as aerobicoxidation, cellsconvert the
energy releasedby the oxidation ("burning") of glucoseor
fatty acidsinto the terminal phosphoanhydridebond of ATp.
r The complete aerobic oxidation of each molecule of glu-
coseproduces six moleculesof CO2 and approximately 30
ATP molecules.The entire process,which starts in the
cytosol and moves into the mitochondrion, can be divided
into four stages:(I) glycolysis to pyruvate in the cytosol,
(II) pyruvate oxidation to C02 in the mitochondrion.
(III) electron transport to generatea proton-motive force
together with conversion of molecular oxygen to wate!
and (IV) ATP synthesis.
r The mitochondrion hastwo distinctmembranes(outerand
inner) and two distinct subcompartments(intermembrane

492 c H A P T E R1 2 | cELLULAR
ENERGETTCS
space between the two membranesand the matrix sur-
rounded by the inner membrane).Aerobic oxidation occurs
in the mitochondrial matrix and on the inner mitochondrial
membrane.
r Each turn of the citric acid cycle releasestwo molecules
of CO2 and generatesthree NADH molecules,one FADH2
molecule. and one GTP.
r In glycolysis (stageI), cytosolic enzymesconvert glucose
to two moleculesof pyruvate and generatetwo molecules
each of NADH and ATP.
r The rate of glucoseoxidation via glycolysisand the citric
acid cycle is regulated by the inhibition or stimulation of
severalenzymes,dependingon the cell'sneed for ATP. Glu-
coseis stored (asglycogenor fat) when ATP is abundant.
r Some of the energy releasedin the early stagesof oxida-
tion is temporarily stored in the reducedcoenzymesNADH
or FADH2, which carry high-energy electrons that subse-
quently drive the electron transport chain (stageIII).
r In the absenceof oxygen (anaerobicconditions),cellscan
metabolize pyruvate to lactate or (in the case of yeast) to
ethanol and CO2, in the processconvertingNADH back to
NAD*, which is necessaryfor continued glycolysis.In aero-
bic conditions (presenceof oxygen),pyruvate is transported
into the mitochondrion, where stagesII through IV occur.
r In stage II, the three-carbon pyruvate molecule is first
oxidized to generateone moleculeeach of CO2, NADH'
and acetyl CoA. The acetyl CoA is then oxidized to CO2 by
the citric acid cycle.
r Neither glycolysis(stageI) nor the citric acid cycle (stage
II) directly use molecularoxygen (02).
r The malate/aspartateshuttle regeneratesthe supply of
cytosolicNAD* necessary for continuedglycolysis.
r Like glucoseoxidation, the oxidation of fatty acids takes
place in four stages.In stageI, fatty acids are converted to
fatty acyl CoA in the cytosol. In stageII, the fatty acyl CoA
is first converted into multiple acetyl CoA moleculeswith
generationof NADH and FADH2. Then, as in glucoseoxi-
dation, the acetyl CoA entersthe citric acid cycle. StagesIII
and IV are identical for fatty acid and glucoseoxidation.
r In most eukaryotic cells, oxidation of short- to long-
chain fatty acids occurs in mitochondria with production
of ATR whereas oxidation of very long chain fatty acids
occurs primarily in peroxisomes and is not linked to ATP
production; the releasedenergy is converted to heat.
Slfl The ElectronTransportchain and
Generationof the Proton-MotiveForce
Most of the energy releasedduring the oxidation of glucose
and fatty acidsto CO2 (stagesI and II) is convertedinto high-
energyelectronsin the reducedcoenzymesNADH and FADH2.
\7e now turn to stageIII, in which the energytransiently stored
in the coenzymesis converted by an electron transport
THEELECTRoNTRANSPORTcHA|NANDGENERAT|oNoFTHEPRoToN-MoT|VEFoRcE
chain, also known as the respiratorychain into the proton-
motive force.'We first describethe logic and componentsof the
electron transport chain and the pumping of protons acrossthe
mitochondrial inner membrane.'Weconcludethe sectionwith a
discussion of the magnitude of the proton-motive force pro-
duced by electron transport and proton pumping. In the fol-
lowing section,we describestageIV, focusing on the structure
of the AIP synthaseand how it usesthe proton-motive force to
synthesizeATP.
StepwiseElectronTransportEfficientlyReleases
t h e E n e r g yS t o r e di n N A D Ha n d F A D H 2
During electron transport' electrons are released from
NADH and FADH2 and eventually transferred to 02, form-
ing H2O according to the following overall reactions:
NADH + H+ + 1/z02 --+ NAD* + H2O,
LG: -52'6 kcal/mol
FADH2 -r 1/z02 --+ FAD + H2O,
L'G : -43.4 kcal/mol
Recall that the conversion of 1 glucosemolecule to CO2 via
the glycolytic pathway and citric acid cycle yields 10 NADH
and-2 FADH2 molecules (seeTable 12-1). Oxidation of
-613 kcal/mol
thesereducedcoenzymeshas a total AGo'of
duction of FAD, which requires lessenergy.
The energy carried in the reduced coenzymescan be re-
leased by oxidizing them. The biochemical challengefaced
by the mitochondrion is to transfer,as efficiently as possible'
the energy releasedby this oxidation into the energy in the
terminal phosphoanhydridebond in ATP.
P,t- * H* + ADP3- -+ATPa- + H2o,
L,G : +7.3 kcal/mol
A relatively simple one-to-onereaction in which reduction of
one coenzyme molecule and synthesis of one ATP occurs
would be terribly inefficient, becausethe AG'' for ATP gen-
eration from ADP and P1is substantially less than for the
coenzymeoxidation and much energywould be lost as heat'
To efficiently recover the energ5 the mitochondrion first
converts the energy of coenzyme oxidation into a proton-
motive force using a seriesof electron carriers' all but one of
which are integral components of the inner membrane'
ElectronTransportin Mitochondrials Coupled
t o P r o t o nP u m P i n g
At severalsites during electron transport from NADH and
FADH2 to C,2,protons from the mitochondrial matrix are
493
 pH electrode
O, solution c
60
8= 40
+P
.=- 20
a)
(J
n
Mitochondrion
EXPERIMENTAL FIGURE12-13Electrontransferfrom NADH
to 02 is coupledto proton transportacrossthe mitochondrial
membrane.lf NADHisaddedto a suspension of mitochondria
depleted of 02, no NADHisoxidizedWhena smallamountof O, is
addedto thesystem (arrow),
thereisa sharprisein theconcentration
of protonsin thesurroundingmediumoutside the mitochondria
pumped acrossthe inner membrane;this generares proton con-
centration and electricalgradientsacrossthe inner membrane
(seeFigure 12-2). This pumping causesthe pH of the mito-
chondrial matrix to becomehigher (i.e.,the H+ concentration
is lower) than that of the intermembranespaceand cytosol.An
electricpotential acrossthe inner membranealso resultsfrom
the pumping of H* outward from the matrix, which becomes
negative with respect to the intermembrane space.Thus free
energy releasedduring the oxidation of NADH or FADH2 is
storedboth as an electricpotential and a proton concentrarion
gradient-collectivelS the proton-motive force-across the
major source of ATP in aerobic nonphotosyntheticcells.
Much evidenceshows that in mitochondria and bacteriathis
processof oxidative phosphorylation dependson generarion
of a proton-motive force acrossthe inner membrane (mito-
chondria) or bacterial plasma membrane, with electron
transport, proton pumping, and ATp formation occurring
simultaneously.In the laboratory, for instance, addition o]
02 and an oxidizable substratesuch as pyruvate or succinare
to isolated intact mitochondria results in a net synthesisof
ATP if the inner mitochondrial membrane is intact. In the
presence of minute amounts of detergents that make the
membrane leaky, electron transport and the oxidation of
these metabolites by 02 still occurs. However, under these
conditions no ATP is made, becausethe proton leak prevents
the maintenance of the transmembrane proton concentra-
tion gradientand the membraneelectricporential.
The coupling between elecrron transporr from NADH
(or FADH2) to 02 and proron rransporr aiross the inner mi-
tochondrial membrane can be demonstrutedexperimentally
494 . cHAprE1
R2 I c E L L U L AER
NERGETtcs
0 60 120 180 240 300
E l a p s etdi m e( s )
(decrease in pH) Thustheoxidation of NADHby 02 iscoupledto the
movement of protons out of the matrixOncethe 02 isdepleted,
the
excess protons slowlymovebackintothe mitochondria (powering
the synthesis of ATP)andthe pHof the extracellularmediumreturns
t o i t si n i t i avl a l u e .
with isolated, intact mitochondria (Figure 12-13). As soon
as 02 is addedto a suspensionof mitochondria in an other-
wise O2-free solution that contains NADH, the medium
outside the mitochondria transiently becomesmore acidic
(increasedproton concentration), becausethe mitochondrial
outer membrane is freely permeableto protons. (Remember
that malate/aspartateand other shuttles can convert the
NADH in the solution into NADH in the matrix.) Once the
02 is depleted by its reduction, the excessprotons in the
medium slowly leak back into the matrix. From analysisof
the measuredpH changein such experiments,one can calcu-
late that about 10 protons are transported out of the matrix
for every electron pair transferred from NADH to 02.
To obtain numbers for FADH2, the above experiment can
be repeated,but with succinateinsteadof NADH as the sub-
strate. (Recall that oxidation of succinateto fumarate in the
citric acid cycle generatesFADH2; see Figure 12-10). The
amount of succinateaddedcan be adiustedso that the amount
of FADH2 generatedis equivalentto the amount of NADH in
the first experiment. As in the first experiment, addition of
oxygen causes the medium outside the mitochondria to
becomeacidic, but lessso rhan with NADH. This is not sur-
prising becauseelectronsin FADH2 havelesspotential energy
(43.4 kcal/mol) than electrons in NADH (52.6 kcal/mole).
and thus it drives the translocation of fewer protons from
the matrix and a smaller changein pH.
ElectronsFlow from FADH2and NADHto 02
T h r o u g hF o u rM u l t i p r o t e i nC o m p l e x e s
I7e now examine more closely the energetically favored
movement of electronsfrom NADH and FADH2 to the final
electron acceptor,02. For simplicity, we will focus our dis-
cussion on NADH. In respiring mitochondria, each NADH
molecule releasestwo electronsto the electron transDort
chain; these electrons ultimately reduce one oxygen atom
(half of an 02 molecule),forming one moleculeof water:

NADH--+NAD* + H* + 2e GROUPS-
PR{]STHETIC
COMP()NENT
PROTEIN
t/rOr--HrO
2e- + 2H' +
NADH-CoQ reductase FMN
As electronsmove from NADH to 02, their potential de- (complex I) Fe-S
clines by 1.14 V, which correspondsto 26.2 kcal/mol of
electronstransferred,or :53 kcal/mol for a pair of elec- Succinate-CoQreductase FAD
(complex II) Fe-S
trons. As noted earlier,much of this energyis conservedin
the proton-motive force generatedacross the inner mito-
CoQH2-cytochrome c reductase Heme by
chondrial membrane.
(complex III) Heme bp1
There are four large multiprotein complexesin the elec-
Fe-S
tron transport chain that span the inner mitochondrial
Heme c1
membrane: NADH-CoQ reductase(complex I, >40 sub-
units), swccinate-CoQreductase(complex II, 4 subunits), Heme c
Cytochrome c
CoQH2-cytochromec reductdse(complexIII, 11 subunits),
and cytochrome c oxidase (complex IV, 13 subunits). Elec- Crru2*
Cytochrome c oxidase (comPlex IV)
trons from NADH flow from complex I to III to IV, bypass- Heme a
ing complex II; electronsfrom FADH2 flow from complex II Lut-
to III to IV, bypassingcomplex I (seeFigure 12-8). Herne a3
Each complex contains several prosthetic groups that
participate in moving electrons.These small nonpeptide or- *-Not included is coenzyme Q, an electron carrier that is not
ganic moleculesor metal ions are tightly and specificallyas- permanently bound to a protein complex.
sociatedwith the multiprotein complexes(Table 12-2). iou*.,.' J. \Xl.De Pierre and L. Ernster, L977, Ann. Reu. Biochem'
46:201..

Heme and the Cytochromes Severaltypesof heme,an iron-

containing prostheticgroup similar to that in hemoglobin
and myoglobin (Figure 12-14a\, are tightly bound (cova-
lently or noncovalently) to a set of mitochondrial proteins
called cytochromes.Each cytochrome is designatedby a letter,
such as A, b, c, or c1. Electron flow through the cytochromesoc-
curs by oxidation and reductionof the Fe atom in the centerof
the hememolecule:
. -->
Fe3* + e- . Fe2*
Becausethe heme ring in cytochromesconsistsof alternating
double- and single-bondedatoms, a large number of reso-
nance hybrid forms exist. These allow the extra electron de-
livered to the cytochrome to be delocalizedthroughout the
heme carbon and nitrogen atoms as well as the Fe ion.
The various cytochromes have slightly different heme
groups and surrounding atoms (called axial ligands), which

ta) (b)
HrC:CH 9H.
II

Protein

tl
CH, H,C
l--l
o2c-cH2 H2c-co2
othercytochromes in theelectron transport chain.All hemesaccept
A FIGURE 12-14 Hemeand iron-sulfurprostheticgroupsin
andrelease oneelectron at a time. (b)Dimeric cluster
iron-sulfur (Fe-S)
the electrontransportchain.(a)Hemeportionof cytochromes bg
of CoQHz-cytochrome c reductase Each Featom is bonded to four S atoms: two are sulfur
inorganic and
andbs,whicharecomponents
(complex ring(yellow)is presentin allhemes two arein cysteine sidechains of the associated proteinAll Fe-S
lll) Thesameporphyrin
Thechemical attached
substituents to the porphyrinring differin the acceptandrelease
clusters oneelectron at a time.

O F T H E P R O T O N - M O T I VFEO R C E
T H A I NA N D G E N E R A T I O N
T R A N S P O RC
THE ELECTRON
495
 generate different environments for the Fe ion. Therefore,
each cytochrome has a different reduction potential, or ten-
dency to accept an electron-an important property dictat-
ing the unidirectional "downhill" electron flow along the
chain. Just as water spontaneously flows downhill from a
higher to lower potential energy state-but not uphill-so
too do electronsflow in only one direction from one heme
(or other prosthetic group) to another due to their differing
reduction potentials. All the cytochromes, excepr cy-
tochrome c, are componentsof integral membranemultipro-
tein complexesin the inner mitochondrialmembrane.
fron-Sulfur Clusters lron-sulfur clustersare nonheme,iron-
containing prosrheticgroups consistingof Fe atoms bonded
both to inorganicS atomsand to S atomson cysteineresiduesin
a protein (Figure I2-l4b). Some Fe aroms in the cluster bear a
*2 charge;othershavea *3 charge.However,the net chargeof
eachFe atom is actuallybetween+2 and *3, becauseelectrons
in their outermost orbitals together with the extra electron de-
livered via the transport chain are dispersedamong the Fe
atoms and move rapidly from one atom to another.Iron-sulfur
clustersacceptand releaseelecffonsone at a time.
form a semiquinone, a charged free radical denoted by
CoQ .. Addition of a secondelectronand two protons (thus
a total of two hydrogen atoms) to CoQ-. forms dihy-
droubiquinone (CoQH2), the fully reducedform. Both Coe
and CoQH2 are solublein phospholipidsand diffuse freely in
the hydrophobic centerof the inner mitochondrial membrane.
This is how it participatesin the electron transport chain-
carrying electronsand protons betweenthe complexes.
As shown in Figure 12-16, CoQ acceptselectronsreleased
from NADH-CoQ reductase(complex I) or succinate-Coe
reductase(complex II) and donates them to CoeH2-
cytochrome c reductase(complex III). Importantly, reduction
and oxidation of CoQ are coupled to pumping of protons.
Ifhenever CoQ acceptselectrons,it does so at a binding site
on the matrix (also called the cytosolic) face of the protein
complex, always picking up protons from the medium there.
\Thenever CoQH2 releasesits electrons,it doesso at a srteon
the intermembranespace(also called the exoplasmic)side of
the protein complex, releasingprotons into the intermem-
brane (or exoplasmic) fluid. Thus transport of each pair of
electronsby CoQ is obligatorily coupled to movement of two
protons from the matrix to the intermembranespacefluid.

Coenzyme Q (CoQ) Coenzyme e (Coe), also called

NADH-CoQ Reductase (Complex l) Electronsare trans-
ubiquinone, is the only small-moleculeelectron carrier in the
ferred from NADH to CoQ by NADH-CoQ reducrase(Figure
chain that is not a protein-bound prosthetic group (Figure
12-16).In bacteriathe massof this complex is about 500 kDa
12-15). It is a carrier of both protons and electrons.The oxi-
(-14 subunits),whereasfor the L-shapedeukaryoticcomplex
dized quinone form of CoQ can accepr a single electron to
it is 1 MDa (14 centraland as many as 32 accessorysubunits).
NAD- is exclusively a two-electron carrier: it accepts or re-
leasesa pair of electronssimultaneously.In NADH-Coe reduc-
o tase (complex I), electronsfirst flow from NADH to FMN
H3co
(flavin mononucleotide), a cofactor related to FAD, then to an
(coo) cH,
ubiquinone itt iron-sulfur clusteq and finally to CoQ. FMN, like FAD, can ac-
( o x i d i z feodr m ) H3CO (CHr-CH:C-CH2)10-H
cept two electronsbut does so one electron at a trme.
o Each transported electron undergoesa drop in potential
e rl
I
of =360 mV, equivalenrto a AG'' of -16.6kcallmol for the
two electrons transported. Much of this releasedenergy is
J used to transport four protons across the inner membrane
o- per molecule of NADH oxidized by the complex I. Those
H3cO CH. 9H'
four protons are distinct from the two protons transferredto
s e m i q u i n o n e( c o e ; )
{ f r e er a d i c a l ) H3CO (CHr-CH:J-CH2)10-H the CoQ in the chemical reaction shown above.
The overall reaction catalyzedby this complex is
U.

t.l NADH + CoQ * 6H*1. --+

zut +
"-
J
OH
(Reduced) (Oxidized)

NAD- * Hti. + CoeH2 + 4H+o,t

(Oxidized) (Reduced)
H3co cH,
Dihydroquinone ?*'
1 999H,) H3CO (CHr-CH:C-CH2)10-H
(fully
reduced
form) Succinate-CoQ Reductase (Complex ll) Succinatedehy-
OH drogenase,the enzyme that oxidizes a molecule of succinate
A FIGURE12-15 Oxidized and reduced forms of coenzyme to fumaratein the citric acid cycle(and in the processgenerares
e
(CoQ),which can carry two protons and two electrons. Because the reduced coenzymeFADH2), is one of the four subunits of
of its long hydrocarbon "tail" of isopreneunits,Coe, alsocalled complex II. In this way the citric acid cycle is physically as well
ubrquinone, is solublein the hydrophobic coreof phospholipidbilayers as functionally linked to the electron transport chain. The two
and is verymobile.Reductionof Coe to the fully reducedform, electronsreleasedin conversionof succinateto fumarate are
eH2
(dihydroquinone), occursin two stepswith a half-reduced free-radical transferredfirst to FAD in succinatedehydrogenase,then to
intermediate ca, l l e ds e m i q u i n o n e
iron-sulfur clusters-regenerating FAD-and finally to Coe,

496 . c H A p r E R1 2 | C E L L U L AERN E R G E T t c s
llll+ Animation:ElectronTransport

at
( a ) F r o mN A D H (b) Fromsuccinate

Intermembranespace
(exoplasmic) zn

4 H-
Exoplasmic
+++
oO

4H-
Cytosolic
Matrix
(cytosolic) 1120+
22H
zn H* H z O
O 2H*
NADH NAD++ H+
NADH-CoOreductase CoOH2-cytochrome c Gytochrome c oxidase Complex lll
{ c o m p l e xl ) reductase (complex lll) (complex lV)
Succinate Fumarate+2 H*

Succinate-CoOreductase
(comPlexll)

FIGURE 12-16 Multiproteincomplexesand mobileelectron matrixspaceduringoxidation of NADHby complex I are

carriersof the electrontransportchain.Electrons (bluearrows) consumed in the formation of water from 02 by complex lV,
flow throughfour majormultiprotein complexes (l-lV) Electron in no netprotontranslocation
resulting fromthesereactions
movement between complexes rsmediated eitherbythe lipid-soluble (b)Pathway fromsuccinate Electrons flow fromsuccinate (via
molecule coenzyme Q (CoQ,oxidized form;CoQHz, reduced form)or FADHz) in complex ll to complex lllandthenlV Electrons released
thewatersoluble proteincytochrome c (cytc),Themultipleprotein duringoxidation of succinate to fumarate in complex ll areused
complexes usethe energyreleased frompassing electronsto pump to reduceCoQ to CoQHz without translocating additional
protons fromthe matrixto the intramembrane space(redarrows) protons,Theremainder of electron transport fromCoQHz
(a)Pathway fromNADHFromNADHelectrons flowto complex l, proceeds bythe samepathwayasfor the NADHpathwayin (a).
thenlllandthenlV A totalof 10 protons aretranslocated perpairof Thusfor everypairof electrons transported fromsuccinate to 02,
electronsthatflowfromNADHto O, Theprotonsreleased intothe sixprotons are translocated by complexes lll and lV

which binds to a cleft on the matrix side of the transmem-
brane portions of complex II (Figure 72-16).
The overall reaction catalyzedby this complex is
Succinate+ CoQ --+fumarate + CoQH2
(Reduced) (Oxidized) (Oxidized) (Reduced)
Although the AGo' for this reactionis negative,the released
energyis insufficientfor proton pumping in addition to re-
duction of CoQ to form CoQH2, which then dissociates
from complex II. Thus no protons are translocateddirectly
acrossthe membraneby the succinate-CoQreductasecom-
plex, and no proton-motive force is generatedin this part of
the respiratorychain. Shortly we will seehow the protons
and electronsin the CoQH2 moleculesgeneratedby com-
plex I and complex II contribute to the generationof the
proton-motive force.
Complex II generatesCoQH2 from succinatevia FAD/
FADH2-mediatedredox reactions.Another setof proteinsin the
matrix and inner mitochondrialmembraneperformsa compa-
rable setof FAD/FADH2-mediated redox reactionsto generate
CoQH2 from fatty acyl CoA. Fatty acyl-CoA dehydrogenase,
which is a water-solubleenzyme,catalyzesthe first stepof the
oxidation of fatty acyl CoA in the mitochondrial matrix (see
Figure 12-12).There are severalfatty acyl-CoA dehydrogenase
enzymeswith specificitiesfor fatty acyl chains of different
lengths.Theseenzymesmediate the initial step in a four-step
processthat removestwo carbons from the fatty acyl group by
oxidizing the carbon in the B position of the fatty acyl chain
(thus the entire processis often referred to as B-oxidation).
Thesereactionsgenerateacetyl CoA, which in turn entersthe
citric acid cycle. They also generatean FADH2 intermediate
and NADH. The FADH2 generated remains bound to the
enzymeduring the redox reaction,as is the casefor complex II.
A water-soluble protein called electron transfer flauoprotein
(ETF) transfersthe high-energy electrons from the FADH2 in
the acyl-CoA dehydrogenaseto electron transfer flauopro-
tein:ubiquinoneoxidor eductase(ETF: QO), a membranepro-
tein that reducesCoQ to CoQH2 in the inner membrane.This
CoQH2 intermixes in the membrane with the other CoQH2
moleculesgeneratedby complexesI and II.
CoQH2-Cytochrome c Reductase(Complex lll) A CoQHz
generatedeither by complex I or complex II (or ETF:QO) do-
nates two electronsto CoQH2-cytochrome c reductase(com-
plex III), regeneratingoxidized CoQ. Concomitantlyit releases
into the intermembranespacerwo protonspreviouslypickedup
on the matrix face, generatingpart of the proton-motive force

NF T H E P R O T O N - M O T I VFEO R C E
T H E E L E C T R OTNR A N S P O RCTH A I NA N D G E N E R A T I OO 497
 (Figure 12-16). Vithin complex III, the releasedelectronsfirst
are transferredto an iron-sulfur cluster within complex III and
then to cltochrome c1 or to two &-type cytochromes (by and
bs, seeQ cycle below). Finally, the two electronsare transferred
sequentially to two molecules of the oxidized form of cy-
tochrome 6, a water-soluble peripheral protein that diffuses in
the intermembranespace.For eachpair of electronstransferred,
the overall reaction catalyzed by the CoQH2-cytochrome c
reductasecomplex is
CoQH2 + 2 Cytc3* *2 H+i. -+ Coe + 4 H+o,t+ 2 Cytc}+
(Reduced) (Oxidized) (Oxidized)
The AG'' for this reaction is sufficiently negative that two
protons in addition to those from CoQH2 are translocated
from the mitochondrial matrix across the inner membrane
for each pair of electronstransferred; this involves the pro-
ton-motive Q cycle, discussedlater. The heme protein cy-
tochrome c and the small lipid-soluble molecule Coe play
similar roles in the electron transport chain in that they both
serveas mobile electron shuttles,transferring electrons(and
thus energy)betweenthe complexesof the elecrronrransporr
chain.
three subunits.The function of the remaining subunits is less
well understood. Bacterial cytochrome c oxidases contain
only the three catalyric subunits. Four moleculesof reduced
cytochrome c bind, one at a time, to the oxidase.An electron
is transferred from the heme of each cytochrome c, first to
the pair of copper ions called Crr^2*,then to the heme in cy-
tochrome a, and next to the Cub2+ and the heme in cy-
tochrome a3 that together make up the oxygen reduction
center. The electrons are finally passedto 02, the ultimate
electron acceptor,yielding 4 H2O, which together with CO2
is one of the end products of the oxidation pathway. Pro-
(Reduced) posed intermediatesin oxygen reduction include the perox-
ide anion (Ort-) and probably the hydroxyl radical iOH.),
as well as unusual complexes of iron and oxygen atoms.
These intermediateswould be harmful to the cell if they es-
caped from the reaction center, but they do so only rarely
(seethe discussionof reactiveoxygen speciesbelow). During
transport of four electrons through the cytochrome c oxi-
dase complex, four protons from the matrix space are
translocatedacrossthe membrane.However, the mechanism
by which theseprotons are translocatedis not known.
For each four electronstransferred, the overall reaction
catalyzed by cytochrome c oxidase is

Cytochrome c Oxidase (Complex lV) Cytochrome c, after 4 C y t c 2 * * 8 H + 1 , + 0 2 - + 4 C y t c 3 ++ 2 H z O + 4H+o,t

being reduced by CoQH2-cytochrome c reductase(complex (Reduced) (Oxidized)
III), is reoxidized as it transports electrons,one at a time, to
cytochrome c oxidase (complex IV) (Figure 12-16). Mito- The poison cyanide, used as a chemical warfare agent,
chondrial cytochrome c oxidases contain 13 different sub- by spies to commit suicide when captured, in gas
units, but the catalytic core of the enzyme consists of only chambers to executeprisoners, and by the Nazis (Zyklon B

(a)
C o m p l e xl l l d i m e r C o m p l e xl V

Supercomplexl/lll2llV
,-
#- gupspsomplexl/lll,
- Complex I
**- ATP synthase
- C o m p l e xl l l d i m e r ( l l l 2 )

- Complex lV

- C o m p l e xl l

EXPERIMENTAL FIGURE l2-17 Electrophoresis and electron extractedfromthe gel,andthe particles werenegatively stained
microscopicimaging identifiesan electrontransportchain with 1% uranylacetate andvisualized by transmissionelectron
supercomplex containingcomplexesl, lll, and lV.(a)Membrane microscopy.lmages of 228 particles
werecombined at a resolution
proteinsin isolated
bovineheartmitochondria weresolubilized wlth a o'f-3 4 nm to generate an averaged imageof the complex viewed
detergent,andthe complexes andsupercomplexes wereseparated fromthesidein the planeof the membrane Approximate locations
by gelelectrophoresisusingthe bluenative(BN)-PAGE method.Each of thecomplex llldimerandcomplex lV areindicated
by dashed
blue-stained bandwithinthe gelrepresentsthe indicated protein ovals;the outlineof complex I isalsoindicatedby a dashed line
complex or supercomplex, with ll12
representing
a dimerof complex lll (white).Scalebaris 10 nm. fAdapted fromE Schaferet al, 2006,]. Biol.
Intensity
of the bluestainisapproximately
proportionalto theamount Chem 2A1Q2): 1537 O-1537 5 l
of complex or supercomplex present(b)Supercomplex l/lllrllV
was

498 C H A P T E R1 2 I CELLULAR
ENERGETICS
gas) for the mass murder of Jews and others, is toxic be-
causeit binds to the heme a3 in mitochondrial cytochrome
c oxidase (complex IV), inhibiting cellular respiration and
therefore production of ATP. Cyanide is one of many toxic
small moleculesthat interfere with energy production in
mitochondria. I
Electron Transport Supercomplexes Over 50 years ago
Britton Chance proposed that electron transport com-
plexes might assembleinto large supercomplexes.Doing
so would bring the complexesinto closeand highly organ-
ized proximity, which might improve the speed and effi-
ciency of the overall process.However, an alternativeview
holding that the complexesbehaved as independententi-
ties diffusing freely in the inner membrane became the
dominant paradigm. During the past several years,
genetic, biochemical, and biophysical studies have pro-
vided very strong evidencefor the existenceof electron
transport chain supercomplexes.These studies involved
relatively new gel electrophoreticmethods called blue na-
tive (BN)-PAGE and colorless native (CN)-PAGE, which
permit separation of very large macromolecular protein
complexes, and electron microscopic analysisof their three-
dimensionalstructures. One such supercomplex contains
one copy of complex I, a dimer of complex III (III2), and
one or more copies of complex IV (Figure 1,2-1,7).The
unique phospholipid cardiolipin (diphosphatidylglycerol)
Cardiolipin
o g6
+lta-o-[-o.-X-.o
do
)
HO<
) -37.8 kcal/mol for transfer of two electrons),the most pos-
o
I itive in the whole series;thus this reaction also tends to pro-
a-o-i-
appears to play an important role in the assembly and
function of these supercomplexes.Generally not observed
in other membranes of eukaryotic cells, cardiolipin has
been observed to bind to integral membrane proteins of
the inner membrane (e.g., complex II). Genetic and bio-
chemical studies in yeast mutants in which cardiolipin
synthesisis blocked have establishedthat cardiolipin con-
tributes to the formation and activity of mitochondrial su-
percomplexes,and thus it has been called the glue that
holds together the electron transport chain, though the
precise mechanism remains to be defined. In addition,
there is evidencethat cardiolipin may influence the inner
membrane'sbinding and permeability to protons and con-
sequentlythe proton-motive force.
T R A N S P O RC
THE ELECTRON
T HAINAND GENERATION
ReductionPotentialsof ElectronCarriersFavor
ElectronFlow from NADHto 02
As we saw in Chapter 2, the reduction potential E fot a par-
tial reduction reaction
= reduced molecule
Oxidized molecule * e- -
is a measureof the equilibrium constant of that partial reac-
tion. lfith the exception of the b cytochromes in the
CoQH2-cytochrome c reductasecomplex, the standard re-
duction potential Eo' of the electron carriers in the mito-
chondrial respiratory chain increasessteadily from NADH
to 02. For instance,for the partial reaction
'NADH
NAD* +H* +2C_ -
-320 mV,
the value of the standard reduction potential is
which is equivalentto a AGo' of + 14.8 kcal/mol for transfer
proceed
of two electrons. Thus this partial reaction tends to
toward the left, that is, toward the oxidation of NADH to
NAD+.
By contrast, the standard reduction potential for the partial
reactron
- cytochromecred(F.t*)
Cytochromeco*(Fe3*) + e -
is +220 mV (AG"' : -5.1 kcal/mol)for transferof one elec-
tron. Thus this partial reaction tends to proceed toward the
*
right, that is, toward the reduction of cytochrome c (Fe3 ) to
cytochrome c (Fe'-).
The final reaction in the respiratory chain, the reduction
of 02 to H2O
2H- + 'lror+ 2e- -+H2O
has a standard reduction potential of +816 mV (AG'' :
ceedtoward the right.
As illustrated in Figure 12-1'8,the steady increasein Eo'
values,and the correspondingdecreasein AGo' values' ofthe
carriers in the electron transport chain favors the flow of
electrons from NADH and FADH2 (generatedfrom succi-
nate) to oxygen.
ExperimentUs s i n gP u r i f i e dC o m p l e x e s
Established Stoichiometryof Proton
the
Pumping
The multiprotein complexesresponsiblefor proton pump-
ing coupled to electron transport have been identified by
selectively extracting mitochondrial membranes with de-
tergents, isolating each of the complexes in nearly pure
form, and then preparing artificial phospholipid vesicles
(liposomes)containing each complex. $7hen an appropri-
ati electrondonor and electronacceptorare added to such
O F T H E P R O T O N - M O T I VFEO R C E
 Redox potential F r e ee n e r g y
tmV) (kcal/mol)
60
NADH-CoO reductase
-400 - (complex l)

NADH NAD++ H+
\.v
2 e- F u m a r a t e+ 2 H +
5U
-200

Succinate-CoO
reductase(complexll)
40

H+
")
Fe-S
H* 30
CoQHr-cytochrome c
Cyt c., r e d u c t a s e( c o m p l e x l l l )

Cyt c
cuu
I 20
t-
*

10

(complex lV) 2 e-

800 tlz Oz + 2H* HzO

FIGURE 12-18Changesin redoxpotentialand free energy potentialto thosewith a higher(morepositive) (leftscale),
potential
duringstepwiseflow of electronsthroughthe respiratory with a correspondingreductionin freeenergy(rightscale)The
chain.Bluearrowsindicateelectron
flow;redarrows, translocation energyreleased aselectronsflowthroughthreeof thecomplexes is
of protons
across
the innermitochondrial
membraneElectrons pass to powerthe pumpingof H* ionsacross
sufficient the membrane,
throughthe multiprotein
complexes fromthoseat a lowerreduction establishinga proton-motiveforce

liposomes,a changein pH of the medium will occur if rhe
embedded complex transporrs protons (Figure 12-19).
Studies of this type indicate that NADH-Coe reductase
(complex I) translocatesfour protons per pair of electrons
transported, whereas cytochrome c oxidase (complex IV)
t r a n s l o c a t e st w o p r o t o n s p e r e l e c t r o n p a i r t r a n s p o r t e d
(or, equivalently,for every two moleculesof cytochrome c
oxidized).
Current evidencesuggeststhat a total of 10 protons are
transportedfrom the matrix spaceacrossthe inner mitochon-
drial membranefor everyelectronpair that is transferredfrom
NADH to 02 (seeFigure 12-1,6).Becausesuccinate-Coe
reductase(complexII) doesnot transport protons and com-
plex I is bypassedwhen the electronscome from succinate-
derived FADH2, only six prorons are rransported acrossthe
membrane for every electron pair that is transferred from
this FADH2 to 02.
The Q CycleIncreasesthe Numberof Protons
Translocatedas ElectronsFlow Through
C o m p l e xl l l
Experiments such as the one depicted in Figure 12-1,9have
shown that four protons are translocated across the mem-
brane per electron pair transported from CoQH2 through
CoQH2-cytochrome c reductase(complex III). Thus this
complex transports two protons per electron transferred,
whereas cytochrome c oxidase (complex IV) transports
only one proton per electrontransferred.An evolutionarily
conservedmechanism, called the Q cycle, accounts for the

s00 c H A P T E R1 2 | CELLULAE
RN E R G E T T C S
(a) into the intermembranespace,but one moleculeof CoQH2 is
regeneratedfrom CoQ at the Q1 site (seeFigure 12-20, bot-
tom).Thus the net result of the Q cycleis that four protons are
Phospholipid translocatedto the intermembranespacefor every two elec-
-../ membrane
trons transported through the CoQH2-cytochrome c reduc-
tasecomplex and acceptedby two moleculesof cytochromec.
t02+ 2 Ht The translocatedprotons are all derived from CoQH2, which
(reduced)
Hzo

2Ht

lntermembrane
space

CoOH2
K*
Valinomycin-bound l2e I

(b)

Matrix
E
a
q)

E
-o-
GoOH2-cytochromec
reductase(complex lll)

At Oo site: 2 CoOH2 + 2 Cyt C+ ---')

012 ( 4 H + , z l e) 2 CoO+ 2 Cyt C+ +2 e +4 H+{outside)
time(min)
Elapsed
(2e I
A EXPERIMENTAL FIGURE12-19 Electrontransfer from
At O; site: CoO + 2 e + 2 H+lmatrix -----+CoOH2
reduced cytochrome c to 02 via cytochrome c oxidase (complex ";6sy ( 2 H ' ' 2 e \
lV) is coupled to proton transport. The oxidasecomplexis
incorporated into liposomes with the bindingsitefor cytochrome c Net O cycle (sum of reactions at Oo and O;):
positioned on the outersurface(a)When 02 and reducedcytochrome CoOH2+ 2 Cyr C'+ 2 H+1661rix +
side)
c are added,electronsare transferred to 02 to form H2Oand protons 12H*,'l t: I CoO + 2 Cyt C+ + 4 H+loutside)
aretransportedfrom the insideto the mediumoutsideof the (2e )
v e s i c l e sA d r u g c a l l e dv a l i n o m y c iwna s a d d e dt o t h e m e d i u mt o
t h r o u g hc o m p l e xl l l t o c y t o c h r o m ec , 4 H "
P e r2 e t r a n s f e r r e d
dissipatethe voltagegradientgeneratedby the translocation of H*, released t o t h e i n t e r m e m b r a n sep a c e
which would otherwisereducethe numberof protonsmovedacross
the membrane(b) Monitoringof the medium'spH revealsa sharp A FIGURE12-20 The Q cycle.The Q cyclebeginswhen a molecule
drop in pH followingadditionof 02 As the reducedcytochromec from the combinedpool of reducedCoQH, in the membranebinds
becomesfully oxidized,protonsleakbackinto the vesicles, and the to the Qo site on the intermembranespace(outel side of the
transmembrane portionof complexlll (steptr) There,CoQHz
p H o f t h e m e d i u mr e t u r n st o i t s i n i t i avl a l u e M e a s u r e m e nst sh o w
releases two protonsinto the intermembrane space(stepEEI) and
that two protonsare transportedper O atom reducedTwo electrons
two electronsand the resultingCoQ dissociate (stepB) One of the
are neededto reduceone O atom, but cytochromec transfersonly
electrons is transported, via an iron-sulfur protein and cytochromec1,
one electron;thus two molecules of Cyt c'* are oxidizedfor eachO
reduced lAdaptedfrom B Reynafarleetal, 1986,J Biol Chem26128254] directlyto cytochromec (stepEE) (Recall that eachcytochromec
shuttlesone electronfrom complexlll to complexlV) The other
electronmovesthroughcytochromes b1 and bs and partiallyreduces
an oxidizedCoQ molecule bound to the second,Qi, siteon the
matrix(inner)sideof the complex,forming a CoQ semiqutnone
two-for-one transport of protons and electronsby complex
anion,Q ' (step4). The processis repeatedwith the bindingof a
I I I ( F i g u r e1 2 - 2 0 ) .
secondCoQH2at the Qo site(stepEt), proton release(stepEE),
The substratefor complex III, CoQH2, is generatedby reductionof anothercytochromec (stepEEI),and additionof the
severalenzymes,includingNADH-CoQ reductase(complex other electronto the Q-'bound at the Qi site(stepZ) There,the
I) and succinate-CoQreductase(complex II), electron trans- additionof two protonsfrom the matrixyieldsa fully reducedCoQHz
fer flauoprotein:ubiquinone oxidoreductase(ETF:QO, dur- moleculeat the Qrsite,which then dissociates (stepsE and 9),
ing B-oxidation), and, as we shall see,by complex III itself. freeingthe Q to bind a new moleculeof CoQ (stepI0) and begin
In one turn of the Q cycle,two moleculesof CoQH2 are ox- the Q cycleover again lAdapted from B Trumpower l99O,J Biol Chem
idizedto CoQ at the Q. siteand releasea total of four protons 265:11409, andE Darrouzet et al, 2001,Trends BiochemSci26:4451

NF T H E P R O T O N - M O T I VFEO R C E
T H E E L E C T R OTNR A N S P O RCTH A I NA N D G E N E R A T I OO 501
obtained its protons from the matrix, as a consequenceof
the reduction of CoQ catalyzedby either NADH-CoQ re,
ductase (complex I) or by CoQH2-cytochrome c reductase
(complex III) (seeFigure 12-16). Although seeminglycum-
bersome, the Q cycle optimizes the numbers of protons
pumped per pair of elecrronsmoving through complex III.
The Q cycle is found in all plants and animals as well as in
bacteria. Its formation at a very early stageof cellular evolu-
tion was likely essentialfor the successof all life-forms as a
way of convertingthe potential energyin reducedcoenzymeQ
into the maximum proton-motive force acrossa membrane.
How are the two electronsreleasedfrom CoQH 2 at the
Qo site directed to different acceptors, either to Fe-S, cy-
tochrome c1 and then cytochrome c (upward in Figure 12-
20) or to cytochrome bL, cytochrome bs, and then CoQ at
the Q1 site (downward in Figure 12-20)?The answer is sim-
ple and depends on a flexible hinge in the Fe-S-containing
protein subunit of complex III. Initially the Fe-Scluster is
close enough to the Qo site to pick up an electron from
CoQH2 bound there. Once this happens, a segment of the
protein containing this Fe-Scluster swings the cluster away
from the Qo site to a position near enough to the heme on
cytochrome c1 for electron transfer to occur. With the Fe-S
subunit in this alternate conformation, the second electron
releasedfrom CoQH2 bound to the Qo site cannot move to
the Fe-Scluster-it is too far away, so it takes an alternative
path open to it via a somewhat less thermodynamically
favored route to cytochrome 61.
The Proton-MotiveForcein Mitochondria
ls Due Largelyto a Voltage GradientAcross
t h e I n n e rM e m b r a n e
One result of the electron transport chain is the generation
of the proton-motive force (pmf), which is the sum of a
transmembraneproton concentration (pH) gradient and
electricpotential, or voltage, gradient. It has beenpossibleto
determine experimentally the relative contribution of the
two componentsto the total pmf. The relative contributions
depend on the permeability of the membrane to ions other
than H+. A significant voltage gradient can develop only if
the membrane is poorly permeable to other cations and to
anions. Otherwise, anions would leak across from the ma-
trix to the intermembrane spacealong with the protons and
prevent a voltage gradient from forming. Similarly cations
leaking acrossfrom the intermembrane spaceto the matrix
(exchangeof like charge) would also short-circuit voltage
gradient formation. Indeed, the inner mitochondrial mem-
brane is poorly permeablero other ions. Thus proron pump-
ing generatesa voltage gradient that makes it energetically
difficult for additional prorons ro move across becauseof
charge repulsion. As a consequence,proton pumping by the
electron transport chain establishesa robust voltage gradient
in the context of a rather small pH gradient.
Becausemitochondria are much too small to be impaled
with electrodes,the electricpotential and pH gradient across
the inner mitochondrial membrane cannot be determined by
direct measurement. Nevertheless,it has been possible to
502 CHAPTER
12 I CELLULAE
RN E R G E T I C S
develop methods to measureindirectly these critical values.
The electricpotential can be measuredby adding radioactive
42K* ions
and a trace amount of valinomycin to a suspen-
sion of respiring mitochondria. Although the inner mem-
brane is normally impermeableto K*, valinomycin is an
ionophore, a small lipid-soluble molecule that selectively
binds a specific ion (in this case, K*) and carries it across
otherwise impermeable membranes.In the presenceof vali-
nomycin, a2K* equilibrates across the innir membrane of
isolated mitochondria in accordancewith the electric poten-
tial: the more negativethe matrix side of the membrane, the
more 42K* will be attracted to and accumulatein the matrix.
At equilibrium, the measuredconcentration of radioac-
tive K+ ions in the matrix, [K6], is about 500 times greater
than that in the surrounding medium, [Ko"J. Substitution of
this value into the Nernst equation (Chapter 11) shows that
the electricpotential E (in mV) acrossthe inner membranein
respiring mitochondria is - 160 mV, with the matrix (inside)
negatlve:
fK'-l
E : - 5 9 l o e # i : - 5 e l o g 5 0 0: - 1 6 0 m V
L N o u rl
Researcherscan measurethe matrix (inside)pH by trap-
ping pH-sensitivefluorescent dyes inside vesiclesformed
from the inner mitochondrial membrane, with the matrix
side of the membrane facing inward. They also can measure
the pH outside of the vesicles(equivalent to the intermem-
brane space)and thus determine the pH gradient (ApH),
which turns out to be -1 pH unit. Since a differenceof one
pH unit representsa tenfold differencein H+ concentrarion,
according to the Nernst equation a pH gradient of one unit
across a membrane is equivalent to an electric potential of
59 mV at 20 'C. Thus, knowing the voltage and pH gradi-
ents, we can calculatethe proton-motive force, pmf, as
^ - (l R T \
-p m f : v q/ - 59 ApH
= ' /H I :
x Ap
\t
where R is the gasconstantof 1.987 call(degree.mol),7 is the
temperature (in degreesKelvin), F is the Faraday constant
123,062 call(V.mol)1, and V is the transmembraneelectric
potential; V and pmf are measuredin millivolts. The electric
potential V acrossthe inner membraneis - 160 mV (negative
inside matrix) and ApH is equivalent to :60 mV. Thus the
total pmf is -220 mV, with the transmembraneelectric po-
tential responsiblefor about 73 percentof the total.
ToxicBy-productsof ElectronTransport
C a n D a m a g eC e l l s
About 1-2 percent of the oxygen metabolized by
aerobic organisms, rather than being converted to
water, is partially reducedto the superoxideanion radical
(02 ). Superoxideis unstable in aqueous biological liquids,
breaking down into especially toxic hydrogen peroxide
(HzOz) and then hydroxyl radicals.Theseand other reactiue
oxygen species(ROS), which contribute to what is often
called cellular oxidatiue stress, can be highly toxic, because
they chemically modify proteins, DNA, and unsaturated
fatty acyl groups in membrane lipids, thus interfering with
normal function. Indeed, ROS are purposefully generated
by body defensecells (e.g.,macrophages)to kill pathogens.
In humans, excessiveor inappropriate generationof ROS
has been implicated in many diverse diseases,including
heart failure, neurodegenerativediseases,alcohol-induced
liver disease,diabetes,and aging.
Although ROS can be generatedby a number of meta-
bolic pathways, the major source of ROS appearsto be the
electron transport chain, in particular mechanismscoupled
to complexes I and III. The semiquinone form of
ubiquinone, CoQ-. (seeFigure 72-15), an intermediateform
of CoQ generatedin the Q cycle, may play a particularly im-
portant role in superoxidegeneration.
To help protect against ROS toxicity, mitochondria
have evolved several defensemechanisms,including the
use of enzymes that inactivate superoxide first by con-
verting it to H2O2 (Mn-containing superoxide dismutase)
a n d t h e n t o H 2 O ( g l u t a t h i o n e p e r o x i d a s e ,w h i c h a l s o
detoxifies the lipid hydroperoxide products formed when
ROS react with unsaturated fatty acyl groups). Cardiac
mitochondria also have catalase(normally only found in
p e r o x i s o m e s )t o h e l p b r e a k d o w n H z O z . T h i s i s n o t s u r -
p r i s i n g , b e c a u s et h e m o s t o x y g e n - c o n s u m i n go r g a n i n
mammals is the heart. In addition, the small molecule an-
tioxidants c-lipoic acid and vitamin E help protect the
mitochondrion from ROS. f
ElectronTransport and Generation of the
Proton-Motive Force
r By the end of the citric acid cycle (stageII), much of the
energy originally present in the covalent bonds of glucose
and fatty acids is converted into high-energy electrons in
the reduced coenzymesNADH and FADH2. The energy
from theseelectronsis used to generatethe proton-motive
force.
r In the mitochondrion, the proton-motive force is gener-
ated by coupling electron flow (from NADH and FADH2
to 02 ) to the uphill transport of protons from the matrix
across the inner membrane to the intermembrane space.
This processtogether with the synthesisof ATP from ADP
and P1driven by the proton-motive force is called oxidative
phosphorylation.
r The flow of electronsfrom FADH2 and NADH to 02 is di-
rectedthrough multiprotein complexes.The four major com-
plexesareNADH-CoQ reductase(complexI), succinate-CoQ
reductase(complex II), CoQH2-cytochrome c reductase
(complex III), and cytochrome c oxidase (complex IV).
r Each complex contains one or more electron-carrying
prostheticgroups: iron-sulfur clusters,flavins, hemegroups,
and copper ions (seeTable 12-2). Cytochrome c, which con-
tains heme, and coenzymeQ (CoQ), a lipid-soluble small
GH E p R O T O N - M O T I V F
H A R N E S S I NT
molecule,are mobile carriersthat shuttle electronsbetween
the complexes.
r ComplexesI, III, and IV pump protons from the matrix
into the intermembrane space.Complexes I and II reduce
CoQ to CoQH2, which carries protons and high-energy
electrons to complex III. The heme protein cytochrome c
carries electrons from complex III to complex IV, which
usesthem to pump protons and reduce molecular oxygen
to water.
r The high-energyelectronsfrom NADH enter the electron
transport chain through complex I, whereasthe high-energy
electronsfrom FADH2 (derivedfrom succinatein the citric
acid cycle) enter the electron transport chain through com-
plex II. Additional electrons derived from FADH2 by the
initial step of fatty acyl-CoA B-oxidation increasethe sup-
ply of CoQH 2 avallable for electron transport.
r \(ithin the inner membrane, electron transport com-
plexes assembleinto supercomplexesheld together by
cardiolipin, a specialized phospholipid. Supercomplex
formation may enhancethe speedand efficiency of gener-
ation of the proton-motive force.
r Each electron carrier acceptsan electron or electron pair
from a carrier with a lesspositive reduction potential and
transfers the electron to a carrier with a more positive re-
duction potential. Thus the reduction potentials of electron
carriers favor unidirectional electron flow from NADH
and FADH2 to 02 (seeFigure 12-1'8).
r The Q cycle allows four protons (rather than two) to be
translocatedper pair of electronsmoving through complex
III (seeFigure 12-20).
r A total of 10 H+ ions are translocated from the matrix
acrossthe inner membrane per electron pair flowing from
NADH to 02 (seeFigure 12-1,6),whereas 6 H* ions are
translocatedper electron pair flowing from FADH2 to 02.
r The proton-motive force is due largely to a voltage gra-
dient across the inner membrane produced by proton
pumping; the pH gradient plays a quantitatively less im-
portant role.
r Reactive oxygen species(ROS) are toxic by-products of
the electron transport chain that can modify and damage
proteins, DNA, and lipids. Specificenzymes(e.g.,glutathi-
none peroxidase, catalase)and small molecule antioxi-
dants (e.g.,vitamin E) help protect againstROS-induced
damaqe.
Harnessingthe Proton-Motive
Processes
Forcefor Energy-Requiring
The hypothesisthat a proton-motive force across the inner
mitochondrial membrane is the immediate source of energy
for ATP synthesiswas proposedin 1'961by PeterMitchell.
Virtually all researchersstudying oxidative phosphoryla-
tion and photosynthesisinitially reiected his chemiosmotic
hypothesis. They favored a mechanism similar to the then
' 503
EO R C EF O R E N E R G Y - R E Q U I R I N
PGROCESSES
well-elucidated substrate-levelphosphorylation in glycoly- Bacterium
sis, in which chemical transformation of a substrate mole-
cule (i.e.,phosphoenolpyruvate) is directly coupled to ATP
synthesis.Despite intenseefforts by a large number of inves- ,1

tigators, however, compelling evidencefor such a direct

mechanismwas neverobserved.
Definitive evidencesupporting Mitchell's hypothesis de-
Plasma
pended on development of techniquesto purify and recon- memorane
stitute organellemembranesand membrane proteins. The
experiment with vesiclesmade from chloroplast thylakoid
membranes(describedin detail below) that contain ATP
synthase, outlined in Figure 12-21, was one of several Mitochondrion
demonstratingthat this protein is an ATP-generatingenzyme Outer Intermembranesoace
and that ATP generation is dependenton proton movement membrane
down an electrochemical gradient.It turns out that the pro- Matrix
,0
tons actually move tbrougD the ATP synthaseas they rra-
Fl
versethe membrane!
,.1 noe + e,

troFr --1 )
lLIr '
:j pH 7.5

attt' '
ai:r::llr'rr"" Chloroplast
a"
T h y l a k o i dm e m b r a n e Outer L i gh t Intermembrane
S o a kf o r s e v e r a m
l inutes membrane space
at pH 4.0
Stroma

HI /---'> Af P
Fl ,t A
uI
illi
,riil, pH 4.0 :;::rpHa.o

Inner
I memDrane
I A d d a s o l u t i o no f p H 8 . 0 T h v l a k o i dm e m b r a n e
II t h a t c o n t a i n sA D P a n d P 1
v A FIGURE 12-22Chemiosmosis in bacteria,mitochondria, and
ADP + P; ADP + P, chloroplasts.Themembrane surface facinga shaded areaisa
ATP cytosolic
face;thesurface facingan unshaded, whiteareaisan
exoplasmicface,Notethatthecytosolic faceof the bacterial plasma
H*
li:i:li:la:l:li:illlia::iiilrrl: membrane, the matrixfaceof the innermitochondrial membrane,
andthestromal faceof thethylakoid membrane areallequivalent
,,,:"' H+ Duringelectron protons
transport, arealwayspumpedfromthe
rlr pH 4.0 .l.,1
pH 8.0 cytosolic
faceto theexoplasmic face,creating a protonconcentration
gradient(exoplasmic face> cytosolic face)andan electric potential
(negativecytosolicfaceandpositive exoplasmic face)across the
EXPERIMENTAL membraneDuringthe synthesis of ATP, protons flow in the reverse
FTGURE 12-21Synthesis of ATpby FeFl (downtheirelectrochemical
direction gradient)throughATPsynthase
dependson a pH gradientacrossthe membrane.lsolated
(FoFr
complex), whichprotrudes in a knobat the cytosolic facein all
chloroplast thylakoid vesicles containing FoF,particles wereequllibrated
CASCS
in the darkwith a buffered solution at pH4.0 Whenthe pH in the
thylakoid lumenbecame 4 0, thevesicles wererapidlymixedwith a
solution at pH8 0 containing ADPandP, A burstof ATpsynthesis As we shall see,the ATP synthaseis a multiprotein com-
accompanied thetransmembrane movement of protons drivenbythe plex that can be subdivided into two subcomplexescalled
i orna d i e n( ltO 4 M v e r s u1sO - 8M ) I n
1 0 , 0 0 0 - f oHl d* c o n c e n t r a t g Fe (containing the transmembraneportions of the com-
similar experiments using"inside-out" preparations of mitochondrial plex) and F1 (containingthe globular portions of the com-
membrane vesicles, an artificially generated membrane electric plex that sit above the membrane and point toward the
potential alsoresulted in ATPsynthesis matrix spacein mitochondria). Thus the ATP synthaseis

504 c H A P T E R1 2 | CELLULAE
RN E R G E T T C S
often also called the FeFl complex; we will use the terms
interchangeably.
T h e M e c h a n i s mo f A T PS y n t h e s i sl s S h a r e d
A m o n g B a c t e r i aM
, itochondria,
and Chloroplasts
Although bacteria lack any internal membranes,aerobic
bacteria nonethelesscarry out oxidative phosphorylation by
the same processesthat occur in eukaryotic mitochondria.
Enzymes that catalyze the reactions of both the glycolytic
pathway and the citric acid cycle are presentin the cytosol of
bacteria; enzymesthat oxidize NADH to NAD* and trans-
fer the electrons to the ultimate acceptor 02 reside in the
bacterialplasmamembrane.
The movement of electronsthrough thesemembranecar-
riers is coupled to the pumping of protons out of the cell. The
movement of protons back into the cell, down their concen-
tration gradient, is coupled to the synthesisof ATP. This gen-
eral processis similar for bacteria and eukaryotes (in both
mitochondriaand chloroplasts)(Figuret2-22). The bacterial
ATP synthases(F6F1complex) are essentiallyidentical in
structure and function to the mitochondrial and chloroplast
ATP synthasesbut are simpler to purify and study.
A primitive aerobic bacterium was probably the progeni-
tor of mitochondria in eukaryotic cells (Figure 12-23). hc-
cording to this endosymbionthypothesis,the inner mitochon-
drial membrane would be derived from the bacterial plasma
membrane with its cytosolic face pointing toward what be-
came the matrix space of the mitochondrion. Similarly in
plants the progenitor'splasma membranebecamethe chloro-
plast'sthylakoid membrane and its cytosolic face pointed to-
ward what becamethe stromal spaceof the chloroplast (de-
scribedin detail below).
In all cases,ATP synthaseis positioned with the globular
F1 domain, which catalyzesATP synthesis,on the cytosolic
face of the membrane, so ATP is always formed on the
cytosolic face of the membrane (seeFigure 1'2-22).Protons
always flow through ATP synthasefrom the exoplasmic to
the cytosolic faceof the membrane, which in the mitochon-
drion is from the intermembrane to the matrix space.This
flow is driven by the proton motive force. InvariablS the
cytosolic face has a negativeelectric potential relative to the
exoplasmic face.
In addition to ATP synthesis,the proton-motive force
acrossthe bacterial plasma membraneis usedto power other
processes,including the uptake of nutrients such as sugars
(using proton/sugar symporters) and the rotation of bacter-
ial flagella. Chemiosmotic coupling thus illustrates an im-
portant principle introduced in our discussionof active
transport in Chapter 1.1.:the membrane potential, the con-
centrdtion gradients of protons (and other ions) across a
membrane, and the phosphoanhydride bonds in ATP are
equiualent and interconuertibleforms of chemical potential
energy.Indeed, AIP synthesisthrough ATP synthasecan be
thought of as active transport ln reverse.
ATPSynthaseComprisesTwo Multiprotein
ComplexesTermedFsand F1
With generalacceptanceof Mitchell's chemiosmoticmechanism,
researchersturned their attention to the structure and operation
of the F6F1complex. The FsFl complex, or AIP synthase,has
two principal components, Fe and F1, both of which are

Eukarvotic
o l a s m am e m b r a n e
Endocytosisof bacterium I
caoableof oxidative Endocytosisof bacterium
phosphorylation capableof photosynthesis

Bacterial
p l a s m am e m b r a n e

B a c t e r i apl l a s m a
m e m b r a n eb e c o m e s
B a c t e r i apl l a s m a
m e m b r a n eb e c o m e s a(-- .{
i n n e rm e m b r a n e
of mitochondrion
i n n e rm e m b r a n e
of chloroplast ,-#r..\
l n n e r m e m b r a n eb u d s
off thylakoid vesicles
M i t o c h o n d r i am
l atrix Thylakoid
memorane
a FIGURE 12-23 Endosymbiont hypothesis for the evolutionary mitochondrion
facethe matrixof the evolving (/eft)or chloroplast
origin of mitochondriaand chloroplasts. Endocytosis of a (ngrht)Budding fromthe innerchloroplast
of vesicles membrane,
bacterium by an ancestral cell(stepII) wouldgenerate
eukaryotic suchasoccurs duringdevelopment of in
chloroplasts contemporary
an organelle with two membranes,theoutermembrane derived wouldgenerate
plants, thethylakoidmembranes with the F1subunit
fromtheeukaryotic plasmamembrane andthe inneronefrom remaining face,facingthe chloroplast
on the cytosolic stroma(step
the bactenal membrane (stepE) TheF1subunitof ATPsynthase, B) Membrane surfacesfacinga shaded areaarecytosolic faces;
localizedto thecytosolic membrane,
faceof the bacterial wouldthen surfacesfacinqan unshaded areaareexoplasmic faces

H A R N E s s I N G T H E P R o T o N - M o T | V E F o R c E F o R E N E R G Y - R E Q U I R | N G P R o c E s 505
sES
 Animation:ProtonTranslocating,
RotaryF-ATPase{lttt
l<-- 100nm ------------>l < FIGURE 12-24 Structureand function of ATPsynthase(the
FeFlcomplex)in the bacterialplasmamembrane.TheFe
membrane-embedded portionof ATPsynthase isbuiltof three
integral membrane proteins: onecopyof a, two copres of b, andon
'1
average0 copies of c arranged in a ringin the planeof the
ATP membrane Twoprotonhalf-channels lieat the interface between
thea subunitandthec ring Half-channel I allowsprotons to move
oneat a timefromthe exoplasmic mediumandbindto aspartate-61
in thecenterof a c subunitnearthe middleof the membraneHalf-
ADP+ P; channel ll (afterrotation of thec ring)permits protons to dissociate
fromtheaspartate andmoveintothecytosolic mediumTheF,
portioncontains threecopies eachof subunits ctandB thatforma
Cytosolic fi static hexamer resting atopthesinglerod-shaped
medium 1 subunit, whichis
Rotates inserted intothec ringof F6.Thee subunitis rigidlyattached to the1
subunitandalsoto several of thec subunitsThe6 subunit
permanently linksoneof theo subunits in the F1complex to the b
s u b u no
i tf F 6T h u st h eF ea a n db s u b u n i tasn dt h eF 16 s u b u n a
i tn d
(oB)3hexamer forma rigidstructure anchored in the membrane
(orange)Duringprotonflow,the c ringandthe attached F1e and1
subunits rotateasa unit(green), causing conformation changes in
Exoplasmic the F1B subunits, leading to ATPsynthesis [Adapred fromM J
medium Rotation of c ring Schnitzer, 2001,Nature 410:878, andP D Boyel1999,Nature 402:247 I
Proton half-channel Proton bound
to aspartate

multimericproteins(Figure12-24).The Fecomponenrcontains
three rypesof integralmembraneproteins,designateda, b, and
c. In bacteriaand in yeastmitochondriathe most common sub-
unit compositionis a1b2c1e, but Fe complexesin animal mito-
chondria have 1,2c subunitsand thosein chloroplastshave 14.
In all casesthe c subunits form a doughnut-shapedring in the
plane of the membrane.The a and two b subunitsare rigidly
linked to one another but not to the ring of c subunits,a critical
feature of the protein to which we will return shortly.
The F1 portion is a water-solublecomplex of five distinct
polypeptideswith the composition ct3B3^yEe that is normally
firmly bound to the F0 subcomplexat the surfaceof the mem-
brane. The lower end of the rodlike ^ysubunit of the F1 sub-
complex is a coiled coil that fits into the centerof the c-subunit
ring of Fe and appearsrigidly attached to it. Thus when the
c-subunit ring rotates, the rodlike "y subunit moves with it.
The F1 e subunit is rigidly attachedto ^yand also forms tight
contactswith severalof the c subunitsof F6.The o.and p sub-
units are responsiblefor the overall globular shapeof the F1
subcomplexand associatein alternatingorder to form a hexa-
mer,ctBctBctB, or (ctB)3,which restsatop the singlelong n7sub-
unit. The F1 6 subunit is permanentlylinked to one of the F1 cr
subunits and also binds to the b subunit of F6. Thus the F6 a
and b subunitsand the E subunit and (oB)3 hexamerof the F1
complex form a rigid structure anchored in the membrane.
The rodlike b subunitsform a "srator" that preventsthe (ctB)3
hexamer from moving while it resrson the 1 subunit, whose
rotation together with the c subunits of F6 plays an essential
role in the ATP synthesismechanismdescribedbelow.
'Sfhen
ATP synthaseis embeddedin a membrane, the F1
component forms a knob that protrudes from the cytosolic
(in the mitochondrion this is the matrix) face. BecauseF1
separatedfrom membranesis capable of catalyzingATP hy-
drolysis (ATP conversion to ADP plus Pi) in the absenceof
the Fe component, it has beencalled the F1 ATPase;however,
its function in the cells is the reverse,ro synthesizeATP. ATP
hydrolysisis a spontaneousprocess(AG < 0); thus energyis
required to drive the AIPase in "reverse" and generateATP.
Rotationof the F1^ySubunit,Driven by Proton
MovementThrough Fs,PowersATPSynthesis
Eachof thethreeB subunits
in theglobularF1portionof the
complete FsFl complex can bind ADP and P; and catalyze
the endergonicsynthesisof ATP when coupled to the flow of
protons from the exoplasmic (intermembrane space in the
mitochondrion) medium to the cytosolic (matrix) medium.
However, the coupling between proron flow and AIP syn-
thesismust be indirect, sincethe nucleotide-bindingsites on
the B subunitsof F1,where ATP synthesisoccurs,are 9-10 nm
from the surface of the mitochondrial membrane. The
most widely acceptedmodel for ATP synthesisby the F6F1
complex-th e binding-change mechanism-posits just such
an indirect coupling (Figure 12-25).
According to this mechanism,energy releasedby the
"downhill" movement of protons through Fe directly pow-
ers rotation of the c-subunit ring together with its attached
1 and e subunits (seeFigure 12-24). The 1 subunit acs as a
cam, or nonsymmetrical rotating shaft, whose rotation
within center of the static (crB)3hexamer of F1 causesit to
push sequentiallyagainsteach of the B subunits and thus
causecyclical changesin their conformations between three

s06 CHAPTER
12 I CELLULAE
RN E R G E T I C S
 sts

ADP
P

Reaction
Rotation (no rotation)
--+
E a

Rotation
--
E .v

I
A?P
Pi

FIGURE 12-25The binding-change mechanism of ATP bound(fromL -+ T ), anda decrease in thebinding of

affinity
synthesisfrom ADPand P;.Thisviewislookingup at F1from the B2subunitfor a previouslyboundATP(fromT --+O),causing
the membrane surface(seeFigure12-24)Asthe1 subunitrotates by releaseof the boundATPStepZ: Withoutadditional rotationthe
120'inthecenter, eachof theotherwise identical F1B subunits ADPandPlin theT site(herethe B3subunit) formATBa reaction
alternates between threeconformational states (O,openwith oval thatdoesnotrequire an inputof additional energy dueto the
representation of the bindingsite;L,loosewith a rectangular binding environment
special in the activesiteof theT stateAt thesame
site;T,tightwith a triangularsite)thatdifferin theirbindingaffinities timea newADPandP1bindloosely to the unoccupied O siteon B2
for ATP,ADP,andP,.Thecyclebegins(upperleft)whenADP StepE: Protonf luxpowersanother120' rotationof the1 subunit,
andP,bindloosely to oneof thethreeB subunits (here,arbitrarily consequent conformational changes in the bindingsites(L-+T,O->
designated Br)whosenucleotide-binding siteisin the O (open) L,T+ O),andrelease of ATPfromP3 Step4: Withoutadditional
conformation Protonfluxthroughthe Foportionof the protein rotationtheADPandP,in theT siteof B1formATBandadditional
powersa 120"rotationof the"ysubunit(relative to thefixedB ADPandP,bindto the unoccupied O siteon B3 Theprocess
subunits) (steptr) Thiscauses the rotating"ysubunit, whichis continues with rotation(stepE) andATPformation(step6) until
asymmetric, to pushdifferentially
againstthe B subunits, resulting the cycleiscomplete, with threeATPshavingbeenproduced for
in a conformational changeandan increase in the bindingaffinity every360"rotationof ry lAdapted fromP Boyer, 1989,FASEB].3:2164;
of the Br subunitfor ADPandP1(fromO -+ L),an increase in the Y Zhouetal, 1997, AcadSciUSA94:10583;
ProcNat'l. andM Yoshida,
bindingaffinityof the B3subunitfor ADPandP1thatwerepreviously E Muneyuki, 2001,Nat Rev.
andT,Hisabori, Mol CellBiol2:669-677l

different states. As schematically depicted in a view of the
bottom of the (ctB)3hexamer's globular structure in Figure
1.2-25,rotation of the 1 subunit relative to the fixed (aB)3
hexamer causesthe nucleotide-bindingsite of each B sub-
unit to cycle through three conformational statesin the fol-
lowing order:
L An O (open) state that binds ATP very poorly and ADP
and P; weakly
2. An L (loose)state that binds ADP and Pi more strongly
but cannot bind ATP
3. A T (tight) state that binds ADP and Pl so tightly that
they spontaneouslyreact and form ATP
In the T state the ATP produced is bound so tightly that
it cannot readily dissociatefrom the site-it is trapped until
another rotation of the ^ysubunit returns that B subunit to the
O state,thereby releasingATP and beginningthe cycle again.
ATP or ADP also binds to regulatory or allostericsiteson the
three ct subunits;this binding modifies the rate of ATP synthe-
sis accordingto the level of AIP and ADP in the matrix, but is
not directly involved in synthesisof ATP from ADP and P;.
Severaltypes of evidencesupport the binding-change
mechanism. First, biochemical studies showed that one of
the three B subunits on isolated F1 particles can tightly bind
ADP and P; and then form ATP, which remains tightly
bound. The measuredAG for this reaction is near zero, indi-
cating that once ADP and Pi are bound to the T state of a B
subunit, they spontaneouslyform AIP. Importantly dissoci-
ation of the bound ATP from the B subunit on isolated F1
particles occurs extremely slowly. This finding suggested
that dissociationof ATP would have to be powered by a con-
formational changein the B subunit, which in turn would be
causedbv proton movement.

PG
H A R N E S S I NTGH E P R O T O N - M O T I VFEO R C EF O R E N E R G Y . R E Q U I R I N ROCESSES 507
 Video: Rotation of Actin Filament Bound to ATP 5
X-ray crystallographicanalysis of the (oB)3 hexamer
yielded a striking conclusion: although the three B subunits
are identical in sequence and overall structure, the
ADP/ATP-binding siteshave different conformations in each
s u b u n i t . T h e m o s t r e a s o n a b l ec o n c l u s i o n w a s t h a t t h e
three B subunits cycle in an energy-dependentreaction be-
tween three conformational states(O, L, T), in which the
nucleotide-bindingsite has substantially different srrucrures.
In other studies,intact F6F1complexeswere treated with
chemical cross-linking agents that covalently linked the "y
and e subunits and the c-subunit ring. The observation that
such treated complexescould synthesizeATP or use ATP to
power proton pumping indicates that the cross-linked pro-
teins normally rotate together.
Finally, rotation of the ^y subunit relative to the fixed
(aB)3 hexamer, as proposed in the binding-changemecha-
nism, was observeddirectly in the cleverexperimentdepicted
in Figure 12-26.In one modification of this experimentin
which tiny gold particles,rather than an actin filament, were
attachedto the 1 subunit, rotation ratesof 134 revolutions
per secondwere observed.Hydrolysis of 3 ATPs, which you
recall is the reversereaction catalyzedby the sameenzyme,is
thought to power one revolution; this result is close to the
experimentally determined rate of ATP hydrolysis by F6F1
complexes:about 400 ATPs per second.In a related experi-
ment, a ^ysubunit linked to an e subunit and a ring of c sub-
units was seento rotate relative to the fixed (cp)j hexamer.
Rotation of the 1 subunit in these experimentswas pow-
ered by ATP hydrolysis,the reverseof the normal process
in which proton movement through the Fs complex drives
rotation of the ry subunit. These observationsestablished
that the 1 subunit, along with the attachedc ring and e sub-
unit, doesindeedrotate,therebydriving the conformational
changesin the B subunits thar are required for binding of
ADP and Pi, followed by synthesisand subsequentrelease
of ATP.
508 c H A P T E R1 2 I CELLULAR
ENERGETTCS
< EXPERf MENTALFIGURE 12-26 lhe rysubunit of the F1
complexrotatesrelativeto the (crg)ghexamer.F' complexes
wereengineered thatcontained with an additional
B subunits His6
sequence, whichcauses themto adhereto a glassplatecoated
with a metalreagent thatbindspolyhistidineThe1 subunitin the
engineered F1complexes waslinkedcovalently to a fluorescently
labeledactinfilament. Whenviewedin a fluorescence microscope,
the actinfilaments wereseento rotatecounterclockwise in discrete
120"stepsin the presence of ATP,poweredby ATPhydrolysis by
the B subunitsfAdapted fromH Noliet al, 199-/
, Nature 386:299,
and
R Yasuda etal. 1998.Cell93 1117I
Number of Translocated Protons Required for ATP
Synthesis A simple calculationindicatesthat the passageof
more than one proton is required to synthesizeone molecule
of ATP from ADP and P1.Although the AG for this reaction
under standardconditions is * 7.3 kcaVmol,at the concentra-
tions of reactantsin the mitochondrion, AG is probably
higher (+10 to *12 kcaVmol).We can calculatethe amount
of free energy releasedby the passageof 1 mol of protons
down an electrochemicalgradient of 220 mY (0.22 V) from
the Nernst equation, setting n : 1 and measuring AE in volts:
A G ( c a l l m o l ): - n F L , E : - ( 2 3 , O 6 2 c a l . V - 1 . m o l 1)AE
: ( 2 3 , 0 5 2 c a 1 . V -r . m o l - 1 ; 1 o . z zV ;
: -5074 callmol, or -5.1 kcal/mol
Sincethe downhill movement of 1 mol of protons releasesjust
over 5 kcal of free energy,the passageof at least rwo protons is
required for synthesisof eachmoleculeof AT? from ADP and P1.
Proton Movement Through F6 and Rotation of the c
Ring Eachcopy of subunitc containstwo membrane-spanning
cr helices that form a hairpin-like structure. An aspartate
residue,Asp61, in the centerof one of thesehelicesis thought to
participate in proton movement. Chemical modification of this
aspartateby the poison dicyclohexylcarbodiimide or its muta-
tion to alaninespecificallyblocks proton movement through F6.
According to one current model, two proton half-channels,I
and II, lie at the interface betweenthe a subunit and c ring (see
Figure 12-24). Protons are thought to move one at a time
through half-channel I from the exoplasmic medium and bind
to the carboxylatesidechain on Asp61 of one c subunit.Bind-
ing of a proton to this aspartatewould result in a conforma-
tional changein the c subunit, causingit to move relative to the
fixed a subunit or equivalentlyto rotate in the membraneplane.
This rotation would bring the adjacent c subunit, with its ion-
ized asparrylsidechain, into channelI, thereby allowing it to re-
ceive the next proton and subsequentlymove relative to the a
subunit.Continuedrotation of the c ring, due to bindingof pro-
tons to additional c subunits,eventuallywould align the first
c subunit containinga protonatedAsp61 with the secondhalf-
channel (II), which is connectedto the cytosol. A positively
chargedside chain of Arg210 in the a subunit has been pro-
posed to interact with the negativelychargedAsp61 and facili-
tate movementof the c subunitsand proton translocation.Once
this occurs,the proton on the aspartylresiduecould dissociate l n n e rm i t o c h o n d r i a l
*
(forming ionized aspartate) and move into the cytosolic H concentration
gradient
medium.
Sincethe 1 subunit of F1 is tighdy attachedto the c ring of
Membrane +
Fe, rotation of the c ring associatedwith proton movemenr Matrix
electric T
causesrotation of the 1 subunit. According to the binding- potential T

changemechanism,a 720" rotation of 1 powers synthesisof

one ATP (seeFigure 12-25). Thus complete rotation of the c
ring by 360" would generatethree ATPs. In E. col1,where Translocationof H-
" d u r i n ge l e c t r o nt r a n s p o r t
the Fo composition is a1b2c1s,movement of 10 protons
drives one complete rotation and thus synthesisof three
ATPs. This value is consistentwith experimentaldata on
.t
' - | Phosphatetransporter
proton flux during ATP synthesis,providing indirect support ^')
for the model coupling proton movementto c-rlng rotatron
depictedin Figure 12-24. The F6 from chloroplastscontains
14 c subunitsper ring, and movementof 14 protons would noet aPPt ATp/ADp
antiportel
fr'
be neededfor synthesisof three ATPs. Why theseotherwise \ / ATP4
ATP4-(
similar FsFl complexes have evolved to have different
H*:ATP ratios is not clear. ADP3-+ HPOo2

ATP-ADPExchangeAcrossthe Inner
M i t o c h o n d r i aM
l e m b r a n el s P o w e r e d
by the Proton-MotiveForce
In addition to powering ATP synthesis,the proton-motive
force acrossthe inner mitochondrial membranepowers the
exchangeof ATP formed by oxidative phosphorylation in-
side the mitochondrion for ADP and P1in the cytosol.This
exchange,which is requiredto supply ADP and Pi substrare
for oxidative phosphorylationto continue, is mediated by
two proteins ^in the inner membrane: a phosphate trans-
porter (HPO4' /OH- antiporter) that mediatesthe import
of one HPO42 coupled to the export of one OH- and an
ATP/AD P antiporter (Figure12-27\.
The ATP/ADP antiporter allows one moleculeof ADP to
enter only if one molecule of ATP exits simultaneously.The
MP/ADP antiporter, a dimer of two 30,000-Da subunits,
makesup 10-15 percentof the protein in the inner membrane,
so it is one of the more abundant mitochondrial proteins.
Functioningof the two antiporterstogetherproducesan influx
of one ADP3- and one P,2- and efflux of one ATPa- together
with one OH . EachOH transportedoutr,vardcombineswith
a proton, translocatedduring electrontransport to the inter-
membranespace,to form H2O. This drivesthe overallreaction
in the direction of AIP export and ADP and P1import.
Becausesome of the protons translocatedout of the mi-
tochondrion during electron transport provide the power
(by combining with the exported OH-) for the MP-ADP
exchange,fewer protons are availablefor ATP synthesis.It is
estimatedthat for every four protons translocatedout, three
are used to synthesizeone ATP molecule and one is used to
power the export of ATP from the mitochondrion in ex-
changefor ADP and P1.This expenditureof energyfrom the
proton concentration gradient to export ATP from the mito-
chondrion in exchangefor ADP and Pi ensuresa high ratio
of ATP to ADP in the cytosol, where hydrolysis of the high-
energy phosphoanhydride bond of ATP is utilized to power
many energy-requlrlng reactlons.
lntermembrane
space
ATP4 + OH
FfGURE'12-27The phosphateand ATP/ADPtransport
systemin the innermitochondrialmembrane.Thecoordinated
actionof two antiporters (purpleandgreen)results in the uptakeof
oneADP3-andoneHPOa2in exchange for oneATPa-andone
hydroxyl,powered by theoutwardtranslocation of oneproton
(mediated bythe proteins transport
of theelectron chain,blue)
duringelectron transportTheoutermembrane isnot shownhere
because it ispermeable to moleculessmallerthan5000Da
Studiesof what turned out to be ATP/ADP antiporter
activity were first recorded about 2000 years ago,
when Dioscoridesdescribeda poisonousherb from the this-
tle Atractylis gummifera, found commonly in the Mediter-
ranean region. The same agent is found in the traditional
Zulu multipurposeherbal remedyimpila (Callilepislaureola).
In Zulu impila means"health," although it has beenassoci-
ated with numerouspoisonings.In 1.962the activeagent in
the herb, atractyloside,which inhibits the ATPiADP an-
tiporter,was shown to inhibit oxidative phosphorylationof
extramitochondrialADP but not intramitochondrial ADP.
This demonstrated the importance of the ATP/ADP an-
tiporter and has provided a powerful tool to study the
mechanismby which this transporterfunctions.
Dioscorides(-AD 40-90) lived near Tarsus'at the time a
province of Rome in southeasternAsia Minor in what is now
Turkey. His five-volume De Materia Medica (The Materials of
Medicine) "on the preparation, properties' and testing of
drugs" describedthe medicinalpropertiesof about 1000 nat-
ural products and 4740 medicinal usagesof them. For ap-
proximately 1600 yearsit was the basicreferencein medicine
from northern Europe to the Indian Ocean,comparableto to-
day'sPhysicians'Desk Referenceas a guide for using drugs' I

H A R N E S S I NTGH E P R O T O N . M O T I VFEO R C EF O R E N E R G Y - R E Q U I R IPNRGO C E S S E S 509

R a t eo f M i t o c h o n d r i aO
l x i d a t i o nN o r m a l l y
D e p e n d so n A D P L e v e l s
If intact isolatedmitochondria are provided with NADH (or a
source of FADH2 such as succinate)plus 02 and P1,but not
ADP, the oxidation of NADH and the reduction of 02 rapidly
cease,becausethe amount of endogenousADP is depletedby
AIP formation. If ADP is then added,the oxidation of NADH
is rapidly restored. Thus mitochondria can oxidize FADH2
and NADH only as long as there is a sourceof ADP and P; to
generateAIP. This phenomenon,termed respiratory control,
occursbecauseoxidation of NADH and succinate(FADH2) is
obligatorily coupled to proton transport acrossthe inner mi-
tochondrial membrane.If the resultingproton-motive force is
not dissipatedduring the synthesisof AIP from ADP and P1
(or for some other purpose),both the transmembraneproton
concentration gradient and the membrane electric potential
will increaseto very high levels.At this point, pumping of ad-
ditional protons acrossthe inner membranerequiresso much
energy that it eventuallyceases,blocking the coupled oxida-
tion of NADH and other substrates.
Brown-FatMitochondriaUsethe Proton-Motive
Forceto GenerateHeat
Broun-fat tissue,whosecolor is due to the presenceof abun,
dant mitochondria, is specializedfor the gineration of heat.
In contrast, tuhite-fat tissue is specializedfor the storage of
fat and contains relatively few mitochondria.
The inner membrane of brown-fat mitochondria con-
tains thermogenin, a protein that functions as a natural
uncoupler of oxidative phosphorylation and generationof
a proton-motive force. Thermogenin, or UCP1.,is one of sev-
eral uncoupling proteins (UCPs) found in most eukaryotes
(but not in fermentative yeasts).Thermogenin dissipatesthe
proton-motive force by rendering the inner mitochondrial
membrane permeableto protons. As a consequencethe en-
ergy releasedby NADH oxidation in the electron transport
chain is converted to heat. Thermogenin is a proton rrans-
porter, not a proton channel, and shuttlesprotons acrossthe
membrane at a rate that is a millionfold slower than that of
typical ion channels(seeFigure 11-3).Thermogeninis simi-
lar in sequenceto the mitochondrial ATP/ADP transporte!
as are many other mitochondrial transporter proteins that
compose the ATP/ADP transporter family. Certain small-
molecule poisons also function as uncouplers by rendering
the inner mitochondrial membrane permeableto protons.
One example is the lipid-soluble chemical 2,4-dinitrophenol
(DNP), which can reversiblybind to and releaseprotons and
shuttle them acrossthe inner membrane from the intermem-
brane spaceinto the matrix.
Environmental conditions regulate the amount of ther-
mogenin in brown-fat mitochondria. For instance, during
the adaptation of rats to cold, the ability of their tissuesto
generateheat is increasedby the induction of thermogenin
synthesis.In cold-adaptedanimals, thermogenin may consti-
tute up to 15 percent of the total protein in the inner mito-
chondrial membrane.
510 c H A P T E R1 2 | CELLULAR
ENERGETTCS
Adult humans have little brown fat, but human infants
have a great deal. In the newborn, thermogenesisby brown-
fat mitochondria is vital to survival, as it also is in hibernating
mammals.In fur sealsand other animalsnaturally acclimated
to the cold, muscle-cellmitochondria contain thermogenin;as
a result, much of the proton-motive force is usedfor generat-
ing heat, thereby maintaining body temperature.
Harnessingthe Proton-Motive Force
for Energy-RequiringProcesses
r Peter Mitchell proposed the chemiosmotic hypothesis
that a proton-motive force acrossthe inner mitochondrial
membrane is the direct sourceof energy for ATP synthesis.
r Bacteria, mitochondria, and chloroplasts all use the
samechemiosmoticmechanismand a similar ATP synthase
to generate ATP.
r ATP synthase(the FeFl complex) catalyzesATP synthesis
as protons flow through the inner mitochondrial membrane
(plasma membrane in bacteria)down their electrochemical
proton gradient.
r Fs contains a ring of 10-14 c subunits that is rigidly
linked to the rod-shaped1 subunit and e subunit of F1. To-
gether they rotate during ATP synthesis.Resting atop the T
subunit is the hexameric knob of F1 [(ctB)3],which pro-
trudesinto the mitochondrial matrix (cytosolin bacteria).
The three B subunits are the sites of ATP synthesis (see
Figure 12-24).
r Movement of protons acrossthe membranevia two half-
channelsat the interface of the F6 a subunit and the c ring
powers rotation of the c ring with its attached F1 e and 1
subunits.
r Rotation of the F1 ^ysubunit, which is inserted in the
centerof the nonrotating (cB)3 hexamerand operateslike
a camshaft, leads to changesin the conformation of the
nucleotide-bindingsites in the three F1 B subunits (see
Figure 12-25). By means of this binding-changemecha-
nism, the B subunits bind ADP and P1,condensethem to
form ATP, and then releasethe ATP. Three ATPs are made
for each revolution made by the assemblyof c, 1, and e
subunits.
r The proton-motive force also powers the uptake of P1
and ADP from the cytosol in exchangefor mitochondrial
ATP and OH , thus reducing some of the energy available
for ATP synthesis.The ATP/ADP antiporter that partici-
pates in this exchangeis one of the most abundant proteins
in the inner mitochondrial membrane.
r Continued mitochondrial oxidation of NADH and the
reduction of 02 are dependent on sufficient ADP being
present in the matrix. This phenomenon, termed respira-
tory control, is an important mechanism for coordinating
oxidation and ATP synthesisin mitochondria.
r In brown fat, the inner mitochondrial membrane con-
tains the uncoupler protein thermogenin, a proton trans-
 porter that dissipatesthe proton-motive force into heat.
Certain chemicalsalso function as uncouplers (e.g.,DNP)
and have the sameeffect, uncoupling oxidative phosphory-
lation from electron transDort.
Photosynthesis
and
Light-Absorbing
Pigments
We now shift our attention to photosynthesis,the second
main process for synthesizingAIP. Photosynthesisin
plants occursin chloroplasts,Iargeorganellesfound mainly in
leaf cells.The principal end products generatedfrom carbon
dioxide and water are two carbohydratesthat are polymers of
hexose(six-carbon)sugars:sucrose,a glucose-fructose disac-
charide(seeFigure2-19), and leaf starch, alarge insolubleglu-
cose polymer that is the primary storage carbohydrate in
higher plants (Figure 1.2-28).Leaf starch is synthesizedand
stored in the chloroplast.Sucroseis synthesizedin the leaf cy-
tosol from three-carbonprecursorsgeneratedin the chloro-
plast; it is transportedto nonphotosynthetic(nongreen)plant
tissues(e.g.,roots and seeds),which metabolizesucrosefor
energy by the pathways described in the previous sections.
Photosynthesisin plants, as well as in eukaryotic single-celled
algae and in several photosynthetic bacteria (e.g., the
cyanobacteriaand prochlorophytes),also generatesoxygen.
The overall reaction of oxygen-generatingphotosynthesis,
6 CO2 + 6 H2O -->6 02 + C6H12O5
is the reverseof the overall reaction by which carbohydrates
are oxidized to CO2 and H2O. In effect, photosynthesisin
chloroplastsproduces energy-richsugars that are broken
down and harvestedfor energy by mitochondria during the
processof cellular respiration.
Although green and purple bacteria also carry out pho-
tosynthesis,they use a processthat does not generateoxy-
gen. As discussedin Section 12.5, detailed analysisof the
photosynthetic systemin thesebacteriahas provided insights
about the first stagesin the more common processof oxy-
gen-generatingphotosynthesis.In this section,we provide an
overview of the stagesin oxygen-generatingphotosynthesis
Glucose
6
cH2oH
5)-o
H ,/.1. \ H
H
o-
HOHHOH
Starch
Ipoly(c1+4 glucose]l
FIGURE 12-28Structureof starch.Thislargeglucose
polymer
andthe disaccharide (seeFigure
sucrose 2-19)arethe principal
end
productsof photosynthesis
Botharebuiltof six-carbonsugars
(hexoses)
and introduce the main components, including the chloro-
phylls, the principal light-absorbing pigments.I
T h y l a k o i dM e m b r a n e si n C h l o r o p l a s t s
Are the Sitesof Photosynthesisin Plants
Chloroplastsare lensshapedwith an approximatediameterof
5 pm and a width of -25 pm, bounded by two membranes,
which do not contain chlorophyll and do not participate di-
rectly in photosynthesis(Figure 12-29). As in mitochondria,
the outer membraneof chloroplastscontains porins and thus
is permeableto metabolites of small molecular weight. The
inner membrane forms a permeability barrier that contains
transport proteins for regulating the movement of metabo-
lites into and out of the organelle.
Unlike mitochondria, chloroplasts contain a third mem-
brane-the thylakoid membrane-on which photosynthesis
occurs. The chloroplast thylakoid membrane is believed t<r
constitute a single sheet that forms numerous small, inter-
connected flattened structures, the thylakoids, which com-
monly are arranged in stacks termed grana (Figure 1'2-29).
The spaceswithin all the thylakoids constitute a single con-
tinuous compartment, the thylakoid lumen. The thylakoid
membranecontains a number of integral membraneproteins
to which are bound severalimportant prosthetic groups and
light-absorbing pigments, most notably chlorophyll. Carbo-
hydrate synthesisoccurs in the stroma, the soluble phasebe-
tween the thylakoid membrane and the inner membrane. In
photosyntheticbacteriaextensiveinvaginationsof the plasma
membrane form a set of internal membranes, also termed
thylakoid membranes,where photosynthesisoccurs.
Threeof the Four Stagesin Photosynthesis
O c c u rO n l y D u r i n gl l l u m i n a t i o n
The photosyntheticprocessin plants can be divided into four
stages(Figure 1.2-30),each localizedto a defined area of the
chloroplast: (1) absorption of light, generation of a high
energyelectron and formation of 02 from H2O; (2) electron
transport leading to reduction of NADP- to NADPH, and
generation of a proton-motive force; (3) synthesisof ATP;
and (4) conversion of CO2 into carbohydrates,commonly
referred to as carbon fixation. All four stagesof photosyn-
thesisare tightly coupled and controlled so as to produce the
amount of carbohydrate required by the plant. All the reac-
tions in stages 1-3 are catalyzed by multiprotein complexes
in the thylakoid membrane.The generationof a pmf and the
useof the pmf to synthesizeAIP resemblestagesIII and IV of
mitochondrial oxidative phosphorylation. The enzymesthat
incorporate CO2 into chemical intermediatesand then con-
vert them to starch are solubleconstituentsof the chloroplast
stroma; the enzymesthat form sucrosefrom three-carbonin-
termediatesare in the cytosol.
Stage 1: Absorption of Light The initial step in photo-
synthesisis the absorptionof light by chlorophyllsattached
to proteins in the thylakoid membranes. Like the heme
component of cytochromes,chlorophylls consist of a por-
phyrin ring attached to a long hydrocarbon side chain
S D L I G H T - A B S O R B I NPGI G M E N T S
P H O T O S Y N T H E SAI N 511
 (Figure 12-37).In contrast to the hemes(seeFigure 12-14),
chlorophylls contain a central Mg2* ion (rather than Fe)
and have an additional five-memberedring. The energy of
the absorbed light ultimately is used to remove electrons
from a donor (water in greenplants), forming oxygen:

Chloroplasts 2H2o!5or+4H++4e

The electronsare transferredto a primary electron dcceptor,

Mesophyll
a quinone designatedQ, which is similar to CoQ in mito-
chondria. ln plants the oxidation of water takes place in a
multiprotein complex calIedphotosystemII (PSII).

Lower
epidermis
Stage 2: Electron Transport and Generation of a Pro-
ton-Motive Force Electronsmove from the quinone pri-
Cuticle
mary electronacceptorthrough a seriesof electroncarriers
until they reach the ultimate electron acceptor,usually the
I n n e rm e m b r a n e : oxidized form of nicotinamide adenine dinucleotide phos-
Chloroplast transportersfor phate (NADP+), reducing it to NADPH. NADP* is identi-
p h o s p h a t ea n d
cal in structure with NAD* except for the presenceof an
Stroma:enzymesthat sucroseprecursors
catalyzeCO, fixation additional phosphategroup. Both moleculesgain and lose
and starchsynthesis electronsin the sameway (seeFigure2-33).lnplants the re-
duction of NADP- takes place in a complex calledphoto-
systemI (PSI). The transport of electronsin the thylakoid
membraneis coupled to the movementof protons from the
stroma to the thylakoid lumen, forming a pH gradient
acrossthe membrane(pHr,-.. ( pHr,ro-"). This processis
T h y l a k o i dm e m b r a n e : analogousto generationof a proton-motiveforce acrossthe
a b s o r p t i o no f l i g h t b y Outer inner mitochondrial membraneand in bacterialmembranes
c h l o r o p h y l ls, y n t h e s i s memDrane:
ofATP4 , NADPH,and I n t e r m e m b r a n e during electrontransport (seeFigure 12-22).
p e r m e a b tl e
o
e l e c t r o nt r a n s p o r t space s m a l lm o l e c u l e s Thus the overall reaction of stages1, and 2 can be sum-
marizedas

riehtrtH*
2H2O +2NADP+ +2NADPH+02

Stage 3: Synthesis of ATP Protons move down their con-

centration gradient from the thylakoid lumen to rhe stroma
through the FeFl complex (ATP synthase),which couplespro-
Granum
ton movement to the synthesisof ATP from ADP and P;. The
chloroplastAIP synthaseworks similarly to the synthasesof
Thylakoid mitochondriaand bacteria(seeFigure 12-25).
memorane
Stage 4: Carbon Fixation The ATP and NADPH generated
by the secondand third stagesof photosynthesisprovide the
'Jir energyand the electronsto drive the synthesisof polymers of
six-carbonsugarsfrom CO2 and H2O. The overall chemical
equailon ls wrltten as
FIGURE 12-29Cellularstructureof a leaf and chloroplast.
L i k em i t o c h o n d r ipal ,a n tc h l o r o p l a satrseb o u n d e d b ya d o u b l e 6 CO2 + 18 ATP4 + 12 NADPH + 12 H2O -->
m e m b r a nsee p a r a t ebdy a n i n t e r m e m b r asnpea c ep h o t o s y n t h e s i s
occuro s n a t h i r dm e m b r a n e c6Hr2o6 + 1g ADp3 + 1g pi'z- + 12 NADP* + 6 H+
t h, et h y l a k o im d embranw e ,h i c hi s
s u r r o u n d ebdy t h e r n n e m r e m b r a naen df o r m sa s e r i eosf f l a t t e n e d
v e s i c l e( tsh y l a k o i dt sh)a te n c l o sae s i n g l ei n t e r c o n n e c tl e
udminal The reactions that generatethe ATP and NADPH used in
s p a c eT h eg r e e nc o l o ro f p l a n t si sd u et o t h e g r e e nc o l o ro f carbon fixation are directly dependenton light energy;thus
c h l o r o p h yal ll,lo f w h i c hi s l o c a l i z et d
o t h et h y l a k o im d embrane stages 1-3 are called the light reactions of photosynrhesis.
A g r a n u mi sa s t a c ko f a d j a c e nt ht y l a k o i dTs h es t r o m ai st h e The reactionsin stage4 are indirectly dependenton light en-
s p a c e n c l o s ebdy t h e i n n e rm e m b r a naen ds u r r o u n d i nt hge ergy; they are sometimescalled the dark reactions of photo-
thylakoidsIPhotomicrograph courtesy of Katherine Esau, University synthesis becausethey can occur in the dark, utilizing the
o f C a l i f o r n i aD, a v i sl supplies of ATP and NADPH generatedby light energy.

512 . c H A p r E R1 2 | CELLULAR
ENERGETIC5
 Stage 4

Carbonfixation,
carbohydratesynthesrs
Sucrose
t Cytosol
outer
rDernbrane

Stage 1 Stage 2 Stage 3

6 COr+ 2 GlYceralde nujln"'t"to'un"
3-phosphate
Light absorption, Electrontra nsport,formation ATP synthesis , (carbonfixation)
g e n e r a t i o no f h i g h - of proton-motiveforce NADp+ H*
energy electron,
02 formation ADP + P1

ATP

/u+
s*"
Thylakoid
HzO 2H++02 membrane lqrnen

A FIGURE 12-30Overviewof the four stagesof photosynthesis. additionalenergyisintroduced by absorption of lightin photosystem
In stage1, lightisabsorbedby light-harvesting complexes(LHC) | (PSl),
to synthesize the high-energy electron carrierNADPHIn
and reaction centerof photosystem ll (PSll)TheLHCstransfer the stage 3, movement of proteinspowered by the proton-motive force
absorbed energyto the reactioncenters, whichuseit, or theenergy drivesthesynthesis of ATPby an FoFrATPsynthaseStages1-3 in
absorbed froma photon,to oxidize
by directly waterto molecular plantstakeplacein thethylakoid membrane of thechloroplast ln
oxygenandgenerate high-energy electrons In stage2, these stage4, in thechloroplast stroma, theenergy storedin NADPH and
electronsmovedownan electron transport chain,whichuseseither ATPisusedto convert C02initiallyintothree-carbon molecules
(Q/QHr)
lipid-soluble or water-soluble(plastocyanin,PC)electron (glyceraldehyde 3-phosphate),a process knownascarbonfixation
to shuttleelectrons
carriers betweenmultiple proteincomplexes Thesemolecules arethentransported to thecytosolof the cellfor
As electrons movedownthe chain,theyrelease energythatthe conversionto hexose sugarsin theformof sucroseGlyceraldehyde
complexes useto generate a proton-motive forceand,after 3-phosphate isalsousedto makestarch withinthechloroplast

However, the reactions in stage 4 are not confined to the

dark; in fact, they occur primarily during illumination.
Chlorophyll
a
C H-, E a c hP h o t o no f L i g h t H a sa D e f i n e dA m o u n t
ll ,;1
cH H (cg of Energy
rlF
Quantum mechanicsestablishedthat light, a form of elec-
tromagnetic radiation, has propertiesof both waves and par-
ticles.\7hen light interactswith matter, it behavesas discrete

< FIGURE 12-31Structureof chlorophylla, the principal

pigmentthat trapslight energy.Electrons aredelocalized among
threeof chlorophyllsa'sfourcentralrings(yellow) andtheatoms
that interconnect a Mg'* ion,ratherthanthe
them.In chlorophyll,
Fe3*ionfoundin heme,sitsat the centerof the porphyrin ringand
an additional iive-membered ring(blue)ts present;otherwise, the
structureof chlorophyll to thatof heme,foundin molecules
issimilar
o-c suchashemoglobin andcytochromes (seeFigure12-14a)Thehydro-
I carbonphytol"tail"facilitatesbindingof chlorophyll to hydrophobic
o cH. cH. regions proteinsTheCH3group(9reen)
of chlorophyll-binding is
PhytolI l- l-
CHr-CH: C- CH2- (CH2-CH2-CH-CHr)3H replaced by a formaldehyde (CHO) group in chlorophyllb.

PHOTOSYNTHEA
SN N IGG M E N T S .
I SD L I G H T - A B S O R B I P 513
packets of energy (quanta) calledphotozs. The energy of a Action spectrum
photon, e, is proportional to the frequency of the light wave: of photosynthesis
, : h ^ 1w
, h e r eb i s P l a n c k ' sc o n s t a n t( 1 . 5 8 x 1 0 - 3 4c a l . s ,o r
6.63 x t0 34;.s1and 1 is the frequencyof the light wave. It C h l o r o p h y lal
is customaryin biology to refer to the wavelengthof the light
wave, tr, rather than to its frequency1. The two are relatedby
the simple equation 'l : c + \, where c is the velocity of light o
(3 x 1010cm/s in a vacuum). Note that photons of shorter c
o
wavelengthhave bigher energies.Also, the energyin 1 mol of -o
60
photons can be denoted by E : Ne, where N is Avogadro's o
o
number (6.02 x 1023moleculesor photons/mol).Thus
@

Nhc
E: N D ^' iv_ o

The energy of light is considerable,as we can calculate for

light with a wavelengthof 550 nm (550 x 10 7 cm), typical 500 600
of sunlight: Wavelength (nm)
- ( 6 . 0 2x 1 0 2 3 p h o t o n s / m o l ) ( 1x. 5180 3 a c a l . s ) (x3 1 0 1 0 c m / s )A EXPERIMENTAL FIGURE
E-- 12-32The rate of photosynthesis is
550x 10 7cm greatestat wavelengthsof light absorbedby three pigments.
: 5 1 , 8 8 1callmol
Theactionspectrum in plants(theabilityof light
of photosynthesis
of differentwavelengths to supportphotosynthesis) isshownin
or about 52 kcal/mol. This is enough energy to synthesize blackTheenergyfromlightcanbe converted intoATPonlyif it
severalmoles of ATP from ADP and Pi if all the energywere canbe absorbed by pigments in thechloroplast.Absorption spectra
used for this purpose. (showing howwelllightof different wavelengths isabsorbed) for
threephotosynthetic pigments presentin the antennas of plant
photosystems areshownin colorComparison of the actionspectrum
PhotosystemsComprisea ReactionCenter
with the individual
absorption spectrasuggests that photosynthesis
and AssociatedLight-HarvestingComplexes at 680 nm isprimarily dueto lightabsorbed by chlorophyll a, at
The absorption of light energyand its conversioninto chem- 650nm,to liqhtabsorbed by chlorophyll
b, andat shorter wave-
ical energy occurs in multiprotein complexes called photo- lengths,to lightabsorbed by chlorophyllsa andb andby carotenoid
systems.Found in all photosynthetic organisms, both eu- pigments, including B-carotene
karyotic and prokaryotic, photosystems consist of two
closely linked components: a reaction center,where the pri- When chlorophyll a (or any other molecule)absorbsvisible
mary events of photosynthesisoccur, and an antenna com- light, the absorbedlight energyraiseselectronsin the chloro-
plex consistingof numerous protein complexes,including phylla to a higher-energy(excited)state.This state differs from
internal antenna within the photosystem proper and exter- the ground (unexcited)state largely in the distribution of the
nal antenna made up of specializedproteins termed light- electronsaround the C and N atoms of the porphyrin ring. Ex-
haruesting complexes (LHCs), which capture light energy cited statesare unstable,and the electronsreturn to the ground
and transmit it to the reaction center (seeFigure 12-30). stateby one of severalcompetingprocesses. For chlorophyll a
Both reaction centers and antennascontain tightly bound moleculesdissolvedin organic solventssuch as ethanol, the
light-absorbing pigment molecules.Chlorophyll a is the princi- principal reactionsthat dissipatethe excited-stateenergy are
pal pigment involved in photosynthesis,being present in both the emissionof light (fluorescenceand phosphorescence) and
reaction centersand antennas.In addition to chlorophyll a, an- thermal emission(heat).Ifhen the samechlorophyll a is bound
tennascontain other light-absorbing pigments: chlorophyll b in in the unique protein environment of the reaction center,dissi-
vascular plants and carotenoids in both plants and photosyn- pation of excited-stateenergy occurs by a quite different
thetic bacteria.Carotenoidsconsistoflong branchedhydrocar- processthat is the key to photosynthesis.
bon chains with alternating single and double bonds; they are
similar in structure to the visual pigment retinal, which absorbs
PhotoelectronTransportf rom Energized
light in the eye. The presenceof various antenna pigments,
which absorb light at different wavelengths,greatly extendsthe
Reaction-Center Chlorophylla Produces
rangeof light that can be absorbedand usedfor photosynthesis. a C h a r g eS e p a r a t i o n
One of the strongestpiecesof evidencefor the involve- The absorption of a photon of light of wavelength =680 nm
ment of chlorophylls and carotenoids in photosynthesisis by one of the two "special-pair" chlorophyll a moleculesin
that the absorption spectrum of thesepigments is similar to the reaction center increasesits energy by 42 kcal/mol (the
the action spectrum of photosynthesis(Figure 72-32). The first excited state).Such an energizedchlorophyll a molecule
latter is a measureof the relative ability of light of different in a plant reaction center rapidly donates an electron to an
wavelengthsto support photosynthesis. intermediate acceptor,and the electron is rapidly passedon

514 . c H A p r E R1 2 | C E L L U L AERN E R G E T t c s
llil+ Animation:Photosynthesis
P r i m a r ye l e c t r o n
Light Reaction
centel
C h l o r o p h y l la
to the primary electron acceptor,quinone Q, near the stro-
mal surfaceof the thylakoid membrane (Figure i2-33). This
light-driven electron transfer,called photoelectron transport,
dependson the unique environment of both the chlorophylls
and the acceptor within the reaction center. Photoelectron
transport, which occurs nearly every time a photon is ab-
sorbed, leavesa positive charge on the chlorophyll a closeto
the luminal surface of the thylakoid membrane (opposite
side from the stroma) and generatesa reduced, negatively
chargedacceptor(Q-) near the stromal surface.
The Q- produced by photoelectron transport is a powerful
reducing agent with a strong tendencyto transfer an electron to
another molecule, ultimately to NADP+. The positively
charged chlorophyll a*, a strong oxidizing agent, attracts
an electron from an electron donor on the luminal surface
to regeneratethe original chlorophyll a. In plants, the oxidiz-
ing power of four chlorophyll a* moleculesis used,by way of
intermediates,to remove four electronsfrom 2 H2O mole-
culesbound to a site on the luminal surfaceto form 02:
2HzO * 4 chlorophylla* --+4 H* + C2+ 4 chlorophyll a
These potent biological reductants and oxidants provide all
the energy neededto drive all subsequentreactions of pho-
tosynthesis:electron transport (stage 2), ATP synthesis
(stage3), and CO2 fixation (stage4).
Chlorophyll a also absorbs light at discretewavelengths
shorter than 680 nm (see Figure 12-32). Such absorption
raisesthe molecule into one of severalexcited states,whose
energiesare higher than that of the first excited state de-
scribedabove,which decayby releasingenergywithin 10-12
seconds(1 picosecond,ps) to the lower-energyfirst excited
state with loss of the extra energy as heat. Becausephoto-
electron transport and the resulting charge separationoccur
only from the first excited state of the reactron-center
chlorophyll a, the quantum yield-the amount of photosyn-
thesisper absorbedphoton-is the samefor all wavelengths
of visible light shorter (and therefore of higher energy)than
680 nm. How closely the wavelength of light matches the
absorption spectraof the pigmentswill determinehow likely
it is that the photon will be absorbed. Once absorbed, the
S t r oonnggr e d u c i n g < FIGURE 12-33 PhotoelectrontransPort,the primaryevent in
a g e nntt( e l e c t r o nro o n o r photosynthesis. Afterabsorption of a photonof light,oneof the
excited special pairof chlorophyll a molecules in the reactioncenter
(/eft)donatesviaseveral intermediates an electronto a looselybound
acceptor molecule, the quinoneQ, on the stromal surfaceof the
ge
thylakoid membrane, creating an essentiallyirreversiblecharge
separation across the membrane (righi fhe electron cannoteasily
returnthroughthe reaction centerto neutralize the positively
rkoid
brane charged chlorophyll a. In plantstheoxidation of waterto molecular
oxygen takes place in a multiprotein complex calledphotosystem ll.
-umen Thecomplex photosystem I usesa similarphotoelectron transport
Strong o> x i ddiizziinngg pathway,but insteadof oxidizing water,it reduces the electron
g e nntt( e
aq 3 l et c t r o na c c e p t o r ) c a r r i eNr A D P - .
photon's exact wavelength is not critical, provided it is ener-
getic enough to push the chlorophyll into the first excited
state.
I n t e r n a lA n t e n n aa n d L i g h t - H a r v e s t i n g
ComplexesIncreasethe Efficiency
of Photosynthesis
Although chlorophyll a moleculeswithin a reaction center
that are involved directly with charge separation and electron
transfer are capableof directly absorbing light and initiating
photosynthesis,they most commonly are energizedindirectly
by energy transferred to them from other light-absorbing and
energy-transferringpigments. These other pigments, which
include many other chlorophyll molecules, are involved with
absorption of photons and passingthe energyto the chlorophyll
a molecules in the reaction center. Some are bound to protein
subunitsthat are consideredto be intrinsic componentsof the
photosystemand thus are called internal antennas;others are
bound to proteinscomplexesthat bind to but are distinct from
the photosystemcore proteins and are called light-harvesting
complexes(LHCs). Even at the maximum light intensity en-
countered by photosynthetic organisms (tropical noontime
sunlight),eachreaction-centerchlorophyll a moleculeabsorbs
only about one photon per second' which is not enough to
support photosynthesis sufficient for the needs of the plant.
The involvement of internal antenna and LHCs greatly in-
creasesthe efficiency of photosynthesis,especiallyat more
typical light intensities,by increasingabsorption of 680-nm
light and by extending the range of wavelengths of light that
can be absorbedby other antennapigments.
Photonscan be absorbedby any of the pigment molecules
in internal antennasor an LHC. The absorbedenergyis then
rapidly transferred(in <10-e seconds)to one of the two
"special-pair" chlorophyll a moleculesin the associatedreac-
tion center, where it promotes the primary photosynthetic
charge separation(Figure 12-33). Photosystemcore protetns
and LHC proteins maintain the pigment moleculesin the pre-
ciseorientation and position optimal for light absorption and
energy transfer,thereby maximizing the very rapid and effi-
cient resonancetransfer of energy from antenna pigments to
N IGG M E N T S .
A INSD L I G H T - A B S O R B I P
PHOTOSYNTHES 515
 (a) Bridging Reaction E n e r g yr e s o n a n c e
L i gh t
c h l o r o p h y l l center tra nsfer
Stroma

(c) Bridging
chlorophyll
d LHC vFrecial-pair
ne cn orophylls

(b) ight

2\
4--'-e-

E:nergy
n e r g yresonance
transfer
.f\

Reaction
Special-pair
center
chlorophylls

A FIGURE 12-34Light-harvesting complexesand lightto oneof two "bridging"chlorophylls (squares,darkgreen)and

photosystems in cyanobacteria and plants.(a)Diagram of the thenceto chlorophyllsin the reaction
center. (b)Three-dimensional
membrane of a cyanobacterium, in whichthe multiprotein
light- organizationof the photosystemI (PSl)
with its LHCsof Pisumsativum
harvesting
complex (LHC) contains 90 chlorophyll
molecules(green) (garden pea)asdetermined by x-raycrystallographyandasseenfrom
and31 othersmallmolecules, allheldin a specific
Aeometric the planeof the membrane Onlythechlorophylls together withthe
arrangement for optimallightabsorption andenergytransferOf the reactioncenterelectron areshown(c)Expanded
carriers viewof the
sixchlorophyll
molecules in the reactioncenter,
two constitutethe reactioncenterfrom(b)rotated90' abouta vertical axislpart(a)adapted
special-pair (ovals,
chlorophylls darkgreen) thatcaninitiate fromW KUhlbrandt,2001,Nature 411:896,
andPJordan etal. 2001,Narure
photoelectrontransportwhenexcited (bluearrow)Resonance 411:909Parts (bandc)basedonthestructural
determination
bvA Ben-Sham
of energy(redarrows)
transfer rapidly funnelsenergyf romabsorbed etal, 2003,Nature426:6301

reaction-centerchlorophylls. Resonanceenergy transfer does
not involve the transfer of an electron. Studieson one of the
two photosystemsin cyanobacteria,which are similar to
those in higher planrs, suggestthat energy from absorbed
light is funneled first to a "bridging" chlorophyll in each
LHC and then to the specialpair of reaction-centerchloro-
phylls (Figurel2-34a). Surprisingly,however,the molecular
structuresof LHCs from plants and cyanobacteriaare com-
pletely different from those in green and purple bacteria,
even though both types contain carotenoids and chloro-
phylls in a clustered arrangement within the membrane.
Figure 12-346 shows the distribution of the chlorophyll
pigments in the plant photosystem I from Pisum satiuum
(gardenpea)togetherwith its peripheralLHCI antenna.The
large number of internal and LHC antenna chlorophylls
surround the core reaction center to permit efficient transfer
of absorbedlight energyto the specialchlorophyllsin the re-
actlon center.
Although LHC antenna chlorophylls can transfer light
energyabsorbedfrom a photon, they cannot releasean elec-
tron. As we've seenalready,this function residesin the two
reaction-centerchlorophylls. To understand their electron-
releasingability, we examine the structure and function of
the reaction center in bacterial and plant photosystemsin the
next sectlon.
Photosynthetic Stages and Light-Absorbing Pigments
r The principal end products of photosynthesisin plants
are molecular oxygen and polymers of six-carbon sugars
(starchand sucrose).
r The light-capturing and ATP-generatingreactions of
photosynthesisoccur in the thylakoid membrane located
within chloroplasts. The permeable outer membrane and
inner membrane surrounding chloroplasts do not partici-
pate directly in photosynthesis(seeFigure 12-29).
r There are four stagesin photosynthesis:(1) absorption
of light, generationof a high energyelectron and formation
of 02 from H2O; (2) electron transport leading to reduc-
tion of NADP* to NADPH, and to generationof a proton-
motive force; (3) synthesisof ATP; and (4) conversion of
CO2 into carbohydrates(carbonfixation).
r In stage 1 of photosynthesis,light energy is absorbed by
one of two "special-pair" chlorophyll a molecules bound

516 c H A P T E R1 2 | CELLULAR
ENERGETTCS
 to reaction-centerproteins in the thylakoid membrane.The The reaction centerof purple bacteriacontains three pro-
energizedchlorophylls donate via intermediatesan electron tein subunits(L, M, and H) locatedin the plasmamembrane
to a quinone on the opposite side of the membrane, creat- (Figure 12-35). Bound to these proteins are the prosthetic
ing a charge separation(seeFigure 12-33). In green plants, groups that absorb light and transport electrons during
the positively charged chlorophylls then remove electrons photosynthesis. The prosthetic groups include a "special
from water, forming molecular oxygen (02). pair" of bacteriochlorophyll a molecules equivalent to the
reaction-centerchlorophyll a moleculesin plants, as well as
r In stage 2, electrons are transported from the reduced
quinone via carriers in the thylakoid membrane until they severalother pigmentsand two quinones,termed Qa and Qs'
reach the ultimate electron acceptor,usually NADP*, re- that are structurally similar to mitochondrial ubiquinone.
ducing it to NADPH. Electron transport is coupled to
Initial Charge Separation The mechanismof chargesepa-
movement of protons across the membrane from the
ration in the photosystemof purple bacteria is identical with
stroma to the thylakoid lumen, forming a pH gradient
that in plants outlined earlier; that is, energy from absorbed
(proton-motiveforce) acrossthe thylakoid membrane.
light is used to strip an electron from a reaction-centerbac-
r In stage 3, movement of protons down their electro- teriochlorophyll a molecule and transfer it, via several dif-
chemical gradient through FeFl complexes (ATP synthase) ferent pigments, to the primary electron acceptor Qs, which
powers the synthesisof ATP from ADP and P,. is loosely bound to a site on the cytosolic membrane face'
r In stage4, the NADPH and ATP generatedin stages2 and
3 provide the energyand the electronsto drive the fixation of
CO2, which resultsin the synthesisof carbohydrates.These
reactionsoccur in the thylakoid stroma and cytosol.
r Associatedwith eachreaction centerare multiple internal
antenna and light-harvestingcomplexes (LHCs), which
contain chlorophylls a and b, carotenoids, and other pig-
mentsthat absorb light at multiple wavelengths.Energy,but
not an electron,is transferredfrom the internal antennaand
LHC chlorophyll moleculesto reaction-centerchlorophylls
by resonanceenergytransfer (seeFigure 12-34).

MolecularAnalysis
of Photosystems
As noted in the previous section,photosynthesisin the green
and purple bacteria does not generateoxygen, whereaspho-
tosynthesisin cyanobacteria,algae,and higher plants does."
This differenceis attributable to the presenceof two types of
photosystem (PS) in the latter organisms: PSI reduces
NADP* to NADPH, and PSII forms 02 from H2O. In con-
trast, the green and purple bacteria have only one type of
photosystem,which cannot form 02. !7e first discussthe sim-
pler photosystem of purple bacteria and then consider the
Accessory
more complicatedphotosyntheticmachinery in chloroplasts.
chlorophyll
Pheophytin
Special-pair
The SinglePhotosystemof PurpleBacteria chlorophyll

Generatesa Proton-MotiveForcebut No 02 FIGURE 12-35Three-dimensional structureof the

photosyntheticreactioncenterfrom the purplebacterium
The three-dimensionalstructuresof the photosynthetic reac-
Rhodobacter spheroides. (Top)IheL subunit(yellow) andM subunit
tion centers have been determined, permitting scientiststo
(gray)
eachformfivetransmembrane crhelices andhavea verysimilar
trace in detail the paths of electronsduring and after the ab-
the H subunit(lightblue)isanchored
overall;
structure to themembrane
sorption of light. Similar proteins and pigments compose transmembrane a helix. A fourthsubunit (notshown) isa
bya single
photosystemsI and II of plants and photosynthetic bacteria. proteinthat bindsto theexoplasmic
peripheral segments of theother
subunits(Bottom) Withineachreaction center, but not easilydistin-
*A very different type of bacterial photosynthesis, which occurs only in guishedin thetop image, isa special pairof bacteriochlorophylla
molecules(green),capable photoelectron
of initiatinq transport;two
certain archaebacteria, is not discussed here because it is very different
accessory (purple);
chlorophylls turopheophytins (darkblue),andtwo
from photosynthesis in higher plants. In this type of photosynthesis, the
plasma-membrane protein bacteriorhodopsin pumps one proton from the quinones,QaandQe(orange). Qeistheprimary electron acceptor
cytosol to the extracellular space for every quantum of light absorbed. duringphotosynthesis.lAfterM H Stowell etal, 1997,Science2T6:8121

M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S ' 517
The chlorophyll thereby acquires a positive charge, and Qg
acquires a negative charge. To determine the pathway tra-
versed by electronsthrough the bacterial reacrion center,
researchersexploited the fact rhat eachpigment absorbslight
of only certain wavelengths,and its absorptron specrrum
changeswhen it possesses an extra electron.Becausethese
electronmovementsare completedin lessthan 1 millisecond
(ms), a specialtechnique calledpicosecondabsorption spec-
troscopy is required to monitor the changesin the absorp-
tion spectraof the various pigmentsas a function of time
shortly after the absorptionof a light photon.
\7hen a preparation of bacterial membrane vesiclesis
exposedto an intensepulse of laser light lasting less than
1 ps,eachreactioncenterabsorbsone photon (Figure12-36).
l-ight absorbedby the chlorophylla moleculesin each reac-
tion center convertsthem to the excited state, and the subse-
quent electron transfer processesare synchronizedin all
reactioncentersin the experimentalsample.\Tithin 4 x 10 12
seconds(4 ps), an electronmoves,possiblyvia the accessory
bacterial chlorophyll as an intermediare,to the pheophytin
molecules(Ph),leaving a positivechargeon the chlorophyll a.
It takes200 ps for the electronto move ro Qe, and then, in the
slowest step, 200 ps for it to move to Qn. This pathway of
electronflow is traced in the left part of Figore 12-36.
Subsequent Electron Flow and Coupled Proton Move-
ment After the primary electronacceptor,Qe, in the bac-
terial reactioncenteracceptsone electron,forming Qs-., it
accepts a second electron from the same reaction-center
chlorophyll following its re-excitation (e.g.,by absorption of
a second photon or transfer of energy from antenna mole-
cules). The quinone then binds two protons from the cy-
tosol, forming the reducedquinone (QHz), which is released
from the reaction center (Figure 12-36). QH2 diffuseswithin
the bacterial membraneto the Qo site on the exoplasmicface
of a cytochrome bc1 electron transport complex similar in
structure to complex III in mitochondria. There it releasesits
tvvo protons into the periplasmicspace(the spacebetweenthe
plasma membrane and the bacterial cell wall). This process
movesprotons from the cytosol to the outsideof the cell, gen-
erating a proton-motive force acrossthe plasma membrane.
Simultaneously,QHr releasesits two electrons,which move
through the cytochrome bcl complex exacrly as depicted for
the mitochondrial complex III (CoQH2-cytochrome c reduc-
tase) in Figure 12-20. The Q cycle in the bacterial reaction
center, like the Q cycle in mitochondria, pumps additional
protons from the cytosol to the intermembrane space,
thereby increasingthe proton-motive force.
The acceptor for electrons transferred through the cy-
tochrome bc1complex is a solublecytochrome,a one-electron
carrier, in the periplasmic space,which is reduced from the
Fe3* to the Fe2* state.The reducedcytochrome(analogousto
cytochromec in mitochondria) then diffusesto a reactloncen-
ter,where it releasesits electronto a positivelychargedchloro-
phyll a-, returning that chlorophyll to the unchargedground
stateand the cytochromero the Fe3* state.This cyclicelectron

Pi +
O c y c l e :a d d i t i o n a l ADP ATP
proton transport
H_

Cytosol

Plasma
membrane

2 photons

++++
Periplasmic
space

Bacterial reaction Cytochrome bct FoFt complex

complex
FIGURE 12-36Cyclicelectronflow in the single (Cente)Afterdiffusingthroughthe membrane andbindingto the
photosystemof purplebacteria.Cyclic electron flow generates Qositeon theexoplasmic faceof thecytochrome bc1complex, eH,
a proton-motive forcebut no O, Bluearrowsindicate flow of donatestwo electronsandsimultaneously givesup two protons to
electrons; redarrowsindicate protonmovement(left)Energy theexternalmediumin the periplasmic space, generatinga proton
absorbed directly fromlightor funneled froman associated LHC electrochemicalgradient(proton-motive force)Electronsare
(notillustrated here)energizes oneof the special-pair chlorophylls transportedbackto the reaction-center chlorophyllviaa soluble
in the reaction centerPhotoelectron transport fromtheenergized cytochrome,whichdiffuses in the periplasmic spaceNotethecyclic
chlorophyll, viaaccessory chlorophyll, pheophytin (ph),andquinone path(blue)of electronsOperation of a Q cyclein thecytochrome
A ( Q i , t o q u i n o nBe ( Q sf)o r m st h es e m i q u i n oen-e a n dl e a v eas bcl complexpumpsadditional protons across the membrane to the
positive chargeon the chlorophyll Following absorption of a second external
medium, asin mitochondria. fAdapted from.]Deisenhofer
and
photonandtransfer of a second electron to the semiquinone, the H M i c h a e l ,1 9 9 1, A n n R e v . C e lBl i o l . 7 : 1I
quinonerapidlypicksup two protons fromthe cytosol to formeH2,

518 c H A P T E R1 2 I CELLULAR
ENERGETTCS
flow generatesno oxygen and no reducedcoenzymes,but it
has generateda proton-motive force.
Electrons also can flow through the single photosystem
of purple bacteria via a linear (noncyclic) pathway. In this
case, instead of the electron removed from reactron-center
chlorophylls by light moving to the cytochrome bcl complex
and then cycling back again via the water-soluble cy-
tochrome to the reaction center.the electron removed from
reaction-centerchlorophyll is eventually transferred to
NAD* (rather than NADP+ as in plants),forming NADH.
As a consequence,an electron from a different source must
be returned to the special-pairchlorophylls if additional
light energyis to be harvestedby photoelectrontransport. In
bacteriausing a linear pathway, the electronsto do this come
from the oxidation of either hydrogen sulfide (H2S)
H2S--+S+2H+ *2e-
to form elementalsulfur (S)or hydrogengas (H2):
H2--+2H- *2e-
These electrons are used to reduce a cytochrome, which in
turn passesan electron to the special-pairchlorophylls in the
reaction center to bring the oxidized reaction-centerchloro-
phyll a back to its ground state. Overall, the linear pathway
resultsin the light-mediatedoxidation of H2S (or H2) and
the reduction of NAD- to NADH. SinceH2O is not the elec-
tron donor, no 02 is formed.
Both the cyclic and linear pathways of electron flow in
the bacterial photosystem generate a proton-motive force.
As in other systems,this proton-motive force is used by the
FeFl complex located in the bacterial plasma membrane to
synthesizeAIP and also to transport molecules across the
membrane against a concentration gradient.
s o n t a i nT w o F u n c t i o n a l l a
C h l o r o p l a s tC
Spatially Distinct Photosystems
In the 1940s, biophysicist R. Emerson discoveredthat the
rate of plant photosynthesisgeneratedby light of wave-
length 700 nm can be greatly enhanced by adding light of
shorter wavelength (higher energy).He found that a combi-
nation of light at, say, 600 and 700 nm supports a greater
rate of photosynthesisthan the sum of the rates for the two
separatewavelengths.This so-calledEmerson effect led re'
searchersto conclude that photosynthesisin plants involves
the interaction of two separatephotosystems,referred to as
PSI and PSII. PSI is driven by light of wavelength 700 nm
or less;PSII,only by shorter-wavelength light (<680 nm).
In chloroplasts, the special-pair reaction-centerchloro-
phylls that initiate photoelectron transport in PSI and PSII
differ in their light-absorptionmaxima becauseof differ-
encesin their protein environments. For this reason, these
chlorophylls are often denoted P680(PSII) and PTes(PSI).
Like a bacterial reaction center, each chloroplast reaction
center is associatedwith multiple internal antenna and light-
harvesting complexes (LHCs); the LHCs associatedwith
PSII and PSI contain different proteins.
The two photosystems also are distributed differently in
thylakoid membranes:PSIIprimarily in stackedregions(grana,
seeFigure 12-29) and PSIprimarily in unstackedregions.The
stacking of the thylakoid membranesmay be due to the bind-
ing properties of the proteins in PSII. Evidence for this distri-
bution came from studiesin which thylakoid membraneswere
gently fragmented into vesiclesby ultrasound' Stacked and
unstacked thylakoid vesicles were then fractionated by
density-gradientcentrifugation. The stacked fractions con-
tained primarily PSII protein and the unstackedfraction PSI.
Finally, and most importantly, the two chloroplast photo-
systems differ significantly in their functions (Figure 12-37):
only PSII splits water to form oxygen' whereas only PSI trans-
fers electronsto the final electron acceptor,NADP*. Photo-
synthesisin chloroplastscan follow a linear or cyclic pathway,
again like green and purple bacteria. The linear pathwaS
which we discussfirst, can support carbon fixation as well as
ATP synthesis.In contrast, the cyclic pathway supports only
ATP synthesisand generatesno reducedNADPH for use in
carbon fixation. Photosyntheticalgaeand cyanobacteriacon-
tain two photosystemsanalogousto those in chloroplasts.
LinearElectronFlow Through Both Plant
Photosystems,PSlland PSl,Generates
a Proton-MotiveForce,02, and NADPH
Linear electron flow in chloroplastsinvolves PSII and PSI in
an obligate series in which electrons are transferred from
H2O to NADP+. The processbegins with absorption of a
photon by PSII, causing an electron to move from a P53s
chlorophyll a to an acceptorplastoquinone(Qs) on the stro-
mal surface (Figure 12-37). The resulting oxidized P5ss-
strips one electron from the relatively unwilling donor H2O,
forming an intermediate in 02 formation and a proton'
which remains in the thylakoid lumen and contributesto the
proton-motive force. After P5s6absorbsa secondphoton, the
y nd
semiquinoneQ-' acceptsa secondelectron and picks up tvvo
protons from the stromal space,generatingQH2. After dif-
fusing in the membrane, QHz binds to the Q" site on a cy-
tochrome b/complex (analogousto bacterialcytochrome bc1
complex and mitochondrial complex III). As in thesesystems'
a Q cycle operates, thereby increasing the proton-motive
force generatedby electron transport. After the cytochrome
bf complex accepts electrons from QH2' it transfers them'
one at a time, to the Cu2+ form of the solubleelectroncarrier
plastocyanin(analogousto bacterial cytochrome c), reducing
it to the Cu* form. Reducedplastocyaninthen diffusesin the
thylakoid lumen, carrying the electron to PSI.
Absorption of a photon by PSI leads to removal of an
electron from the reaction-centerchlorophyll a,P7ss (Figure
1,2-37).The resulting oxidized P706* is reduced by an elec-
tron passedfrom the PSII reaction centervia the cytochrome
bf complex and plastocyanin' Again, this is analogousto sit-
uation in mitochondria, where cytochrome c acts as a single
electron shuttle from complex III to complex IV (seeFigure
12-1.6).The electron taken up at the luminal surfaceby Pzoo
energizedby photon absorption moveswithin PSIvia several
carriers to the stromal surface of the thylakoid membrane,
A N A L Y S I SO F P H O T O S Y S T E M S O
MOLECULAR 519
 NADP+ H+

O c y c l e :a d d i t i o n a l NADPH T;i
p r o t o nt r a n s p o r t H + Ferrr loxi)
Fe-S
Stroma
AU+

Thylakoid
membrane

2 photons photons
\

++++ O2-evolving Plastocya

ocvanrn
Lumen comprex
o
' 680 D
| 700
chlorophyll cn orophyll
HzO 2 H'+ 1lz
Cytochrome bf PSI reaction FsFt complex
PSll reaction center complex center
A FIGURE 12-37Linearelectronflow in plants,which requires cytochrome bf complex translocates protons
additional across the
both chloroplastphotosystems PSIand PSll.Bluearrowsindicate membrane to thethylakoid lumen,increasingthe proton-motive
flow of electrons;
redarrowsindicate protonmovementLHCsare f orce (Right)In the PSIreactioncenter,eachelectronreleased f rom
not shown(Left)ln the PSllreactioncenter,
two sequential light- light-excited
P76s chlorophylls
movesviaa series of carriers
in the
induced excitations
of thesameP6s6 chlorophylls
resultin reduction reaction centerto thestromal surface,wheresoluble ferredoxin (an
of the primaryelectronacceptorQato eHz Onthe luminal side Fe-Sprotein) transfers the electron
to ferredoxin-NADP+ reductase
of PSll,
electronsremoved fromH2Oin thethylakoid lumenare (FNR)Thisenzyme usesthe prostheticgroupflavinadenine dinu-
transferredto P6se+,
restoringthe reactron-center
chlorophyllsto cleotide(FAD) anda protonto reduce NADP+, formingNADPHpToo+
the groundstateandgenerating Oz Genter)Thecytochrome bf isrestoredto itsgroundstateby addition of an electroncarried from
complex thenacceptselectronsfromQH2,coupled to the release PSllviathecytochrome bf complexandplastocyanin, a soluble
of two protonsintothe lumenOperation of a e cyclein the electron carrier.

where it is acceptedby ferredoxin, an iron-sulfur (Fe-S)pro- 'S7hen

tein. Electrons excited in PSI can be transferred from ferre-
doxin via the enzymeferredoxin-NADP+ reductase(FNR).
This enzyme uses the prosrhetic group FAD as an electron
carrierto reduceNADP*, forming, togetherwith one proton
picked up from the stroma, the reduced molecule NADPH.
FeFl complexesin the thylakoid membraneuse the pro-
ton-motive force generatedduring linear electron flow to
synthesizeATP on the stromal side of membrane.Thus this
pathway exploits the energy from multiple photons ab-
sorbed by both PSII and PSI and their anrennasro generare
both NADPH and ATP in the stroma of the chloroDlast.
where they are utilized for CO2 fixation.
A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e
L u m i n a lS u r f a c eo f t h e p S l lR e a c t i o nC e n t e r
Somewhat surprisingly,the structure of the pSII reaction
center,which removeselectronsfrom H2O to form 02, r€-
semblesthat of the reactioncenrerof photosyntheticpurple
bacteria,which does not form ()2. Like the bacterial reac-
tion center,the PSII reactioncentercontainstwo molecules
of chlorophyll a (P5s6),as well as two other accessory
chlorophylls,two pheophytins,rwo quinones(e6 and es),
and one nonheme iron atom. These small molecules are
bound to two proteinsin PSII,calledD1 andD2, whose se-
quencesare remarkably similar to the sequencesof the L
and M subunitsof the bacterialreactioncenrer,attestingto
their common evolutionary origins (see Figure I2-35).
PSII absorbs a photon with a wavelength of <680
nm, it triggersthe loss of an electronfrom a P5semolecule,
€leneratingP.so*. As in photosyntheticpurple bacteria,the
electron is transported rapidly, possibly via an accessory
chlorophyll, to a pheophytin, then to a quinone (Qa), and
then to the primary electron acceptor, Q", or the outer
(stromal)surfaceof the thylakoid membrane(Figures12-37
and 12-38).
The photochemically oxidized reaction-centerchloro-
phyll of PSII, P5s0*, is the strongesl biological oxidant
known. The reduction potential of P6s6* is more positive
than that of water, and thus it can oxidize water to generate
02 and H* ions. Photosyntheticbacteria cannot oxidize
water becausethe excited chlorophyll a* in the bacterial
reaction center is not a sufficiently strong oxidant. (As noted
earlier,purple bacteria useH2S and H2 as electron donors to
reduce chlorophyll a* in Iinear electron flow.)
The splitting of H2O, which provides the electrons for
reduction of P5se* in PSII, is catalyzedby a three-protein
complex, the oxygen-euoluingcomplex,located on the lumi-
nal surfaceof the thylakoid membrane.The oxygen-evolving
complex contains four manganese(Mn) ions as well as
bound Cl and Ca2* ions (Figure 12-38);this is one of the
very few casesin which Mn plays a role in a biological sys-
tem. These Mn ions together with the three extrinsic pro-
teins can be removed from the reaction center by treatment
with solutions of concentrated salts; this abolishes ()2 for-
mation but does not affect light absorption or the initial
stagesof electron transport.

520 c H A P T E R1 2 | CELLULAE
RN E R G E T T C S
 PSll reaction whether the formation of 02 depends on a single PSII or
centef multiple ones acting in concert. The results indicated that a
D1 D2
l
single PSII must lose an electron and then oxidize the oxy-
Stroma gen-evolvingcomplex four times in a row for an 02 mole-
Or< cule to be formed.
Fe
Thylakoid Manganeseis known to exist in multiple oxidation states
membrane with from two to five positive charges.Indeed, spectroscopic
studiesshowed that the bound Mn ions in the oxygen-evolv-
Special-pair ing complex cycle through five different oxidation states'
4 photons chlorophylls Se-Sa.In this S cycle, a total of two H2O moleculesare split
into four protons, four electrons'and one 02 molecule. The
Thylakoid electronsreleasedfrom H2O are transferred, one at a time,
lumen
O2-evolving via the Mn ions and a nearby tyrosineside chain on the D1
comprex subunit to rthereaction-centerPnro*, where they regenerate
the reducedlchlorophyll, P536ground state. The protons re-
leasedfrom H2O remain in the thylakoid lumen.
2H2O 4H*+02

FIGURE12-38 Electronflow and 02 evolution in chloroplast Herb:icidesthat inhibit photosynthesisnot only are
P5ll.The PSllreactioncenter,comprisingtwo integralproteins,D1 very limportant in agriculture but also have proved
and D2, special-pair chlorophylls (Poeo), and other electroncarriers,is useful in dissectingthe pathway of photoelectron trans-
associated wrth an oxygen-evolving complexon the luminalsurface port in plants. One such classof herbicides,the s-triazines
Boundto the threeextrinsicproteins(33,23, and 17 kDa)of the (e.g., atrazine),binds specificallyto the D1 subunit in the
oxygen-evolving complexare four manganeseions(Mn, red),a Ca2* PSII reaction center, thus inhibiting binding of oxidized
ion (blue),and a Cl ion (yellow)Thesebound ionsfunctionin the
Qs to its site on the stromal surfaceof the thylakoid mem-
splittingof H2Oand maintainthe environmentessential for high
brane. \7hr:n added to illuminated chloroplasts,s-triazines
r a t e so f 0 2 e v o l u t i o nT y r o s i n e - 1 6( Y
1 1 6 1 ) o ft h e D 1 p o l y p e p t i d e
causeall downstreamelectroncarriersto accumulatein the
conductselectronsfrom the Mn ionsto the oxidizedreaction-center
oxidized form, sinceno electronscan be releasedfrom PSII.
chlorophyll(Poeo-), reducingit to the groundstateP6s6[Adapted from
In atrazine-resistant mutants, a singleamino acid changein
C Hoganson andG Babcock, 199-l, Science 277:1953I
D1 rendersit unable to bind the herbicide,so photosynthe-
sis proceeds at normal rates. Such resistant weeds are
The oxidation of two molecules of H2O to form 02 re-
prevalentand presenta major agricultural problem. I
quires the removal of four electrons, but absorption of each
photon by PSII results in the transfer of iust one electron. A
simple experiment, described in Figure 12-39, resolved C e l l sU s eM u l t i p l e M e c h a n i s mtso P r o t e c t
Against Damagefrom ReactiveOxygen Species
During PhotoelectronTransport
As we saw earlier in the caseof ROS generationby the mito-
chondrion, ATP generation via the electron transport chain
!
a

brings with it potential deleteriousside effects.The same is

o
o_ true for the chloroplast. Even though the PSI and PSII photo-
n
0) systemswith their associatedlight-harvestingcomplexesare
remarkably efficient at converting radiant energy to useful
0)
chemical energy in the form of ATP and NADPH, they are
not perfect. Depending on the intensity of the light and the
physiologicconditions of the cells,a relativelysmall-but sig-
.tifi."ttt-u-ount of energy absorbed by chlorophylls in the
123456789101112
light-harvestingantennasand reactionscentersresultsin the
F l a s hn u m b e r
chlorophyll being converted to an activated state called
A E X P E R I M E N T AFLI G U R E1 2 - 3 9 A s i n g l e P S l la b s o r b s a "triplet" chlorophyll. In this state' the chlorophyll can trans-
photon and transfers an electron four times to generate fer some of its energyto molecular oxygen (02), converting it
o n e 0 2 . D a r k - a d a p t e cdh l o r o p l a s tws e r e e x p o s e dt o a s e r i e so f
from its normal, relatiuely unreactive ground state, called
c l o s e l ys p a c e d s, h o r t( 5 p s ) p u l s e so f l i g h t t h a t a c t i v a t e dv i r t u a l l y
triplet oxygen (3Oz) to a very highly reactive (ROS) singlet
a l l t h e P 5 l l si n t h e p r e p a r a t i o nT h e p e a k si n O 2 e v o l u t i o no c c u r r e d 1Or. If the 1O2 is not quickly quenchedby react-
state form,
a f t e r e v e r yf o u r t h p u l s e ,i n d i c a t i n gt h a t a b s o r p t i o no f f o u r tOr "scau.nger molecules," it will react
p h o t o n sb y o n e P S l li s r e q u i r e dt o g e n e r a t ee a c h0 2 m o l e c u l e
ing with specialized
Because the dark-adapted c h l o r o p l a s tws e r e i n i t i a l l yi n a p a r t i a l l y with and usually damagenearby molecules'This damagecan
r e d u c e ds t a t e ,t h e p e a k si n 0 2 e v o l u t i o no c c u r r e da f t e r f l a s h e s3 , suppressthe efficiencyof thylakoid activity and is calledpDo-
7 , a n d 1 1 l F r o mJ B e r ge t a l , 2 0 0 2 ,E r o c h e m i s t r y ,e5dt h, W H toiihibitior. Carotenoids (polymers of unsaturatedisoprene
F r e e m aann dC o m p a nl y groups, including beta-carotene,which gives carrots their

M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S . 521
 Photoinhibition Recovery
(2400pE m-1 s 1)
( 2 0p E m - 1 s 1 )
a
-60 -30 0 30 60 90
T i m e( m i n )
orange color) and o-tocopherol (a form of vitamin E) are hy-
drophobic small moleculesthat play important roles as 1O2
quenchersto protect plants. For example,inhibition of toco-
pherol synthesis in the unicellular green alga Chlamy-
domonas reinhardtii by the herbicidepyrazolynatecan result
in greaterlight-inducedphotoinhibition. To help further limit
the potential damagein light-harvestingantennas,carotenoid
molecules siphon off energy from the dangerous triplet
chlorophyll, thus preventing 102 formation.
Under intenseillumination, photosystempSII is especially
prone to generating tO2, whereas PSI will produce other
ROS, including superoxide, hydrogen peroxide, and hy-
droxyl radicals. The D1 subunit in the pSII reacion cenrer
(seeFigure 12-38) is, even under low light conditions, sub-
jected to almost constant 1O2-mediateddamage. The dam-
aged reaction center moves from the grana to the unstacked
regionsof the thylakoid, where the D1 subunit is degradedby
a proteaseand replaced by newly synthesizedD1 proteins in
what is called the D1 protein damage-repaircycle.The rapid
replacementof damagedD 1, which requiresa high rate of b 1
synthesis,helps the PSII recover from photoinactivation and
maintain sufficient activity. The experiment in Figure 12-40
showsthat an important component in the damage-repaircy-
cle is the chaperoneprotein HSP7OB(seeChapter 3), which
522 CHAPTER
12 I CELLULAR
ENERGETICS
< EXPERIMENTAT FIGURE 12-40ThechaperoneHSP7OB
helps
PSllrecoverfrom photoinhibitionafter exposureto intense
fight. Theunicellular greenalgaChlamydomonas reinhardtiiwas
geneticallymanipulated sothatit hadabnormally highor low levels
of thechaperone proteinHSP7OB. Thehigh,low,andnormalstrains
werethenexposed light(2400pE m 2 s-1)for
to high-intensity
60 minutes to inducephotoinhibition followedbyexposure to
low light(20pE m-2 s-1)forup to 150minutes. Theeffects of
photoinhibition lightandtheabilityof pSilto
bythe high-intensity
recover fromthe photoinhibition weremeasured usingfluorescence
spectroscopy to determinePSllactivityTheabilityof the cellsto
recover PSllactivitydepends on the levelsof HSP7OB-the more
HSP7OB available,
the morerapidthe recovery-dueto HSp7OB
protection of the PSllreaction
centers that hadwithstood1Or-induced
D1subunit damage. [FromSchrodaetal, 1999,PlantCetl1l:t 165]
binds to the damagedPSII and helps prevent loss of the other
components of the complex as the D1 subunit is replaced.
The extent of photoinhibition can depend on the amounr of
HSP7OBavailableto the chloroplasts.
CyclicElectronFlow Through PSIGenerates
a Proton-MotiveForcebut No NADPHor 02
As we've seen,electronsfrom reduced ferredoxin in PSI are
transferred to NADP* during linear electron flow, resulting
in production of NADPH (seeFigure 12-37).In some cir-
cumstancescells must generaterelative amounts of ATP and
NADPH that differ from those produced by linear electron
flow (e.g., greater ATP-Io-NADPH ratio). To do this, they
120 150 photosyntheticallyproduce ATP from PSI without concomi-
tant NADPH production. This is accomplishedusing a PSII-
independent process called cyclic ph otop h osph orylation. In
this processelectronscycle between PSI, ferredoxin, plasto-
quinone (Q), and the cytochrome b/complex (Figure 1.2-41);
thus no net NADPH is generated,and there is no need to
oxidize water and produce 02. There are two distinct cyclic
electron flow pathways: the NAD(P)H dehydrogenase(Ndh)-
dependent (shown in Figure 1,2-41) and Ndh-independent
pathways. Ndh is an enzyme complex very similar to the
mitochondrial complex I (see Figure 12-16) that oxidizes
NADPH or NADH while reducing Q to QH2 and contribut-
ing to the proton motive force by transporting protons. Dur-
ing cyclic electron flow, the substrate for the Ndh is the
NADPH generated by light absorption by PSI, ferredoxin,
and ferredoxin-NADP reductase (FNR). The QH2 formed
by Ndh then diffusesthrough the thylakoid membraneto the
Q" binding site on the luminal surfaceof the cytoch rome bf
complex. There it releasestwo electronsto the cytochrome
b/ complex and two protons to the thylakoid lumen, gener-
atrng a proton-motive force. As in linear electron flow, these
electronsreturn to PSI via plastocyanin. This cyclic electron
flow is similar to the cyclic processthat occurs in the single
photosystemofpurple bacteria (seeFigure 12-36). A e cycle
operatesin the cytochrome bf complex during cyclic electron
flow, leading to transport of two additional protons into the
lumen for each pair of electrons transported and a greater
proton-motive force.
 NADP+ H' /Ferredoxin\
+

s9,/ P i+
NADPH ADP
O c y c l e :a d d i t i o n a l
NADPH NADP*+Ht protontransport Hi Ferre oxtn
="-S_,,

Stroma

Thylakoid
membrane photon

++++ Plastocyanin
Lumen AU+ D
| 700
chlorophyll

Cytochrome bf PSI reaction F6F1complex

NAD(P)Hdehydrogenase
(Ndh) complex center

beingusedto f ixcarbon-isoxidizedby Ndh Thereleased electrons

A FIGURE 12-41 Cyclicelectronflow in plants,which generates
aretransferred (Q)withinthe membrane
to plastoquinone to generate
a proton-motiveforce and ATPbut no oxygen or net NADPH.
(Ndh)-dependent pathway for cyclic QH2,whichthentransfers to the cytochrome
the electrons bf complex,
ln the NAD(P)H-dehydrogenase
electrons In a thento plastocyanin,and back
finally to PSl,as isthe casefor the
electronflow,lightenergyisusedby PSIto transport
cycleto generatea proton-motiveforceandATPwithoutoxidizing f low pathway(seeFigure12-37)
linearelectron
waterTheNADPH formedviathe PS|/ferredoxin/FNR-insteadof

In Ndh-independent cyclic electron floq the mechanism to electron flow. The distribution of LHCII betweenPSI and
of which has not yet beencompletely defined, electronsfrom
the ferredoxin are used to reduceQ, either via a hypothetical
membrane-associated ferredoxin:plastoquinoneoxidoreduc-
tase (FQR) or via the Q1site, which is part of the Q cycle in
the cytochromeb/complex.

RelativeActivitiesof PhotosystemsI
and ll Are Regulated
In order for PSII, which is preferentially located in the
stacked grana)and PSI,which is preferentially located in the
unstacked thylakoid membranes,to act in sequenceduring
linear electron flow, the amount of light energy deliveredto
the two reaction centersmust be controlled so that eachcen-
ter activates the same number of electrons. This balanced flow in state2 (Figure 1'2-42).
condition is called state 1 (Figure 12-42).If the two photo- Regulating the supramolecular organization of the
systems are not equally excited, then cyclic electron flow photoryst.-s in plants thus has the effect of directing
occurs in PSI and PSII becomeslessactive (state2). Varia- th.- to*atd ATP production (state 2) or toward genera-
tions in the wavelengthsand intensitiesof ambient light (as
a consequentof the time of day, clouds, etc.) can changethe
relative activation of the two photosystems'potentially up-
setting the appropriate relative amounts of linear and cyclic
electron flow necessaryfor production of optimal ratios of
ATP and NADPH. chapter.
One mechanismfor regulating the relative contributions
of PSI and PSII,in responseto varying lighting conditions and
thus the relative amounts of linear and cyclic electron flow,
Molecular Analysis of Photosystems
entails redistributing the light-harvestingcomplex LHCII
between the two photosystems.The more LHCII associated In the single photosystemof purple bacteria, cyclic elec-
with a particular photosystem,the more efficiently that sys- on flow fiom light-excited, special-pairchlorophyll a
tem will be activatedby light and the greater its contribution moleculesin the reaction center generatesa proton-motive

O 523
M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S
 State 1, linear electron flow
P S Im e m b r a n ed o m a i n s
(unstacked)

P S l lm e m b r a n ed o m a i n s
( s t a c k e )d

T h y l a k o i dm e m b r a n e
NADPH
ADP + P;
e

Plastocyanin
ATP
svnthase

State 2, cyclic electron flow

Thylakoid membrane

0
Stroma

Lumen
ATP

A FIGURE 12-42Phosphorylation of LHCIIand the regulationof isunbalanced (eg , too muchviapsll),LHCII becomes phosphorylated,
finearversuscyclicelectronflow.(Top)ln normalsunlight, pSIand drssociatesfromPSll, anddiffusesintotheunstacked membranes,
PSllareequallyactivated,
andthephotosystems areorganized in state whereit associates with PSIanditspermanently associated
LHCI. In
1 ln thisarrangement,light-harvesting
complex ll (LHCll)
isnot thisalternativesupramolecular (state
organtzation 2),mostof the
pirosphorylated withthe psrrreaction
andistightlyassociated centerin absorbed lightenergyistransferred to psl,supportingcyclic
electron
thegranaAsa result, pSllandpSlcanfunctionin parallelin linear flowandATPproduction butno formation of NADPH andthusno CO2
eleclronflow (Bottom)when lightexcitation
of thetwo photosvstems fixatron.
[AdaptedfromFA.Wollman, 2OOj, EMBO J.20:3623.]

force, which is used mainly to power ATp synthesisby the
FeF, complex in the plasmamembrane(seeFigure 12-36).
r Plants contain two photosystems,pSI and pSII, which
have diff'erentfunctions and are physically separatedin the
thylakoid membrane. PSII splits H2O into 02, and pSI
reduces NADP+ to NADPH. Cyanobacteria have two
analogousphotosystems.
In chloroplasts,light energyabsorbedby light-harvesting
rnplexes(t.HCs) is transferredto chlorophyll a moleculei
the reactioncenters(Pos6in pSII and pr,16in pSI).
r Electronsflow through PSII via the samecarriersthat are
presentin the bacterialphotosystem.In contrastto the bac_
rerial system,photochemicallyoxidized p680+in pSII is
re€eneratedro P6ssby electronsderived from the splitting
rrf FI2O with evolution of 02 (seeFigure 72-37, left).
r [n linear electronflow, photochemicallyoxidized proo* in
I)Sl is reduced, regeneratingpzoo, by electrons transferred
frorn PSll via the cytochrome b/ complex and soluble plas_
tocyanin. Electronsreleasedfrom pTeefollowing excitation
of PSI are transported via severalcarriers u[imately to
NADP' , generaringNADPH (seeFigure 72-37 rigbt).
, green leaves.In addition to CO2 fixation-incorpora-
-f
r he absorption of light by pigmentsin the chloroplastcan
generirtetoxic reacive oxygen species(ROS), including sin_
glet crxygen,rOr, and hydrogenperoxide, HzOz.Small mol_
ecule scavengersand antioxidant enzymeshelp to protect
againstROS-induceddamage;however,singletoxygendam-
ageto the D1 subunit of PSIIstill occurs,causingphotoinhi-
bition. An HSP70 chaperone helps PSII recover from the
damage.
r In contrast to linear electron flow, which requires both
PSII and PSI, cyclic electron flow in plants involves only
PSI.In this pathwaS neither ner NADPH nor 02 is formed
although a proton-motive force is generated.
r Reversiblephosphorylation and dephosphorylationof
the light-harvesting complex II control the functional or-
ganization of the photosynthetic apparatus in thylakoid
membranes. State 1 favors linear electron flow, whereas
state2 favors cyclic elecron flow (seeFigure 12-42).
CO2MetabolismDuring
Photosynthesis
Chloroplasts perform many metabolic reactions in
tion of gaseousCO2 into small organic moleculesand then
sugars-the synrhesisof almost all amino acids. all fattv
acids and carotenes, all pyrimidines, and probably ail

524 c H A P T E R1 2 | cELLULAR
ENERGETTCS
 cH"-o- Po"H
I
cHr-o-Po3H o cHr-O- PO3H- HrO H-C-OH
I ill ____!___>
I
O:C:O c:o o c-c-oH c:o
I I
H-C-OH c:o o-
T

H-C-OH H-C-OH
I - I
cH2-o- Po3H cH2-O-PO3H c:o
l
H-C-OH
I
cHr-o-Po3H
co' intermediate
tnzvme'bound
l,'3i'.l,ii."on"r" ir-i:Tltl:$[:T""
a F I G U R E l 2 - 4T3h e i n i t i a rl e a c t i o n o rf u b i s c o t h a t f i x e s c o 2 f i v e - c a r b o n s u g a r r i b u l o s e l , 5 - b i s p hTohs e
pph raot e
ductsaretwo
into organiccompounds.In thisreaction, catalyzed by ribulose 1,5- molecules of 3-phosphoglycerate
bisphosphate carboxylase (rubisco), CO2condenses with the

purines occurs in chloroplasts.However, the synthesisof 10 molecules(30 C atoms) are convertedto 6 moleculesof
,,rg"r, from CO2 is the most extensivelystudied biosynthetic ribulose 1,5-bisphosphate(Figure 12-44, top) ' The fixation
p"Ih*"y in plani cells. !7e first considerthe unique pathway, of six CO2 molecules and the net formation of two glycer-
known as the Calvin cycle (after discovererMeivin Calvini, aldehyde3-phosphatemoleculesrequire the consumption of
that fixes CO2 into thr..-."rbon compounds, powered by 18 ATPs and1,2 NADPHs, generatedby the light-requiring
energy ,el."rJ during ATP hydrolysis and oxidation of processes of photosynthesis.
NADPH.
Synthesisof SucroseUsing FixedCO2
RubiscoFixesCO2in the ChloroplastStroma ls Completedin the Cytosol
The enzyme ribulose 1,5-bisphosphatecarboxylase, or ru- After its formation in the chloroplast stroma, glyceraldehyde
bisco, fixes CO2 into precursor molecules that are subse-
quently convertedinto carbohydrates.Rubisco is locatedin
the stromal spaceof the chloroplast.This enzymeadds CO2
to the five-carbonsugar ribulose 1,5-bisphosphate to form
two moleculesof the three-carbon-containing3-phospho-
glycerate (Figure 72-43). Rubisco is a large enzyme (:500
kDa) composed of eight identical large and eight identical
small subunits.One subunit is encodedin chloroplastDNA;
the other, in nuclear DNA. Becausethe catalytic rate of ru-
bisco is quite low, many copies of the enzyme are neededtcl
fix sufficient CO2. Indeed, this enzyme makes up almost 50
percentof the chloroplastprotein and is believedto be the
most abundantprotein on earth.
When photosynthetic algae are exposed to a brief pulse
1ac-labeledCO2 and the cellsare then quickly disrupted,
of
3-phosphoglycerate is radiolabeledmost rapidly, and all the Liqht and Rubisco Activase Stimulate
radioactivityis found in the carboxyl group. Because(iO:, rs
C5,
Lv f ixation
initially incorporated into a three-carboncompound, the
Calvin cycleis also calledthe C3 pathway of carbe'nfixatron
(Figure 12-44).
The fate of 3-phosphoglycerateformcd by rubisco is
complex: some is converted to hexosesincorporated into
starch or sucrose,but some is used to regenerateribulose
Calvin cycleenzymes..Because protons are transportedfrom
1,5-bisphosphate.At least nine enzymesare required to
photoelectrotl
regenerare ribulose1,5-bisphosphate from 3-ph<-rspLoglycer- the stroma into the thvlakoid lumen during
transport (seeFigure 1)'37),the pL1of-fhe the stroma increascs
atl euantitatively fo, .u.ry 12 moleculer-o63-phospho-
fr.r,,, =7 in the ri"rrii t<i =8 in the ligirt' increased activity
gly...it. g..r.r",.b by rubisco (a total of 36 C atorns), 2 of
of severalCalvin cvcle enzymesat the higher pI{ promotes
them (6 C atoms) are converredto 2 moleculesof giy.*r-
(and later to t hexose), whereas (,O2 fixation inthe light'
aldehyde 3-phosphate

D U R I N GP H O T O S Y N T H E S I S
CO, METABOLISM 525
 CO, O:C:O

o cHroH
I
c-o- c:o
I I
<----ry
6ADP co2 FtxATtoN 12 ATP
H-C-OH
I
H-C-OH
cH2- oPo32-
I
6 ATp___/f (cALVtNCYCLEI H-C-OH
12ADP 3-Phosphoglycerate I
ofififfi",":sc 1 2 1 , 3 - B i s p h o s p h o g l y c e r -a t 3
eC o
cH2-oPO32-
Ribulose

+ P <--{
\r enzymes
12NADPH

1 2N A D P *
c-oPo"2-
t-
5-phosphate

i7 H-C-OH
cH2-oPO32-
N-r t^ I
.,0 Glyceraldehyde: .'.' G l y c e r a l d e h y d-e r u cH2- oPo3'- c:o
'" 3C
3-phosphate 3-phosphate 1,3-Bisphosphoglycerate I
H-C-OH
H I
I H-C-OH
c:o I
,- G l y c e r a l d e h y d e 2 ^ I cH2-oPO32-
3-phosphate H-C-OH Ribulose
I 1,5-bisphosphate
2P, cH2- oPo32-
Stroma Glyceraldehyde3-phosphate
Phosphate-
triosephosphate Inner chloroplast membrane
antiportprotein
Cytosol
2P,

=3c
,3.',#::1f:lyd"
J
'' 6c
f3-Tl;?".ohat"=
cH2-o-PO32-
J" I
c:o
, f3-Tl;?",ohat":
6G HO-C-H
I

I
,r-7 H-C-OH
I
H-C-OH
I
' fliil','o"h.,":
u" cH2-o-Po32-
Fructose
1,6-bisphosphate

I sucRor ,|

..
f-'J,i315n..": 1 5':;:oss;at" : 6c

OH
2
OH OH
1 UDP-glucose Fructose
6-phosphate
Glucose 1-phosphate

: 1r.
' 3-l"il:iin",u
t
'l'-> P' OH OH
Sucrose: 12C Sucrose6-phosphate

526 o c H A p r E R1 2 | C E L L U L AERN E R G E T t c s
< FIGURE 12-tt4The pathwayof carbonduring and Mg2* concentrations' The activating reaction entails co-
photosynthesis.(Top)Sixmolecules of C02areconverted intotwo valent addition of CO2 to the side-chainamino group of lysine
moleculesof glyceraldehyde 3-phosphate. Thesereactions,which in the active site, forming a carbamate group that then binds a
theCalvincycle,
constitute occurin the stromaof thechloroplast Via Mg2* ion required for activity. Under normal conditions' how-
the phosphate/triosephosphate someglyceraldehyde
antiporter, 3-
ever, with ambient levels of CO2, the reaction is slow and
phosphate istransported to thecytosolin exchange for phosphate
usually requires catalysis by rubisco actiuase,an enzyme that
(Bottom)In the cytosol,an exergonic seriesof reactionsconverts
simultaneouslyhydrolyzesATP and usesthe energyto attach a
glyceraldehyde 3-phosphate to fructose1,6-bisphosphate.Two
CO2 to the lysine.Rubiscoactivasealso accelerates an activat-
moleculesof f ructose
1,6-bisphosphate areusedto synthesizeoneof
ing conformational change in rubisco (inactive-closed to
the disaccharidesucroseSomeglyceraldehyde 3-phosphate(not
u.iiu.-op.n.d state). The regulation of rubisco activase by
shownhere)isalsoconverted to aminoacidsandfats,compounds
for
essential plantgrowth thioredoxin is, at leastin part in some species,responsiblefor
rubisco'slight/redox sensitivity'Furthermore, rubisco activase's
activity is sensitiveto the ratio of ATP:ADP. If that ratio is low
A stromal protein called thioredoxin (Tx) also plays a (relatively high ADP), then the activase will not activate ru-
role in controlling some Calvin cycle enzymes.In the dark, bisco (and so the cell will expend less of its scarceATP to fix
thioredoxin contains a disulfide bond; in the light, electrons carbon).Giventhe key role of rubiscoin controlling energyuti-
are transferred from PSI, via ferredoxin, to thioredoxin, re- lization and carbon flux-both in an individual chloroplast
ducing its disulfide bond: and, in a sense,throughout the entire biosphere-it is not sur-
prising that its activity is tightly regulated.
PSI

sH Photorespiration,Which Competeswith
z--\s
(Tx)l Photosynthesis,ls Reducedin PlantsThat
\__As SH
Fix CO2by the C4PathwaY
Reducedthioredoxin then activatesseveralCalvin cycle en- As noted above, rubisco catalyzesthe incorporation of CO2
zymesby reducing disulfide bonds in them. In the dark, when
thioredoxin becomesreoxidized,theseenzymesare reoxidized
and so inactivated. Thus these enzymesare sensitiveto the
redox stateof the stroma, which in turn is light sensitive-an
elegantmechanismfor regulatingenzymaticactivity by light.
Rubisco is one such light/redox-sensitiveenzyme,although of the two-carbon compound phosphoglycolate' The first
its regulation is very complex and not yet fully understood. Ru- (carbon-fixing) reaction is favored when the ambient CO2 con-
bisco is spontaneouslyactivatedin the presenceof high CO2 centration is relatively high' whereasthe secondis favored when

o
--> ---> ---> Sugars
|
H-C-OH
n u - n pv , n 2
vr ,2 v3

3-Phosphoglycerate

CH,OH
Glycolate

3-Phosphoglycerate Phosphoglycolate

Phosphoglycolate viaa complexsetof reactions

is recycled that take
FfGURE12-45 C}2fixation and photorespiration.These
placein feroxisomes andmitochondria, aswellaschloroplasts The
competing pathways arebothinitiated 1,5-bisphosphate
by ribulose
C02 netresult:for everytwo molecules of phosphoglycolateformed by
carboxylase(rubisco),
andbothutilizeribulose1,5-bisphosphate.
pathwayIl, isfavoredby highc02 andlow02 pressures; photorespiration(fourc atoms), onemolecule of 3-phosphoglycerate
fixation,
isultimatelyformed andrecycledandonemolecule of c02 islost'
photorespiration,pathway[, occursat lowc02 anorrgno,
pressures(thatis,undernormalatmospheric conditions)

o 527
c o 2 M E T A B O L I S MD U R I N GP H O T O S Y N T H E S I S
 CO2 is low and 02 relatively high. The pathway initiated by
the second reaction with 02 is called photorespiration-a
processthat takes place in light, consumes02, and converts
react and form distincr products with the same initial
enzyme/ribulose 1,5-bisphosphate intermediate.
Excessivephororespiration could become a problem for
plants in a hot, dry environment, becausethey must keep the
gas-exchangepores (stomata) in their leavesclosed much of
the time to prevent excessiveloss of moisture. As a conse-
quence,the CO2 level inside the leaf can fall below the K- of
rubisco for CO2. Under theseconditions, the rate of photo-
synthesisis slowed, photorespiration is greatly favored, and
the plant might be in danger of fixing inadequateamounts of
CO2. Corn, sugarcane,crabgrass,and other plants that can
grow in hot, dry environmentshave evolved a way to avoid
this problem by utilizing a two-step pathway of CO2 fixa-
tion in which a CO2-hoarding stepprecedesthe Calvin cycle.
The pathway has been named the Ca pathway because
[14C]CO2 labeling showed that the first iadioactive mole-
culesformed during photosynthesisin this pathway are four-
carbon compounds, such as oxaloacetateand malate, rather
than the three-carbonmoleculesthat initiate the Calvin cvcle
(C3 pathway).
The Ca pathway involves two types of cells: mesophyll
cells, which are adjacent to the air spacesin the leaf interior,
and bundle sheath cells, which surround the vascular tissue
and are sequesteredaway from the high oxygen levelsto which
mesophyllcellsare exposed(Figure 12-46a).In the mesophyll
cellsof Ca plants,phosphoenolpyruvate, a three-carbonmole-
cule derived from pyruvate, reacts with CO2 to generate

V a s c u l a rb u n d l e < FfGURE'12-46Leatanatomy of Coplantsand the Co

( x y l e m ,p h l o e m )
pathway.(a)In C+plants, bundlesheath cellslinethevascular bundles
containing thexylemandphloemMesophyll cells,
whichareadjacent
to the substomal airspaces,
canassimilate CO, intofour-carbon
molecules at low ambientC02anddeliver it to the interiorbundle
sheathcells.Bundlesheathcellscontainabundant chloroplasts and
arethesitesof photosynthesis andsucrose synthesis Sucrose is
carried to the restof the plantviathe phloemIn C3plants, which
lackbundlesheathcells,theCalvincycleoperates in the mesophyll
a' cellsto fix COz(b)Thekeyenzyme in the Capathwayis phospho-
ra enolpyruvate
o! carboxylase,whichassimilates CO,to formoxaloac-
etatein mesophyll cellsDecarboxylationof malateor otherCo
e
Mesophyll intermediates in bundlesheathcellsreleases COr,whichenters
cells thestandard Calvincycle(seeFigure12-44,top)
I
n Bundle
rl sheath
cells

Epidermis

Chloroplast

Mesophyll cell o
f; lreoen o Bundle sheath cell
c-o +H+ NADP+ I ., co
l \ / 1 ---------\--z------ I
9H" 9H, CH,
| 1'
c o H-C-OH H-C-OH
I I co, + c"'y:l|
c-o c_o-
lltt
Og o
Oxaloacetate Malate Malate
NADP-

co2+ co2 9Ht nu cH.

il ^ Pyruvate- ;"3
.- C - O-PO"'- phosphate I' N A D P H+ H -
| -- dikinase v-v c:o
u -u
.-^t-----__---Z---|
c_o
I
/ \ c-o
oYt olt
Phosphoe?otpyruvate d o
PP, P, Pyruvate Pyruvate

528 CHAPTER
12 I CELLULAR
ENERGETICS
oxaloacetate,a four-carbon compound (Figure 12-46b). The in this regulation as does the regulation of the activity of
enzyme that catalyzes this reaction, p h o sph o enolpyruuate rubisco by rubisco activase.
carboxylase, is found almost exclusively in Ca plants and un- r ln C3 plants, a substantial fraction of the COz fixed by
Iike rubisco is insensitiveto 02. The overall reaction from the Calvin cycle can be lost as the result of photorespira-
pyruvate to oxaloacetateinvolves the hydrolysis of one ATP tion, a wasteful reaction catalyzed by rubisco that is fa-
and has a negativeAG. Therefore,CO2 fixation will proceed vored at low CO2 and high 02levels (seeFigure 1'2-45).
even when the CO2 concentration is low. The oxaloacetate
r In Ca plants, CO2 is fixed initially in the outer mesophyll
formed in mesophyll cells is reduced to malate, which is
cells by reaction with phosphoenolpyruvate.The four-
transferred by a special transporter to the bundle sheath
carbon moleculesso generatedare shuttled to the interior
cells, where the CO2 releasedby decarboxylation entersthe
bundle sheath cells, where the CO2 is releasedand then
Calvin cycle (Figure12-46b).
used in the Calvin cycle.The rate of photorespiration in Ca
Becauseof the transport of CO2 from mesophyllcells,
plants is much lower than in C3 plants'
the CO2 concentration in the bundle sheathcellsof Ca plants
is much higher than it is in the normal atmosphere.Bundle
sheathcellsare also unusualin that they lack PSIIand carry
out only cyclic electron flow catalyzedby PSI, so no ()2 is
evolved.The high CO2 and reduced02 concentrationsin the
Although the overall processesof photosynthesisand mito-
bundle sheath cells favor the fixation of CO2 by rubisco to
form 3-phosphoglycerateand inhibit the utilization of ribu- chondrial oxidation are well understood, many important
lose 1,5-bisphosphate in photorespiration. details remain to be uncoveredby a new generationof scien-
For example,little is known about how complexesI and
In contrast, the high 02 concentrationin the atmosphere tists.
favors photorespiration in the mesophyll cells of C3 plants IV in mitochondria couple proton and electronmovementsto
(pathway2 in Figure 1,2-45\;as a result,as much as 50 percent createa proton-mottve force. SimilarlS although the binding-
changemechanismfor ATP synthesisby the F6F1complex is
of the carbon fixed by rubiscomay be reoxidizedto CO2 in C-3
plants. Ca plants are superior to C3 plants in utilizing the now generally accepted,we do not understand how confor-
mational changesin each B subunit are coupled to the cycli-
availableCO2, sincethe Ca enzymephosphoenolpyruvatecar-
cal binding of ADP and P;,formation of ATP, and then release
boxylasehas a higher affinity for CO2 than doesrubisco in the
of ATP. In addition, many questionsremain about the precise
Calvin cycle.However, one ATP is consumedin the cyclic Ca
process (to generate phosphoenolpyruvatefrom pyruvate); mechanismof action of transport proteins in the inner mito-
chondrial and chloroplast membranesthat play key roles in
thus the overall efficiencyof the photosyntheticproduction of
oxidative phosphorylation and photosynthesis.
sugarsfrom NADPH and ATP is lower than it is in C3 plants'
\fe now know that releaseof cytochrome c and other
which use only the Calvin cycle for CO2 fixation. Nonethe-
less,the net rates of photosynthesisfor Ca $rasses,such as
corn or sugarcane,can be two to three times the ratesfor oth-
erwise similar C3 grasses,such as wheat, rice, or oats, owing
to the elimination of lossesfrom photorespiration.
Of the two carbohydrate products of photosynthesis,
connectionsbetween energy metabolism and mechanisms
starch remains in the mesophyll cells of C3 plants and the
bundle sheafcellsin Ca plants. In thesecells,starch is sub-
jected to glycolysis, mainly in the dark, forming ATR
NADH, and small moleculesthat are usedas building blocks
for the synthesisof amino acids, lipids, and other cellular
constituents.Sucrose,in contrast, is exported from the pho-
tosynthetic cells and transported throughout the plant.

and inexpensivefood to all who need it.

CO2Metabolism During Photosynthesis
r In the Calvin cycle,CO2 is fixed into organic moleculesin KeyTerms
a seriesof reactionsthat occur in the chloroplaststroma.The
initial reaction, catalyzedby rubisco, forms a three-carbon aerobic oxidation 479 chemiosmosis480
intermediate. Some of the glyceraldehyde3-phosphate chlorophylls511
ATP synthase504
generatedin the cycle is transported to the cytosol and con-
Calvin cycle525 chloroplasts479
verted to sucrose(seeFigure 72-44).
carbon fixation 511 citric acid cycle479
r The light-dependent activation of several Calvin cycle
cellularrespiration485 cytochromes495
enzymesand other mechanismsincreasesfixation of COz
in the light. The redox state of the stroma plays a key role Ca pathway 528 electron carriers487

K E YT E R M S 529
 electron transport chain 481 photosynthesis479
endosymbiont photosystems514
hypothesis485 proton-motiv e force 48 0
fermentation 485
Q cycle500
FeFl complex 505
reactive oxygen species502
glycolysis481
respiratory control 51 0
mitochondria 479 rubisco 525
oxidative phosphorylation substrate-level
481 phosphorylation 481
photoelectron transport 51 5 thylakoids511
photorespiration 528 uncouplers5-10
Reviewthe Concepts
1. The proton-motive force (pmf) is essentialfor both mi-
tochondrial and chloroplast function. \fhat produces the
pmf, and what is its relationship to ATp?
2. The mitochondrial inner membrane exhibits all of the
fundamental characteristicsof a typical cell membrane, but
it also has severalunique characteristicsthat are closely as-
sociatedwith its role in oxidative phosphorylation. What are
these unique characteristics?How does each contribute to
the function of the inner membrane?
3. Maximal production of ATP from glucose involves the
reactions of glycolysis,the citric acid cycle, and the electron
transport chain. \fhich of these reactions requires 02, and
why? \Which, in certain organisms or physiological condi-
tions, can proceed in the absenceof 02?
4. Describe how the elecrronsproduced by glycolysis are
deliveredto the electron transport chain. What would be the
consequencefor overall ATp yield per glucosemolecule if a
mutation inactivated this delivery system?rVhat would be
the,longer-termconsequencefor the activity of the glycolytic
pathway?
5. Mitochondrial oxidation of fatty acids is a major source
of ATP, yet fatty acids can be oxidized elsewhere.Sfhat or-
ganelle, besidesthe mitochondrion, can oxidize fatty acids?
\What is the fundamental
difference between oxidaiion oc-
curring in this organelleand mitochondrial oxidation?
6. Each of the cytochromes in the mitochondria contains
prosthetic groups. \fhat is a prosthetic group? Vhich type
of prosthetic group is associatedwith the cytochromes?
'What
property of the various cytochromesensuresunidirec-
tional electron flow along the electron transport chain?
7. It is estimatedthat eachelecron pair donated bv NADH
leadsto the synthesisof approximateiy three AIp molecules,
whereas each electron pair donated by FADH2 leads to the
synthesisof approximately two ATp molecules.\7hat is the
underlying reason for the difference in yield for electrons
donated by FADH2 versusNADH?
8. Much of our understanding of ATp synthaseis derived
from researchon aerobic bacteria.\fhat makes theseorgan_
isms useful for this research?Where do the reactions of gly_
colysis,the citric acid cycle, and the electron t.urrrpo.t .li"i.,
530 . cHAprE1
R2 | C E L L U L E
ANRE R G E T t c s
occur in theseorganisms?Where is the pmf generatedin aer-
obic bacteria?rVhat other cellular processesdepend on the
pmf in theseorganisms?
9. An important function of the mitochondrial inner mem-
brane is to provide a selectivelypermeable barrier to the
movement of water-solublemoleculesand thus generatedif-
ferent chemical environments on either side of the mem-
brane. However, many of the substratesand products of ox-
idativephosphorylarionare water solubleunJ rnurt crossthe
inner membrane. How does this transport occur?
10. The Q cycle plays a major role in the elecrron ffansport
chain of mitochondria, chloroplasts, and bacteria. What is the
function of the Q cycle, and how does it carry out this func-
tion? Sfhat electron transport componentsparticipate in the e
cycle in mitochondria, in purple bacteria, and in chloroplasts?
11. \frite the overall reaction of oxygen-generatingphoto-
synthesis.Explain the following statement:the 02 generated
by photosynthesisis simply a by-product of the pathway's
generationof carbohydratesand ATP.
12. Photosynthesiscan be divided into multiple stages.
'What
are the stagesof photosynthesis,and where does each
occur within the chloroplast?Vhere is the sucroseproduced
by photosynthesisgenerated?
13. The photosystemsresponsiblefor absorption of light en-
ergy are composed of two linked components, the reaction
center and an antenna complex. What is the pigment com-
position and role of each in the processof light absorption?
IThat evidenceexists that the pigments found in thesecom-
ponents are involved in photosynthesis?
14. Photosynthesisin green and purple bacteria does not
produce 02. 'Why?How can theseorganismsstill usephoto-
'S7hat
synthesisto produce ATP? moleculesserveas electron
donors in theseorganisms?
15. Chloroplasts conrain two photosystems.Sfhat is the func-
tion of each?For linear electron flow, diagram the flow of elec-
trons from photon absorption to NADPH formation. Ifhat
does the energy stored in the form of NADPH synthesize?
16. The Calvin cycle reactionsthar fix CO2 do not function
in the dark. What are the likely reasonsfor this? How are
thesereactionsregulated by light?
17. Rubisco,which may be the most abundant protein on
earth, plays a key role in the synthesisof carbohydrates in
organismsthat usephotosynthesis.What is rubisco. where is
it located, and what function does it serve?
Analyzethe Data
A proton gradient can be analyzed with fluorescent dyes
whose emission-intensityprofiles depend on pH. One of the
most useful dyes for measuring the pH gradient acrossml-
tochondrial membranes is the membrane-impermeant,
water-solublefluorophore 2',7' -bis-(2-carboxyethyl)-5(6)-
carboxyfluorescein(BCECF). The effect of pH on the emis-
sion intensity of BCECE,excited at 505 nm. is shown in the
accompanyingfigure. In one studg sealedvesiclescontaining
this compound were prepared by mixing unsealed,isolated
inner mitochondrial membraneswith BCECF; after resealing
of the membranes,the vesicleswere collected by centrifuga-
tion and then resuspendedin nonfluorescentmedium.
a. When thesevesicleswere incubatedin a physiological
buffer containingNADH, ADR Pi, and 02, the fluorescenceof
BCECF trapped insidegradually decreasedin intensity. What
does this decreasein fluorescentintensity suggestabout this
vesicularpreparation?
b. How would you expect the concentrationsof ADP,
P1,and 02 to changeduring the courseof the experiment de-
scribedin oart a? Whv?
q The Electron Transport Chain and Generation of the Proton-
0)
c
c Babcock,G. 1,999.How oxygen is activatedand reducedin
q respiration.Proc. Nat'l. Acad. Sci.USA 96:12971-12973.
q 'l'997
E nature'smodular, multipurposestructures.Science27 7 :65 3-6 5 9 .
tJJ
450 500 550 600 650
W a v e l e n g t(hn m )
c. After the vesicleswere incubated in buffer contain-
ing ADP, P;, and 02 for a period of time, addition of dinitro-
phenol caused an increasein BCECF fluorescence.In con-
trast, addition of valinomycin produced only a small
transient effect. Explain thesefindings.
d. Predict the outcome of an experiment performed as
describedin part a if brown-fat tissuewas used as a source
of unsealed,isolated inner mitochondrial membranes. Ex-
plain your answer.
References
First Steps of Glucose and Fatty Acid Catabolism: Glycolysis
and the Citric Add Cycle
'W.
Berg,J., J. Tymoczko, and L. Stryer.2002. Biochemistry,Sth ed.
H. Freemanand Company,chaps.16 and 1,7.
Canfield,D. E. 2005. The early history of atmosphericoxygen:
homageto Robert M. Garrels.Ann. Reu.Earth Planet.Sci.33:1.-36.
Chan, D.C. 2005. Mitochondria: dynamic organellesin disease,
aging, and development.Cell 125(7l:'1.241-1.2 52.
Depre, C., M. Rider, and L. Hue. 1998. Mechanismsof control
of heart glycolysis.Eur. J. Biochem.258:277:290.
Eaton, S., K. Bartlett, and M. Pourfarzam.1.996.Mammalian
mitochondrial beta-oxidation. Biochem./. 320( Part 2\:345-S57.
Fersht,A. 1999. Structureand Mechanismin Protein Science:A
Guide to Enzyme Catalysisand Protein Folding. W. H. Freemanand
Comoanv.
Fothergill-Gilmore,L. A., and P. A. Michels. 1993. Evolution of
glycolysis.Prog. Biophys. Mol. Biol. 59:105-135.
Guest,J. R., and G. C. Russell.1'992.Complexesand complexi-
ties of the citric acid cycle in Escherichia coli. Curr. Top. Cell Reg.
t t
JJiZJ
1 ) i 1 / ?
t-Za / .
Krebs, H. A. 1,970.The history of the tricarboxylic acid cycle.
Perspect.Biol. Med. l4:154-1'70.
Nelson, D. L., and M. M. Cox. 2000. LehningerPrinciplesof
Biochemistry.Worth, chaps.1'4-1'7,1'9.
Pilkis, S. J., T.H. Claus,I. J. Kurland, and A. J. Lange. 1'995.
5-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic
signalingenzyme.Ann. Reu.Biochem. 64:799-83 5.
Rasmussen,B., and R. Wolfe. 1999. Regulationof fatty acid
oxidation in skeletalmuscle.Ann. Reu.Nutrition 19:463-484.
Velot, C., M. Mixon, M. Teige,and P. Srere.1997. Model of a
quinary structure betweenKrebs TCA cycle enzymes:a model for
the metabolon.Biochemistry36:1'4271-14276.
lWanders,R. and H. R. !(aterham.2006. Biochemistryof
J.,
mammalianperoxisomesrevisited.Annu. Reu.Biocltem. 75 :295-332.
Motive Force
Beinert,H., R. Holm, and E. Miinck . . kon-sulfur clusters:
Brandt, U.2006. EnergyConvertingNADH:quinone oxidore-
ductase(complexll. Ann. Reu.Biochem. 75:165-187.
Brandt, U., and B. Trumpower. 1'994.Theprotonmotive Q cyclein
mitochondria and bacteria.Crit. Reu.Biochem.Mol. Biol.29:165-197.
Darrouzet.E.. C. Moser. P. L. Dutton, and F. Daldal. 2001.
Larqe scaledomain movementin cytochromebc1: a new devicefor
elecirontransfer in proteins.TrendsBiochem. Sci.26:445451.
Grigorieff, N. 1999. Structureof the respiratoryNADH:ubiquinone
oxidoreductase(complexl). Curr. Opin. Struc.Biol.9:476483.
Hosler,J. P.,S. Ferguson-Miller,,and D. A' Mills. 2006. Energy
transduction:proton transferthrough the respiratorycomplexes.
Ann. Reu.Biochem.7 5 :165-L87.
Michel, H., J. Behr,A. Harrenga,and A. Kannt. 1'998.Cy-
tochrome c oxidase.Ann. Reu.Biophys. Biomol. Struc.27:329-356.
Mitchell, P. 1'979.Keilin's respiratorychain conceptand its chemios-
motic consequences.Science206:11'48-'L'1, 59. (Nobel PrizeLecture.)
Ramirez,B. E., B. Malmstrom, J. R' \7inkler, and H. B. Gray.
1995. The currentsof life: the terminal electron-transfercomplex of
respiration.Pro c. N at' L A cad. Sci. USA 92:1'L949-1'1,95 1,'
Ruitenberg,M., et al. 2002. Reduction of cytochrome c oxidaseby
a secondelectronleadsto proton translocation.Nature 417:99-102.
Saraste,M. 1'999.Oxidative phosphorylationat the fin de
sldcle.Science 283 :l 48 8-1'49 2.
Schafer.E.. et al. 2005. Architectureof activemammalian respi-
ratory chain supercomplexe s. J . Biol. Chem. 281(22):15 370-1 5 375.
Schultz. B.. and S. Chan. 2001. Structuresand proton-pumping
strategiesoi mitochondrial respiratoryenzymes.Ann. Reu.Biophys'
Biomol. Struc.3O:23-65.
Sheeran,F. L., and S. Pepe.2006.Energy deficiencyin the failing -
heart: linking increasedreactiveoxygen speciesand disruption 9f gxida-
tive phospholylation rate. Biochim. Biopbys.Acta. 1757(5-6\:543-5 52'
Tsukihara,T., et al. 1996.The whole structureof the 13-subunit
oxidized cytochromec oxidaseat 2.8 A. Science272:1136-'l'L44'
\falker, J. E. !995. Determinationof the structuresof respira-
tory enzyme-complexes from mammalian mitochondtia. Biochim.
B iophy s. Acta 127| :221-227.
'Wallace,
D. C. 2005. A mitochondrial paradigm of metabolic
and degenerativediseases, aging, and cancer:a dawn for evolution-
ary medicine.Ann. Reu.Genet. 39:359-407.
.
REFERENCES 53,I
 Xia, D., et al. 1997. Crystal structureof the cytochromebc1
complex from bovine heart mitochondria.Science277:60-66.
Zaslavsky,D., and R. Gennis.2000. Proronpumpingby cy-
tochronreoxidase:progressand postulates.Biochim. Bktphys. Acta
I458:164-179.
M.Zhang, E. Mileykovskaya,and W. Dowhan.2005. Cardiolipin
is essentialfor organizationof complexesIII and IV into a supercom-
plex in intactyeastmirochondria.J. Biol. Chem.280(331:2948t9408.
Zhang,Z., et al. 1998.Electrontransferby domarnmovenlenr
in cytochromebc1. Nature 392:677-684.
Harnessing the Proton.Motive Force for
Energy-Requiring
Processes
Aksimentiev, A., L A. Balabin.R. I{. Fillingame.and K.
Schulten.2004. Insightsinro the molecularmelhanisrnof rotation
in the F6 sectorof ATP synthase.Bxtphys.J.86(3)t1332-1344.
Biancher,M. A., J. Hullihen,P. Pedersen, and M. Arnzel.1998
The 2.8 A structureof rat liver F1-ATPase: confieurationof a criti-
cal irrtermediate in ATP synrhesis/hydrolysis. Proc. Nat'1.Acad. Sci.
U S A9 . 5 : 1 1 0 6 5 -110 7 0 .
Bover,P.D.1997. The AT'Psynrhase-asplendidrrolecularma-
chine.Ann. Reu.Biochem.66:7L7-749.
Capaldi,R., and R. Aggeler.2002. Mechanismof the FeF,-typeATp
synthase-a biologicalrotary motor.TrendsBiochem.Sci.27:1 54-160.
Elsron,T., lL Wang,and G. Osrer.1998.Energytransductionin
ATP synthase. Nature 391:-510-512.
Hinkle, P. C. 2005. P/O ratiosof mitochondrialoxidativephos-
phorylation. Biochim. Biophys.Acta. 1706(1-2):1,-11.
Kinosita,K., et al. 1998.Fr-ATPase: a rotary motor madeof a
s i n g l em o l e c u l eC. e l l 9 3 : 2 1 - 2 4 .
Klingenberg,M., and S. Huang. 1999.Structureand functionof
the uncoupling protein from brown adipose rissue.Biochim. Bkt-
p h y s .A c t a l 4 l 5 : 2 7l - 2 9 a .
Nury, H., et al.. 2006. Relationsberweenstructureand function of
the mitochondrialADP/ATP carrter.Ann. Reu.Biocbem.T5:713-741.
Tsunoda,S.,R. Aggeler,M. Yoshrda,and R. Capaldi.2001.
Rotationof the c subunitoligomerin fully functirtnil FoF1ATp
synthase.Proc. Nat'1.Acad. Sci.USA 98:898-902.
Vercesi,A. 8., et al. 2006. Plant uncoupling mitochondrial
proteins.Ann. Reu.Plant Biol. 57:383404.
Yasuda,R., et al. 2001. Resolutionof distinctrotarionalsubstens
by submillisecond kineticanalysisof F1-ATPase. Nature 4lO:898-904.
Photosynthetic pigments
Stagesand Light-Absorbing
_ Ben-Shem, A., F Frolow,and N. Nelson.2003. Crystalsrructure
of plant phorosystem l. Nature 426(6967):630-635.
Blankenship,R. E. 2002. Molecular Mechanismsof photosltn-
t/resls.Blackwell.
_ Deisenhofer,J., and'1. J. R. Norris, eds. 1993. Tbe photosynthetic
ReactionCenter,vols. and 2. Academicpress.
McDermott, G., et al. 1995.Crystalstructureof an integral
membranelight-harvestingcomplex from photosyntheticbaiteria.
Natwre364:517.
Nelson, N., and C. F. Yocum. 2006. Structureand function of
photosystems I and II. Ann. Reu.Plant Biol. 57:521-565.
Prince,R. 1996. Photosynthesis: the Z-schemerevisited.Trends
Bic;chem. Sci.21:121-122.
Wollman, F. A. 2001. Statetransitionsrevealthe dynamicsand
flexibiliry of rhe photosynrheticappararus.EMBO I. 2:,0t3623-3630.
MolecularAnalysisof Photosystems
Allen, J. F. 2002. Photosynthesisof A fp-electrons! proron
pumps, rotors, and poise.Cell 110:273-276.
_ Aro, E. M., I. Virgin, and B. Andersson.1993.photoinhibirion
ot pbotosystemII: Ltactivation, protein damage,and turnover.
Biochim.Biophys.Acta Lt43:1|3-1 34.
532 CHAPTER
12 I CELLULAR
ENERGETICS
Deisenhofer,J., and H. Michel. 1989. The photosyntheticreac-
tion centerfrom the purple bacteriumRbodopseudomonasuiridis.
Science245t1463-1473. (Nobel Prize Lecture.)
Deisenhofer,I., and H. Michel. 1991. Structuresof bacterial
photosynthetic reactioncenters.Ann. Reu.Cell Biol.7:1-23.
Dekker,J. P., and E. J. Boekema.2005. Supramolecularorgani-
zarion of thylakoid membraneproteins in green plants.Biochim.
B iophys. A cta. 1706('1. -21:1,2-39.
Finazzi,G. 2005. The centralrole of the greenalga Chlamy-
domondsreinhardtii in revealingthe mechanismof statetransitions
J. Exp. Bot. 56(411):383-388.
Haldrup, A., P.Jensen,C. Lunde, and H. Schelle r. 2001..Balance
of power: a view of the mechanismof photosyntheticstaterransi-
tions. TrendsPlant Sci.6:301-30.5.
Hankamer,B., J. Barber,and E. Boekema.1.997.Structureand
membraneorganizationof photosystemII from green plants.Ann.
Reu.Plant Physiol. Plant Mol. Biol.48:641-672.
Heathcote,P.,P. Fyfe, and M. Jones.2002. Reactioncentres:the
structureand evolution of biological solar power. TrendsBiochem.
Sci.27:79-87.
Horton, P.,A. Ruban, and R. Walters.1,996.Regulationof light
harvestingin green plants.Ann. Reu.Plant Physiol. Plant Mol. Biol.
47:655-684.
Joliot, P.,and A. Joliot. 2005. Quantificarion of cyclic and linear
flows in plants.Proc. Natl. Acad. Sci.USA 102(131:49134918.
Jordan, P.,et al. 2001 Three-dimensionalstructureof cyanobac-
terialph()tosystem I at 2.5 A resolution.Nature 411:909-917.
KUhlbrandr,\7. 2001. Chlorophyllsgalore.Nature 411:896-898.
Martin, J. L., and M. H. Vos. 1992. Femtosecondbiology.Ann.
Reu.Biophys. Biomol. Struc.2l:'1,99-222.
Penner-Hahn,J. 1998. Structural characterizationof the Mn site in
the photosynthericoxygen-evolvingcomplex. Struc. Bonding 90:'1,-36.
Tommos, C., and G. Babcock.1998. Oxygen production in na-
ture: a light-drivenmetalloradicalenzymeprocess.Acc. Chem. Res.
3l:18-25.
CO2Metabolism
DuringPhotosynthesis
Buchanan,B. B. 1991,.Regulationof CO2 assimilationin oxy-
genic photosynthesis:the ferredoxin/thioredoxinsystem.Perspective
on its discovery,presentstatus,and future development.Arch.
Biochem. Biophys.288: | -9.
Gutteridge,S., and J. Pierce.2006. A unified rheory for the basis
of the limitations of the primary reacrionof photosyntheticCO2 fix-
ation: was Dr. Panp;loss right?Proc. Natl. Acad. Sci.USA
103:7203-7204.
Portis,A. 1992. Regulationof ribulose 1,S-bisphosphate car'
boxylase/oxygenase activity.Ann. Reu.Plant Pbysiol.Plant Mol.
Biol.43:415437.
Rawsthorne,5.1992. Towardsan understanding of C3-Capho-
tosynthesis. Essays Biocbem.27:135-146.
Rokka, A.,I. Zhang, and E.-M. Aro. 2001. Rubrscoactrvase: an
enzymewith a remperature-dependent dual function? Plant l.
25:463-472.
Sage,R., and J. Colemana.2001,. Effectsof low atmosphericCO,-
on plants:more than a thing of the past.TrendsPlant Sci.Z:18-24.
Schneider,G., Y. Lindqvisr, and C. I. Branden.1992. Rubisco:
structureand mechanism.Ann. Reu.Biophys. Biomol. Struc.
2l:119-153.
Tcherkez.,G. G., G. D. Farquhar,and T. J. Andrews. 2006.De-
spite slow catalysisand confusedsubstrarespecificity,all ribulose
bisphosphatecarboxylasesmay be nearly perfectly optimized..proc.
Natl. Acad. Sci.USA 103(19):7246-7251.
'Wolosiuk,
R. A., M. A. Ballicora,and K. Hagelin.1993.The
reductivepentosephosphatecycle for photosyr-rthetic CO2 assimila-
tion: enzymemodulation.FASEB[. 7:622-637.
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i
SECRETORY
PATHWAY
FIGURE 13-1 Overviewof major protein-sorting the matrixor stromal space, respectivelyOtherproteins aresortedto
pathwaysin eukaryotes. All nuclear-encodedmRNAs are othersubcompartments of theseorganellesby additional sorting
translated on cytosolic
ribosomesRight(nonsecretory pathways). stepsNuclear proteins enterandexitthroughvisible poresin the
Synthesis of proteins
lacking an ERsignalsequence iscompleted nuclearenvelopeLeft(secretory pathway):Ribosomes synthesizing
on freeribosomes (step[) Thoseproteins thatcontainno nascent proteinsin the secretorypathwayaredirected to the rough
targeting sequence arereleased intothe cytosol
andrematn tnere endoplasmic reticulum (ER)byan ERsignalsequence (pink;steps[,
(stepE). Proteins with an organelle-specific
targeting
sequence Z) Aftertranslation iscompleted on the ER,theseproteins can
(pink)firstarereleasedintothecytosol (stepZ) butthenare moveviatransport vesicles to the Golgicomplex (stepB) Further
imported intomitochondria, chloroplasts,peroxisomes,
or the sortingdeliversproteins eitherto the plasmamembrane or to
nucleus (stepsB-E) Mitochondrial andchloroplastproteins lysosomes (stepsEE, @ ) Theprocesses underlying the secretory
typicallypassthroughtheouterandinnermembranes to enter pathway(stepsB, El, shadedbox)arediscussed in Chapter'l4

well as in the plasma membrane. Targeting to the ER gener-
ally involves nascent proteins still in the process of being
synthesized.Once translocatedacross the ER membrane,
proteins are assembledinto their native conformation by
protein-folding catalystspresenrin the lumen of the ER. This
process is monitored carefullS and only after their folding
and assemblyis complete are proteins permitted to be trans-
ported out of the ER to other organelles.Proteins are also
modified in various ways after translocation into the ER.
These modifications can include addition of carbohydrate
groups, stabilization of protein structure through disulfide
bond formation, and specificproteolytic cleavages.Proteins

534 CHAPTER
13 | M O V T N GP R O T E t NtSN T O M E M B R A N E A
SN D O R G A N E L L E S
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uI 'sllrJ reulre rltporJued qtrlr\ sluaurradxa 3ur1age1-aspd ueuo soJuenbesyeu8rs'dleurg 'suratord roldore.r Jeqlo PUP
dq parepnnla lsrrJ ara.t uralord drotarcas e yo srsoqlu.,{s saruonbasleu8rs.raqlola.{ uo spuedapSurlrosqons lelleue8ro
erJtreryedlelerpaurur rnJJo lugt stuo^ato aruanbesaq1 le8rel eqr ulqll^/r luaurl.reduof,qns e qf,EOr ol requnt
'demqredsrqt Surdpnrsur pesndlapm uoaq o^Er{daqt 'acuep pauos dlluanbasqnse:e sutelord aruos AIV ;o stsdlo;pdq
-unge ]eer8 ur (r31ogpue UE ''3'a) de,vrqred,{rorarcasaql
;o sE qJns ssarord e]qero^E dllecrle8reuauE ol uollelolsuerl
selleue8roeql urEtuor sylec.{rotonas qJns asnpJJfl'ourlsoturar{l Surldnor dq pa,rerqcr dllensn sl 'useldoldf, oql olul lno
ot ppel terlt selnDnp olur pelerres aft wql sarudzuaarrrtsa8rp >1ceqSurp{s lnoqtll\ 'alyeue8touP olul uralo.rdE Jo raJsuerl
'aluelsur.ro;'s11arreurle
IEJeAes Jo sertrluenba8rel ozrsaqluds Ieuonrarrprun erll redepq euerquou aqr q8nolqr ssed ol
JrlpeJf,ued'surelo.rdcr;rcadsto slunoup a8rel ;o uorloJJosJoI ureto.rdeqr s.^oll€ l:-tll Puueq) uotlo)olsuotT to PUDIauos
pazqenedsare sller yo seddruretJet '(suretordxrJlpru relnllel ol porreJsuertsI ulel{f, utalo.rd aq} loldof,er Surpuodsa:ror
-erxa ''3'a) suralord dlarrerrE eterfossllel aqr qlr/v\palJEJOluIseq acuanbasleu3rs u Sututeluoc utalord
Jo ile g8noqlly
rardeqr 3urmo11o1 or{t ur possorppesr trodsue.rl p aluo 'surta8rul 1o dlnr;roeds aql suralo8 ecuonbasleu8ts
.seJuenbas
alJrse^1o ssecordaql 'UA eql retue ,(aqrse pJrJrporueJEpue e ur pepof,uouorleuroJur JI{} reqt Sut.rnsuesnql
p1o;suralo.rdaorl 't'€I uorlf,osur 'oroq roprsuof,osle olN rol leu8rs ;o spur>1cr;rcedsol dyuo PuIq rpgr sutalord roldacar
-deqc srqr ur possnlsrpsallaue8rorer{to eqr ot suratord 3ur
Jo Jo les e serrrer allaue8ro qcea'sap4f,ad lpuBrsto sacuanbas
-la8.retol Jppurs ,{ylecgsrueqceru eJEsessacordesor{lesnpJoq Su4a7nx-a4wdn pe11etoslr aru esaqr :(1-E1 arn8rg eas)
'euerqruetu 'sptce ourtue
UE er{l olur peuasur aq UEJsura}o.rdJruos A\oq saruanbespu8rs su dlporraua8 u,vloul 05-02
';1eslt utelord eqr
pue UA aql ot pareSretere suratord ,(rolarcas.{\oq ssnf,srp ;o seruenbes urqtl.ry\dllensn ;o ocuanb
a.\\ relder{f,srr.{lJo suorlreso,v\l tsrrJeqt uI 'selrrse,r;o Sursn; -es prle ouILuE eql ulqll.ry\poPorua sI uolleullsop a11aue3
pue Surppnqq8norqr of,EJrnsIIef ro
(seuososll 'r31o5 aql ot -ro relnrrued e ot utelo.rd v le?rct ol uoIlEIuroJuI agt let{l
trodsuerl uretord (t) pue'uerunl UE erp eprsuruou€f,l;Iporu 'ef,uelsurro; 1r,roo1A{ou el( 'salleur8ro JelnllaleJlul snoIJEA
pue Surploy urelord (7) 'aue.rqurau UA eqt ssorre uou aqt IIE ol Surl.rosutoto:d uralo8 susru?qJalullseq elues el{l
-EJolsuertpue srsaqtudsuralord (1) :sdarsaarqt se,rlo,rurdezn 'suo'Er',reuosarrdsep
r'gr u^ oqst^"U ttf,iJi"ilirq*U*,
-qled drolerJaseqf rusrupqreu lrseq eql 'suralo.td{.toptt
Jo 's;edeltq
-as sE dle^IlJellof, sulelord asaqr ol raJar o.&\tlIfIIduIS roJ pelelndrueru ,(1aa:;aq uEf, stuolsds orlll uI qcn5
'(4rt 't-tt arn8rg) saurososdlpue '€1og 'UA aq] Jo euprq prdrl lenr;rup otul palerod.rocut sluauodtuoo urelo.rd peg
-ruoru eqt .ro acrds -rrnd aql Sursnpetntrlsuorer ueoq e^Pq IIOI eqi ut sassalo-rd
leurrunl snoanbe Jrll ur aprserler{l surel
-ord pue suralord patorJesSurt.ros pue Surzrsaqtuds ro1 delr uorlprolsuErt rolpur eql Jo duetu
'uorltPPP uI 'seuerg
-qled drolerces eruesaql .{llerluasseesn slleJ rrrod.re4na -uer.u rElnllerPrlur lueraJJlpeql Jo I{lPe ssorf,?uollef,olsuerl
11y
ro; pa.unber stueuodulof, relnllel eqr Jo dueru pet;ttuept
ouerquawul aql ssor)vsurolord a,reqstsrSolorq11ar'sdatsuorleJolsuerl relncrlred elnJexJ ol
elqeun sluulnlu Surd;rruaprJoJ suooJf,srtleuaS Pu€ spoqreur
'sa1nr
fto1ene5lo uorle)otsue4l$l
uorlecr;rrnd leJlrueqlolg Jo uolleulqruoo e Sursn
-elour
llprus pup suol ot reureq e se .radepqaql Surldnrsrp
'sarod realonu q8norqr snolJnu eql lnoqtr^r euErqtueluP ssoJrepelerolsuErl eg Plnol salnJalou
to tno pue olur surato.rd
'satuosrxoradpue 'slseld uretord a8rel dlezrrreler,vloqpue auerglueu cr;nads euo dluo
;o uodsue.rt agt JaAoJa.n tlleurg
-orolql 'errpuoqcolnu ol suratord;o Surta8reteqursep ueql ot potrerrp eq plnor utalo.rdua,l,t8e .ttoq :3urg;eq attnb era.'rn
'JoIJ
ar16 de.nqred drolanas ar{t retue deql se suralo.rd ot Jnf,f,o d11erlrurssocord Surlros-utelord srql Jo saJnlee' oA/rI
-elur slr olur peloru Jo eueJqueu o1laue8roJI{l olul pelJesul
leqt suortplrtrpou leuorlelsuerl-1sodaql Surpnlcur'Ug eqt ol
surato;dJo Surla8relreloJ e.taterdeqr egr 1o rred lsrrJ eqr uI dlluanbesqnspue sellaueSrorrlnllef,Erlul Jo seuerqlueu eql
ol pala8rel are sutalord ^\oq euluexa a.vrleldeqc
:,#l""lrr",
ieueJqureu eql ssoJJEJaJsuEJl
'V -leferlur Jo seuprqrueruaql ol Surla8rel utalo.rdruor; .{lluec
Ieuortrerrprunse^rrp wql KBnua Jo eJrnos eql sr reql1 -r;ru8rs sJeJlp lr dlleousrueqlatu asnef,agrordeqr lxJu JI{l
'I
isuretuopuroto.rdpJplo; ur Surlros uratord Peseq-olllsa^ssnf,slPe11.'@oqPapvqs
atepouurofJe lr llr,ry\lo 'alels peplotun ue ur dluo q8norqr -91 arn8rg aes)raqloue to euerqrueu aql qllltl osn; uaql pue
sseduer suralord leql ,{orreu os Ieuueql eqt sr 'relnrrlred e1laue3.ro auo Jo auergueu oqr tuo{ Pnq 13qrself,Ise^IIPtus
u1 i.radelq oueJqurou oqt ssorf,€suralordJo reJsuprts.^aol]E Jo uorlfe aqr dq dezrrqrBd ,(rolercaseqr 3uo1epelrodsuerl a.re
'r31o5 eql sI uolleullsoP
wql Puupq) uotlr2?olsup4eqr Jo ernlrnrts eq] sr rEqlN 't af,Erns lyar ro 'aruosos.,{1 IBUI}esoq,,la
 s l ' l ' l l N V 9 u o c N V s 3 N V U S l A l l Ao l _ N t s N E I o U d 9 N t n o N | €t Ull_dVH) 9€S
u a e q r o t a u o s o q r r e q l s t J e r t p u r a t o J d t u e J s E ua q l u r 'ouErqujeur
uE aqt ssorf,Epepeerql sr
af,uonbas Ieu8rs UA enprser-0€ot -9I e
'losotdr eqt ur seruos uratord .{rolarcaslualseu E Jo snurrurel-N er{l ,ra,oqpue sur8
-ogrJ eeJJuo sur8aquratord d.rola.rras e ;o srsaqtudsJaUV -ag srsaqludsrraqt reue .{llroqs surato.rddrotertes se pazruSo
-rar ere saprtdaddlod .4orl Jo uortsanbeqt pasrersrqt se qtns
ul aql ol sulaloJd Alolelra5 luo)seN sle6tel
stueruuodxg 'sallrsel UE ]o ueunl oql un{tr^^ dn pue sauros
a ) u a n b o sl e u O l Sl e u l u r a l - N ) t q o q d o r p f HV -oqrr aseqt ,{g parrpord seprrdad.,tlod eqt (auerquetu Ug
aql Jo arpJ rrlosotdr aqr or punoq sauosoqu uo pazrsaqtu.{s
g3 qbnoraq] +o uauJnloql ol luole^tnbo'soLlosol)tu.l eql optsul
aresurelordepeu Ilnnauaq] ]eq] solp)tpur6urpurlsrql luablelepqlrm
are surato.rdd.rola.rcasq8noqlle tEql elertsuoruap 'asealord
e qlr.^apeluen ore sller palaqey-as1nd Luo{ palelosr seruos
]uaurlparl{q pa,{otlsep}slt}st auetquiau Ieuloso.l)truoq} }o lar.lleq
-orrnu qrrq.{\ ur 'g-91 ernSrg ur percrdapstuaurr.radxaeq1
ifrlrqeauradeql 1rIluo eseelotdeq1{q palseOrp ele sou.]osolltuJeq}
'(eprslnoeqt uo saruosoqu)
q]lM paler)osse surelordfuoleltespalaqeloql luebtalepe +o o)uosqe IJaJttetur eql ur puno; teql se
ro a)uosordaq] ur oseo]oldp q1^^paleol] aje soLuoso.t)Lll perltnd eq1
16raldeql) uorlebnltlua>luerpet6-,{1suep asol)nspue let}uola+}tp
]o uorleurqulo) e Iq ulaq+u.tot1peletedaseq ue) pue sa;leuebro
snouprqurolr roqlo ueq] {lrsuep1ue,4onq teleetOqfnul e aneq I opeled9 1o,4sapno;(e)upd] a)uanbasleu6rsX3
sotuosor)rLU 'solxosoqu punoq ue 6urureluotaprided{1od uralord6uunp uJnln)t}al
aneq{eq} esne>egsau;osol.l/upalle) e 1osrsaq}u,{s
sal)rsa^ lleursolut g3 qOnoteq16uueaqspue oue..tqLUaLU euseldeq1 >rLuseldopue aq] ol po]rnr)ar1a6seulosoqupunoq-ouelqulon
6uunlrer,r'pezruabouoqole ueq] sllo)oql poloqelaulo)aqsurelotd le)rluoprare seulosoqutrlosolI: aarJpue punoq-auelqulou]eqt
pezrsaqluris{lnneu{;uo }eLllos spt)eoutL|epalaqelotpej q}tMoLUt} oloN Ul eq1uo srsoqlu,{s )r}puloq)S(q)
ure}ord1o uorleluasa.rder
+ar.rq e Jo] palpqn)ura.resllo) srsaqlu{s ta11e
{;poqs uaLUnl !l aql o} surelordrtolerlesuouloqlo lo trlosol{t 6urzrsaqlu{s ole asaLl}
pozrle)ol eresurelotdfuolettes lpq] alellsuoLuapslueutuadxa 6urlaqe1 Alqeunserd1luapr^o oJesoLL]osoqt.t
(aal])porl)eueun-ouelquta[! Mo]
'Ul oql regua suralo.rdtuo1a,oe5
€-€t tunglJ lvl_NllAjlUldXJ < v sourosoqupoqle]le-auelquaLu uo pau]lo]ole pue pe]oj)os0q o1
arp lla) ,roadi{tsrq}{q pezrseqlu{s surelotdoq} +o }solA llo).reut)e
urolojd Alololsas u ralord Joleloas
rrleer>uede ur g3 q6noraq] o] poq)e1]esoLuosoql1o qder6ottrut
1 o u o r l s a b r po p ;o uor1se616
t uoj])oll (e) 'Ul q6nor aql lo arnpnlts Z-€l lunglJ V
t t
AI
t
t
:tr4 L r ' rt
t #
t uolunlH3
+
auerqursruul
aulosoou
losol^C
P"qrntiV
luebrelep
qllM leall s l r un qn s
leurosoqru
saruosoqu
poqcelle qu,v\
saruoso,tctli^l
(q)
' rrrrl9'6
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f f i nl^ $'sE'
v{$ Qqr
w
I , ;:. i , ...,'., l, :r,:.., '.$,1'1
suerquror.rJ
uf uaurnl uf losol^3
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rau€ saurl ruOrOtJIPlE sarnlxlu uoIlJEer egl or PepPP
aqt uo rotderor dUS eql ol Surpurqlq auerqueru UE Jql oJeA\ satuosolcnu tsJuJurrradxeJSaI{l uI 'JnJJo ol uoll
ol xalduor auosoqrr-uratordluacseueql sle8rel urqt dUS -pJolsuErtrot JOProut luasa.rdJq lsntu sJtuosoJf,Iulqllqr^
lxaldruor a3.re1e Sururol 'lrunqns Ipuosoqrr a8.reyaql se le srsaqtudsutatord 1o a8els astrard oql eullurataP ol
IIO,,\\ sp uretord tuerseu E ur aJuanbesleu8rsUE or{rqtoq or peuSrsapere,ry\ sluorurradxaluanbasqn5'(y-E1 orn8rg) srnc
sputq dlruetsuerlteql alrrtred uralordoalonuoqrrrrlosotdc -f,o seruosoJf,rru eql olur lJodsuerl urolord ou 'PolalduoJ
p sl dUS eqJ 'ouprqtuetu oql ur parelol 'roldere.r sr srseqtudsuralord ralJE IUOls.,(s eqr 01 POPp?ere sotuosorr
UE
str pue (495) oprlred uorlruSorar-leu8rs eql arp 8ur -rr.u 'srsoqtudsuralord Surrnp luesald erE saluosorf,rluoql
JI
-la8ret srql ur stuauodruoc .{a4 o,uo oql 'arar{t uaqt }a8-ret dluo peloruoJ acuanbesI€UBISsll sEL{Pue seluosoJJltu
;r
lsntu tusruEqceru uortruSorerecuanbas-leu8rs e'auerqueru olur pelerodJorursr uretoJd.{rolarcaslecrd& P lPql POlEJls
relnllor ror{lo due rltr^{ tou tnq euerqr.uau UE Jqr qtr,lr -uouep urotsds slql qll.&\ sluaruuedxe IEIIIuI 'sof,uonb
uorterJosse ur pazrsaqludsa;e sureto.rddrolarces acurg -as
leu8rs UE Jo alpJ pup uollrunJ aql PaIJITEIIa^Pq
sotuosoqrJPunoq u^\o Jlaql ;o paddrrrs soluosorflul Pue
surslord6urz^;o.rpAH-dIg o^ I ^q 'ulelord drolaroase Surpocue 'tualsdsSutzrseqluds
VNUtu
p a l e r l r u ls l u o r l e ) o l s u e 4l e u o t l e l s u e J l o ) -uratord rerJ-lloJ e 3utzrlr1n salPnts IerllueqJoIfl
.auerqrue{u
ua eql
'uorl9Jolsu€Jl sE ol uretord eqt Sulla8r€l ro; elqrsuodsardreutqceuraql qll^{
IeuorlEIsuPJlof,
ol peJlelerssaro.rd€ 'oLuosoqrJ agr or punoq ylrlssr urato-rd sefuenbas 1eu3rs;o uollf,eJolul eql Jot IefIIIJJ SI lEr{l elIS
(tuarseu)pazrsaqruds dlaralduocur aqt elrr{1\^. sur8aquorunl Surpurq € ruroJ soJuenbasleu8rs gE Jo erof, aql uI senplsor
UE aql olur sutatord drotarros lsolu Jo trodsueJl aLItsnql rrqoqdorpdq aqt snqJ 'uaunl Ug aqr ol pererolsuerl
'(97-y an3rg aas) auosogrr er{t ur sr uralo;d tllosoldr pelllpotu P qlns 'rrqoqdorpdlJ PUE
]auupr{re urqlr^.\porrnq
';;o pe^eell aq 3uo1 dlluarcrttns sI acuanbasPaPPBel{l Papl^ord 'saJuenbas
Ilrls arp sprlB ounuE os Jo 0t lxeu eqt pue
'auosoqrr aql prre ourrue Ieullurol-N PaPpB tltl./lr surolord rrlosol,{r
llr./!\ralel teqt eouanbasleu8rsaqr Surpnlrur
ruorj aPnrlord os .ro sprrE ourruE0t rsrrJer{l 'lurod srqt tv parnpord e^Eq srel{rruasal'sanbruqlJrVNCI lueulqulorer
'uatunl Sursn 'ueunl eql otul aueJqlueul Ug oql ssorJ ol alqeun
IeruosoJJrrueqt ur pozrletol oq ol uraloJdf.rolanas (aJuanbasuofE
patalduor egl ro; ropro ur reqraSotprlull ere sprru ourrup '1osol.{ceql uI suleruarurelord
PorJrpou oql
os ro 0/ lsrrJ eqr eroteq popp€ 0q tsntu seuosorf,rr.ulEr{l sy 'aruanbesleu8rse se uorlJunJ ol uralord e to snulluJel-N
pJ,ln.oqs sluarurrodxeasaql 'un8aq peq srseqtu.(suratord aqr;o dlrlrqeaqt qsIIoqEupr uolletntu dq eror rtqoqdorpdq
oql otur spne ouILuEpa8reqc to uoltrnporlul eql ro aruanb
-as yeu8rse ruort spIJE oultue crqoqdotpdq el{l Jo Iero^as
(etuelsul roC 'uollJun; rIaIJrroJ
to uortalep orlcads oqt IEII
-uassosr seouanbas leu8rs gg Jo eror crqoqdorpdq eq1
acuenoes 'sllor ur
;eubrs1o le^ourorpue PunoJsurol
ecuenbes;eubrs ourosoJcr.lr olur urelo.rd;o -old at{l uI luasard tou dyyensne:r sacuanbespu
,,elnleru,,
l n o q i l Mu r e q c u o o s u e r ll e u o r l e l s u e r l o c -8rs snqr lauosoqrr oqt uo Surle8uola
uraloroarnleN llrls sr 1r alrqzrtutalo-ld
eqt urorJ pe^po]Jst oluanbasluu8ls aql'surelord drolercas
'(a.ror
lsoru roJ 'uotuluoJ ur JIDII a,req .,{aqlJSLi!\Jeqlolnq
aql) senprsarcrqoqdorpfq U-g Jo qllorls snonulluoJ E
ot tuarelpe splfe oultue pa8reqc dle.ttlrsodeJotu Jo euo uIEl
-uol IIE suralo:d drolarcasluarotllP Jo sef,uenbesleu8rs aq1
'pazrsaqtuds ag ol uralord eqr;o tred lsrlt eql 'uralord aqt 1o
snurrural-N erp lp patelol sr dllurrdll aruenbasleu8rsgg uy
'({tt't-tt arn8rgeas)auerquraruUfl aqr ssorreeprrdaddlod
:srsorllu^suralordoor]-lloJ (q)
lueserdsaurosorcrLU
Sur.r,rorBoql to uolleJolsuEJl saleIlIuI Pue auPJqluetu
ecuanbes;eubrs
JOleAOUrAr OU
:soLuosolcrLU olul
u o r l e r o o r o c uorN (q)stsaqlu^s uralotd6uttnpluesardeleseuosolltu.t ;t {1uo
a>uanbes leu6ts s1tsasolpueeuelqulaLu al)lsenaql ssol)epale)olsuel]
srlnq (e)saulosol)tul]o a)uasqe stutalold
oql ul pozlsaqluAs
fuoler>esaql pappestutalotd,to1er>es e 6utpoluaVNUUr q)lqMo]
>r1oso1b
ser-ul{zue puei11g'dIV'sVNU]'saulosoqll 6uturel
leuot1>un;
-uot uels{soarJ-llo) e urlno paule)stsrsaqlu,is (g-g;etn6t3eas)
t souerqurauJ Ul o] luelentnbo oal,-auosoqll
aleq)tqM'sottlosor)ll.lJ jo
6urrrno;;e
uorielosr 'seurosoqtJ
]o
pelPl)osse uleqlsdtlls'suot.*661
solelaq) q)lqM'vlcl q]rMsaLuosol)lul +o]uoulleo{ uol}elsuej}
sauerq urour oi peldnorsrsauiosol)tu.t olut sutelotd,fuolallas+ouollelolsuell
eLuosorcrurppv ln))o
]eq}alerlsuoruep siuer-utladxeaaj]-lloff ;snoauel;nu!s
luasardsourosoruluou lsrseqluAsurelo.rdoorl-lloo (e) uorlelsueJl pue uolle)olsue4 t-€l lun9lJ'lvINltNluldxl <
 s r r ' E N V 9 U O C N V S t N V U S I A 3 I AO t _ N tS N E I O U d 9 N t n O N I tr UtIdVHf 8ES
seop auerqueu UE eqt ur roldalar dUS eqr ot punoq ser1 ue loldaf,or dUS eql r{lr^\ 8ur>llop dg auerqruou UA eqr
xalduror euosoqrr/urerlf,luaJseu/d1s oqt rarle dlug .UA aql o1 xaldruor eruosoqrr-urpr{f, tuJtseu ogl s8ur.rqdUS ar{J
otul uortef,olsueJtro1 3uo1oot SunuocaqruoJJurer{f luef,seu 'uetunl
UE ar{t olur uortef,olsuerl uralo.rdro; perrnba.re.re
eqt slue.tard euosoqrr aart or{l qtr.a pue uralord d.rota.rcas ro atuosoqu eqr qrr.,talJpratur dUS aqt ur seprrdaddlodraqrg
e Jo ulel{r luef,seuaqr i{roq qrl. dus aql uorlreretur 13r{l 'auprqrueu pue suretord
Jo UE ar{l ol uroql sla8rel ,,(1o,rrna1as
stseSSns Surpur;slql'(t-gI arn8rgees)uralord eralduroceql f.role.rrasluerseu;o sleu8rsleurr.urot-Ncrqoqdorpdqeql qtr^
'luasqe ere seurosorf,rr.uueq.v\ slf,protur
;o srsaqludsSurrrqrqurdqareqr tpqt UOIJsnoSoleueue ureluof,or rq8noqr sl tgd to
uralord d.rola;case ;o uorte8uole s,4^olsdUS Jo acuasardeql uor8arorqoqdo-rp,(q eqI'(eS-tI orn8rg)sureql eprstrqoqdo.rp
1{1snor,r,ardpaqrrtsep tuetsdsuorlplsuprt aa{-llel eqt uJ -dq dq paurl sr of,€'rnsrouur asoq^\
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uorlp)olsuBJl utalojd ut €l)tupd uotltu6olcH lBUStSaLll l0 uorl3unj pue ajnl)njls :lselpod
ll[]
FocusAnimation:Synthesisof Secetedand Membrane-Bound
Proteins
mRNA SRP

Signal
sequence

SRPreceptor
GDP+ Pi

Translocon Translocon -tl I l

(closed) (open) , t-aLa
\tt\k
ti....F
a/ a \
sat
aa a f

Folded
protein

FIGURE 13-6 Cotranslational translocation. Steps[, [: polypeptide chainStepE: Asthe polypeptide chainelongates, it
Oncethe ERsignalsequence emerges fromthe ribosome, it isbound passes throughthetranslocon channel intothe ERlumen,where
by a signal-recognition
particle (SRP).
StepS: TheSRpdelivers the thesignalsequence iscleaved by signalpeptidaseandisrapidly
ribosome/nascent polypeptide complex to the SRpreceptor in the ER degradedStep6: Thepeptide chaincontinues to elongate as
membrane. Thisinteractionisstrengthened by bindingof GTpto the mRNAistranslated towardthe 3' end Because the ribosome
boththe SRP anditsreceptor.Step4: Transfer of the ribosome/nascent isattached to thetranslocon, thegrowingchainisextruded
polypeptideto thetranslocon leadsto openingof thistranslocation throughthetranslocon intothe ERlumenStepsfl, S: Once
channel andinsertionof the signalsequence andadjacent segment translationiscomplete, the ribosome is released,
the remainder
of the growingpolypeptide intothe centralpore Boththe SRpand of the proteinisdrawnintothe ERlumen,thetranslocon closes,
SRPreceptor,oncedissociated fromthetranslocon, hydrolyze their andthe oroteinassumes itsnativefoldedconformation.
boundGTPandthenarereadvto initiate theinsertion of another

SRP releasethe nascent chain, allowing elongation to con-
tinue at the normal rate. Thus the SRPand SRPreceptor not
only help mediate interaction of a nascentsecretoryprotein
with the ER membrane but also act together to permit elon-
gation and synthesis of complete proteins only when ER
membranesare present,
Passageof Growing PolypeptidesThrough
the Transloconls Driven by EnergyReleased
D u r i n gT r a n s l a t i o n
Once the SRP and its receptor have targeted a ribosome
synthesizinga secretoryprotein to the ER membrane, the
ribosome and nascentchain are rapidly transferredto the
translocon, a protein-lined channel within the membrane.
As translation continues, the elongating chain passesdi-
rectly from the large ribosomal subunit into the central
pore of the translocon. The 50S ribosomal subunit is
aligned with the pore of the translocon in such a way that
the growing chain is never exposed to the cytoplasm and is
prevented from folding until it reaches the ER lumen
( F i g u r e1 3 - 5 ) .
The translocon was first identified by mutations in the
yeast gene encoding Sec61o,which causeda block in the
translocation of secretoryproteins into the lumen of the
ER. SubsequentlSthree proteins called the Sec61complex
were found to form the mammalian translocon: Sec51cr,
an integral membrane protein with 10 membrane-span-
ning ct helices,and two smaller proteins, termed Sec61B
and Sec51"y.Chemical cross-linking experiments demon-
strated that the translocating polypeptide chain comes
into contact with the Sec61a protein in both yeast and

NF S E C R E T O RPYR O T E I N A
T R A N S L O C A T I OO S C R O s sT H E E R M E M B R A N E 539
 ArtificialmRNA The translocon must be able to allow passageof a wide
variety of polypeptide sequenceswhile remaining sealedto
small moleculessuch as ATP and amino acids. Furthermore,
to maintain the permeability barrier of the ER membrane in
the absenceof a translocating polypeptide, there must be
some way to regulate the translocon so that it is closed in its
Cytosol default state, opening only when a ribosome-nascentchain
complex is bound. A high-resolution structure of the Sec61
complex from the archaebacteriumMethanococcusian-
naschii was recently determined by x-ray crystallograph5
Sec61o
which suggestshow the translocon preservesthe integrity of
Microsomal
membrane Cross- the membrane(Figure13-8).The 10 transmembranehelices
linking of Sec6lcr form a central channel through which the
agent translocating peptide chain passes.A constriction in the
middle of the central pore is lined with hydrophobic
Nascent
protein isoleucine residues that may form a gasket around the
Microsomal translocating peptide. In addition, the structural model of
lumen
NH: the Sec61 complex (which was isolated without a translo-
cating peptide and therefore is presumed to be in a closed
conformation) reveals a short helical peptide plugging the
EXPERIMENTAL FIGURE 13-7 Sec61cis a translocon central channel. Biochemical studies of the Sec51complex
component.Cross-linking experiments showthatSec6lcrisa have shown that the peptide that forms the plug undergoes
translocon component that contactsnascentsecretory proteinsas
a significant conformational change during active translo-
theypassintothe ERlumen.An mRNAencoding the N-terminal
cation, and researchersthink that once a translocating
70 aminoacidsof the secreted oroteinorolactin wastranslated in
(seeFigure13-4b). peptide enters the channel, the plug peptide swings away to
a cell-freesystem containing microsomes The
mRNAlackeda chain-termination codonandcontained onelysine allow translocation to proceed.
codon,nearthe middleof the sequence. Thereactions contained a As the growing polypeptide chain entersthe lumen of the
chemically modifiedlysyl-tRNA in whicha light-activated cross- ER, the signal sequenceis cleavedby signal peptidase,which
linkingreagent wasattached to the lysine sidechainAlthoughthe is a transmembraneER protein associatedwith the translo-
entiremRNAwastranslated, the completed polypeptidecouldnot con (seeFigure 13-6). Signalpeptidaserecognizesa sequence
be released fromthe ribosome withouta chain-termination codon on the C-terminal side of the hydrophobic core of the signal
andthusbecame"stuck"crossing the ERmembrane. Thereaction peptide and cleavesthe chain specifically at this sequence
mixtures thenwereexposed to an intense light,causingthe nascent once it has emergedinto the luminal spaceof the ER. After
chainto become covalentlyboundto whatever proteinswerenear the signal sequencehas been cleaved,the growing polypep-
it in thetranslocon. Whentheexperiment wasperformed using tide moves through the translocon into the ER lumen. The
microsomes frommammalian cells,the nascent chainbecame translocon remains open until translation is completed and
covalently linkedto Sec61oDifferent versions of the prolactin
mRNA
the entire polypeptide chain has moved into the ER lumen.
werecreated sothatthe modified lysineresidue wouldbe placedat
Electron microscopy of the Sec61complex isolated from
different distances fromthe ribosome; cross-linkingto Sec61c was
the ER of eukaryotic cells reveals that three or four copies
observed onlywhenthe modifiedlysine waspositioned withinthe
translocation channel[Adapted fromT.A Rapoport, 1992,Science of Sec51acoassemblein the plane of the membrane.The
258:931, andD Gorlich andT.A Rapoport, 1993, Cell75:615]1 functional significanceof this association between translo-
con channels is not understood at this time. but the
oligomerization of translocon channels may facilitate the
mammalian cells, confirming its identity as a translocon association between the translocon, signal peptidase, and
component (Figure 1 3-7). other luminal protein complexes that participate in the
lfhen microsomes in the cell-free translocation system translocation process.
were replaced with reconstituted phospholipid vesiclescon-
taining only the SRP receptor and Sec61complex, nascent ATPHydrolysisPowersPost-translational
secretory protein was translocated from its SRP/ribosome
Translocationof SomeSecretoryProteins
complex into the vesicles.This finding indicates that the
SRP receptor and the Sec61 complex are the only ER- in Yeast
membrane proteins absolutely required for translocation. In most eukaryotes,secretoryproteins enter the ER by co-
Becauseneither of these can hydrolyze ATP or otherwise translationaltranslocation.In yeast,however,some secretory
provide energyto drive the translocation, the energyderived proteins enter the ER lumen after translation has been com-
from chain elongation at the ribosome appears to be suffi- pleted. In such post-translational translocation, the translo-
cient to push the polypeptide chain acrossthe membrane in cating protein passesthrough the sameSec61translocon that
one direction. is used in cotranslational translocation. However, the SRP

540 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 (a) Side view within the ER lumen. Like other members of the Hsc70
famlIy, BiP has a peptide-binding domain and an ATPasedo-
main. These chaperonesbind and stabilize unfolded or par-
tially folded proteins(seeFigure3-15).
The current model for post-translationaltranslocation of
i
a protein into the ER is outlined in Figure 13-9. Once the N-
; terminal segmentof the protein enters the ER lumen, signal
peptidase cleavesthe signal sequencejust as in cotransla-
tional translocation (step [). Interaction of BiP.ATP with
the luminal portion of the Sec53complex causeshydrolysis
of the bound ATR producing a conformational change in
BiP that promotes its binding to an exposed polypeptide
chain (step [). Sincethe Sec63complex is located near the
;n
translocon, BiP is thus activated at sites where nascent
PIug
polypeptidescan enter the ER. Certain experimentssuggest
that in the absenceof binding to BiP, an unfolded polypep-
tide slides back and forth within the translocon channel.
(b) Top view
P o r er i n g Such random sliding motions rarely result in the entire
polypeptide'scrossingthe ER membrane. Binding of a mole-
cule of BiP.ADP to the luminal portion of the polypeptide
prevents backsliding of the polypeptide out of the ER. As
Lateralexit further inward random sliding exposesmore of the polypep-
to lipid
tide on the luminal side of the ER membrane. successive
bilayer
binding of BiP.ADP moleculesto the polypeptide chain acts
as a ratchet, ultimately drawing the entire polypeptide into
the ER within a few seconds(stepsB and 4). O" a slower
time scale,the BiP molecules spontaneouslyexchangetheir
bound ADP for ATP, leading to releaseof the polypeptide,
Plug which can then fold into its native conformation (steps E
removeo and 6). The recycled BiP.ATP then is ready for another in-
EXPERIMENTAL FIGURE 13-8 Structureof a bacterialSec61 teraction with Sec53.BiP and the Sec53complex are also re-
complex.Thestructure of thedetergent-solubilizedSec61 complex quired for cotranslational translocation. The details of their
fromthearchaebacterium M jannaschl (alsoknownasthe Secy role in this process are not well understood, but they are
complex) wasdetermined byx-raycrystallography(a)A sideview thought to act at an early stageof the processsuch as thread-
showsthe hourglass-shaped channelthroughthe centerof the pore ing the signal peptide into the pore of the translocon.
A ringof isoleucineresiduesat theconstrictedwaistof the poremay The overall reaction carried out by BiP is an important
forma gasketthat keepsthe channel sealedto smallmoleculeseven example of how the chemical energy releasedby the hydrol-
asa translocatingpolypeptide passesthroughthechannelWhenno ysis of ATP can power the mechanical movement of a pro-
translocatingpeptideispresent, thechannel isclosedby a short tein across a membrane. Bacterial cells also use an ATP-
plug,Thisplugisthoughtto moveout of thechannel
helical during driven process for translocating completed proteins across
translocation Inthisviewthefronthalfof proteinhasbeenremoved
the plasma membrane. In bacteria the driving force for
to bettershowthe pore.(b)A viewlookingthroughthe centerof the
translocation comes from a cytosolic ATPaseknown as the
channel showsa region(onthe leftside)wherehelices mayseparate
SecA protein. SecA binds to the cytoplasmic side of the
allowinglateralpassage of a hydrophobictransmembrane domain
intothe lipidbilayer. translocon and hydrolyzes cytosolic AIP. By a mechanism
[AdaptedfromA R Osborne etal, 2005.Ann Rev.
CellDev.Biology21:529l that is not well understood, the SecA protein pushes seg-
ments of the polypeptide through the membrane in a me-
chanical cvcle coupled to the hvdrolvsis of ATP.
and SRP receptor are not involved in post-translational
translocation, and in such casesa direct interaction between
the translocon and the signal sequenceof the completedpro-
tein appears to be sufficient for targeting to the ER mem- Translocation of Secretory Proteins Across
brane. In addition, the driving force for unidirectional t h e E RM e m b r a n e
translocation acrossthe ER membraneis provided by an ad- r Synthesisof secretedproteins, integral plasma-membrane
ditional protein complex known as rhe Se;63 complex and a proteins, and proteins destinedfor the ER, Golgi complex,
member of the Hsc70 family of molecular chaperonesknown or lysosome begins on cytosolic ribosomes,which become
as BiP.The tetrameric Sec63complex is embeddedin the ER attachedto the membraneof the ER, forming the rough ER
membrane in the vicinity of the translocon. whereas BiP is (seeFigure 13-1, left).

T R A N S L O C A T I OO S C R O S ST H E E R M E M B R A N E
NF S E C R E T O RPYR O T E I N A 541
 Translocating
polypeptide
chain

Translocon

ER lumen
NHs*
Cleaved BiP
s i gn a l (bound
sequenc to ATP)

FIGURE 13-9 Post-translational

translocation.Thismechanism
isfairlycommonin yeastandprobably occurs in higher
occasionally
eukaryotes Smallarrowsinsidethetranslocon random
represent
polypeptide
slidingof thetranslocating inwardandoutwardSuccessive
bindingof BiP.ADP to entering
segmentsof the polypeptide
prevents
the chainfromslidingout towardthecytosol[See K E Matlacketal,
1997 . Science
277:938I

r The ER signal sequenceon a nascent secretory protein
consistsof a segmentof hydrophobic amino acids, gener-
ally locatedat the N-terminus.
r In cotranslational translocation, the signal-recognition
particle (SRP) first recognizesand binds the ER signal
sequenceon a nascent secretoryprotein and in turn is
bound by an SRP receptor on the ER membrane, thereby
targeting the ribosome/nascentchain complex to the ER.
r The SRP and SRP receptor then mediate insertion of the
nascent secretoryprotein into the translocon (Sec61com-
plex). Hydrolysis of two moleculesof GTP by the SRP and
its receptor drive this docking processand causethe disso-
ciation of SRP(seeFigures13-5 and 13-6).As the ribosome
attached to the translocon continues translation, the un-
folded protein chain is extruded into the ER lumen. No ad-
ditional energy is required for translocation.
r The translocon contains a central channel lined with
hydrophobic residues that allows transit of an unfolded
protein chain while remaining sealedto ions and small
hydrophilic molecules.In addition, the channel is gated so
that it only is open when a polypeptide is being trans-
located.
r In post-translational translocation, a completed secre-
tory protein is targetedto the ER membrane by interaction
of the signal sequencewith the translocon. The polypeptide
chain is then pulled into the ER by a ratcheting mechanism
that requires ATP hydrolysis by the chaperone BiP, which
stabilizesthe enteringpolypeptide (seeFigure 13-9). In bac-
teria, the driving force for post-translational translocation
comes from SecA,a cytosolic ATPasethat pushespolypep-
tides through the translocon channel.
r In both cotranslational and post-translationaltransloca-
tion, a signal peptidasein the ER membranecleavesthe ER
signal sequencefrom a secretoryprotein soon after the N-
terminus entersthe lumen.
lnsertionof Proteinsinto
the ERMembrane
In previous chapters we have encountered many of the vast
array of integral (transmembrane)proteins that are present
throughout the cell. Each such protein has a unique orien-
tation with respectto the membrane'sphospholipid bilayer.

542 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
Integral membrane proteins located in the ER, Golgi, and
lysosomes and also proteins in the plasma membrane,
which are all synthesizedon the rough ER, remain embed-
ded in the membrane in their unique orientation as they
move to their final destinationsalong the same pathway
followed by soluble secretory proteins (see Figure 13-1,
left).During this transport, the orientation of a membrane
protein is preserved;that is, the samesegmentsof the pro-
tein always face the cytosol, whereas other segments
always face in the opposite direction. Thus the final orien-
tation of these membrane proteins is establishedduring
their biosynthesison the ER membrane.In this secrion,we
first seehow integral proteins can interact with membranes
and then examine how severaltypes of sequences,collec-
tively known as topogenic sequences,direct the membrane
insertion and orientation of various classesof integral pro-
teins. Theseprocessesoccur via modifications of the basic
mechanism used to translocatesoluble secretoryproteins
acrossthe ER membrane.
SeveralTopologicalClassesof Integral
MembraneProteinsAre Synthesized
on the ER
The topology of a membraneprotein refers to the number of
times that its polypeptide chain spansthe membrane and the
orientation of these membrane-spanning segmentswithin
the membrane.The key elementsof a protein that determine
its topology are membrane-spanningsegmentsthemselves,
which usually are ct-helicescontaining 20-25 hydrophobic
amino acids that contribute to energeticallyfavorable inter-
actions within the hydrophobic interior of the phospholipid
bilayer.
Most integral membrane proteins fall into one of the
four topological classesillustrated in Figure 13-10. Topo-
logical classesI, II, and III comprise single-passproteins,
which have only one membrane-spanninga-helical seg-
ment. Type I proteins have a cleaved N-terminal ER signal
sequenceand are anchored in the membrane with their
hydrophilic N-terminal region on the luminal face (also
known as the exoplasmic face) and their hydrophilic C-
terminal region on the cytosolic face. Type II proteins do
not contain a cleavableER signal sequenceand are oriented
with their hydrophilic N-terminal region on the cytosolic
face and their hydrophilic C-terminal region on the exo-
plasmic face (i.e., opposite to type I proteins). Type III pro-
teins have the same orientation as type I proteins but do not
contain a cleavablesignal sequence.These different topolo-
gies reflect distinct mechanismsused by the cell to establish
the membrane orientation of transmembrane segments,as
discussedin the next section.
The proteins forming topological classIV contain two or
more membrane-spanning segments and are sometimes
called multipass proteins. For example, many of the mem-
brane transport proteins discussedin Chapter 11 and the
numerous G protein-coupled receptors covered in Chapter
15 belong to this class. A final type of membrane protein

Cytosol
coo NHst

ii,,ltla
itirir i
Exoplasmic
space Cleaved
(ERor Golgi s i gn aI
sequence
lumen; NHs'
cell exterior)

Type I Type ll Type lll Type lV N Hg*

LDL receptor Asialoglycoprotein Cytochromep450 G protein-coupledreceptors GPI-linkedprotein

InfluenzaHA orotein receptor Glucosetransporters
Transferrinreceptor Plasminogen
Insulinreceoto'. Voltage-gatedCa2+channels activator receptor
Growth hormone Golgi galactosyltransferase A B C s m a l l m o l e c u l ep u m p s Fasciclinll
receptor Golgi sialyltransferase CFTR(Cl-) channel
Sec61
A FIGURE 13-10ERmembraneproteins.Fourtopological classes typelV proteinshavemultiple
transmembrane a helicesThetypelV
of integral
membrane proteinsaresynthesizedon the roughERas topologydepicted to thatof G protein-coupled
herecorresponds
wellasa fifthtypetethered to the membrane by a phospholipid receptors:
seveno helices, on theexoplasmic
the N-terminus sideof
anchor. Membrane proteinsareclassified
bytheirorientationin the the membrane, andthe C-terminus side.Othertype
on the cytosolic
membrane andthetypesof signals theycontainto directthemthere lV proteins
mayhavea differentnumberof helicesandvarious
In classes
l-lv the hydrophobic segmentsof the proteinchainformo of the N-terminus
orientations andC-terminus. E Hartmann
[See etal,
helicesembedded in the membrane bilayer;
the regionsoutsidethe 1989,ProcNat'lAcad.SciUSA86:5786,andC.A Brown andS,D,Black,
membrane arehvdrophilic andfold intovariousconformations. All 1989,JBiol Chem254:44421

N F P R O T E I NISN T OT H E E R M E M B R A N E
I N S E R T I OO 543
lacks a hydrophobic membrane-spanningsegment alto-
gether; instead, these proteins are linked to an amphipathic
phospholipid anchor that is embedded in the membrane
(Figure 13-10, right).
Internal Stop-Transfer and Signal-Anchor
S e q u e n c eD s e t e r m i n eT o p o l o g y
o f S i n g l e - P a sPsr o t e i n s
\Ve begin our discussionof how membraneprotein topology
is determined with the membrane insertion of integral pro-
teins that contain a single,hydrophobic membrane-spanning
segment.Two sequencesare involved in targeting and orient-
ing type I proteins in the ER membrane,whereastype II and
type III proteins contain a single,internal topogenicsequence.
As we will see,there are three main types of topogenic se-
quencesthat are used to direct proteins to the ER membrane
and to orient them within it. We have alreadybeenintroduced
to one, the N-terminal ER signalsequence.The other two, in-
troduced here, are internal sequencesknown as stop-transfer
anchor sequencesand signal-anchorsequences.
Type I Proteins All type I transmembraneproteins possess
an N-terminal signal sequencethat targetsthem to the ER as
well as an internal hydrophobic sequencethat becomesthe
membrane-spanninga helix. The N-terminal signal sequence
on a nascenttype I protein, like that of a secretoryprotein, ini-
tiates cotranslationaltranslocationof the protein through the
combined action of the SRP and SRP receptor.Once the N-
terminus of the growing polypeptide entersthe lumen of the
ER, the signalsequenceis cleaved,and the growing chain con-
tinues to be extruded across the ER membrane. However,
unlike the casewith secretoryproteins,when the sequenceof
approximately 22 hydrophobic amino acidsthat will become
a transmembrane domain of the nascent chain enters the
translocon,it stopstransferofthe protein through the channel
(Figure 13-11). The structure of the Sec61complex suggests
that the channelmay be able to open like a clamshell,allow-
ing the hydrophobic transmembranesegmentof the translo-
cating peptide to move laterally betweenthe protein domains
constituting the translocon wall (seeFigure 13-8). \Whenthe
peptide exits the translocon in this manner, it becomesan-
chored in the phospholipid bilayer of the membrane.Because
of the dual function of sucha sequenceto both stop passageof
the polypeptidechain through the transloconand to becomea
hydrophobic transmembranesegment in the membrane bi-
layer,it is called a stop-transferanchor sequence.
Once translocation is interrupted, translation continues
at the ribosome, which is still anchored to the now unoccu-
pied and closedtranslocon. As the C-terminus of the protein
chain is synthesized,it loops out on the cytosolic side of the
membrane.When translationis completed,the ribosome is

Cytosol

Open
translocon

'l,.il
ji;i i
lr,!t rl

'fr " Nascent Stop-transfer

polypeptide
Signal ancnor
chain sequence
peptidase
Cleaved
ER lumen s i gn aI
sequence
NHs
NHs
NH:
FIGURE 13-11Positioning type lsingle-pass proteins. the translocon subunits andbecomes anchored in the phospholipid
Step[: Afterthe ribosome/nascent chaincomplex becomes bilayer.
At thistime,thetranslocon probably closesStepS: As
associated
with a transloconin the ERmembrane, the N-terminal synthesis continues, theelongating chainmayloopout intothe
signal
sequence iscleavedThisprocess occursbythesamemechanism cytosolthroughthe smallspacebetweenthe ribosome andtranslocon
astheonefor solublesecretoryproteins (seeFigure13-6)Steps2, Step6: Whensynthesis rscomplete, the ribosomal subunits are
B: Thechainiselongated untilthe hydrophobic stop-transfer
anchor released intothe cytosol, leaving the proteinfreeto diffusein the
sequenceissynthesized andentersthe translocon, whereit m e m b r a n[eS e e HD o e t a,l1 9 9 6C, e l8l 5 : 3 6 a9 n, d WM o t h e s e t,a1l9 9 7 ,
prevents
the nascent chainfromextruding fartherintothe ERlumen Cell89:523l
Step@: Thestop-transferanchorsequence moveslaterallybetween

544 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
releasedfrom the translocon and the C-terminus of the
newly synthesizedtype I protein remains in the cytosol.
Support for this mechanism has come from studies in
which cDNAs encoding various mutant receptorsfor human
growth hormone (HGH) are expressedin cultured mam-
malian cells. The wild-type HGH receptor, a typical type I
protein, is transported normally to the plasma membrane.
However, a mutant receptor that has charged residues in-
serted into the single a-helical membrane-spanningsegment
or that is missingmost of this segmentis translocatedentirely
into the ER lumen and is eventually secretedfrom the cell as
a soluble protein. These kinds of experimentsestablishthat
the hydrophobic membrane-spanningo helix of the HGH re-
ceptor and of other type I proteins functions both as a stop-
transfer sequenceand a membrane anchor that preventsthe
C-terminus of the protein from crossingthe ER membrane.
Type ll and Type lll Proteins Unlike type I proteins, type II
and type III proteins lack a cleavableN-rerminal ER signalse-
quence.Instead, both possessa single internal hydrophobic
signal-anchorsequencethat functions as both an ER signal
sequenceand membrane-anchorsequence.Recall that type II
and type III proteins have opposite orientations in the mem-
brane (seeFigure 13-10); this differencedependson the ori-
entation that their respectivesignal-anchorsequencesassume
within the translocon.The internal signal-anchorsequencein
type II proteins directs insertion of the nascentchain into the
ER membrane so that the N-terminus of the chain facesthe
cytosol, using the sameSRP-dependentmechanismdescribed
for signal sequences(Figure 13-l2a). However, the internal
signal-anchorsequenceis not cleayedand moves laterally be-
tween the protein domains of the translocon wall into the
phospholipid bilayer, where it functions as a membrane
anchor.As elongationcontinues,the C-terminal region of the
growing chain is extruded through the transloconinto the ER
lumen by cotranslationaltranslocation.
In the case of type III proteins, the signal-anchor se-
quence, which is located near the N-terminus, inserts the
nascent chain into the ER membrane with its N-terminus
facing the lumen, in the opposite orientation of the signal
anchor in type II proteins. The signal-anchor sequenceof
type III proteins also functions like a stop-transfer sequence
and prevents further extrusion of the nascentchain into the
ER lumen (Figure 13-12b). Continued elongation of the

(a) (b)
E
Nascent
polypeptide
chain
NHs*
NH:*
Cposol E E coo

mRNA +
+
+
i,ri:j;ii:l:,1
ilttffi
1ru,|i
Signal- NH : *
ER lumen a n c n o r
sequence

coo
FIGURE 13-12Positioningtype ll and type ilt single-pass translocon andanchors the chainin the phospholipid bilayer.
proteins.(a)Typell proteinsStep[: Afterthe internal signal- StepB: Onceproteinsynthesis iscompleted, theC-terminus of the
anchorsequence issynthesized on a cytosolic ribosome, it isbound polypeptide is released intothe lumen,andthe ribosomal subunits
by an SRP(notshown), whichdirects the ribosome/nascent chain arereleased intothe cytosol(b)Typelll proteins. Step[: Assembly
complex to the ERmembrane Thisissimilar to targeting of soluble isby a similar pathway to thatof typell proteins exceptthatpositively
secretory proteins exceptthatthe hydrophobic signalsequence is charged residues on theC-terminal sideof thesignal-anchor sequence
not located at the N-terminus andis notsubsequently cleavedThe causethetransmembrane segment to be oriented withinthetranslocon
nascent chainbecomes oriented in thetranslocon with itsN-terminal with itsC-terminal portionoriented to the cytosol andthe N-terminal
portiontowardthecytosolThisorientation isbelieved to be mediated s i d eo f t h ep r o t e i inn t h eE Rl u m e nS t e p [s, B : C h a i ne l o n g a t i o n
by the positively charged residues shownN-terminal to thesignal- of theC-terminal portionof the proteiniscompleted in the cytosol,
anchorsequence StepE:As the chaintselongated andextruded andribosomal subunits arereleased. [See M Spiess andH F Lodish,
i n t ot h el u m e nt,h ei n t e r n asli g n a l - a n c hmoorv e lsa t e r a lol yu to f t h e 1986, Cell 44:177, and H Do et al , 1996, Ce//85:369 l

N F P R O T E I NISN T OT H E E R M E M B R A N E
I N S E R T I OO 545
chain C-terminal to the signal-anchor/stop-transfer
sequenceproceeds as it does for type I proteins, with the
hydrophobic sequence moving laterally between the
translocon subunits to anchor the polypeptide in the ER
membrane(seeFigure 13-11).
One of the features of signal-anchor sequencesthat ap-
pears to determine their insertion orientation is a high den-
sity of positively chargedamino acids adjacentto one end of
the hydrophobic segment.For reasonsthat are not well un-
derstood,thesepositively chargedresiduestend to remain on
the cytosolic side of the membrane, not traversing the mem-
brane into the ER lumen. Thus the position of the charged
residuesdictates the orientation of the signal-anchorse-
quencewithin the translocon as well as whether or not the
rest of the polypeptide chain continues to pass into the ER
lumen: type II proteins tend to have positively charged
residues on the N-terminal side of their signal-anchor se-
quence,orienting the N-terminus in the cytosol and allowing
passageof the C-terminal side into the ER (Figure 1.3-L2a),
whereas type III proteins tend to have positively charged
residues on the C-terminal side of their signal-anchor se-
quence,inserting the N-terminus into the translocon and re-
stricting the C-terminus to the cytosol (Figure 13-1,2b).
A striking experimental demonstration of the impor-
tance of the flanking charge in determining membrane ori-
entation is provided by neuraminidase,a type II protein in
the surface coat of influenza virus. Three arginine residues
are located just N-terminal to the internal signal-anchorse-
quencein neuraminidase.Mutation of thesethree positively
charged residues to negatively charged glutamate residues
causes neuraminidase to acquire the reverse orientation.
Similar experimentshave shown that other proteins, with ei-
ther type II or type III orientation, can be made to "flip"
their orientation in the ER membrane by mutating charged
residuesthat flank the internal signal-anchorsegment.
M u l t i p a s sP r o t e i n sH a v eM u l t i p l eI n t e r n a l
TopogenicSequences
Figure 13-13 summarizesthe arrangementsof topogenic
sequencesin single-passand multipass transmembrane
proteins. In multipass (type IV) proteins, each of the
membrane-spanninga helices acts as a topogenic sequence
in the ways that we have already discussed:they can act to
direct the protein to the ER, to anchor the protein in the
ER membrane, or to stop transfer of the protein through
the membrane. Multipass proteins fall into one of two
types depending on whether the N-terminus extends into
the cytosol or the exoplasmicspace(e.g.,the ER lumen, cell
exterior). This N-terminal topology usually is determined

< FIGURE 13-13Topogenic sequences determine

orientationof ERmembraneproteins.Topogenic
sequences areshownin red;soluble, hydrophilic
portionsin blue Theinternal topogenic sequences form
transmembrane cthelices thatanchorthe proteins or
segments of proteinsin the membrane(a)TypeI proteins
containa cleaved signalsequence anda singleinternal
STA= Internalstop-transferanchorsequence
SA = Internalsignal-anchorsequence
stop-transferanchor(STA). (b, c)Typell andtypelll
proteinscontaina singleinternal signal-anchor (SA)
sequence. Thedifference in theorientation of these
proteinsdepends largely on whetherthereisa high
(a) Type I N H 3 +- coo densityof positivelycharged aminoacids(+ + +) on
Signal the N-termrnal sideof the SAsequence (typell)or on
STA
sequence (typelll).(d,e)
the C-terminal sideof the SA sequence
Nearlyall multipassproteins lacka cleavable signal
sequence, asdepicted in theexamples shownhere.
( b ) T y p el l NH:* coo TypelV-Aproteins, whoseN-terminus facesthe cytosol,
+++ containalternating typell S sequences andSTA
SA
sequences TypelV-Bproteins, whoseN-terminus faces
the lumen,beginwith a typelllSAsequence followedby
alternatingtypell SAand STAsequences Proteins of
(c) Type lll NHs* coo (oddor
+++ eachtypewith differentnumbersof ct helices
SA even)areknown

Cvtosol Lumen
(d)Type lV-A NHs* COO
SA STA SA STA

Lumen Cytosol Lumen Cytosol Lumen Cytosol Lumen Cytosol

(e) Type lV-B NHqt COO
+++ +++
STA SA STA SA STA

546 C H A P T E R1 3 I MOVINGPROTEINS
INTO MEMBRANES
AND ORGANELLES
by the hydrophobic segment closest to the N-terminus
and the charge of the sequencesflanking it. lf a type IV
protein has an euen number of transmembrane cr helices,
both its N-terminus and C-terminus will be oriented to-
ward the same side of the membrane (Figure 13-13d).
Conversely, if a type IV protein has an odd number of a
helices, its two ends will have opposite orientations
( F i g u r e1 3 - 1 3 e ) .
Type lV Proteins with N-Terminus in Cytosol Among
the multipass proteins whose N-terminus extends into the
cytosol are the various glucose transporters (GLUTs) and
most ion-channel proteins, discussedin Chapter 11. In these
proteins, the hydrophobic segmentclosestto the N-terminus
initiates insertion of the nascent chain into the ER mem-
brane with the N-terminus oriented toward the cytosol; thus
this a-helical segment functions like the internal signal-
anchor sequenceof atype II protein (seeFigure 13-12a).As
the nascent chain following the first a helix elongates, it
moves through the translocon until the secondhydrophobic
a helix is formed. This helix prevents further extrusion of
the nascentchain through the translocon;thus its function is
similar to that of the stop-transferanchor sequencein a type I
p r o t e i n ( s e eF i g u r e1 3 - 1 1 ) .
After synthesisof the first two transmembranecl helices,
both ends of the nascentchain face the cytosol and the loop
between them extends into the ER lumen. The C-terminus
of the nascentchain then continues ro grow into the cytosol,
as it does in synthesis of type I and type III proteins. Ac-
cording to this mechanism,the third a helix acts as another
type II signal-anchor sequenceand the fourth as another
stop-transferanchor sequence(Figure 13-13d). Apparently,
once the first topogenic sequenceof a multipass polypeptide
initiates associationwith the translocon, the ribosome re-
mains attached to the translocon, and topogenic sequences
that subsequentlyemerge from the ribosome are threaded
into the translocon without the need for the SRP and the
SRP receptor.
Experiments that use recombinant DNA techniques to
exchangehydrophobic cr heliceshave provided insight into
the functioning of the topogenic sequencesin type IV-A mul-
tipass proteins. Theseexperimentsindicate that the order of
the hydrophobic a helicesrelative to each other in the grow-
ing chain largely determineswhether a given helix functions
as a signal-anchor sequenceor stop-transfer anchor se-
quence. Other than its hydrophobicitS the specific amino
acid sequenceof a particular helix has little bearing on its
function. Thus the first N-terminal a helix and the subse-
quent odd-numbered ones function as signal-anchor se-
quences,whereas the intervening even-numberedhelices
function as stop-transferanchor sequences.
Type lV Proteins with N-Terminus in the Exoplasmic
Space The large family of G protein-coupled receptors,all
of which contain seventransmembranect helices,constitute
the most numerous type IV-B proteins, whose N-terminus
extends into the exoplasmic space.In theseproteins, the hy-
drophobic ct helix closestto the N-terminus often is followed
by a cluster of positively charged amino acids, similar to a
type III signal-anchorsequence(seeFigure 1,3-1,2b)'. As a re-
sult, the first a helix inserts the nascent chain into the
translocon with the N-terminus extending into the lumen
(seeFigure 13-13e).As the chain is elongated,it is inserted
into the ER membrane by alternating type II signal-anchor
sequencesand stop-transfer sequences,as just describedfor
type IV-A proteins.
A PhospholipidAnchor TethersSomeCell-
SurfaceProteinsto the Membrane
Somecell-surfaceproteins are anchored to the phospholipid
bilayer not by a sequenceof hydrophobic amino acids but by
a covalently attached amphipathic molecule, glycosylphos-
phatidylinositol (GPI) (Figure 1.3-1.4aand Chapter 10).
These proteins are synthesizedand initially anchored to the
ER membrane exactly like type I transmembrane proteins,
with a cleavedN-terminal signal sequenceand internal stop-
transfer anchor sequencedirecting the process (see Figure
13-11).However,a short sequenceof amino acidsin the lu-
minal domain, adjacent to the membrane-spanningdomain,
is recognizedby a transamidaselocated within the ER mem-
brane. This enzymesimultaneously cleavesoff the original
stop-transferanchor sequenceand transfersthe luminal por-
tion of the protein to a preformed GPI anchor in the mem-
brane (Figure 1.3-'1.4b).
\X/hy change one type of membrane anchor for an-
other? Attachment of the GPI anchor, which results in re-
moval of the cytosol-facing hydrophilic domain from the
protein, can have severalconsequences. Proteinswith GPI
anchors, for example, can diffuse relatively rapidly in the
plane of the phospholipid bilayer membrane. In contrast,
many proteins anchored by membrane-spanningct helices
are impeded from moving laterally in the membrane
becausetheir cytosol-facing segments interact with the
cytoskeleton. In addition, the GPI anchor targets the
attachedprotein to the apical domain of the plasma mem-
brane in certain polarized epithelial cells, as we discussin
C h a p t e r1 4 .
The Topologyof a MembraneProteinOften
Can Be Deducedfrom lts Sequence
As we have seen,various topogenic sequencesin integral
membrane proteins synthesizedon the ER govern interac-
'When
tion of the nascent chain with the translocon. scien-
tists begin to study a protein of unknown function, the
identification of potential topogenic sequenceswithin the
corresponding gene sequencecan provide important clues
about the protein's topological class and function. Suppose,
for example, that the gene for a protein known to be required
for a cell-to-cell signaling pathway contains nucleotide
sequencesthat encodean apparent N-terminal signal sequence
and an internal hydrophobic sequence.These findings sug-
gest that the protein is a type I integral membrane protein
NF P R O T E I NISN T OT H E E R M E M B R A N E
I N S E R T I OO 547
(a)
O = Inositol
drophilic amino acids negative values. Although different
scalesfor the hydropathic index exist, all assignthe most
I = Glucosamine
= positive valuesto amino acids with side chains made up of
tO Mannose
=
NH, P1-'o.01''o"thanolamine
mostly hydrocarbon residues(e.g.,phenylalanineand me-
P o o*
thionine) and the most negativevalues to charged amino
acids (e.g., arginine and aspartate).The second step is to
% identify longer segmentsof sufficient overall hydrophobic-
Fattyacyl chains L POo PO4-+ NH3+ ity to be N-terminal signal sequencesor internal stop-
transfer sequences and signal-anchorsequences. To accom-
plish this, the total hydropathic index for each successive
Hvdrophobic Polar NHg*
segmentof 20 consecutiveamino acids is calculatedalong
J
the entire length of the protein. Plots of these calculated
values againstposition in the amino acid sequenceyield a
(b) hydropathy profile.
GPI Figure 13-15 shows the hydropathy profiles for three
Cytosol transamidase different membrane proteins. The prominent peaks in such
coo plots identify probable topogenicsequences as well as their
position and approximate length. For example, the hy-
dropathy profile of the human growth hormone receptor
revealsthe presenceof both a hydrophobic signal sequence
. NHs*
at the extreme N-terminus of the protein and an internal hy-
Preformed drophobic stop-transfersequence(Figure 13-15a). On the
G P Ia n c h o r basis of this profile, we can deduce, correctly, that the hu-
Precursor man growth hormone receptor is a type I integral mem-
NlH3* protein NHs* brane protein. The hydropathy profile of the asialoglyco-
protein receptor, a cell-surface protein that mediates
Mature GPI-linked
ER lumen protein removal of abnormal extracellular glycoproteins, reveals a
FIGURE 13-14GP|-anchored proteins.(a)Structure of a gly- prominent internal hydrophobic signal-anchor sequence
cosylphosphatidylinositol (GPl)fromyeast. Thehydrophobic portion but gives no indication of a hydrophobic N-terminal signal
of the molecule iscomposed of fattyacylchains, whereas the polar sequence(Figure 13-15b). Thus we can predict that the
(hydrophilic) portionof the molecule iscomposed of carbohydrate asialoglycoproteinreceptor is a type II or type III membrane
residues andphosphate groupsIn otherorganisms, boththe length protein. The distribution of charged residueson either side
of theacylchains andthe carbohydrate moieties mayvarysomewhat of the signal-anchorsequenceoften can differentiate be-
fromthe structure shown(b)Formation of GPI-anchored proteins in tween these possibilities since positively charged amino
the ERmembrane Theproteinissynthesized andinitially inserted acids flanking a membrane-spanning segment usually are
i n t ot h eE Rm e m b r a naess h o w ni n F i g u r 1e 3 - 11 .A s p e c i f ti cr a n s a m i - oriented toward the cytosolic face of the membrane. For
dasesimultaneously cleaves the precursor proteinwithinthe instance,in the caseof the asialoglycoproteinreceptor,ex-
exoplasmic-facing domain,nearthe stop-transfer anchorsequence amination of the residuesflanking the signal-anchor se-
(red),andtransfers the carboxyl groupof the newC-terminus to the quence reveals that the residues on the N-terminal side
terminal amrnogroupof a preformed GPIanchor[See C Abeijon and
carry a net positive charge, thus correctly predicting that
C B Hirschberg,1992,TrendsBioch Sec mi 1 7 : 3 2 , a n d K K o d u k u l ae t a l ,
1992, Proc Nat'|.Acad Sci USA89:49821
this is a type II protein.
The hydropathy profile of the GLUT1 glucose trans-
porter, a multipass membrane protein, shows the presence
of many segmentsthat are sufficiently hydrophobic to be
membrane-spanninghelices(Figure 13-15c).The complex-
and therefore may be a cell-surfacereceptor for an extracel- ity of this profile illustrates the difficulty both in unambigu-
lular ligand. ously identifying all the membrane-spanningsegmentsin a
Identification of topogenic sequences requiresa way to multipass protein and in predicting the topology of individ-
scan sequencedatabasesfor segmentsthat are sufficiently ual signal-anchorand stop-transfer sequences.More so-
hydrophobic to be either a signal sequenceor a transmem- phisticated computer algorithms have been developed that
brane anchor sequence.Topogenicsequencescan often be take into account the presenceof positively charged amino
identified with the aid of computer programs that generare acids adjacent to hydrophobic segmentsas well as the
a hydropathy profile for the protein of inrerest. The first length of and spacing between segments.Using all this in-
step is to assign a value known as the bydropathic index to formation, the best algorithms can predict the complex
each amino acid in the protein. By convention, hydropho- topology of multipass proteins with an accuracy of greater
bic amino acids are assigned positive values and hy- than 7 5 percent.

548 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 ( a ) H u m a ng r o w t h h o r m o n er e c e p t o r( t y p el )

3
z
1
0
-1
-2
-3
N-terminus 100 200 300 400 C-terminus

( b )A s i a l o g l y c o p r o t e ri ne c e p t o r( t y p el l ) ( c ) G L U T(tt y p el V )

4 S i g n a l - a n c h osre q u e n c e 4

2 z
1 1
0 0
-1 r'i;.:'1+i i ...,, -1
-z -z

100 200 100 200 400

A E X P E R I M E N TFA
IGL U R1E3 - 1 5H y d r o p a t h p
yrofiles. portions; values,
negative polarportions
relatively of the protein
Hydropathy profilescanidentifylikelytopogenic sequences in Probable
topogenic sequencesaremarked.Thecomplex profiles
for
integral
membrane proteinsTheyaregenerated by plottingthetotal (typelV)proteins,
multipass suchasGLUT1 in part(c),oftenmustbe
hydrophobicity of eachsegment of 20 contiguous aminoacidsalong supplemented to determrne
with otheranalyses the topology of
the lengthof a proteinPositive valuesindicate relativelyhydrophobic theseoroteins

Finally, sequencehomology to a known protein may chain and the presenceof adjacent positively charged
permit accurateprediction of the topology ol a newly dis- r e s i d u e s( s e eF i g u r e1 3 - 1 3 ) .
covered multipass protein. For example, the genomesof
r Somecell-surfaceproteins are initially synthesizedas type
multicellularorganismsencodea very large number of mul-
I proteins on the ER and then are cleavedwith their luminal
tipass proteins with seventransmembranecr helices.The
domain transferredto a GPI anchor (seeFigure t3-14).
similaritiesbetweenthe sequences of theseproteinsstrongly
suggestthat all have the sametopology as the well-studied r The topology of membraneproteinscan often be correctly
G protein-coupled receptors,which have the N-terminus predicted by computer programs that identify hydrophobic
orientedto the exoplasmicsideand the C-terminusoriented topogenicsegmentswithin the amino acid sequenceand gen-
to the cytosolicside of the membrane. eratehydropathyprofiles(seeFigure13-15).

ProteinModifications, Folding,
Insertion of Proteins into the ER Membrane
and QualityControlin the ER
r Integralmembraneproteinssynthesized on the rough ER
fall into four topological classesas well as a lipid-linked Membrane and soluble secretoryproteins synthesizedon the
t y p e ( s e eF i g u r e1 3 - 1 0 ) . rough ER undergo four principal modifications before they
reachtheir final destinations:(1) covalentaddition and pro-
r Topogenic sequences-N-terminal signal sequences,in- cessing of carbohydtates (glycosylation) in the ER and
ternal stop-transferanchor sequences, and internal signal- Golgi, (2) formation of disulfidebondsin the ER, (3) proper
anchor sequences-direct the insertion and orientation of folding of polypeptide chains and assemblyof multisubunit
nascentproteinswithin the ER membrane.This orientarion proteins in the ER, and (4) specific proteolytic cleavagesin
is retained during transport of the completedmembrane the ER, Golgi, and secretory vesicles.Generally speaking,
p r o r e i nt o i r s f i n a l d e s t i n a t i o n . these modifications promote folding of secretory proteins
r Single-passmembrane protelns contarn one or two into their native structures and add structural stability to
topogenic sequences.
In multipassmembraneproteins, each proteins exposed to the extracellular environment. Modifi-
ct-helicalsegmentcan function as an internal topogenic cations such as glycosylation also allow the cell to produce a
sequence,depending on its location in the polypeptide vast array of chemically distinct moleculesat the cell surface

IN THEER
. L D I N GA, N D Q U A L I T YC O N T R O L
M O D I F I C A T I O NFSO
PROTEIN 549
that are the basis of specific molecular interactions used in Glc G l c N A c= N - A c e t y l g l u c o s a m i n e
cell-to-cell adhesionand communication.
One or more carbohydrate chains are added to the vast ma- I
Glc
Man = Mannose
Glc= Glucose
jority of proteinsthat are synthesizedon the rough ER; indeed,
glycosylationis the principal chemicalmodification to most of
these proteins. Proteins with attached carbohydrates are
I
Glc
= Conserved
= Variable

known as glycoproteins. Carbohydrate chains in glycoproteins

may be attached to the hydroxyl group in serineand threonine
residuesor to the amide nitrogen of asparagine.Theseare re-
ferred to as Olinked oligosaccharidesand Nlinked oligosac-
charides,respectively.The various types of O-linked oligosac-
charides include the mucin-type O-linked chains (named after
abundantglycoproteinsfound in mucus)and the carbohydrate
modifications on proteoglycansdescribedin Chapter 19. O-
Iinked chains typically contain only one to four sugar
residues,which are added to proteins by enzymesknown as
glycosyltransferases, located in the lumen of the Golgi com-
plex. The more common Nlinked oligosaccharides are larger
and more complex,containingseveralbranchesin mammalian
cells. In this section we focus on N-linked oligosaccharides,
whose initial synthesisoccurs in the ER. After the initial N-gly-
cosylationof a protein in the ER, the oligosaccharidechain is
modified in the ER and commonly in the Golgi as well.
Disulfide bond formation, protein folding, and assembly
of multimeric proteins, which take place exclusively in the
rough ER, also are discussedin this section. Only properly
folded and assembledproteins are transported from the
rough ER to the Golgi complex and ultimately to the cell
surface or other final destination. Unfolded, misfolded, or
partly folded and assembledproteins are selectivelyretained
in the rough ER. Sile consider several features of such
"quality control" in the latter part of this section.
As discussedpreviously,N-terminal ER signal sequences
are cleaved from secretory proteins and type I membrane
proteins in the ER. Someproteins also undergo other specific
proteolytic cleavagesin the Golgi complex or secretoryvesi-
cles. We cover thesecleavages,as well as carbohydrate mod-
ifications that occur primarily or exclusively in the Golgi
complex, in the next chapter.
A PreformedA/-LinkedOligosaccharide ls Added
t o M a n y P r o t e i n si n t h e R o u g hE R
Biosynthesisof all N-linked oligosaccharidesbeginsin the
rough ER with addition of a preformed oligosaccharide
precursorcontaining 14 residues(Figure 13-t6). The struc-
ture of this precursor is the same in plants, animals, and
single-celledeukaryotes-a branchedoligosaccharide,con-
taining three glucose(Glc), nine mannose(Man), and two
N-acetylglucosamine(GlcNAc) molecules, which can be
written as GIcaMane(GlcNAc)2. Once added to a protein,
this branchedcarbohydrate srructureis modified by addition
or removal of monosaccharidesin the ER and Golgi com-
partments. The modifications to N-linked chains differ from
one glycoprotein to another and differ among different or-
ganisms,but a core of 5 of the 14 residuesis conservedin the
structures of all N-linked oligosaccharideson secretoryand
membrane proteins.
Man Man Man
I II II
Man Man Man
Man
I Ma n
*in
GlcNAc
GlcNAc
II
N H s *. . - X - A s n - X - ( S e r / T h r ) - ' . . C O O -
A FIGURE 13-16Commonprecursorof N-linkedoligosac-
charides. This14-residue precursor of tV-linkedoligosaccharides
is
addedto nascent proteins in the roughERSubsequent removal and
in somecases addition of specific sugarresidues occurin the ERand
GolgicomplexThecoreregion, composed of fiveresidues high-
lightedin purple,is retained in allN-linkedoligosaccharides The
(Asn)residues
precursor canbe linkedonlyto asparagine thatare
separated by oneaminoacid(X)froma serine (Ser)
or threonine(Thr)
on the carboxylside.
Prior to transfer to a nascent chain in the lumen of the
ER, the precursor oligosaccharideis assembledon a mem-
brane-attached anchor called dolichol phosphate, a Iong-
chain polyisoprenoid lipid (Chapter 10). After the first sugar,
GlcNAc, is attached to the dolichol phosphate by u py-
rophosphate bond, the other sugarsare added by glycosidic
bonds in a complex set of reactions catalyzed by enzymes
attached to the cytosolic or luminal faces of the rough ER
membrane (Figure L3-1.7).The final dolichol pyrophospho-
ryl oligosaccharideis oriented so that the oligosaccharide
portion facesthe ER lumen.
The entire 14-residueprecursor is transferred from the
dolichol carrier to an asparagine residue on a nascent
polypeptide as it emergesinto the ER lumen (Figure 13-18,
step E). Only asparagineresidues in the tripeptide se-
quencesAsn-X-Ser and Asn-X-Thr (where X is any amino
acid except proline) are substratesfor oligosaccharyltrans-
ferase, the enzyme that catalyzes this reaction. Two of the
three subunits of this enzyme are ER membrane proteins
whose cytosol-facingdomains bind to the ribosome,localiz-
ing a third subunit of the transferase,the catalytic subunit,
near the growing polypeptide chain in the ER lumen. Not all

550 CHAPTER
13 I M O V T N GP R O T E | NtSN T O M E M B R A N E A
SN D O R G A N E L L E S
 Gytosol
5 GDP
a
4 GDP 3 UDP
4GDP O 3 UDP l
'flin
I\ ,/

a- ^.
e
t\l
I
Dolichol
phospha

I = N-Acetylglucosamine
Completed
O = Mannose precursor
A - Glucose
ER lumen

FIGURE 13-17Biosynthesis of the oligosaccharide precursor. theseven-resrdue dolichol pyrophosphorylintermedrateisflipped

Dolichol phosphate isa strongly hydrophobic lipid,containing 75-95 face(stepZl),the remaining
to the luminal four mannose andall
c a r b o na t o m st,h a t i s e m b e d d eidn t h e E Rm e m b r a n e
T.w o threeglucose residues areaddedoneat a time(steps5, 6). In the
tV-acetylglucosamine (GlcNAc) andfivemannose residues areadded thesugarto be addedisfirsttransferred
laterreactions, froma
oneat a timeto a dolichol phosphate on the cytosolic faceof the ER nucleotide-sugarto a carrier phosphate
dolichol on thecytosolicface
membrane (stepsIl-B) Thenucleotide-sugar donorsin theseand of the ER;thecarrieristhenflippedto the luminalface,wherethe
laterreactions aresynthesized in thecytosolNotethatthefirstsugar sugaristransferred to the growrngo|gosaccharide,afterwhichthe
residue isattached to dolichol by a high-energy pyrophosphate "empty"carrier isflippedbackto the cytosolic C Abeijon
face.[After
linkageTunicamycin, whichblocks thefirstenzyme in thispathway, andC B Hirschberg,1992,Trends BiochemSci17:32]1
inhibits thesynthesis of allN-linked oligosaccharides in cellsAfter

Asn-X-Ser/Thr sequencesbecome glycosylated,and it is not
possible to predict from the amino acid sequencealone
which potential N-linked glycosylation sites will be modi-
fied; for instance, rapid folding of a segment of a protein
containing an Asn-X-Ser/Thr sequencemay prevent transfer
of the oligosaccharide precursorto it.
Immediately after the entire precursor, GlcaMane
(GlcNAc)2, is transferred to a nascent polypeptide, three
different enzymes, called glycosidases,remove all three
glucoseresiduesand one particular mannoseresidue (Fig-
u r e 1 3 - 1 8 ,s t e p sZ - 4 ) . T h e t h r e eg l u c o s er e s i d u e sw
, hich
are the last residuesadded during synthesisof the precursor

Dol

To
cts-
E Golgi

(Man)a(GlcNAc)z

ER lumen

Dol = Dolichol o = Mannose

I = N-Acetylglucosamine a = Glucose

FIGURE 13-18Additionand initial processing of N-linked glucose residues(stepB), andfinallyonemannose residue(step4)

oligosaccharides.Inthe roughERof vertebrate cells,the areremoved. Re-additionof oneglucoseresidue(stepl[) playsa
GlcrMann(GlcNAc)2 precursor istransferred
fromthe dolicholcarrier foldingof manyproteins
rolein the correct in the ER,asdiscussed
to a susceptible
asparagine residue on a nascentproteinassoonas later[SeeR Kornfeldand 1985,Ann
S Kornfeld, Rev.Biochem 45:631,
and
the asparagine
crossesto the luminalsideof the ER(step[) In M S o u s a a n d AJ P a r o d i1, 9 9 5 , E M B O l 1 4 i 4 1 9 6 )
threeseparate firstoneglucose
reactions, residue(stepEl),thentwo

, N D Q U A L I T YC O N T R O LI N T H E E R
P R O T E I NM O D I F I C A T I O N SF,O L D I N G A 551
on the dolichol carrier, appear to acr as a signal that the
oligosaccharideis complete and ready to be transferredto
a protern.
O l i g o s a c c h a r i dSei d eC h a i n sM a y P r o m o t e
F o l d i n ga n d S t a b i l i t yo f G l y c o p r o t e i n s
The oligosaccharidesattachedto glycoproteinsservevarious
functions. For example, some proteins require N-linked
oligosaccharidesin order to fold properly in the ER. This
function has been demonstrated in studies with the antibi-
otic tunicamycin, which blocks the first step in the formation
of the dolichol-linked oligosaccharideprecursor and there-
fore inhibits synthesisof all N-linked oligosaccharidesin
cells (seeFigure 13-17).In the presenceof tunicamycin,the
hemagglutinin precursor polypeptide (HAe) is synthesized,
but it cannot fold properly and form a normal trimer; in this
case,the protein remains,misfolded, in the rough ER. More-
over, mutation of a particular asparaginein the HA sequence
to a glutamine residue prevents addition of an N-linked
oligosaccharideto that site and causesthe protern to accu-
mulate in the ER in an unfolded state.
In addition to promoting proper folding, N-linked
oligosaccharides also confer stability on many secretedgly-
coproteins.Many secretoryproteins fold properly and are
transported to their final destination even if the addition of
all N-linked oligosaccharides is blocked,for example,by tu-
nicamycin. However, such nonglycosylatedproteins have
been shown to be lessstablethan their glycosylatedforms.
For instance,glycosylatedfibronectin, a normal component
of the extracellularmatrix, is degradedmuch more slowly
by tissueproteasesthan is nonglycosylatedfibronectin.
Oligosaccharideson certain cell-surfaceglycoproteins
also play a role in cell-cell adhesion. For example, the
plasma membrane of white blood cells (leukocytes)con-
tains cell-adhesionmolecules(CAMs) that are extensively
glycosylated.The oligosaccharidesin these moleculesin-
teract with a sugar-binding domain in certain CAMs
found on endothelial cells lining blood vessels.This inter-
action tethers the leukocytesto the endothelium and as-
sists in their movement into tissuesduring an inflamma-
t o r y r e s p o n s et o i n f e c t i o n ( s e e F i g u r e 1 , 9 - 3 6 ) .O t h e r
c e l l - s u r f a c eg l y c o p r o t e i n s p o s s e s so l i g o s a c c h a r i d es i d e
chains that can induce an immune response.A common
example is the A, B, O blood-group antigens,which are
O-linked oligosaccharidesattached to glycoproteins and
glycolipids on the surface of erythrocytes and other cell
t y p e s ( s e eF i g u r e 1 0 - 2 0 ) .
D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e d
b y P r o t e i n si n t h e E RL u m e n
In Chapter 3 we learned that both intramolecular and
intermolecular disulfide bonds (-S-S-) help stabilize the
tertiary and quaternary structure of many proteins. These
covalent bonds form by the oxidative linkage of sulfhydryl
552 CHAPTER
13 | M O V T N Gp R O T E t N |SN T O M E M B R A N E A
SN D O R G A N E L L E S
groups (-SH), also known as thiol groups, on two cysteine
residuesin the same or different polypeptide chains. This
reaction can proceed spontaneouslyonly when a suitable
oxidant is present.In eukaryotic cells, disulfide bonds are
formed only in the lumen of the rough ER; in bacterial
cells, disulfide bonds are formed in the periplasmic space
between the inner and outer membranes.Thus disulfide
bonds are found only in secretoryproteins and in the exo-
plasmic domains of membraneproteins. Cytosolic proteins
and organelleproteins synthesizedon free ribosomeslack
disulfide bonds and depend on other interactionsto stabi-
lize their structures.
The efficient formation of disulfide bonds in the lumen
of the ER dependson the enzymeprotein disulfide isomerase
(PDI), which is presentin all eukaryotic cells.This enzymeis
especiallyabundant in the ER of secretorycells in such or-
gans as the liver and pancreas,where large quantities of pro-
teins that contain disulfide bonds are produced. As shown in
Figure t3-1.9a, the disulfide bond in the active site of PDI
can be readily transferred to a protein by two sequential
thiol-disulfide transfer reactions.The reducedPDI generated
by this reaction is returned to an oxidized form by the action
of an ER-residentprotein, called Ero1, which carriesa disul-
fide bond that can be transferredto PDI. Erol itself becomes
oxidized by reaction with molecular oxygen that has dif-
fused into the ER.
In proteins that contain more than one disulfide bond,
the proper pairing of cysteineresiduesis essentialfor nor-
mal structure and activity. Disulfide bonds commonly are
formed between cysteinesthat occur sequentially in the
amino acid sequencewhile a polypeptide is still growing
on the ribosome. Such sequential formation, however,
sometimesyields disulfide bonds between the wrong cys-
teines.For example,proinsulin, a precursor to the peptide
hormone insulin, has three disulfide bonds that link cys-
teines 1 and 4, 2 and 6, and 3 and 5. In this case,a disul-
fide bond that initially formed sequentially(e.g., between
cysteinesI and 2) would have to be rearranged for the
protein to achieveits proper folded conformation. In cells,
the rearrangementof disulfide bonds also is acceleratedby
PDI, which acts on a broad range of protein substrates,
allowing them to reach their thermodynamicallymost sta-
ble conformations (Figure 13-19b). Disulfide bonds gener-
ally form in a specificorder, first stabilizingsmall domains
of a polypeptide, then stabilizing the interactions of more
distant segments;this phenomenon is illustrated by the
folding of influenza HA protein, discussedin the next
sectron.
C h a p e r o n eas n d O t h e r E RP r o t e i n sF a c i l i t a t e
F o l d i n ga n d A s s e m b l yo f P r o t e i n s
Although many denatured proteins can spontaneouslyre-
fold into their native state in vitro, such refolding usually
requires hours to reach completion. Yet new soluble and
membraneproteins produced in the ER generallyfold into
 ( a ) F o r m a t i o no f a d i s u l f i d eb o n d

Reduced
substrate Oxidized
prorern substrate
protein

( b ) R e a r r a n g e m e notf d i s u l f i d eb o n d s

.
'Reduced -qr.l zSH
t "' '
i -
PDI ic \Q. - Reduced ""
PDI -SH
$z

e_
t"

,n.o,.,.i.',oj"i
il rHi'J
o""o, .",.,."L':[,l,ilXto"o.
FIGURE 13-19Actionof proteindisulfideisomerase (PDl).PDI thesubstrate thenreacts withthisintermediate,forminga disulfide
formsandrearranges disulf
idebondsviaan activesitewithtwo bondwithinthesubstrate proteinandreleasing reduced PDIPDl,in
closely
spaced cysteineresiduesthatareeasily interconvertedbetween turn,transferselectrons bondin the luminalprotein
to a disulfide
the reduceddithiolformandtheoxidized disulfideform Numbered Ero1,thereby regenerating theoxidized formof PDl.(b)Reduced PDI
redarrowsindicate thesequence of electrontransfers Yellowbars cancatalyze rearrangement of improperlyformeddisulfidebondsby
represent bonds(a)Intheformation
disulfide of disulfide
bonds,the similar transfer
thiol-disulfide reactions.Inthiscase,reducedPDIboth
ionized(-S )form of a cysteinethiolin thesubstrate proteinreacts andis regenerated
initiates in the reactionpathway.Thesereactions
withthedisulfide(9-S) bondin oxidized PDIto forma disulfide- arerepeated untilthe moststableconformation of the proteinis
bondedPDI-substrate proteinintermediate A second ionizedthiolin achieved [SeeMM LylesandH F.Gilbert,
1991
,Biochemistry30t6l9l

their proper conformation within minutes after their syn-
thesis. The rapid folding of these newly synthesizedpro-
teins in cells dependson the sequentialaction of several
proteins present within the ER lumen. We have already
seen how the molecular chaperone BiP can drive post-
translationaltranslocationin yeastby binding fully synthe-
sized polypeptidesas they enter the ER (seeFigure 13-9).
BiP can also bind transiently to nascentchains as they en-
ter the ER during cotranslationaltranslocation.Bound BiP
is thought to preventsegmentsof a nascentchain from mis-
folding or forming aggregates,thereby promoting folding
of the entire polypeptide into the proper conformation.
P r o t e i n d i s u l f i d e i s o m e r a s e( P D I ) a l s o c o n t r i b u t e s t o
proper folding, becausecorrect 3-D conformation is stabi-
l i z e db y d i s u l f i d eb o n d si n m a n y p r o t e i n s .
As illustrated in Figure 1,3-20,two other ER proteins,
the homologous lectins (carbohydrate-binding proteins)
cdlnex,in and calreticulin, bind selectively to certain N-
linked oligosaccharideson growing nascent chains. The
ligand for thesetwo lectins, which contains a single glucose
residue, is generatedby a specific glucosyltransferasein the
ER lumen (seeFigure 1,3-18,step Ed). This enzyme acts
only on polypeptide chains that are unfolded or misfolded,
and in this respectthe glucosyltransferase acts as one of the
primary surveillance mechanismsto ensure quality control
of protein folding in the ER. Binding of calnexin and cal-
reticulin to unfolded nascent chains marked with glucosy-
lated N-linked oligosaccharidesprevents aggregation of
adjacent segmentsof a protein as it is being made on the
ER. Thus calnexin and calreticulin, like BiP' help prevent
premature, incorrect folding of segmentsof a newly made
protern.
Other important protein-folding catalysts in the ER Iu-
men are peptidyl-prolyl isomerases,a family of enzymesthat

IN THEER
, L D I N GA, N D Q U A L I T YC O N T R O L
M O D I F I C A T I O NFSO
PROTEIN 553
 (al Oligosaccharyl
transferase
Dolichol
oligosaccharide M e m b r a n e - s p anni n g Luminal
s helix crhelix
Cytosol

ER lumen I
HAotrimer

Completed
H A sm o n o m e r

> FIGURE 13-20Hemagglutinin folding and assembly. (b)

(a)Mechanism of (HAo) trimerassembly Transtent bindingof the
chaperone BiP(stepIE) to the nascent chainandof two lectins,
calnexin andcalreticulin, to certainoligosaccharide chains (steplE)
promotes properfoldingof adjacent segments. A totalof seven
N-linked oligosaccharide chains areaddedto theluminal portionof the
nascent chainduringcotranslational translocation, andPDIcatalyzes
theformation of sixdisulfide bondspermonomerCompleted HA6
monomers areanchored in the membrane by a singlemembrane-
i nst h el u m e n( s t e p[ )
s p a n n i nagh e l i xw i t ht h e i rN - t e r m i n u
Interaction of threeHAechains with oneanother, initially viatheir
transmembrane crhelices, apparently triggers formation of a long
stemcontaining onecthelixfromthe luminalpartof eachHAs
polypeptide Finally, interactions between thethreeglobular heads
occur,generating a stableHA6trimer(stepB) (b)Electron micrograph
of a complete influenza virionshowing trimers of HAprotein
protruding asspikes fromthesurface of theviralmembrane (a)
[part
SeeU Tatuetal, 1995, EMBO J 14:1340, andD Hebert elal, 1997, J Cell
Biol.139:613 Part(b) ChrisBjornberg/PhotoResearchers,
Inc l

acceleratethe rotation about peptidyl-prolyl bonds at pro-

line residuesin unfolded segmentsof a polypeptide:

o*^ 2,
Rotation about - Prolyl Many important secretory and membrane proteins syn-
peptide bond .U
,/-
t11
thesizedon the ER are built of two or more polypeptide sub-
units. In all cases,the assemblyof subunits constituring these
I
multisubunit (multimeric) proteins occurs in the ER. An im-
lzu\
O' NH portant class of multimeric secretedproteins is the im-
\
crs munoglobulins, which contain two heavy (H) and two light
trans
(L) chains,all linked by intrachaindisulfidebonds. Hemag-
Such isomerizations sometimesare the rate-limiting step in glutinin (HA) is another multimeric protein that provides a
the folding of protein domains. Many peptidyl-prolyl iso- good illustration of folding and subunit assembly(Figure 13-
merasescan catalyzethe rotation of exposedpeptidyl-prolyl 20). This trimeric protein forms the spikes that protrude
bonds indiscriminately in numerous proteins, but some have from the surface of an influenza virus particle. The HA
very specificprotein substrates. trimer is formed within the ER of an infected host cell from

554 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 Hacl < FIGURE 13-21The unfolded-protein response'lre1,a
transmembrane proteinin the ERmembrane, hasa bindingsite
f o r B i Po n i t sl u m i n adlo m a i nt h d o c o n t a i nas
; ec y t o s o l i c m a i n
specific RNAendonuclease. Il: Step Accumulating unfolded
Spliced
Hacl mRNA proteins in the ERlumenbindBiPmolecules, releasing themfrom
monomeric lrel Dimerization of lrel thenactivates its
endonuclease activityStepsE, B: Theunspliced mRNA
precursor encoding thetranscription factor Hacl is cleaved by
E n d o n u c l e a s e - cH
uat cl dimericlre1,andthe two exons are joined to form functional
Hacl mRNACurrent evidence indicates thatthisprocessing
occurstn thecytosol, althoughpre-mRNA processing generally
Unspliced
Hacl mRNA occursin the nucleusStep4: Hacl is translated into Hacl
protein, whichthenmoves back into the nucleus and activates
transcription of genesencoding several protein-folding catalysts'
U Ruegsegger etal, 2001, Cell'lO7:103;A Bertolottietal' 2000'
[See
Nature andP Walter,1997,Ceil
CellBiol 2:326;andC Sidrauski
9 0 : 1 0 3l1

lrel dimer

folding of normal proteins by preventing their aggregation

and binding to irreversibly misfolded proteins'
Both mammalian cellsand yeastsrespond to the presence
Unfoldedproteins Unfoldedproteins
w i t h B i Pb o u n d

three copiesof a precursor protein termed HA6, which has a

single membrane-spanningct helix. In the Golgi complex,
each of the three HAe proteins is cleaved to form two
polypeptides, HA1 and HA2; thus each HA molecule that
eventually resideson the viral surfacecontains three copies
of HAi and threeof HA2 (seeFigure3-10).The trimer is sta-
bilized by interactions between the large exoplasmic do-
mains of the constituent polypeptides,which extend into the
ER lumen; after HA is transported to the cell surface,these
domains extend into the extracellular space' Interactions
between the smaller cytosolic and membrane-spanning
portions of the HA subunits also help stabilize the trimeric
protein. Studieshave shown that it takesjust 10 minutesfor
proteins that assistin protein folding' . -
the HAs polypeptidesto fold and assembleinto their proper
Mammalian cells contain an additional regulatory
trimeric conformation.
pathway that operatesin responseto unfolded proteins in
in. En. In this pathway, accumulation of unfolded pro-
l m p r o p e r l yF o l d e dP r o t e i n si n t h e E RI n d u c e
Expressionof Protein-FoldingCatalysts
Vild-type proteins that are synthesizedon the rough ER
cannot exit this compartment until they achievetheir com-
pletely folded conformation. Likewise, almost any mutation
ih"t p..u..tts proper folding of a protein in the ER also
blocks movement of the polypeptide from the ER lumen or
membrane to the Golgi complex. The mechanismsfor re-
taining unfolded or incompletely folded proteins within the
F i g u r e s1 6 - 3 6 a n d 1 6 - 3 8 ) .
ER probably increase the overall efficiency of folding by
keeping intermediateforms in proximity to folding catalysts,
which are most abundant in the ER. Improperly folded pro- A hereditaryform of emphysemaillustratesthe detri-
teins retained within the ER generallyare seenbound to the mental effects that can result from misfolding of
ER chaperonesBiP and calnexin. Thus theseluminal folding proteins in the ER. This disease is caused by a point
catalvsts perform two related functions: assisting in the -rrt"tion in ct1-antitrypsin,which normally is secretedby

IN THEER O 555
, L D I N GA, N D Q U A L I T YC O N T R O L
M O D I F I C A T I O NFSO
PROTEIN
 the cell altogether by degradation in the proteasome(see
U n a s s e m b l eodr M i s f o l d e dp r o t e i n si n t h e Figure 3-29).
ERAre Often Transportedto the Cytosol
for Degradation
Protein Modifications, Folding, and euality Control
in the ER
r All N-linked oligosaccharides,which are bound to as_
paragineresidues,contain a core of three mannoseand two
N-acetylglucosamineresidues and usually have several
branches (seeFigure 13-16). O-linked olisosaccharides.
proteases were never found. More recent studies have
which are bound to serineor threonine resid-ues, g.rr.r-
shown that misfolded membrane and secretoryprorelns are "..
ally short, often containing only one to four ,rlg"r..ridrl...
recognized by specific ER membrane proteins and are tar_
geted for transport from the ER lumen into the cytosol, by r Formarion of all N-linked oligosaccharidesbegins with
a processknown as dislocation or letrotrlnslocat.ion. assemblyof a conserved14-residuehigh-mannose precur_
The dislocationof misfoldedproteins out of the ER de_ sor on dolichol, a lipid in the membrane of the rough ER
pends on a set of proteins located in the ER membrane and (seeFigure 73-17). After this preformed oligosaccharideis
in the cytosol that perform three basic functions. The first transferred to specific asparagine residues of nascent
polypeptide chains in the ER lumen, three glucoseresidues
and one mannoseresidueare removed(seeFigure 13-1g).
r Oligosaccharideside chains may assist in the proper
folding of glycoproteins, help protect the mature p.ot"irrc
from proteolysis,participate in cell-celladhesion, fr.rrr._
tion as antigens. ".rd
r Disulfide bonds are added to many secretory proteins
and the exoplasmic domain of membrane proteins in the
ER. Protein disulfide isomerase(pDI), presentin the ER lu_
men, catalyzesboth the formation and the rearrangement
of disulfide bonds (seeFigure 13-19).
r The chaperoneBiP, the lectins calnexin and calreticulin,
and peptidyl-prolyl isomeraseswork together ro ensure
proper folding of newly made secreroryand membrane
proteins in the ER. The subunits of multimeric proteins
also assemblein the ER.
r Only properly folded proteins and assembledsubunits
are transported from the rough ER to the Golgi complex in
vesicles.
r The accumularion of abnormally folded proteins and
unassembledsubunits in the ER can induce increasedex_
pression of ER protein-folding catalystsvia the unfolded_
protern response(seeFigure 13-21).
r Unassembledor misfolded proteins in the ER often are
transported back to the cytosol, where they are degradedin
the ubiquitin/proteasomepathway.

556 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 Sortingof Proteinsto
and Chloroplasts
Mitochondria
In the remainder of this chapter, we examine how proteins
synthesizedon cytosolic ribosomes are sorted to mitochon-
dria, chloroplasts, peroxisomes,and the nucleus (seeFigure
Proteins encoded by mitochondrial DNA or chloroplast
13-1).In both mitochondriaand chloroplastsan internallu-
DNA are synthesizedon ribosomes within the organelles
men called the matrix is surrounded by a double membrane,
and directed to the correct subcompartmentimmediately af'
and internal subcompartments exist within the matrix. In
ter synthesis.The majority of proteins located in mitochon-
contrast, peroxisomesare bounded by a single membrane
dria and chloroplasts, however, are encoded by genesin the
and have a single luminal matrix compartment. Becauseof
nucleus and are imported into the organellesafter their syn-
these and other differences,we consider peroxisomessepa-
thesis in the cytosol' Apparently, over aeons of evolution'
rately in the next section. Likewise, the mechanism of pro-
much of the geneticinformation from the ancestralbacterial
tein transport into and out of the nucleus differs consider-
DNA in these endosymbiotic organellesmoved' by an un-
ably from sorting to mitochondria and chloroplasts; this is
known mechanism, to the nucleus' Precursor proteins syn-
d i s c u s s eidn t h e l a s t s e c t i o n .
thesizedin the cytosol that are destinedfor the matrix of mi-
In addition to being bounded by two membranes'mito-
tochondria or the equivalent space in chloroplasts, the
chondria and chloroplastsshare similar types of electron
stroma, usually contain specificN-terminal uptake-targeting
transport proteins and use an F-classATPase to synthesize
sequencesthat specifybinding to receptor proteins on the or-
ATP (seeFigure 12-2). Remarkably,gram-negativebacteria
ganelle surface. Generally, this sequenceis cleaved once it
also exhibit thesecharacteristics.Also like bacterial cells' mi-
i.a.hes the matrix or stroma. CIearlS theseuptake-targeting
tochondria and chloroplastscontain their own DNA, which
sequencesare similar in their location and general function
encodesorganellerRNAs, tRNAs, and someproteins (Chap-
to ihe signal sequencesthat direct nascentproteins to the ER
ter 6). Moreover, growth and division of mitochondria and
lumen. Although the three types of signalsshare some com-
chloroplasts are not coupled to nuclear division. Rather,
mon sequencefeatures, their specific sequencesdiffer con-
these organellesgrow by the incorporation of cellular pro-
siderably,as summarizedin Table 13-1.
teins and lipids, and new organellesform by division of pre-
In both mitochondria and chloroplasts, protein import
existing organelles.The numerous similarities of free-living
requiresenergy and occurs at points where the outer and in-
bacterialcellswith mitochondria and chloroplastshave led
nei organellemembranesare in closecontact' Becausemito-
scientiststo hypothesize that these organelles arose by the
chondiia and chloroplasts contain multiple membranesand
incorporation of bacteria into ancestraleukaryotic cells,

ORGANETTE
TARGTT LOCATII]N WITHIN
OFSEOUENCE OFSEOUEI|CE
BEM()VAL
PROTTIN OTSEOUEIICE
NATURE

Endoplasmic reticulum N-terminus Yes Core of 6-12 hydrophobicamino acids,

(lumen) often precededby one or more basicamino
acids (Arg, Lys)

Mitochondrion (matrix) N-terminus Yes Amphipathichelix, 20-50 residuesin length,

with Arg and Lys residueson one sideand
hydrophobicresidueson the other

Chloroplast (stroma) N-terminus Yes No commonmotifs;generallyrich in Ser'

Thr, and smallhydrophobicresiduesand
poor in Glu and AsP

No at extreme
PTSl signal(Ser-Lys-Leu)
Peroxisome (matrix) C-terminus(mostproteins)
N-terminus(few proteins) C-terminus;PTS2signalat N-terminus

Nucleus (nucleoplasm) Varies No Multipledifferentkindsla commonmotif

includesa short segmentrich in Lys and Arg
residues

subcompartments'
"Different or additional sequencestarget proteins to organelle membranes and

AND CHLOROPLASTS
SO M I T O C H O N D R I A
557
S O R T I N GO F P R O T E I N T
 > EXPERIMENTAL FIGURE 13-22lmport Uptake-
of mitochondrialprecursorproteinsis targeting
assayedin a cell-freesystem.Inside sequence h_\{L/
mitochondria, proteins areprotected from
the actionof proteases
t
suchastrypsinWhen
no mitochondria arepresent, mitochondrial Add energized
proteinssynthesized in thecytosol Mitochondrial
are prolern
yeasr
degraded by addedproteaseproteinuptake mitochondria
occurs onlywith energized (respiring)
mitochondria, whichhavea proton
electrochemicalgradient (proton-motive Yeast mitochondrial Proteintaken up Proteinsseques-
force)acrossthe innermembrane proteinsmade by into mitochondria; tered within
The
cytoplasmicribosomes uptake-targeting mitochondria
imported proteinmustcontainan appropriate
in a cell-freesystem sequenceremoved are resistantto
uptake-targetingsequence Uptakealso and degraded trypsin
requiresATPanda cytosolic extractcontaininq
chaperone proteins thatmarntain the ..a
precursor .
proterns in an unfolded .a.
conformation Trypsin , &
Thisassay hasbeenusedto 't' '.e '
studytargeting sequences andotherfeatures j
.r'' r
of thetranslocationprocess t'. a'
'a a.
Uptake-targeting -..3 -
sequenceano
mitochondrial
protein degraded

membrane-limited spaces,sorting of many proteins to their
correct location often requires the sequentialaction of two
targetlng sequencesand two membrane-boundtranslocation
A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c e s
D i r e c tP r o t e i n st o t h e M i t o c h o n d r i aM
l atrix
pathicity of matrix-targeting sequencesis critical to their
function.
The cell-free assay outlined in Figure 13-22 has been
widely used in studies on the import of mitochondrial pre-
cursor proteins. In this system, respiring (energized)mito_
chondria extractedfrom cellscan incorporate mitochondrial
precursor proteins carrying appropriate uptake-targetingse_
quencesthat have been synthesizedin the absenceof mito-
chondria. Successfulincorporation of the precursor into the

translocation of secretoryproteins into the ER, which gener_

ally occurs only when microsomal (ER-derived)-.rnbn"rr.,
are present during synthesis(seeFigure 13-4).

Mitochondrial matrix-targeting sequencesare thought to
assume an a,helical conformation in which positively
charged amino acids predominate on one side ofthe helix
M i t o c h o n d r i aP
l r o t e i nl m p o r t R e q u i r e s
Outer-MembraneReceptorsand Translocons
in Both Membranes
Figure 13-23 presentsan overview of protein import from
the cytosol into the mitochondrial matrix, the route into the
mitochondrion followed by most imported proteins. \Wewill
discussin detail each step of protein transport into the ma-
trix and then consider how some proteins subsequentlyare
'
targeted to other compartments of the mitochondiion.
After synthesisin the cytosol, the soluble precursors of
mitochondrial proteins (including hydrophobic integral
membrane proteins) inreract directly with the mitochondrial
membrane. In general, only unfolded proteins can be im_
ported into the mitochondrion. Chaperoneproteins such as

558 ' c H A p r E R1 3 I M o v r N Gp R o r E r NrsN T oM E M B R A N A

ENs Do R G A N E L L E '
 coo < FIGURE 13-23Proteinimport intothe
mitochondrialmatrix.Precursor proteins
synthesized on cytosolic ribosomes are
maintained in an unfolded or partially folded
ATP Hsc70
stateby bound chaperones, such as
A D P+ P ; (steptr) Aftera precursor proteinbindsto
an importreceptor neara siteof contactwith
the innermembrane (stepZ), it istransferred
Cytosolic
Hsc70 intothe general importpore(stepB) The
translocating protein thenmovesthroughthls
c h a n n ealn da n a d j a c e nc th a n n ei nl t h ei n n e r
-'.'./ Matrix-targeting membrane (stepsZl, E). Notethat
sequence translocation occurs at rare"contactsites"
at whichthe inner and outermembranes
NHs* of thetranslocating
appearto touch.Binding
proteinbythe matrixchaperone Hsc70and
subsequent ATPhydrolysis by Hsc70helps
driveimportintothe matrixOncethe
uptake-targeting sequence is removed by a
lmport General
i m p o r tp o r e matrixprotease andHsc70isreleased from
recepror
(Tom 20122\ (Tom40) the newlyimported protein(step6), it folds
intothe mature, activeconformation within
Cytosol +\ (step of some proteins
the matrix Z) Folding
depends on matrixchaperonins [See
Outer membrane and
G Schatz, 1996, J BiolChem271:31763,
el al , 1997,Ann Rev.CellDevelBiol
N Pfanner
13:25l

Matrix
Hsc70
ADP + P;
Matrix
processlng
protease

Cleaved
targeting
seq uence

cytosolic Hsc70 keep nascentand newly made proteins in an
unfolded stateso that they can be taken up by mitochondria'
This processrequiresATP hydrolysis' Import of an unfolded
mitochondrial precursor is initiated by the binding of a mito-
chondrial targeting sequenceto an import receptor in the
outer mitochondrial membrane. These receptors were first
identified by experimentsin which antibodiesto specificpro-
teins of the outer mitochondrial membranewere shown to in-
hibit protein import into isolated mitochondria. Subsequent
genetic experiments, in which the genes for specific mito-
chondrial outer-membraneproteins were mutated, showed
that specificreceptorproteinswere responsiblefor the import
of different classesof mitochondrial proteins. For example,
N-terminal matrix-targeting sequencesare recognized by
Tom20 and Tom22. (Proteinsin the outer mitochondrial
membrane involved in targeting and import are designated
Tom oroteins for /ranslocon of the outet membtane.)
The import receptorssubsequentlytransfer the precursor
proteins to an import channel in the outer membrane' This
ch".tn.l, composed mainly of the Tom40 protein, is known
^s the general import pore becauseall known mitochondrial
compartments
precursor proteins gain accessto the interior 'Sfhen
ft tn. -itt.hondrion through this channel' purified
and incorporated into liposomes, Tom40 forms a transmem-
brane channel with a pore wide enough to accommodatean
unfolded polypeptideihain. The general import pore forms
a largely p"tti". channel through the outer mitochondrial
-.*L.".t., and the driving force for unidirectional transport
into mitochondria comesfrom within the mitochondrion' In
the caseof precursorsdestinedfor the mitochondrial matrix,
transfer through the outer membrane occurs simultaneously
with transfer through an inner-membranechannel composed
of the Tim23 and1iml7 proteins. \Tim standsfor translo-
con of the lnner membrane.)Translocation into the matrix

O 559
SO M I T O C H O N D R I A N D C H L O R O P L A S T S
S O R T I N GO F P R O T E I N T
 thus occurs at "contact sites" where the outer and inner
membranesare in close proximitv.
drial membraneby interactingwith transmembraneprotein
Tim44. This interaction stimulatesATp hydrolysis by matrix
Hsc70, and together these two proteins are thought to
power translocationof proteinsinto the matnx.
Some imported proteins can fold into their final, active
conformation without further assistance.Final folding of
many matrrx proteins, however, requires a chaperonin. As
discussedin Chapter 3, chaperoninproteins aciively facili-
tate protein folding in a processthat dependson ATp. For in_
stance,yeastmutantsdefectivein Hsc60, a chaperoninin the
mitochondrial matrix, can import matrix proteins and
cleavetheir uptake-targerrngsequencenormally, but the im-
ported polypeptidesfail to fold and assembleinto the native
tertiary and quaternarystructures.
S t u d i e sw i t h C h i m e r i cP r o t e i n sD e m o n s t r a t e
l m p o r t a n tF e a t u r e so f M i t o c h o n d r i a lm p o r t
Dramatic evidencefor the ability of mitochondrial matrix-
targettng sequencesto direct import was obtained with
chimeric proteins produced by recombinant DNA tech-
niques. For example, the matrix-targeting sequenceof alco-
hol dehydrogenasecan be fused to the N-terminus of dihy-
drofolate reductase(DHFR), which normally residesin the
cytosol. In the presenceof chaperones,which prevent the C-
terminal DHFR segmentfrom folding in the cytosol, cell-free
translocation assaysshow that the chimeric protein is trans-
ported into the marrix (Figure 13-24a). The inhibitor
methotrexate,which binds tightly ro the acive site of DHFR
and greatly stabilizes its folded conformation, renders the
chimeric protein resistant to unfolding by cytosolic chaper-
ones. \il1'hentranslocation assaysare performed in the pres-
ence of methotrexate, the chimeric protein does not iom-
pletely enter rhe matrix. This finding demonstratesthat a
precursor must be unfolded in order to traverse the imoort
poresin the mitochondrialmembranes.

(a)
(b) Bound (cl
methotrexate
inhibitor
Cytosol
Outer
F o l d e d-
DHFR
Cytosol

Outer membrane
Intermembrane
space
Intermembrane
space
NHr*
.d9"
.s9'
. ((.9.
\$$o

Cleaved
Mitochondrial targeting
matrix sequence
o'2 P'm
Spacerr"qr"n.i / , ,

EXPERIMENTAL FTGURE 13-24 Experimentswith chimeric transport channels, a stabletranslocation intermediate, with the
proteins elucidate mitochondrial protein import. These targeting sequence cleaved off, isgenerated in the presence of
experiments show that a matrix-targeting
sequencealonedirects methotrexate, asshownhere.(c)TheC-terminus of thetranslocation
intermediate in (b)canbe detected by incubating the mitochondria
with antibodies thatbindto the DHFR segment, followedby gold
particlescoatedwith bacterial proteinA, whichbindsnonspecificallv
to antibody molecules (seeFigure9-21).An electron micrograph of a
sectioned sample reveals goldparticles(redarrowhead) boundto the
translocationintermediate at a contactsrtebetween the innerand
outermembranes Othercontactsites(blackarrows) alsoareevident
energizem d i t o c h o n d r iaan d t h e m a t r i x _ t a r g e t isnigg n atl h e n i s (a)and(b)adapted
IParts fromJ Rassow et al, 1990,FEBS Letters
2Tstigo
removed.(b) When the C-terminusof the chimericproteinis locked Part(c) from M SchweigereI al , j987, I CellBiol. 105:235,courtesyof
in the foldedstateby bindingof methotrexate, translocalonrs W N e u o e r tl
blocked lf the spacersequenceis long enoughto extendacrossboth

560 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 Additional studies revealed that if a sufficiently long
spacer sequenceseparatesthe N-terminal matrix-targeting
sequenceand DHFR portion of the chimeric protein, then in
the presenceof methotrexate a translocation intermediate
that spansboth membranescan be trapped if enough of the
polypeptideprotrudes into the matrix to preventthe polypep-
tide chain from sliding back into the cytosol, possibly by sta-
bly associatingwith matrix Hsc70 (Figure 13-24b).In order
for such a stable translocation intermediateto form, the
spacer sequencemust be long enough to span both mem-
branes;a spacerof 50 amino acids extendedto its maximum
possiblelength is adequateto do so. If the chimeracontains
a shorter spacer-say, 35 amino acids-no stable transloca-
tion intermediateis obtained becausethe spacercannot span
both membranes.These observationsprovide further evi-
dence that translocated proteins can span both inner and
outer mitochondrial membranes and traverse these mem-
branesin an unfolded state.
Microscopic studiesof stabletranslocation intermediates
show that they accumulateat siteswhere the inner and outer
mitochondrial membranes are close together, evidencethat
precursor proteins enter only at such sites (Figure 1,3-24c).
The distancefrom the cytosolic face of the outer membrane
to the matrix face of the inner membrane at these contdct
sitesis consistentwith the length of an unfolded spacer se-
quence required for formation of a stable translocation in-
termediate.Moreover, stabletranslocation intermediatescan
be chemically cross-linkedto the protein subunits that com-
prise the translocation channelsof both the outer and inner
membranes.This finding demonstratesthat imported pro-
teins can simultaneouslyengagechannels in both the outer
and inner mitochondrial membrane, as depicted in Figure
13-23. Since roughly 1000 stuck chimeric proteins can be
observedin a typical yeast mitochondrion, it is thought that
mitochondria have approximately 1000 general import
pores for the uptake of mitochondrial proteins.
Three EnergyInputs Are Neededto lmport
P r o t e i n si n t o M i t o c h o n d r i a
As noted previously and indicated in Figure 13-23, ATP hy-
drolysis by Hsc70 chaperoneproteins in both the cytosol and
the mitochondrial matrix is required for import of mitochon-
drial proteins. Cytosolic Hsc70 expends energy to maintain
bound precursor proteins in an unfolded statethat is compe-
tent for translocationinto the matrix. The importance of ATP
to this function was demonstratedin studiesin which a mito-
chondrial precursor protein was purified and then denatured
(unfolded) by urea. \7hen tested in the cell-free mitochon-
drial translocation system,the denatured protein was incor-
porated into the matrix in the absenceof AIP. In contrast'
import of the native, undenaturedprecursorrequired ATP for
the normal unfolding function of cytosolic chaperones.
The sequentialbinding and ATP-drivenreleaseof multi-
ple matrix Hsc70 moleculesto a translocatingprotein may
simply trap the unfolded protein in the matrix. Alterna-
tively, the matrix Hsc70, anchoredto the membraneby the
Tim44 protein, may act as a molecular motor to pull the
S O R T I N GO F P R O T E I N T
protein into the matrix (seeFigure 13-23).In this case'the
lunctions of matrix Hsc70 andTim44 would be analogous
to those of the chaperone BiP and Sec63 complex, respec-
tivelS in post-translational translocation into the ER lumen
( s e eF i g u r e1 3 - 9 ) .
The third energy input required for mitochondrial
protein import is a H+ electrochemicalgradient, or proton-
motive force, acrossthe inner membrane' Recall from Chap-
ter 12 that protons are pumped from the matrix into the
intermembrane space during electron transport, creating
transmembranepotential across the inner membrane' In
general,only mitochondria that are actively undergoing res-
piration, and thereforehave generateda proton-motive force
acrossthe inner membrane' are able to translocateprecursor
proteins from the cytosol into the mitochondrial matrix'
ir.u,-.n, of mitochondria with inhibitors or uncouplers of
oxidative phosphorylation' such as cyanide or dinitrophe-
nol, dissipatesthis proton-motive force. Although precursor
proteins still can bind tightly to receptorson such poisoned
mitochondria, the proteins cannot be imported, either in in-
tact cells or in cell-freesystems,even in the presenceof ATP
and chaperone proteins. Scientistsdo not fully understand
how the proton-motive force is used to facilitate entry of a
p...rr.roi p.otein into the matrix. Once a protein is partially
inserted into the inner membrane' it is subjectedto a trans-
M u l t i p l eS i g n a l sa n d P a t h w a y sT a r g e tP r o t e i n s
t o S u b m i t o c h o n d r i aCl o m p a r t m e n t s
Unlike targeting to the matrix, targeting of proteins to the in-
termembrane space,inner membrane, and outer membrane
of mitochondria generally requires more than one targetlng
sequenceand occurs via one of severalpathways' Figure 13-
25 summarizes the organization of targeting sequencesin
proteins sorted to different mitochondrial locations.
lnner-Membrane Proteins Three separatepathways are
known to target proteins to the inner mitochondrial mem-
brane. One pathway makes use of the same machinery that
is used for targeting of matrix proteins (Figure 13-26, path
A). A cytochrome oxidase subunit called CoxVa is a protein
transported by this pathway' The precursor form of CoxVa'
which contains an N-terminal matrix-targeting sequence
recognizedby the Tom20l22 import receptor,is transferred
through the generalimport pore of the outer membrane and
the inier-membrane Tim23l17 translocation complex' In ad-
dition to the matrix-targeting sequence'which is cleaved
during import, CoxVa contains a hydrophobic stop-transfer
s.qr'r*ce. A. the protein passesthrough theTim23ll'7 chan-
.r.i th. stop-transfer sequenceblocks translocation of the
AND CHLOROPLASTS O
SO M I T O C H O N D R I A
561
 Location lmported Locations of targeting sequences < FIGURE 13-25Targetingsequences in
of imported protein in preprotein
protein imported mitochondrialproteins.Most
mitochondrial proteins havean N-terminal
Cleavageby matrix-targeting sequence (pink)thatissimilar
matrix protease but not identical in different proteins
proteins.
Matrix Alcohol
d e h y d r o g e n a slel l
*.*T**" destined for the innermembrane,
rntermembrane space,
the
or the outermembrane
Matrix-targeting
haveoneor moreadditional targeting
sequence Mature protein sequences thatfunctionto directthe proteins
to theselocations by severaldifferent
Inner pathways. Theletteredpathways correspond
Cleavageby Hydrophobic
membrane matrix protease stop-transfersequence to thoseillustrated in Figures 13-26and
Cytochrome 13-27 [See W Neupert, 1997,Ann Rev.Biochem
PathA o x i d a s es u b u n i t 66:863l
CoxVa

Cleavageby Internalsequences
matrix protease recognizedby Oxal
ATP
Path B synthase
s u b u n i t9

Internalsequencesrecognized
by Tom70 receptor andTrm22 complex
ADP/ATP
Path c
anttporter

Intermembrane Firstcleavageby Secondcleavageby protease

space matrix

PathA Cytochromeb2
Intermembrane-space-targeting
sequence

Targetingsequencefor
the generalimport pore
path B Cytochromec
\--...P\-..^t
h e m el y a s e

Outer Stop-transferand outer-memorane

membrane localizationsequence

Porin (P70)

C-terminus across the inner membrane. The membrane-
anchored intermediate is then transferred laterally into the
bilayer of the inner membrane much as type I integral mem-
brane proteins are incorporated into the ER membrane (see
F i g u r e1 3 - 1 1 ) .
matrix via the Tom40 andTim23l17 channels.After cleav_
age of the matrix-targeting sequence,the protein is inserted
into the inner membrane by a processthai requires interac_
tion with Oxal and perhaps other inner-membraneproteins
(Figure 13-26, parh B). Oxal is related to a bacterialprotein
involved in inserting some inner-membraneproteins in bac-
teria. This relatednesssuggeststhat Oxal may have de-
scendedfrom the translocation machinery in the endosymbi-
otic bacterium that eventually became the mitochondrion.
However, the proteins forming the inner-membranechannels
in mitochondria are not related to the proteins in bacterial
translocons. Oxal also participates in ihe inner-membrane
insertion of certain proteins (e.g., subunit II of cytochrome
oxidase) that are encoded by mitochondrial DNA and syn-
thesizedin the matrix by mitochondrial ribosomes.
The final parhway for insertion in the inner mitochon-
drial membrane is followed by multipass proteins that

562 . c H A p r E lR3 | M o v t N Gp R o r E t Nt sN T oM E M B R A NAENsD O R G A N E L L E S

 Path A Path B

Stop-transfer Matrix-targeting
sequence sequence

NHs

NHs*

Tom40 Tom22 Tom20

Cytosol

Outer
membrane

lntermembrane
space

Ttm23l17

; €
A

Mitochondrial Assembled
protein
matrix

Cleaved
matrix-targeting
SeQu€nCeS-_--_-

playsa rolesimilar to itsrolein the importof solublematrixproteins

A FIGURE 13-26Threepathwaysto the innermitochondrial
with differenttargeting (seeFigure13-23). Proteinsdelivered by pathwayC containtnternal
membranefrom the cytosol.Proteins
viadifferentpathways. sequences that arerecognized by the Tom70/fom22 importreceptor;
sequences aredrrected to the innermembrane
a different inner-membrane translocation channel(Tim22154)isused
In allthreepathways, proteins crossthe outer membrane viathe
A andB in thispathway. Two intermembrane (Tim9
proteins andTim10)
Tom40general importpore Proteins deliveredby pathways
containan N-terminal matrix-targetingsequence thatis recognized facilitatetransfer betweenthe outerandinnerchannelsSeethe
importreceptor in theoutermembrane Although text for discussion [SeeR E Dalbey andA Kuhn,2000,AnnRevCell
bytheTom20/22
boththesepathways usetheTim23/1 7 inner-membrane channel, DevelBiol16:5l,andNPfannerandAGeissler,2OOl,NatureRevMolC
theydifferin thatthe entireprecursor proteinentersthe matrixand Biol 2:339l
thenisredirected to the innermembrane in pathwayB MatrixHsc70

contain six membrane-spanning domains, such as the

ADP/ATP antiporter. Theseproteins, which lack the usual
N-terminal matrix-targeting sequence'contain multiple
internal mitochondrial targeting sequences.After the in-
ternal sequencesare recognizedby a secondimport recep-
tor composed of outer-membrane proteins Tom70 and
Tom22, the imported protein passesthrough the outer
membranevia the generalimport pore (Figure 13-26, path
C). The protein then is transferredto a secondtransloca-
tion complex in the inner membrane composedof the
Tim22 and Tim54 proteins. Transfer to the Tim22l54 protein into the inner membrane.

. 563
SO M I T O C H O N D R I A N D C H L O R O P L A S T S
S O R T I N GO F P R O T E I N T
 Intermembrane-Space Proteins Two pathways deliver
cytosolicproteinsto the spacebetweenthe inner and outer mi-
tochondrial membranes.The major pathway is followed by
proteins, such as cytochrome b2, whose precursorscarry rwo
different N-terminal targeting sequences,both of which ulti-
mately are cleaved.The most N-terminal of the rwo seouences
is a matrix-targeting sequence,which is removed by th. -a-
import pore without involvement of any inner-membrane
translocationfactors (Figure 13-27, path B). Sincetranslo-
cation through the Tom40 general import pore does not
seemto be coupled to any energeticallyfavorable process
such as hydrolysis of ATP or GTP, the mechanism rhat
drives unidirectional translocation through the outer mem-
brane is unclear. One possibility is that cytochrome c heme
lyase passively diffuses through the outlr membrane and
then is trapped within the intermembrane spaceby binding
to another protein that is deliveredto that location by one
of the translocationmechanismsdiscussedpreviously.

Outer-Membrane Proteins Many of the proteinsthat re-

proteolytic cleavage,this pathway is similar to that of inner-
membraneproteins suchas CoxVa (seeFigure 13-26,parh A).
Cytochrome c heme lyase, the enzyme responsiblefor
the covalent attachment of heme to cytochrome c, illus-
trates a secondpathway for targetingto the intermembrane
space.In this pathway, the imported protein does not con_
tain an N-terminal matrix-targetingsequenceand is deliv_
ered directly to the intermembranespace via the general
side in the mitochondrial outer membrane, including the
Tom 40 pore itself and mitochondrial porin, have a B-barrel
structure in which antiparallel strandsform the hydrophobic
transmembranesegmentssurrounding a central channel.
Such proteins are incorporated into the outer membrane by
first interacting with the general import pore, Tom40, and
then they are transferred to a complex known as the SAM
(sorting and assembly machinery) complex, which is com-
posedof at leastthree outer membraneproteins.presumably
it is the very stable hydrophobic nature of B-barrel proteins

Path A Path B
Intermembrane-space-targeti
ng I n t e r m e mD r a n e-space-targeting
sequence
Matrix-targeting
sequence Protein
/,
'NHs* -*..- NHs

Tom22
Tom20

Tim23l17
Mitochondrial
matrix
Cleaved

3:'J'J;'.T'"''"n
FfGURE 13-27 Two pathwaysto the mitochondrial theproteinon the intermembrane-space
sideof the membrane
intermembrane space.pathway A, the majoronefor delivery
of Thereleasedproteinthenfoldsandbindsto itshemecofactor within
proteins
fromthe cytosol to the intermembrane space,issimilar
to the intermembrane spacePathwayB involvesdirectdelivery
to the
pathway A for delivery
to the innermembrane (seeFigure13_26) intermembrane spacethroughtheTom40general importporein the
Themajordifference isthatthe internaltargeting sequencein outermembrane. [SeeR E Dalbey
andA Kuhn,2OOO, Ann Rev.
Cett
proteins
suchascytochrome b2destined for the intermembrane Devel Biol 16:51; N PfannerandA Geissler,2001, Nature Rev.Mol. CettBiot.
spaceisrecognized by an inner-membrane protease. whichcleaves 2 : 3 3 9 ;a n d K D i e k e r te t a l , 1 9 9 9 ,p r o c N a t , l A c a d S c i U S A 9 6 : 1 1 1 5 2 ] |

564 CHAPTER
13 | M O V T N GP R O T E t NtSN T O M E M B R A N E A
SN D O R G A N E L L E S
that ultimately causesthem to be stably incorporated into
the outer membrane, but precisely how the SAM complex
facilitatesthis processis not known.
Targetingof ChloroplastStromalProteins
l s S i m i l a rt o l m p o r t o f M i t o c h o n d r i a l
Matrix Proteins
Among the proteins found in the chloroplast stroma are the
enzymesof the Calvin cycle,which function in fixing carbon
dioxide into carbohydratesduring photosynthesis(Chap-
ter 12). The large (L) subunit of ribulose 1,5-bisphosphate
carboxylase(rubisco) is encodedby chloroplast DNA and
synthesizedon chloroplastribosomesin the stromal space.
The small (S) subunit of rubisco and all the other Calvin
cycle enzymesare encodedby nuclear genesand transported
to chloroplastsafter their synthesisin the cytosol.The precur-
sor forms of these stromal proteins contain an N-terminal
stromal-impolt sequence(seeTable 13-1).
Experiments with isolated chloroplasts, similar to those
with mitochondria illustrated in Figure 1'3-22, have shown
that they can import the S-subunitprecursor after its synthe-
sis. After the unfolded precursor entersthe stromal space,it
binds transiently to a stromal Hsc70 chaperone and the
N-terminal sequenceis cleaved. In reactions facilitated by
Hsc60 chaperoninsthat reside within the stromal space,
eight S subunits combine with the eight L subunits to yield
the active rubisco enzyme.
The generalprocessof stromal import appearsto be very
similar to that for importing proteins into the mitochondrial
matrix (seeFigure 1,3-23).At least three chloroplast outer-
membrane proteins, including a receptor that binds the
stromal-import sequenceand a translocationchannelprotein'
and five inner-membraneproteins are known to be essential
for directing proteins to the stroma. Although theseproteins
are functionally analogousto the receptor and channel pro-
teins in the mitochondrial membrane,they are not structurally
homologous. The lack of sequencesimilarity between these
chloroplast and mitochondrial proteins suggeststhat they
may have arisen independentlyduring evolutton.
The available evidencesuggeststhat chloroplast stromal
proteins, like mitochondrial matrix proteins, are imported in
the unfolded state. Import into the stroma dependson ATP
hydrolysis catalyzedby a stromal Hsc70 chaperone whose
function is similar to that of Hsc70 in the mitochondrial ma-
trix and BiP in the ER lumen. Unlike mitochondria, chloro-
plasts do not generatean electrochemicalgradient (proton-
motive force) across their inner membrane. Thus protein
import into the chloroplast stroma appears to be powered
s o l e l yb y A T P h y d r o l y s i s .
ProteinsAre Targetedto Thylakoidsby
MechanismsRelatedto TranslocationAcross
t h e B a c t e r i aIln n e r M e m b r a n e
In addition to the double membranethat surroundsthem,
chloroplasts contain a series of internal interconnected
S O R T I N GO F P R O T E I N T
membranous sacs, the thylakoids (seeFigure 12-29). Pro-
teins localized to the thylakoid membrane or lumen carry
out photosynthesis.Many of theseproteins are synthesized
in the cytosol as precursorscontaining multiple targeting se-
quences.For example, plastocyanin and other proteins des-
tined for the thylakoid lumen require the successiveaction
of two uptake-targetingsequences. The first is an N-terminal
stromal-import sequence that directs the protein to the
stroma by the same pathway that imports the rubisco S sub-
unit. The second sequence targets the protein from the
stroma to the thylakoid lumen' The role of these targeting
sequences has been shown in experiments measuring the
uptake of mutant proteins generated by recombinant DNA
techniques into isolated chloroplasts. For instance' mutant
plastocyanin that lacks the thylakoid-targeting sequence
but contains an intact stromal-import sequenceaccumu-
lates in the stroma and is not transported into the thylakoid
Iumen.
Four separatepathways for transporting proteins from
the stroma into the thylakoid have been identified. All four
pathways have been found to be closely related to analo-
golls t."ntport mechanismsin bacteria' illustrating the close
evolutionary relationship between the stromal membrane
and the bacterial inner membrane. Transport of plasto-
cyanin and related proteins into the thylakoid lumen from
the stroma occurs by a chloroplast SRP-dependentpathway
that utilizes a translocon similar to SecY,the bacterial ver-
sion of the Sec61complex (Figure 1'3-28,left). A second
pathway for transporting proteins into the thylakoid lumen
inuolues a protein related to bacterial protein SecA, which
uses the energy from ATP hydrolysis to drive protein
translocation through the SecY translocon. A third path-
way, which targets proteins to the thylakoid membrane, de-
pends on a protein related to the mitochondrial Oxal pro-
iein and the homologous bacterial protein (see Figure
13-26,path B). Someproteinsencodedby chloroplastDNA
and synthesizedin the stroma or transported into the
stroma from the cytosol are inserted into the thylakoid
membrane via this pathwaY.
Finally, thylakoid proteins that bind metal-containing
cofactors follow another pathway into the thylakoid lumen
(Figure 13-28, rigbt). The unfolded precursorsof thesepro-
teiis are first targeted to the stroma' where the N-terminal
stromal-import sequenceis cleavedoff and the protein then
folds and bittdt itt cofactor' A set of thylakoid-membrane
proteins assistsin translocating the folded protein and
tound cofactor into the thylakoid lumen, a processpowered
by the pH gradient normally maintained across the thy-
lakoid membrane.The thylakoid-targeting sequencethat di-
rects a protein to this pH-dependent pathway includes two
closely spacedarginine residuesthat are crucial for recogni-
tion. Baiterial cells also have a mechanismfor translocating
folded proteins with a similar arginine-containing sequence
across the inner membrane. The molecular mechanism
whereby theselarge folded globular proteins are transported
across the thylakoid membrane is currently under intense
srudy.
SO M I T O C H O N D R I A N D C H L O R O P L A S T S
555
 < FIGURE 13-28Transporting proteins
Thylakoid-targeting
sequence
coo to chloroplastthylakoids.Twoof the
Plastocya
nin
Precursor
four pathways for transporting proteins
COO
Stromal-import --..-
fromthecytosol to thethylakoid lumenare
sequence shownhere In thesepathways, unfolded
precursors aredelivered to thestromavia
NrLr
thesameouter-membrane proteins that
Hat
importstromal-localized proteins. Cleavage
Toc Toc
complex of the N-terminal stromal-import sequence
complex
Cytosol by a stromalprotease thenreveals the
thylakoid-targeting sequence (steptr)
Outer membrane
At thispointthetwo pathways diverge.
ln the SRP-dependent pathway (/efr),
plastocyanin andsimilar proteins arekept
Intermembrane unfolded in the stromal spaceby a setof
space
chaperones (notshown)and,directed by
thethylakoid-targeting sequence, bindto
I n n e rm e m b r a n e proteins thatareclosely related to the
, ,) r,..r, Tic bacterial SRP, SRPreceptor, andSecY
comprex translocon, whichmediate movement into
Stroma
the lumen(stepZ). Afterthe thylakoid-
targeting sequence is removed in the
""\ :-.-.,** RR Metal-binding thylakoid lumenbya separate endoprotease,
"".*' g'"auudrnoon
sequence theproteinfoldsintoitsmatureconformation
Cleavedimporl
(stepB) In the pH-dependent pathway
Fl (nght),metal-binding
E,[\
sequence proteins fold in the
/ ,t- I SRP-dependent
.' stroma,andcomplexredoxcofactors
Chloroplast pathway are
SRP added(stepZ). Twoarginine residues (RR)
Bound at the N-terminus of thethylakoid-
metal targeting sequence anda pHgradient
tons
across the innermembrane arereouired for
transport of thefoldedproteinintothe
thylakoid lumen(stepg). Thetranslocon
in thethylakoid membrane iscomposed of
at leastfourproteins related to proteins in
thebacterial rnnermembrane. Thethylakoid
targeting sequence containing thetwo
Chloroplast arginine residues iscleaved in thethylakoid
SRPreceptor
':,!.) lumen(step4) [See R Datbey andC Robinson,
RR.b.-- 'j.:
1999,TrendsBiochem Sci24:1j,R E Dalbey
andA Kuhn,2000, Ann Rev. CellDevelBiol.
Mature
Mature 1 5 : 5 1 ;a n d C R o b i n s oann d A B o l h u i s2.0 0 1 .
metal-binding
plastocyani
n protein NatureRev.Mol CellBiol 2:350)

Translocation in mitochondria occurs at sites where the

Sorting of Proteins to Mitochondria and Chloroplasts
r Most mitochondrial and chloroplast proteins are en_
coded by nuclear genes,synthesizedon cytosolic ribosomes,
and imported post-translationallyinto the organelles.
r Cytosolic chaperonesmaintain the precursors of mito_
chondrial and chloroplast proteins in an unfolded state.
Only unfolded proteins can be imported into the organelles.
outer and inner membranesof the organellesare close to-
gether.
r Proteins destined for the mitochondrial matrix bind to
receptors on the outer mitochondrial membrane and then
are transferred to the general import pore (Tom40) in the
outer membrane.Translocationoccursconcurrentlythrough
the outer and inner membranes,driven bv the Droton-
motive force acrossthe inner membraneand ATp hyirolysis
by the Hsc70 ATPasein the matrix (seeFigure 13-23).
r Proteins sorted to mitochondrial destinationsother than
the matrix usually contain two or more targeting sequences,
one of which may be an N-terminal matrix-targeting sequence
(seeFigure 13-25).

556 CHAPTER
13 I M O V T N Gp R O T E t N |SN T O M E M B R A N E A
SN D O R G A N E L L E S
r Somemitochondrial proteins destinedfor the intermem- many different peroxisomal matrix proteins bear a sequence
brane spaceor inner membrane are first imported into the of this type, known as peroxisomal-targetingsequenceL' or
matrix and then redirected; others never enter the matrix simply PTS1.
but go directly to their final location. The pathway for import of catalaseand other PTSI-
bearing proteins into the peroxisomal matrix is depicted in
r Protein import into the chloroplast stroma occurs
Figure 13-29. The PTSl binds to a soluble carrier protein in
through inner-membrane and outer-membrane transloca-
the cytosol (Pex5), which in turn binds to a receptor in the
tion channelsthat are analogousin function to mitochon-
peroxisomemembrane(Pex14).The solubleand membrane-
drial channels but composed of proteins unrelated in se-
associatedperoxisomal import receptors appear to have a
quenceto the correspondingmitochondrial proteins.
function analogous to that of the SRP and SRP receptor in
r Proteins destined for the thylakoid have secondarytar- targeting proteins to the ER lumen, except that the soluble
geting sequences.After entry of these proteins into the ptsf -bindlng protein functions post-translationally.The pro-
stroma,cleavageof the stromal-targetingsequences reveals tein to be imported then moves through a multimeric
the thylakoid-targetingsequences.
r The four known pathways for moving proteins from the
chloropiast stroma to the thylakoid closely resemble
translocation acrossthe bacterial inner membrane (seeFig-
ure 13-28). One of these systemscan translocatefolded
protelns.

Proteins
Sortingof Peroxisomal
Peroxisomesare small organellesbounded by a single mem-
brane. Unlike mitochondria and chloroplasts, peroxisomes
lack DNA and ribosomes.Thus all peroxisomalproteinsare
encoded by nuclear genes,synthesizedon ribosomes free in
the cytosol, and then incorporated into preexisting or newly
generatedperoxisomes.As peroxisomesare enlargedby ad-
dition of protein (and lipid), they eventuallydivide, forming
new ones,as is the casewith mitochondria and chloroplasts.
The size and enzyme composition of peroxisomes vary Pex14
considerablyin different kinds of cells. However, all peroxi-
somescontain enzymesthat use molecular oxygen to oxidize
various substratessuch as amino acids and fatty acids,
breaking them down into smaller components for use in
biosyntheticpathways.The hydrogenperoxide (H2O2)gen-
erated by theseoxidation reactionsis extremely reactiveand E
potentially harmful to cellular componentsl however, the
Peroxisomal
peroxisome also contains enzymes,such catalase,that effi-
matrix protein
ciently convert H2O2 into H2O. In mammals,peroxisomes
are most abundant in liver cells,where they constitute about
1 to 2 percent of the cell volume. A FIGURE 13-29PTS1directedimportof peroxisomalmatrix
proteins.Step[: Catalase andmostotherperoxisomal matrix
proteinscontaina C-terminal PTSluptake-targeting sequence (red)
CytosolicReceptorTargetsProteinswith thatbindsto the cytosolic receptor Pex5. Step Z: Pex5 with the
a n S K LS e q u e n c ea t t h e C - T e r m i n uisn t o boundmatrixproteininteracts withthe Pex'14 receptor located on
t h e P e r o x i s o m aM
l atrix the peroxisome membrane. StepB: Thematrixprotein-Pex5
complex isthentransferred to a setof membrane proteins (Pex'l0,
The import of catalaseand other proteins into rat liver per- thatare necessary for translocation into the
Pexl2, andPex2)
oxisomes can be assayedin a cell-freesystemsimilar to that peroxisomal matrixby an unknownmechanism. Step4: At some
used for monitoring mitochondrial protein import (seeFig- point,eitherduringtranslocation or in the lumen,Pex5dissociates
ure 73-22). By testing various mutant catalaseproteins in fromthe matrixproteinandreturns to the cytosol, a process that
this system, researchersdiscovered that the sequenceSer- involves IhePex2/10112 complex andadditional membrane and
Lys-Leu (SKL in one-lettercode) or a related sequenceat the cytosolic proteins not shown.Notethatfoldedproteins canbe
C-terminus was necessaryfor peroxisomal targeting. Fur- imported intoperoxisomes andthatthetargeting sequence ls not
ther, addition of the SKL sequenceto the C-terminus of a removed in the matrix[See P E Purdue andP B Lazarow,2001 , Ann
normally cytosolicprotein leadsto uptake of the alteredpro- CellDevel
Rev. Biot17:101, S Subramani et al, 2000,Ann RevBrcchem
tein by peroxisomes in cultured cells. All but a few of the 59:39a9n ; d V D a m maanid SS u b r a m a n i , ,2C0e0l1l 0 5 : 1 817

LROTEINS .
S O R T I N GO F P E R O X I S O M AP 567
 translocation channel while still bound to pex5, a feature identified more than 20 genesthat are required for peroxi-
that differs from protein import into the ER lumen. At some some biogenesis.I
stage either during or after entry into the matrix, pex5 dis-
sociatesfrom the peroxisomal matrix protein and is recycled Studieswith peroxisome-assembly mutants have shown that
back to the cytoplasm. In conrrasr to the N-terminal uptake- different pathwaysare usedfor importing peroxisomalmatrix
targetlng sequenceson proteins destined for the ER lumen, proteins versusinserting proteins into the peroxisomal mem-
mitochondrial matrix, and chloroplast stroma, the pTSl se- brane. For example, analysis of cells from some Zellweger
quence is not cleaved from proteins after their entry into a patients led to identification of genesencoding the putative
peroxisome. Protein import into peroxisomesrequires ATp translocation channel proteins Pex10, Pex12, and pex2.
hydrolysis, but it is not known how the energyreliased from Mutant cells defectivein any one of these proteins cannot
ATP is used to power unidirectional translocation acrossthe rncorporate matrix proteins into peroxisomes;nonetheless,
peroxisomal membrane. the cellscontain empty peroxisomesthat have a normal com-
The peroxisome import machinery, unlike mosr sysrems plement of peroxisomal membraneproteins (Figure 13-30b).
that mediate protein import into the ER, mitochondria, and
chloroplasts, can translocate folded proteins across the
membrane. For example, catalaseassumesa folded confor-
mation and binds to heme in the cytoplasm before traversing Stainedfor Stainedfor
( a ) W i l d - t y p ec e l l s PMPTO catalase
the peroxisomal membrane. Cell-free studies have shown
that the peroxisome import machinery can transport large Catalase PMP70
macromolecular objects, including gold particles of about 9
nm in diameter,as long as they have a pTSl tag attached to
them. However, peroxisomal membranes do nor appear to
contain large stablepore structures,such as the nuclear Dore
o, .o 9' O" , '
describedin the next section. The fundamental mechanism
of peroxisomal matrix protein translocation is not well un- Peroxisome
derstood but is a topic under active investigation. Some of
(b) Pex12mutants (deficient
the mechanisms under consideration include the idea that
in matrix protein import)
peroxisomal membrane proteins pex10, pex12, and pex2
may assembleto form a relatively large transmembrane
channel with a gated opening of about 10-15 nm (for refer-

(c) Pex3 mutants (deficient

peroxisomal matrix and Pex5 would be releasedback into i n m e m b r a n eb i o g e n e s i s )
the cytosol to complete another round of cargo import.
A f e w p e r o x i s o m am
l a r r i x p r o r e i n ss u c h - a st h i o l a s ea r e
synthesized as precursors with an N-terminal uptake-
targetingsequenceknown as PTS2.These proteins bind to
a different cytosolic receptor prorein, but oiherwise import
is thought to occur by the same mechanismas for piSl-
containing proteins.

P e r o x i s o m aM
l e m b r a n ea n d M a t r i x p r o t e i n s EXPERIMENTAL FTGURE 13-30Studiesrevealdifferent
pathwaysfor incorporationof peroxisomalmembraneand
Are Incorporatedby Different pathways matrixproteins.Cellswerestained with fluorescent antibodiesto
Autosomal recessivemutations that cause defective PMP70, a peroxisomal membrane protein,or with fluorescenr
peroxisome assemblyoccur naturally in the human antibodies to catalase,
a peroxisomal matrixprotein, thenviewed
population. Such defectscan lead to severedeveloomental in a fluorescent microscope.(a)In wild-typecells,bothperoxisomal
defectsoften associatedwith craniofacialabnormaiities.In membrane andmatrixproteins arevisible
asbrightfociin numerous
Zellweger syndrome and related disorders, for example. peroxisomal bodies(b)In cellsfroma pex12-deficient patient,
t h e t r a n s p o r to f m a n y o r a l l p r o t e i n si n t o t h e p e r o x i s o m a l catalase isdistributeduniformly throughout the cytosol,whereas
PMP70 is localized
normallyto peroxisomal bodies(c)In cellsfrom
matrix is impaired: newly synthesizedperoxisomal en_
a Pex3-deficient patient,peroxisomal membranes cannotassemble,
zymes remain in the cytosol and are eventually degraded.
andasa consequence peroxisomal bodiesdo notform.Thusboth
Genetic analysesof cultured cells from different Zellweser
catalase andPMP70 aremis-localized to thecytosol[Courtesyof
patients and of yeast cells carrying similar mutations hive
Stephen Gould, JohnsHopkinsUniversitvl

568 CHAPTER
13 I M O V I N GP R O T E I NISN T O M E M B R A N E A
SN D O R G A N E L L E S
 Precu
rsor
membrane
Peroxisomal Peroxisomal
memDrane ghost PTSl-bearing
proteins matrix protein
PTS2-bearing
matrix protein
PMPTO
13-31 Modelof peroxisomal biogenesis and
FIGURE
division.Thefirststagein the de novoformation of peroxisomes
of peroxisomal membrane proteins intoprecursor
the incorporation
for
membranes derived fromthe ER Pex19 actsasthe receptor
sequences Pex3andPexl6arerequired for the
membrane-targeting
properinsertion of proteins intotheformingperoxisomal membrane
l n s e r t i oonf a l l o e r o x i s o mmael m b r a nper o t e i npsr o d u c eas
Mutations in any one of threeother geneswere found to block
insertion of peroxisomalmembraneproteins as well as import
of matrix proteins (Figure 13-30c). These findings demon-
strate that one set of proteins translocatessoluble proteins
into the peroxisomalmatrix but a different set is required for
insertion of proteins into the peroxisomalmembrane.This sit-
uation differs markedly from that of the ER, mitochondrion,
and chloroplast, for which, as we have seen,membranepro-
teinsand solubleproteinssharemany of the samecomponents
for their insertion into theseorganelles.
Although most peroxisomes are generated by division
of preexisting organelles,these organellescan arise de novo
by the three-stageprocessdepicted in Figure 1'3-31'.In this
case, peroxisome assembly begins in the ER' At least two
peroxisomal membrane proteins, Pex3 and Pex16, are in-
serted into the ER membrane by the mechanismsdescribed
in Section13 .2. Pex3 and Pex16 recruit additional peroxiso-
mal proteins such as Pex19 forming a specializedregion of
the ER membrane that can bud off of the ER to form a per-
oxisomal precursor membrane. Analysis of mutant cells re-
vealed that Pex19 is the receptor protein responsiblefor tar-
geting of peroxisomal membrane proteins' whereas Pex3
and Pex15 are necessaryfor their proper insertion into the
membrane. These three proteins are thought to be responsi-
ble for peroxisomal membrane protein assemblyin mature
peroxisomesas well as during the de novo formation of new
peroxisomes.The insertion of peroxisomal membrane pro-
teins generatesmembranesthat have all the componentsnec-
essaryfor import of matrix proteins, leading to the forma-
tion of mature, functional peroxisomes.Division of mature
peroxisomes,which largely determinesthe number of perox-
isomeswithin a cell, dependson still anotherprotein,Pex11.
Overexpressionof the Pex11 protein causesalatge increase
in the number of peroxisomes,suggestingthat this protein
controls the extent of peroxisome division. The small perox-
isomesgeneratedby division can be enlargedby incorpora-
tion of additional matrix and membrane proteins via the
same pathways describedpreviously'
Mature peroxisome
Catalase
peroxisomalghost,whichiscapable of importing protetns
targeted
rs to the matrixThe pathways for importing PTSl- and PTS2-bearing
matrixproteinsdifferonlyin the identity receptor
of thecytosolic
(Pex5 andPex7,respectively)thatbindsthetargeting sequence(see
Figure13-29)Complete incorporation yields
of matrixproteins a
matureperoxisome. Theproliferationof peroxisomes division
requires
of matureperoxisomes, a process thatdepends on the Pexl1 protein'
Sorting of PeroxisomalProteins
r All peroxisomal proteins are synthesizedon cytosolic
ribosomes and incorporated into the organelle post-
translationally.
r Most peroxisomal matrix proteins contain a C-terminal
PTS1 taigeting sequence;a few have an N-terminal PTS2
targeting sequence.Neither targeting sequenceis cleaved
after import.
r All proteins destinedfor the peroxisomal matrix bind to
a cytosolic carrier protein, which differs for PTSI- and
PTS2-bearingproteins' and then are directed to common
import receptor and translocation machinery on the perox-
isomal membrane (seeFigure 13-29).
r Translocation of matrix proteins acrossthe peroxisomal
membrane dependson ATP hydrolysis. Many peroxisomal
matrix proteins fold in the cytosol and traverse the mem-
brane in a folded conformation, which is different than for
protein import into organellessuch as the ER' mitochon-
drion, and chloroplast.
r Proteinsdestinedfor the peroxisomal membranecontain
different targeting sequencesthan peroxisomal matrix pro-
teins and are imported by a different pathway'
r Unlike mitochondria and chloroplasts, peroxisomescan
arise de novo from precursor membranesprobably derived
from the ER as well as by division of preexistingorganelles
( s e eF i g u r e1 3 - 3 1 ) .
TransPortinto and out
of the Nucleus
The nucleus is separatedfrom the cytoplasm by two mem-
branes,which form the nuclearenvelope(seeFigure 9-1)' The
nuclear envelopeis continuous with the ER and forms a part
. 569
T R A N S P O R ITN T O A N D O U T O F T H E N U C L E U S
 of it. Transport of proteins from the cytoplasm into the nu-
cleus and movement of macromolecules,including mRNps,
tRNAs, and ribosomal subunits, out of the nucleus occur
through nuclearpores,which span both membranesof the nu-
clear envelope. Import of proteins into the nucleus shares
some fundamental features with protein import into other
organelles. For example, imported nuclear proteins carry
specific targeting sequencesknown as nuclear localization
sequences, or NLSs. However, proteins are imported into the
nucleusin a folded state,and thus nuclear import differs fun-
damentally from protein translocationacrossthe membranes
of the ER, mitochondrion, and chloroplast,where proteins are
unfolded during translocation.In this section we discussthe
main mechanism by which proteins and some ribonuclear
proteins such as ribosomesenter and exit the nucleus.\Wewill
also discusshow mRNAs and other ribonuclear protein com-
plexesare exported from the nucleusby a processthat differs
mechanisticallyfrom nuclear protein import.
L a r g ea n d S m a l lM o l e c u l e sE n t e ra n d L e a v et h e
Nucleusvia NuclearPoreComplexes
Numerous pores perforate the nuclearenvelopein all eukary-
otic cells. Each nuclear pore is formed from an elaborate
structure termed the nuclear pore complex (NPC), which is
one of the largest protein assemblagesin the cell. The total
massof the pore structureis 60-80 million Da in vertebrates,
which is about 15 times larger than a ribosome. An NpC is
made up of multiple copies of some 30 different proreins
called nucleoporins. Electron micrographs of nuclear pore
complexesreveal a roughly octagonal,membrane-embedded
structure from which eight approximately 1O0-nm-longfila-
ments extend into the nucleoplasm(Figure 13-32). The distal
ends of thesefilaments are joined by the terminal ring, form-
ing a structure called the nuclear basket. The membrane-
embeddedportion of the NPC is also attacheddirectly to the
nuclear lamina, a network of lamin intermediate filaments
that form a meshwork extending over the inner surface of the
nuclear envelope (seeFigure 20-1,6). Cytoplasmic filaments
extend from the cytoplasmic side of the NPC into the cytosol.
Ions, small metabolites, and globular proreins up to
2040 kDa can passivelydiffuse through the central aqueous
region of the nuclear pore complex. However, large proteins
and ribonucleoprotein complexescannot diffuse in and out
of the nucleus. Ratheq these macromolecules are actively
transported through the NPC with the assistanceof soluble
transporter proteins that bind macromoleculesand also in-
teract with nucleoporins.

(b)

Cytoplasm Cytoplasmic
filaments

O u t e rn u c l e a r
memDrane

Nu c l e a r
envelope

I n n e rn u c l e a r
membrane
Nucleoplasm
N u c l e a rl a m i n a

Nuclearbasket

T e r m i n a rl i n g

A FIGURE 13-32Nuclearporecomplex.(a)Nuclear envelopes nucleoplasmic

faceshowsthe nuclear
basket thatextendsfromthe
mrcrodissected
fromthe largenucleiof Xenopusoocytes
visualized membrane portion.(b)Cutawaymodelof the porecomplex. [part(a)
by fieldemission
in-lens
scanningelectronmicroscopy.
Iop:Vrewof fromV DoyeandE Hurt,1997,
CurrOpinCellBiol9:401;
courtesy
of M W
the cytoplasmic
facerevealsoctagonalshapeof memorane_ G o l d b e r ga n d T . D A l l e n P a r t( b ) a d a p t e df r o m M p R o u ta n d J D A t c h i s o n .
embedded portionof nuclear
porecomplexes Bottom:Viewof the 2001, J Biol. Chem. 276:165931

570 ' c H A p r E R1 3 | M o v r N Gp R o r E r N sr N T oM E M B R A N EAsN D o R G A N E L L E '

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 The mechanismfor import of cytoplasmiccargo proteins
mediated by an importin is shown in Figure 13-35 (the gen-
eral mechanismis the samefor either a monomeric or dimeric
importin). Freeimportin in the cytoplasm binds to its cognate
NLS in a cargo protein, forming a bimolecwlar cargo com-
plex. The cargo complex then translocatesthrough the NPC
channel as the importin B subunit interacts with a class of
nucleoporins called FG-nucleoporins. These nucleoporins,
which line the channel of the nuclear pore complex and also
are found in the nuclear basket and the cytoplasmic fila-
ments, contain multiple repeats of short hydrophobic se-
quencesrich in phenylalanine (F) and glycine (G) residues
(FG-repeats).The hydrophobic FG-repeatsare thought to oc-
cur in regionsof extended,otherwisehydrophilic polypeptide
chains that fill the central transporter channel and in some
way allow the relatively hydrophobic importin complexesto
traverse the channel efficiently while excluding unchaper-
oned hydrophilic proteins larger than 20-40 kDa.
'Sfhen
the cargo complex reachesthe nucleoplasm, the
importin interacts with Ran.GTP, causing a conformational
changein the importin that decreasesits affinity for the NLS,
releasing the cargo protein into the nucleoplasm. The im-
portin-Ran.GTP complex then diffuses back through the
NPC. Once the importin-Ran.GTP complex reachesthe
cytoplasmic side of the NPC, Ran inreracts with a specific
GTPase-actiuatingprotein (Ran-GAP) that is a component
of the NPC cytoplasmic filaments. This stimulates Ran to
hydrolyze its bound GTP to GDP, causing it to convert to a
conformation that has low affinity for the importin, so that
the free importin is releasedinto the cytoplasm, where it can
participate in another cycle of import. Ran.GDP travels back
through the pore to the nucleoplasm,where it encountersa
specificgwaninenucleotide-exchange factor (Ran-G EF) that
causesRan to releaseits bound GDP in favor of GTP. The
net result of this seriesof reactionsis the coupling of the hy-
drolysis of GTP to the transfer of an NlS-bearing protein
from the cytoplasm to the nuclear interior, thus providing a
driving force for nuclear transport.
The import complex travels through the pore by diffusion,
a random process.Yet transport is unidirectional.The direc-
tion of transport is a consequenceof the rapid dissociation of
the import complex when it reachesthe nucleoplasm.As a re-
sult, there is a concentration gradient of the importin-cargo
complex across the NPC: high in the cytoplasm, where the
complex assemblesand low in the nucleoplasm,where it disso-
ciates.This concentration gradient is responsiblefor the unidi-
rectional nature of nuclear import. A similar concentration
gradient is responsiblefor driving the importin in the nucleus
back into the cytoplasm. The concentration of the importin-
Ran.GTP complex is higher in the nucleoplasm,where it as-
sembles,than on the cytoplasmicsideof the NPC, where it dis-
sociates.Ultimately the direction of the transporr processesis
dependenton the asymmetric distribution of the Ran-GEF and
the Ran-GAP. Ran-GEF in the nucleoplasm maintains Ran in
the Ran.GTP state,where it promotesdissociationof the cargo
complex. Ran-GAP on the cytoplasmic side of the NPC

Ra n ' G D P R a n . G T P lmportin < FIGURE 13-35 Nuclearimport.

ti
M e c h a n i sf m r p o rot f " c a r g o "
o r n u c l e ai m
\ra./ proteins. In the cytoplasm (boftorn), a free
D T
P importinbindsto the NLSof a cargoprotein,
forminga bimolecular cargocomplex. ln the
caseof a basicNLS,theadapterprotein
importino bridges the NLSandimportinp,
forminga trimolecular cargocomplex (not
shown). Thecargocomplex diffuses through
the NPCby interacting with successive FG-
nucleoporins ln the nucleoplasm, interaction
of Ran.GTP withthe imoortincauses a
conformational change thatdecreases its
affinityfor the NLS,releasing the cargoTo
supportanothercycleof import,the importin-
Ran'GTP complex istransported backto the
cytoplasm. A GTPase-accelerating protein
(GAP) associated with thecytoplasmic
filaments of the NPCstimulates Ranto
hydrolyze the boundGTPThisgenerates a
conformational changecausing dissociation
fromthe importin, whichcantheninitiate
anotherroundof importRan.GDP isreturned
t o t h en u c l e o p l a swmh,e r ea g u a n i n e
nucleotide-exchange factor(GEF) causes
release of GDPandrebindinq of GTP

572 CHAPTER
13 | M O V T N GP R O T E t NtSN T O M E M B R A N E A
SN D O R G A N E L L E S
converts Ran.GTP to Ran.GDP, dissociatingthe importin-
Ran.GT? complex and releasingfree importin into the cytosol.
ExportinsTransportProteinsContaining
N u c l e a r - E x p oS
r ti g n a l so u t o f t h e N u c l e u s
A very similar mechanism is used to export proteins, tRNAs,
and ribosomal subunits from the nucleus to the cytoplasm.
This mechanisminitiallv was elucidatedfrom studiesof certain
ribonuclear protein complexes that "shuttle" between the nu-
cleus and cytoplasm. Such "shuttling" proteins contain a
nuclear-export signal /NES/ that stimulates their export from
the nucleusto the cytoplasmthrough nuclearpores,in addition
to an NLS that results in their uptake into the nucleus.Experi-
ments with engineered hybrid genes encoding a nucleus-
restricted protein fused to various segmentsof a protein that
shuttles in and out of the nucleus have identified at least three
different classesof NESs: a leucine-rich sequencefound in PKI
(an inhibitor of protein kinaseA) and in the Rev protein of hu-
man immunodeficiency virus (HIV), as well as tvvo sequences
identified in fwo different heterogeneousribonucleoprotein
particles (hnRNPs). The functionally significant structural fea-
tures that specifynuclearexport remain poorly understood.
The mechanismwhereby shuttling proteins are exported
from the nucleus is best understood for those containing a
leucine-richNES. According to the current model, shown in
Figure 13-36a, a specificexportin, or nuclear-exportreceptor,
in the nucleus,called exportin 1, first forms a complex with
Ran.GTP and then binds the NES in a cargo protein. Binding
of exportin 1 to Ran.GTP causesa conformational changein
exportin 1 that increasesits affinity for the NES so that a tri-
molecular cargo compler is formed. Like importins, exportin
1 interacts transiently with FG-repeatsin FG-nucleoporins
and diffusesthrough the NPC. The cargo complex dissociates
when it encountersthe Ran-GAP in the NPC cytoplasmicfila-
ments, which stimulates Ran to hydrolyze the bound GTI
shifting it into a conformation that has low affinity for ex-
portin 1. The releasedexportin 1 changesconformation to a
structurethat has low affinity for the NES, releasingthe cargo
into the cytosol. The direction of the export processis driven
by this dissociationof the cargo from exportin 1 in the cyto-
plasm, which causesa concentration gradient of the cargo
complex acrossthe NPC that is high in the nucleoplasmand
low in the cytoplasm.Exportin 1 and the Ran'GDP are then
transportedback into the nucleusthrough an NPC.
By comparing this model for nuclear export with that in
Figure 13-35 for nuclear import, we can seeone obvious dif-
ference:Ran.GTP is part of the cargo complex during export
but not during import. Apart from this difference,the two
transport processesare remarkably similar.In both processes,
associationof a transport signal receptor with Ran'GTP in
the nucleoplasmcausesa conformational changethat affects
its affinity for the transport signal. During import, the inter-
action causesreleaseof the cargo, whereasduring export, the
interaction promotes associationwith the cargo. In both ex-
port and import, stimulation of Ran'GTP hydrolysis in the
cytoplasm by Ran-GAP producesa conformational changein
Ran that releasesthe transport signal receptor. During nu-
clear export, the cargo is also released.Importins and ex-
portins both are thought to diffuse through the NPC channel
by successiveinteractions with FG-repeatsin FG-nucleoporins.
Localization of the Ran-GAP and -GEF to the cytoplasm and
nucleus,respectivelSis the basisfor the unidirectional trans-
port of cargo proteins acrossthe NPC.
In keeping with the similarity in function of importins
and exportins, the two types of transport proteins are highly
homologous in sequenceand structure. The entire family is
called the importin B familS or karyopherins. There are'l'4
karyopherins in yeastand more than20 in mammalian cells.
The NESs or NLSs to which they bind have been determined
for only a fraction of them. Remarkably, some individual
karyopherins function as both an importin and an exportin.
A similar shuttling mechanismhas beenshown to export
other cargoes from the nucleus. For example, exportin-t
functions to export tRNAs. Exportin-t binds fully processed
tRNAs in a complex with Ran'GTP that diffuses through
NPCs and dissociateswhen it interacts with Ran-GAP in
the NPC cytoplasmic filaments, releasingthe IRNA into the
cytosol. A Ran-dependentprocess is also required for the
nuclear export of ribosomal subunits through NPCs once
the protein and RNA components have been properly as-
sembledin the nucleolus. Likewise, certain specific mRNAs
that associate with particular hnRNP proteins can be
exported by a Ran-dependentmechanism.
Most mRNAsAre Exportedfrom the Nucleusby
a R a n - l n d e p e n d e nMt e c h a n i s m
Once the processingof an mRNA is completedin the nucleus,
it remains associatedwith specifichnRNP proteins rn a mes-
sengerribonuclear protein complex, or mRNP. The principle
transporter of mRNPs out of the nucleus is the nRNP ex-
porter, a heterodimericprotein composedof a large subunit
called nwclearexport factor 1 (NXFI) or TAP and a small
subunit, nuclear export transporter L (Nxtl). The large sub-
unit binds to nuclear mRNPs through cooperative interac-
tions with the RNA and other mRNP adapter proteins that
associatewith nascentpre-mRNAs during transcription elon-
gation and pre-mRNA processing.It seemslikely that multi-
ple TAPNxtl mRNP exportersbound along the length of an
nRNP assistin its export. TAPAJxII acts like a karyopherin
in the sensethat both subunits interact with the FG-domains
of FG-nucleoporins, allowing them to diffuse through the
central channel of the NPC. Tap also binds reversiblyto the
protein Gle2, which in turn binds a nucleoporin in the nu-
clear basket, presumably positioning the mRNP for export
through the nuclear pore. A nucleoporin in the cytoplasmic
filaments of the NPC is also required for mRNP export. This
nucleoporin binds an RNA helicasethat is proposed to func-
tion in the dissociationof the mRNP exporter and other hn-
RNP proteins from the mRNP as it reachesthe cytoplasm.
The TAP/Nxt1 mRNP exporters do not appear to inter-
act with Ran, and thus the unidirectional transport of
mRNA out of the nucleusrequiresa sourceof energyother
than GTP hydrolysis by Ran. As the mRNP complex is
transported through an NPC, the proteins associatedwith
INTOAND OUT OF THE NUCLEUS
TRAN5PORT . 573
(a)
R a n . G D P R a n . G T P Exportin1
< FIGURE 13-36Ran-dependent and
Ran-independent nuclearexport.(a)Ran-
dependent mechanism for nuclear exportof
D T
P cargoproteins containing a leucine-rich nuclear-
Cargo complex exportsignal(NES)In the nucleoplasm, the
proteinexportin1 bindscooperatively to the
NESof thecargoproteinto betransported
andto Ran.GTP Aftertheresulting cargo
complexdiffuses throughan NPCviatransient
interactions with FGrepeats in FG-nucleoporins,
T
P the Ran-GAP associated with the NPCcyto-
plasmic filaments stimulates conversion of
Ran.GTP to Ran.GDP Theaccompanying
conformational changein Ranleadsto dis-
sociation of the complexTheNES-containing
cargoproteinis released intothe cytosol,
whereas exportin1 andRan.GDP are
transported backintothe nucleus through
N P C sR a n - G Ei nFt h en u c l e o p l a tshme n
stimulates conversion of Ran.GDP to Ran.GTP.
(b)Ran-independent nuclear exportof mRNAs
Theheterodimeric TAP/Nxt1 complex bindsto
mRNA-protein complexes (mRNPs) in the
u ' nucleusAssociation of TAP/Nxt1 with
* a\ components of the NPCdirecttheassociated
GEF
mRNP to the centralchannel of the pore.An
P P
RNAhelicase (Dbp5)located on thecyto-
plasmic sideof the NPCisthoughtto provide
the drivingforceby hydrolysis for movingthe
(b) mRNP throughthe pore Thehelicase also
freesthe mRNAfromTAPandNxtl proteins,
whicharerecycled backintothe nucleus by
the Ran-dependent importprocess depicted in
F i g u r 1e 3 - 3 5

Trcnscription

Re-import

it are exchanged for another set of proteins in the cyto-
plasm, a processcalled mRNP remodeling (Figure 13-36b).
Severalnuclear mRNP proteins dissociatefrom the mRNp
before it reachesthe cytoplasmic side of the NpC. These re-
main in the nucleus, where they bind to newly synthesized
nascentpre-mRNA. Other nuclear mRNP proteins, includ-
ing the TAPA{xt1 mRNP exporter, are exported through
NPCs into the cytoplasm. Once they reach the cytoplasmic
side of the NPC, they dissociate from the mRNp with the
help of the RNA helicase,Dbp5, which associateswith cy-
toplasmic NPC filaments. Recall that RNA helicasesuse
the energy derived from hydrolysis of ATP to move along
RNA molecules,separatingdouble-strandedRNA chains
and dissociatingRNA-protein complexes(Chapter4). This
leads to the simple idea that Dpb5, which associateswith
the cytoplasmic side of the nuclear pore complex, acts as
an ATP-driven motor to move mRNP complexes through
the nuclear pore.

574 . c H A p r E R1 3 | M o v r N Gp R o r E r N sr N T o M E M B R A N EASN D o R G A N E L L E s
 After remodelingis completed,the TAP and Nxtl proteins
that have beenstrippedfrom the mRNA by Dbp5 helicaseare
imported back into the nucleus by an importin, where they
can function in the export of another mRNP. Consequently,
thesenuclear mRNP proteins shuttle between the nucleus and
cytoplasm,carrying mRNPs through NPCs (Figure 13-36b).
Transport into and out of the Nucleus
r The nuclear envelope contains numerous nuclear pore
complexes(NPCs), large, complicated structurescomposed
of multiple copies of 30 proteins called nucleoporins (see
Figure 1,3-32).FG-nucleoporins,which contain multiple
repeatsof a short hydrophobic sequence(FG-repeats),line
the central transporter channel and play a role in transport
of all macromoleculesthrough nuclear pores.
r Transport of macromolecules larger than 20-40 kDa
through nuclear pores requires the assistanceof proteins
that interact with both the transported molecule and FG-
repeatsof FG-nucleoporins.
r Proteins imported to or exported from the nucleus con-
tain a specificamino acid sequencethat functions as a nu-
clear-localization signal (NLS) or a nuclear-export signal
(NES). Nucleus-restrictedproteins contain an NLS but not
an NES, whereasproteins that shuttle betweenthe nucleus
and cytoplasm contain both signals.
r Severaldifferent types of NES and NLS have been iden-
tified. Each type of nuclear-transport signal is thought to
interact with a specific receptor protein (exportin or im-
portin) belonging to a family of homologous proteins
termed karyopherins.
r A "cargo" protein bearing an NES or NLS translocates
through nuclear pores bound to its cognatereceptorprotein
(karyopherin), which also interacts with FG-nucleoporins.
Importins and exportins are thought to diffuse through the
channel, filled with a hydrophobic matrix of FG-repeats.
Both transport processesalso require participation of Ran,
a monomeric G protein that exists in different conforma-
tions when bound to GTP or GDP.
r After a cargo complex reachesits destination (the cyto-
plasm during export and the nucleusduring import), it dis-
sociates,freeing the cargo protein and other components.
The latter then are transportedthrough nuclearpores in the
reversedirection to participate in transporting additional
moleculesof cargo protein (seeFigures13-35 and 13-36).
r The unidirectional nature of protein export and import
through nuclear pores results from localization of the Ran
guanine nucleotide-exchangefactor (GEF) in the nucleus
and of Ran GTPase-activatingprotein (GAP) in the cyto-
plasm. The interaction of import cargo complexeswith the
Ran-GTP in the nucleoplasmcausesdissociationof the
complex, releasingthe cargo into the nucleoplasm(seeFig-
ure 13-35). Export cargo complexesdissociatein the cyto-
plasm when they interact with Ran'GAP localized to the
NPC cytoplasmic filaments (seeFigure 13-36).
r Most mRNPs are exported from the nucleus by a het-
erodimeric mRNP exporter that interacts with FG-repeats
of FG-nucleoporins.The direction of transport (nucleusto
cytoplasm) may result from the action of an RNA helicase
associatedwith the cytoplasmic filaments of the nuclear
pore complexes.
As we have seen in this chapter, we now understand many
aspectsof the basic processesresponsiblefor selectively
transporting proteins into the endoplasmic reticulum (ER),
mitochondrion, chloroplast, peroxisome, and nucleus. Bio-
chemical and genetic studies, for instance, have identified
cis-actingsignal sequencesresponsiblefor targeting proteins
to the correct organellemembrane and the membrane recep-
'We
tors that recognize these signal sequences. also have
learnedmuch about the underlying mechanismsthat translo-
cate proteins across organelle membranes and have deter-
mined whether energyis usedto push or pull proteins across
the membrane in one direction, the type of channel through
which proteins pass, and whether proteins are translocated
in a folded or an unfolded state. Nonetheless'many funda-
mental questions remain unanswered' including how fully
folded proteins move acrossa membrane and how the topol-
ogy of multipass membrane proteins is determined.
The peroxisomal import machinery provides one exam-
ple of the translocation of folded proteins. It not only is ca-
pable of translocating fully folded proteins with bound co-
factors into the peroxisomal matrix but can even direct the
import of a large gold particle decoratedwith a (PTS1)per-
oxisomal targetingpeptide.Someresearchershave speculated
that the mechanismof peroxisomal import may be related to
that of nuclear import, the best-understoodexample of post-
translational translocation of folded proteins. Both the per-
oxisomal and nuclear import machinery can transport folded
moleculesof very divergentsizes,and both appear to involve
a component that cycles between the cytosol and the or-
ganelle interior-the Pex5 PTS1 receptor in the caseof per-
oxisomal import and the Ran-importin complex in the case
of nuclear import. However, there also appear to be crucial
differencesbetweenthe two translocation processes.For ex-
ample, nuclear pores representlarge, stable macromolecular
assembliesreadily observedby electron microscopy,whereas
analogousporelike structureshave not been observedin the
peroxisomal membrane. Moreover' small molecules can
readily pass through nuclear pores, whereas peroxisomal
membranesmaintain a permanent barrier to the diffusion of
small hydrophilic molecules.Taken together,these observa-
tions suggestthat peroxisomalimport may require an entirely
new type of translocation mechanism.
The evolutionarily conservedmechanismsfor translocat-
ing folded proteins acrossthe cytoplasmicmembraneof bac-
terial cells and across the thylakoid membrane of chloro-
plasts also are poorly understood.A better understandingof
all of theseprocessesfor translocatingfolded proteins across
SRT H E F U T U R E
P E R S P E C T I VFEO 575
a membrane will likely hinge on future development of in translational translocation into the endoplasmic reticulum
vitro translocation systemsthat allow investigatorsto define (ER); (b) post-translationaltranslocation into the ER; (c)
the biochemical mechanisms driving translocation and to translocation across the bacterial cytoplasmic membranel
identify the structuresof trapped translocationintermediates. and (d) translocation into the mitochondrial marnx.
Compared with our understandingof how soluble proteins 2. Translocation into most organellesusually requires the
are translocatedinto the ER lumen and mitochondrial matrix, activity of one or more cytosolic proteins. Describethe basic
our understanding of how cis-acting sequencesspecify the function of three different cytosolic factors required for
topology of multipass membrane proteins is quite elementary. translocation into the ER, mitochondria, and peroxisomes,
For instance,we do not know how the translocon channel ac- respectively.
commodatespolypeptidesthat are oriented differently with re-
3. Describethe typical principles usedto identify topogenic
spect to the membrane, nor do we understand how local sequenceswithin proteins and how thesecan be used to de-
polypeptide sequencesinteract with the translocon channel
velop computer algorithms. How does the identification of
both to set the orientation of transmembranespansand to sig-
topogenic sequenceslead to prediction of the membrane
nal for lateral passageinto the membrane bilayer. A better un-
arrangementof a multipass protein? What is the importance
derstanding of how the amino acid sequencesof membrane
of the arrangement of positive chargesrelative to the mem,
proteins can specify membrane topology will be crucial for de-
brane orientation of a signal-anchorsequence?
coding the vast amount of structural information for membrane
4. The endoplasmicreticulum (ER) is an important site of
proteins contained within databasesof genomic sequences.
"quality control" for newly synthesizedproteins. \7hat is
A more detailed understanding of all translocation
meant by "quality control" in this context? I7hat accessory
processesshould continue to emergefrom genetic and bio-
proteins are typically involved in the processingof newly syn-
chemical studies, both in yeasts and in mammals. These
thesizedproteins within the ER? Cells generallydegradeER-
studies will undoubtedly reveal additional key proteins in- 'Sfhere
exit-incompetent proteins. within the cell does such
volved in the recognition of targeting sequencesand in the
degradation occur and what is the relationship of the Sec61
translocation of proteins across lipid bilayers. Finally, the
protein translocon and p97 to the degradationprocess?
structural studies of translocon channels will likely be ex-
tended in the future to reveal at resolutions on the atomic 5. Temperature-sensitive yeast mutants have been isolated
scalethe conformational statesthat are associatedwith each that block each of the enzymatic stepsin the synthesisof the
step of the translocation cycle. dolichol-oligosaccharideprecursor for N-linked glycosyla-
tion (seeFigure 1,3-1,7). Proposean explanation for why mu-
tations that block synthesis of the intermediate with the
KeyTerms structure dolichol-PP-(GlcNAc)2Man5 completely prevent
addition of N-linked oligosaccharide chains to secretory
biomolecular cargo post-translational proteins, whereas mutations that block conversion of this
complex 572 translocation 540 intermediate into the completed precursor-dolichol-PP-
cotranslational Ran protein 5Z1 (GlcNAc)2ManeGlca-allow the addition of N-linked
translocation 537 signal-anchorsequence544 oligosaccharidechains to secretoryglycoproteins.
dislocation 555 signal-recognitionparticle 6. Name four different proteins that facilitate the modifi-
dolichol phosphate 550 (sRP)
537 cation and/or folding of secretoryproteins within the lumen
exportin 573 of the ER. Indicate which of theseproteins covalently modi-
signal (uptake-targeting)
fies substrateproteins and which brings about only confor-
FG-nucleoporins572 sequences535
mational changesin substrateproteins.
generalimport pore 559 stop-transferanchor
7. Becauseyou are interestedin studying how a particular
hydropathy profile 548 sequence544
secretoryprotein folds within the ER, you wish to determine
importins 571 topogenic sequences543 whether BiP binds to the newly synthesizedprotein in ER ex-
karyopherins 574 topology of a membrane tracts. You find that you can isolate some of the newly syn-
protein 543 thesizedsecretoryprotein bound to BiP when ADP is added
molecular chaperones541
translocon 539 to the cell extract but not when ATP is added to the extract.
Nlinked
trimolecular cargo Explain this result basedon the mechanism for BiP binding
oligosaccharides550 1
complex J,/J
F - ^
to substrateproteins.
nuclear pore complex
(NPC)570 unfolded-protein 8. Describewhat would happen to the precursor of a mito-
response555 chondrial matrix protein in the following types of mitochon-
O-linked
drial mutants: (a) a mutation in the Tom22 signal receptor,
oligosaccharides550 (b) a mutation in the Tom70 signal receptor, (c) a mutation
in the matrix Hsc70, and (d) a mutation in the matrix signal
Review the Concepts peptidase.
9. Describe the similarities and differences between the
7. Describethe sourceor sourcesof energyneededfor uni- mechanismof import into the mitochondrial matrix and the
directional translocation across the membrane in (a) co- chloroplast stroma.

576 CHAPTER
13 | M O V T N GP R O T E t NtSN T O M E M B R A N E A
SN D O R G A N E L L E S
10. Design a set of experimentsusing chimeric proteins,
composed of a mitochondrial precursor protein fused to di-
hydrofolate reductase(DHFR), that could be used to deter-
mine how much of the precursor protein must protrude into
the mitochondrial matrix in order for the matrix-targeting
sequenceto be cleaved by the matrix-processing protease
(seeFigure 1,3-24).
11. Protein targeting to both mitochondria and chloro-
plasts involves the sorting of proteins to multiple sites
within the respectiveorganelle.Briefly list thesesites.Tak-
ing the mitochondrion as an example and the proteins
ADP/ATP anti-porter and cytochrome b2 as the specific
cases,compareand contrast the extent to which a common
mechanism is used for the site-specifictargeting of these
two protelns.
12. Peroxisomescontain enzymesthat use molecular oxy-
gen to oxidize various substrates,but in the processhydro-
gen peroxide forms and must be degraded.lfhat is the name
of the enzyme responsiblefor the breakdown of hydrogen
peroxide to water and what mechanismand associatedpro-
teins allow for its import into the peroxisome?
13. Supposethat you have identified a new mutant cell line
that lacks functional peroxisomes.Describe how you could
determine experimentally whether the mutant is primarily
defective for insertion/assemblyof peroxisomal membrane
protelns or matrlx protelns.
14. Evidencethroughout Chapter 13 revealsthat specific
motifs within polypeptidesare necessaryto direct or tar-
get these proteins acrossmembranesand into organelles.
The nuclear import of proteins having a molecular mass
more than approximately 40 kDa is no different, and they
must be actively imported through nuclear pore com-
plexes.What is the name given to the amino acid sequence
that allows the selective transport of macromolecular
cargo proteins into the nucleus?Name three proteins that
are required for this import and briefly describehow they
function.
'Sfhy
15. is localization of Ran-GAP in the nucleusand Ran-
GEF in the cytoplasm necessaryfor unidirectional transport
of cargo proteins containing an NES?
Analyze the Data
Imagine that you are evaluating the early stepsin transloca-
tion and processingof the secretoryprotein prolactin. By us-
ing an experimental approach similar to that shown in Fig-
ure 1.3-7,you can usetruncated prolactin mRNAs to control
the length of nascentprolactin polypeptidesthat are synthe-
sized.\fhen prolactin mRNA that lacks a chain-termination
(stop) codon is translated in vitro, the newly synthesized
polypeptide ending with the last codon included on the
mRNA will remain attached to the ribosome, thus allowing
a polypeptide of defined length to extend from the ribosome.
You have generateda set of mRNAs that encodesegmentsof
the N-terminus of prolactin of increasing length, and each
mRNA can be translated in vitro by a cytosolic translation
extract containing ribosomes,tRNAs, aminoacyl-tRNA syn-
thetases,GTP, and translation initiation and elongation fac-
'When
tors. radiolabeled amino acids are included in the
translation mixture, only the polypeptide encoded by the
addedmRNA will be labeled.After completion of translation,
each reaction mixture was resolved by SDS poly-acrylamide
gel electrophoresis,and the labeled polypeptides were iden-
tified by autoradiography.
a. The autoradiogram depicted below shows the re-
sults of an experiment in which each translation reaction
was carriedout eitherin the presence(+) or the absence(-)
of microsomal membranes.Basedon the gel mobility of pep-
tides synthesizedin the presenceor absenceof microsomes,
deducehow long the prolactin nascentchain must be in or-
der for the prolactin signal peptide to enter the ER lumen
and to be cleaved by signal peptidase. (Note that micro-
somes carry significant quantities of SRP weakly bound to
the membranes.)
I
I -
!l - {l!'!**
o
-t
1f'lil!t
-
--
^X --
ar) -O
N;
+- -+
70 90 110 130 150
50
Sizeof mRNA(in codons)
b. Given this length, what can you conclude about the
conformational state(s)of the nascentprolactin polypeptide
when it is cleavedby signal peptidase?The following lengths
will be useful for your calculation: the prolactin signal se-
quenceis cleavedafter amino acid 31; the channel within the
,iboro-. occupied by a nascentpolypeptide is about 150 A
long; a membrane bilayer is about 50 A thick; in polypep-
tides with an a-helical conformation, one residue extends
1.5 A, whereas in fully extended polypeptides, one residue
extendsabout 3.5 A.
c. The experimentdescribedin part (a) is carried out
in an identical manner except that microsomal membranes
are not present during translation but are added after
translation is complete. In this case none of the samples
shows a difference in mobility in the presenceor absence
of microsomes. lfhat can you conclude about whether pro-
lactin can be translocated into isolated microsomes post-
translationally?
d. In another experiment, each translation reaction
was carried out in the presenceof microsomes,and then the
microsomal membranes and bound ribosomes were sepa-
rated from free ribosomes and soluble proteins by centrifu-
gation. For each translation reaction' both the total reaction
(T) and the membrane fraction (M) were resolved in neigh-
boring gel lanes. Basedon the amounts of labeled polypep-
tide in the membrane fractions in the autoradiogram de-
picted beloq deduce how long the prolactin nascent chain
A N A L Y Z ET H E D A T A 577
must be in order for ribosomesengagedin translation to en- Tsai, B., Y. Ye, and T. A. Rapoport.2002. Retro-translocation
gage the SRP and thereby become bound to microsomal of proteins from the endoplasmicreticulum into the cytosol. Nature
Reu.Mol. Cell Biol. 3:246-255 .
membranes.
Sorting of Proteins to Mitochondria and Chloroplasts
l.
I Koehler,C. M. 2004. New developmentsin mitochondrial as-
I
sembly.Ann. Reu.Cell Deu. Biol.20:309-335.
0)
-- -- Dolezal,P.,V. Likic, J.Tachezy,and T. Lithgow 2006. Evolution
_o .9
.Uto of the molecular machinesfor protein import into mitochondria.
oa Science313:31.4-31.8.
dt>
N; Dalbey,R. E., and A. Kuhn. 2000. Evolutionarily relatedinser-
tion pathways of bacterial,mitochondrial, and thylakoid membrane
TMTMTMTMTMTM proteins.Ann. Reu.Cell Deuel. Biol. 16:51-87.
50 70 90 110 130 Matouschek,A., N. Pfanner,and S7.Voos. 2000. Protein un-
150
folding by mitochondria:the Hsp70 import motor. EMBO Rept.
S i z eo f m R N A( i nc o d o n s ) l:404410.
Neupert,'$7.,and M. Brunner.2002.The protein import motor
of mitochondria.Nature Reu.Mol. Cell Biol.3:555-565.
References Rapaport, D. 2005. How doesthe TOM complex mediateinser-
tion of precursorproteins into the mitochondrial outer membrane?
I. Cell Biol. 17l:479423.
Translocation of Secretory Proteins Across Robinson,C., and A. Bolhuis.2001. Protein targeringby the
the ER Membrane twin-argininetranslocationpathway.Nature Reu.Mol. Cell Biol.
2:350-356.
Egea,P. F., R. M. Srroud,and P. Walter.2005. Targetingpro-
teins to membranes:structureof the signalrecognitionparticle. Soll, J., and E. Schleiff.2003. Protein import into chloroplasts.
Curr. Opin. Struct. Biol. 75:213-220. Nature Reu.Mol. Cell Biol.5:198-208.
Osborne,A. R., T. A. Rapoport, and B. van den Berg.2005. Truscott, K. N., K. Brandner,and N. Pfanner.2003.Mecha-
Protein translocationby the Sec51/SecY channel.Annu. Reu.Cell nismsof protein import into mitochondria. Curr. Biol.
D e u .B i o l . 2 l : 5 2 9 - 5 5 0 . 13:R325-R337.
'Wickner,
S7.,and R. Schekman.2005. Protein rranslocation
acrossbiological membranes.Science310:L452-1456. Sorting of Peroxisomal Proteins
Dammai, V., and S. Subramani.2001. The human peroxisomal
Insertion of Proteins into the ER Membrane targetingsignalreceptor,Pex5p,is translocatedinto the peroxisomal
Englund, P.T.1993. The structureand biosynthesisof glyco- matrix and recycledto the cytosol. Cell 105:1,87-1,96.
sylphosphatidylinositolprotein anchors.Ann. Reu.Biochem.. Gould, S. J., and C. S. Collins. 2002. Opinion: peroxisomal-pro-
62:121-1.38. tein import: is it really that complex?Nature Reu.Mol. Cell Biol.
Goder,V., and M. Spiess.2001. Topogenesisof membranepro- 3:382-389.
teins: determinantsand dynamics.FEBSLett. 504:87-93. Gould, S.J., and D. Valle. 2000. Peroxisomebiogenesisdisor-
Mothes, I7., et al. 1997. Molecular mechanismof membrane ders:geneticsand cell biology. TrendsGenet. 16:340-345.
protein integrationinto the endoplasmicreticulum. Cel/ Hoepfner,D., D. Schildknegt,I. Braakman,P. Philippsen,and
89:523-53J. H. F. Tabak. 2005. Contribution of the endoplasmicreticulum to
peroxisomeformation.Cell 122:85-9 5.
von Heijne, G. 1999. Recentadvancesin the understandingof
membraneprotein assemblyand structure.Q. Reu.Biophys. Purdue,P. E., and P. B. Lazarow.2001. Peroxisomebiogenesis.
32:285-307. Ann. Reu.Cell Deuel. Biol. 17:701-752.
Subramani,S., A. Koller, and W. B. Snyder.2000. Import of per-
Protein Modifications, Folding, and euality Control oxisomal matrix and membraneproteins.Ann. Reu.Biochem.
in the ER 69:3994].8.

. Helenius,A., and M. Aebi. 2004. Rolesof Nlinked glycansin
the endoplasmicreticulum. Annu. Reu.Biochem. 73:1019-1049.
Kornfeld, R., and S. Kornfeld. 1985. Assemblyof asparagine-
. import. Curr. Opin. Struct. Biol. ll:703-71,5.
linked oligosaccharides. Ann. Reu.Biochem. 45:631,-6G4.
'Walter.
Patil, C., and P. 2001. Intracellularsignalingfrom the
endoplasmicreticulum to the nucleus:the unfolded protein response
in yeastand mammals. Curr. Opin. Cell Biol.73:349-355.
Meusser,B., C. Hirsch, E. Jarosch,and T. Sommer.2005.
ERAD: the long road ro destruction.Natwre Celt Biol.7:766-772.
Sevier,C. S., and C. A. Kaiser.2002.Formation and transfer of
disulphidebonds in living cells.Nature Reu.Mol. Cetl Biot.
3:836-847.
. .Silberstein,S., and R. Gilmore. 1995. Biochemistry,molecular
brology,and geneticsof the oligosaccharyltransferase. FASEB/.
10:849-85 8.
Trombetta,E. S., and A. J. Parod. 2003. Quality control and
protein folding in the secretorypathway.Annu. Reu.Cell Deu. Biol.
19:649-676.
Transport into and out of the Nucleus
Chook, Y. M., and G. Blobel. 2001. Karyopherinsand nuclear
Cole, C. N., and J. J. Scarceili.2006. Transport of messenger
RNA from the nucleusto the cytoplasm.Curr. Opin. Cell Biol.
18299-306.
Johnson,A. \f., E. Lund, and J. Dahlberg.2002. Nuclear export
of ribosomalsubunits.TrendsBiochem.Sci.27:580-585.
Ribbeck, K., and D. Gorlich. 2001. Kinetic analysisof translo-
cation through nuclearpore complexes.EMBO J. 20:1,320-1330.
Rout, M. P., and J. D. Aitchison. 2001. The nuclearpore com-
plex as a transport machine.J. Biol. Chem. 276:76593-1,6596.
Schwartz,T. U. 2005. Modularity within the architectureof the
nuclearpore complex. Curr. Opin. Struct. Biol. L5:221-226.
Suntharalingam,M., and S. R. Wente.2003. Peeringthrough
the pore: nuclearpore complex structure,assembly,and function.
Deu. Cell.4:775-789.

578 CHAPTER
13 I M O V T N GP R O T E t NtSN T O M E M B R A N E A
S N DO R G A N E L L E S
 CHAPTER

TRAFFIC,
VESICULAR
AND
SECRETION,
ENDOCYTOSIS
Scanningelectronmicrographshowingthe formationof clathrin-
coatedvesicles faceof the plasmamembrane
on the cytosolic
Washington
fJohnHeuser, School
University of Medicine
]

I n the previouschapter we explored how proteins are tar-
I g.ted to and translocatedacrossthe membranesof several
I different intracellular organelles,including the endoplasmic
reticulum, mitochondria and chloroplasts,peroxisomes,and
the nucleus. In this chapter we turn our attention to the
secretory pathway and the mechanismsthat allow soluble
and membrane proteins to be deliveredto the plasma mem-
brane and the lysosome. Ii7e will also discuss the related
processesof endocytosisand autophagy,which deliver pro-
teins and small moleculesfrom either outsidethe cell or from
the cytoplasmto the interior of the lysosomefor degradation.
Soluble and membrane proteins slated to function at the
cell surfaceor in the lysosomeare transported to their final
destination via the secretorypathway. Proteins delivered to
the plasma membrane include cell-surfacereceptors, trans-
porters for nutrient uptake, and ion channelsthat maintain
the proper ionic and electrochemicalbalance across the
plasma membrane.Solublesecretedproteins follow the same
pathway to the cell surface as plasma membrane proteins,
but instead of remaining embedded in the membrane, se-
creted proteins are releasedinto the aqueous extracellular
environment in soluble form. Examples of secretedproteins
are digestive enzymes, peptide hormones, serum proteins,
and collagen. As describedin Chapter 9, the lysosomeis an
organelle with an acidic interior that is generally used for
degradation of unwanted proteins and the storage of small
molecules such as amino acids. AccordinglS the types of
proteins delivered to the lysosomal membrane are subunits
of the V-classproton pump that pumps H* from the cytosol
into the acidic lumen of the lysosomeas well as transporters
to releasesmall molecules stored in the lvsosome into the
cytoplasm. Soluble proteins delivered by this pathway
include lysosomal digestiveenzymessuch as proteases'gly-
cosidases,phosphatases,and lipases.
In contrast to the secretorypathway' which is generally
used to deliver newly synthesizedmembrane proteins to their
correct address,the endocyticpathway is usedto take up sub-
stancesfrom the cell surfaceinto the interior of the cell. The
endocytic pathway is used to take up certain nutrients that
are too large to be transported acrossthe plasma membrane
by one of the transport mechanismsdiscussedin Chapter 1 1.
For example, the endocytic pathway is utilized in the uptake
of cholesterolcarried in LDL particlesand iron atoms carried
by the iron-binding protein transferrin. In addition, the endo-
cytic pathway can be usedremove receptorproteins from the
cell surface as a way to down-regulate their activity.
OUTLINE
14.1 Techniquesfor Studyingthe Secretory
Pathway 580
14.2 MolecularMechanismsof VesicularTraffic 586
14.3 EarlyStagesof the SecretoryPathway 592
597
14.4 Later Stagesof the SecretoryPathway
14.5 Endocytosis
Receptor-Mediated 506
14.6 DirectingMembraneProteinsand Cytosolic
Materialsto the LYsosome 612

579
 A single unifying principle governs all protein trafficking
in the secretoryand endocytic pathways: transport of mem-
brane and soluble proteins from one membrane-bounded
compartmentto another is mediatedby transport vesiclesthat
collect " cargo" proteins in buds arisingfrom the membraneof
one compartment and then deliver thesecargo proteins to the
next compartment by fusing with the membraneof thar com-
partment. Importantly as transport vesiclesbud from one
membrane and fuse with the next, the same face of the mem-
brane remains oriented toward the cytosol. Therefore once a
protein has been insertedinto the membraneor the lumen of
the ER, the protein can be carried along the secretorypath-
way moving from one organelle ro the next without being
translocatedacrossanother membraneor altering its orienta-
tion within the membrane. Similarly, the endocytic pathway
usesvesicletraffic to transport proteinsfrom the plasmamem-
brane to the endosomeand lysosomeand thus preservestheir
orientation in the membraneof theseorganelles.Figure 14-1
outlinesthe major routes for protein trafficking in the cell.
Reducedto its simplest elements,the secretorypathway
for delivery of newly synthesized proteins ro the plasma
membrane or the lysosomeoperatesrn two stages.The first
stage takes place in the rough endoplasmic reticulum (ER),
as describedin Chapter 13. Newly synthesizedsoluble and
membrane proteins are translocatedinto the ER, where they
fold into their proper conformation and receivecovalent
modifications such as N-linked and O-linked carbohydrates
and disulfide bonds. Once newly synthesizedproteins are
properly folded and have received their correct modifica-
tions in the ER lumen, they progressro the second stage of
the secretory pathway, rransport through the Golgi. In the
ER, secretory proteins are packaged into anterograde
(forward-moving) transport vesicles.Thesevesiclesfusewith
each other to form a flattened membrane-boundedcompart-
ment known as the cis-Golgi cisterna. Certain proteins,
mainly ER-localizedproteins, are retrieved from the cis-
Golgi to the ER via a different set of retrograde (backward-
moving) transport vesicles.A new cls-Golgi cisternawith its
cargo of proteins physically moves from the cis position
(nearestthe ER) to the trans position (farthest from the ER),
successivelybecoming first a medial-Golgi cisterna and then
a trans-GoIgi cisterna.This process,known as cisternalmat-
uration, does nor involve the budding off and fusion of an-
terograde transport vesicles.During cisternal maturation,
enzymesand other Golgi-residentproteins are constantly be-
ing retrieved from later to earlier Golgi cisternae by retro-
grade transport vesicles,thereby remaining localized to the
cis-, medial-, or trans-Golgi cisternae.As secreroryprorelns
move through the Golgi, they can receivefurther modifica-
tions to linked carbohydratesby specific glycosyl trans-
ferasesthat are housed in the different Golgi comparrmenrs.
Proteinsin the secretorypathway that are destinedfor the
plasma membrane or lysosome eventually reach a complex
network of membranes and vesiclestermed the trans-Golgi
network (TGN). The TGN is a major branch point in the se-
cretory pathway, and through a process known as protein
sorttng, a protein can be loaded into one of at leastthree dif-
ferent kinds of vesiclesthat bud from the TGN. After buddine
580 C H A P T E R1 4 I V E S T C U L ATRR A F F t CS, E C R E T T OA
NN, D ENDOCYTOS|S
from the trans-Golgi network, the first type of vesicleimmedi-
ately moves to and fuses with the plasma membrane in a
processknown as exocytosis,thus releasingits contentsto the
exterior of the cell while the membraneproteinsfrom the vesi-
cle becomeincorporatedinto the plasmamembrane.In all cell
types,at leastsome proteins are loaded into such vesiclesand
secretedcontinuouslyin this manner.The secondtype of vesi-
cle to bud from the trans-Golgi network, known as secretory
vesicles,are stored inside the cell until a signal for exocytosis
causesreleaseof their contents at the plasma membrane.
Among the proteins releasedby such regulatedsecretionare
peptide hormones (e.g.,insulin, glucagon,ACTH) from vari-
ous endocrinecells,precursorsof digestiveenzymesfrom pan-
creatic acinar cells, milk proteins from the mammary gland,
and neurotransmittersfrom neurons.The third type of vesicle
that buds from the trans-Golgi network is directed to the
lysosome,an organelleresponsiblefor the intracellular degra-
dation of macromolecules, and to lysosome-like storage
organellesin certain cells.Secretoryproteinsdestinedfor lyso-
somes are first transported by vesiclesfrom the trans-Golgi
network to a compartment usually called the late endosome;
proteins then are transferred to the lysosome by direct fusion
of the endosomewith the lysosomalmembrane.
Endocytosis is related mechanistically to the secretory
pathway. In the endocytic pathway, vesiclesbud from the
plasma membrane, bringing membrane proteins and their
bound ligands into the cell (see Figure 14-1). After being
internalized by endocytosis,some proteins are transported
to lysosomesvia the late endosome,whereasothers are recy-
cled back to the cell surface.
In this chapter we first discusshow our knowledge of the
secretorypathway and endocytosishas expandedthrough ex-
perimental techniques.Then we focus on the generalmecha-
nisms of membrane budding and fusion. We will seethat al-
though different kinds of transport vesiclesutilize distinct sets
of proteins for their formation and fusion, all vesiclesuse the
same generalmechanismfor budding, selectionof particular
setsof cargo molecules,and fusion with the appropriatetarget
membrane.The following two sectionsshow how coordina-
tion betweenparticular vesicletrafficking stepscan maintain
the identity (i.e.,a stableset of residentproteins) of the differ-
ent compartments along the secretory pathway and how
cargo selectionby vesiclesis usedto sort proteins to different
intracellular locations.Next we will turn our attention to the
endocytic pathway to examine how endocytosisis used to
transport macromoleculesfrom the extracellularenvironment
into the cell interior. Finally we will examine the variety of
ways that membrane proteins and macromoleculesfrom the
cell interior are transportedto the lysosomefor degradation.
Im Techniques
for Studying
the SecretoryPathway
The key to understandinghow proteins are transporred
through the organelles of the secretory pathway has been
to develop a basic description of the function of transporr
vesicles.Many components required for the formation and
llll+ overviewAnimation:ProteinSecretion

< FIGURE 14-1 Overviewof

the secretoryand endocytic
pathwaysof protein sorting.
pathway:Synthesis of
z Regulated / tr Secretory
proteins bearing an ERsignal
secretion sequence iscompleted on the
/
roughERIl, andthe newlymade

ffi Secretory
vesicle

Sortingto
polypeptide chains
intothe ERmembrane
intothe lumen(Chapter
Someproteins
areinserted
or crossit
13).
(e.g.,ERenzymes
\
E lysosomes
or structural proteins) remain
withinthe ERTheremainder are
packaged intotransPort vesicles
Late
trans- El that budfromthe ERandfuse
Transport endosome
Golgi to form newcr's-Golgi cisternae
network vesicle
Missorted ER-resident proteins
andvesicle membrane proteins
that needto be reusedare
retrieved to the ERbYvesicles
E that budfromthe cts-Golgi
f andfusewith the ER.Eachcts-

.gr
e Golgicisterna,
content,physically
with itsProtein
movesfrom
trans- the crsto the fransfaceof the
Golgi Golgicomplex @ by a
nonvesicular process called
cisternal maturation. Retrograde
transport vesicles E moveGolgi-
medial-
resident proteins to the ProPer
Golgi
Retrograde Golgi compartment. In allcells,
tr transportfrom
l a t e rt o e a r l i e r
certainsoluble
the cellsurface
proteins moveto
in transPort
Golgi cisternae
vesiclesEl andaresecreted
continuously (constitutive
secretion). In certaincelltYPes,
somesoluble proteins arestored
in secretory vesiclesZ andare
released onlyafterthe cell
crs€olgi receives an appropriate neuralor
network hormonal signal(regulated
secretion). Lysosome-destined
membrane andsoluble Proteins,
B u d d i n ga n d f u s i o no f \ whicharetransported in vesicles
!

_grj
ER-to-Golgi
TOrmcrs-(Jorgl
,"*-"\
vesiclesto

^/
ffi E RetrogradeGolgi-to-ER
transoort
that budfromthe trans-Golgi
E, firstmoveto the late
endosome andthento the
lysosomeEndocytic PathwaY:
*ao-& sl*ed
-f." Membrane andsoluble
\,,,,,' ER lumen extracellular proteins takenup in
g vesiclesthatbudfromthe Plasma
membrane I alsocanmoveto
the lysosome viathe endosome

Rough ER Proteinsvnthesison bound ribosomes;

I transportof proteins
co-translational
into or acrossER membrane

SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y
T E C H N I Q U EF 581
fusion of transport vesicleshave been identified in the past
decadeby a remarkable convergenceof the geneticand bio-
chemical approachesdescribedin this section.All studies of
intracellular protein trafficking employ some method for as-
saying the transport of a given protein from one compart-
ment to another. \7e begin by describing how intracellular
protein transport can be followed in living cells and then
considergeneticand in vitro systemsthat have proved useful
in elucidating the secretorypathway.
Transportof a ProteinThroughthe Secretory
PathwayCan Be Assayedin Living Cells
The classicstudiesof G. Paladeand his colleaguesin the 1960s
first established the order in which proteins move from or-
ganelleto organelle in the secretoryp"ih*"y. Theseearly stud-
ies also showed that secretoryproteins are never releasedinto
the cytosol, the first indication that transported proteins are al-
ways associatedwith some type of membrane,boundedinter-
mediate. In theseexperiments,which combined pulse-chasela-
beling (seeFigure 3-39) and autoradiography,radioactively
labeledamino acidswere injectedinto the pancreasof a ham-
ster.At different times after injection, the animal was sacrificed
and the pancreatic cells were chemically fixed, sectioned,and
subjectedto autoradiographyto visualizethe location of the
radiolabeled proteins. Becausethe radioactive amino acids
were administered in a short pulse, only those proteins synthe-
sized immediately after injection were labeled, forming a dis-
tinct group, or cohort, of labeled proteins whose transport
could be followed. In addition, becausepancrearicacinar cells
are dedicatedsecretorycells, almost all of the labeled amino
Throughthe Secretorypathway
Video:Transportof VSVG-GFP
0 min
Plasma
membrane
A EXPERIMENTAL FIGURE 14-2 proteintransportthroughthe
secretorypathway can be visualizedby fluorescence microscopy
of cellsproducinga GFP-tagged membraneprotein.Culturedcells
weretransfected witha hybridgeneencoding theviralmembrane
glycoproteinVSVG proteinlinkedto the genefor greenfluorescent
protein(GFP).A mutantversion of theviralgenewasusedsothat
newlymadehybridprotein (VSVG-GFP) in theERat 40 .C
isretained
but isreleased
for transportat 32 "C.(a)Fluorescencemicrographsof
cellsjustbeforeandat two timesaftertheywereshiftedto the lower
temperature.Movement of VSVG-GFP fromthe ERto the Golqiand
582 C H A P T E R1 4 | VES|CULAR
T R A F F t CS, E C R E T | O N
A,N D E N D O C Y T O S t S
acids in thesecells are incorporated into secretoryproteins, fa-
cilitating the observation of transported proteins.
Although autoradiographyis rarely usedtoday to localize
proteins within cells, these early experiments illustrate the
two basic requirementsfor any assayof intercompartmental
transport. First, it is necessaryto label a cohort ofproteins in
an early compartment so that their subsequent transfer to
Iater compartments can be followed with time. Second,it is
necessaryto have a way to identify the compartment in which
a labeled protein resides.Here we describetwo modern ex-
perimental procedures for observing the intracellular traffick-
ing of a secretoryprotein in almost any type of cell.
In both procedures, a gene encoding an abundant mem-
brane glycoprotein (G protein) from vesicular stomaritis virus
(VSV) is introduced into cultured mammalian cells either by
transfection or simply by infecting the cells with the virus. The
treated cells, even those that are not specializedfor secretion,
rapidly synthesizethe VSV G protein on the ER like normal
cellular secretoryproteins. Use of a mutant encoding a temper-
ature-sensitiveVSV G protein allows researchersto turn subse-
quent transport of this protein on and off. At the restrictive
temperature of 40 'C, newly made VSV G protein is misfolded
and therefore retained within the ER by quality-control mech-
anismsdiscussedin Chapter 13, whereasat the permissivetem-
peratureof 32"C, the protein is correctly folded and is trans-
ported through the secretorypathway to the cell surface.This
clever use of a temperature-sensitivemutation in effect defines
a protein cohort whose subsequenttransport can be followed.
In two variations of this basic procedure, transport of
VSV G protein is monirored by different techniques.Studies
using both of these modern trafficking assaysand Paladet
1 8 0m i n
Bts
5
ll 10
v
I
thF
Time(min)
finallyto thecellsurface occurredwithin180minutes. Thescale
baris 5 p.m (b)Plotof the levelsof VSVG-GFp in the endoplasmic
reticulum (ER),Golgi,andplasma membrane (pM)at different
timesaftershiftto lowertemperature. Thekinetics of transport
fromoneorganelle to anothercanbe reconstructed from
computer analysisof thesedata.Thedecreasein totalfluorescence
thatoccurs at latertimesprobablyresults
fromslowinactivation of
GFPfluorescence IFrom Jennifer
Lippincott-Schwartz
androret
H i r s c h b e r gM, e t a b o l i s mB r a n c h N
, a t i o n aIl n s t i t u t eo f C h i l dH e a l t ha n d
H u m a nD e v e l o o m e nI t
early experimentsall came to the same conclusron:In mam-
malian cellsvesicle-mediatedtransport of a protein molecule
from its site of synthesison the rough ER to its arrival at the
plasma membrane takes from 30 to 60 minutes.
Microscopy of GFP-LabeledVSV G Protein One approach
for observing transport of VSV G protein employs a hybrid
genein which the viral geneis fused to qhegene encodinggreen
fluorescent protein (GFP), a naturally fluorescent protein
(Chapter 9). The hybrid geneis transfectpdinto cultured cells
by techniques described in Chapter 5. \(hen cells expressing
the temperature-sensitiveform of the hybrid protein (VSVG-
GFP) are grown at the restrictive temperature, VSVG-GFP ac-
cumulatesin the ER, which appearsas a lacy network of mem-
branes when cells are observedin a fluorescentmicroscope.
When the cells are subsequentlyshifted to a permissive tem-
perature, the VSVG-GFP can be seento move first to the mem-
branes of the Golgi apparatus,which are denselyconcentrated
at the edge of the nucleus, and then to the cell surface (Figure
1.4-2a).By analyzing the distribution of VSVG-GFP at different
times after shifting cells to the permissivetemperature' re-
searchershave determined how long VSVG-GFP residesin
each organelle of the secretorypathway (Figure 14-2b).
Cis-Golgi
(a)
Mannose
trimming
+
Extract
slycoprotein
I I
Detection of Compartment-Specific Oligosaccharide
Modifications A second way to follow the transport of
secretoryproteins takes advantageof modifications to their
carbohydrate side chains that occur at different stagesof the
secretorypathway. To understand this approach, recall that
many secretoryproteins leaving the ER contain one or more
copies of the N-linked oligosaccharide Mans(GlcNAc)2,
which are synthesizedand attached to secretoryproteins in
the ER (seeFigure 13-18). As a protein moves through the
Golgi complex, different enzymeslocalized to the cis-, me-
dial-, and trans-Golgi cisternae catalyze an ordered seriesof
reactionsto thesecore Mans(GlcNAc)z chains, as discussed
in a later section of this chapter. For instance,glycosidases
that residespecificallyin the cis-Golgi compartment sequen-
tially trim mannose residuesoff the core oligosaccharideto
yield a "trimmed" form Man5(GlcNAc)2. Scientistscan use
a specializedcarbohydrate-cleavingenzymeknown as endo-
glycosidase D to distinguish glycosylated proteins that
remain in the ER from those that have entered the cis-
Golgi: trimmed cls-Golgi-specific oligosaccharides are
cleaved from proteins by endoglycosidaseD, whereas the
core (untrimmed) oligosaccharidechains on secretory pro-
teins within the ER are resistant to cleavageby this enzyme
(Figure 1'4-3a\.Becausea deglycosylatedprotein produced
by endoglycosidaseD digestion moves faster on an SDS gel
(b) Timeat 32 'C (min) 0 5 1 0 1 5 2 0 3 0 4 5 6 0
(ER)Resistant._
i
(cls-Golgi)Sensitive
(c)

1.0
o
(Man)a(GlcNAc)z (Man)s(GlcNAc), 'i 3 o.e
aO

:'6
D
rreatwithendoslycosidase
I I v> 0.6

Eo0.4

ti>
No cleavage, Cleavage, EE 0.2
L O

endoglycosidase D-resistant endoglycosidaseD-sensitive rC

o
o
0
t = N-Acetylglucosamine
O = Mannose T i m e( m i n )

A EXPERIMENTAL FIGURE 14-3 Transportof a membrane processed in thecrs-Golgibut notfromproteins in the ER.(b)SDSgel
glycoproteinfrom the ERto the Golgicanbe assayedbasedon electrophoresisof thedigestionmixtures resolves the resistant,
D. Cellsexpressing a uncleaved (slower-migrating)and sensitive,cleaved (faster-migrating)
sensitivityto cleavageby endoglycosidase
VSVG protein(VSVG) werelabeled with a pulse formsof labeled VSVGAsthiselectrophoretogram shows,initiallyall
temperature-sensitive
aminoacidsat the nonpermissive
of radioactive temperature sothat of theVSVGwasresistant to digestion,butwith timean increasing
fractionissensitive reflecting
to digestion, proteintransported fromthe
labeled proteinwasretarnedin the ERAt periodic timesaftera return
fromcells ERto theGolgi and processedthere.In control cells kept at 40'C, only
to the permissive
temperature of 32"C,VSVGwasextracted
with endoglycosidaseD.(a)Asproteins moveto thec/s- slow-moving, VSVGwasdetected
digestion+esistant after60 minutes
anddigested
Mans(GlcNAc)2 istrimmed (notshown).(c)Plotof the proportion of VSVGthat issensitive to
Golgifromthe ER,thecoreoligosaccharide
compartment digestion,derived fromelectrophoreticdata, reveals the time courseof
to Mans(GlcNAc)2 byenzymes in the crs-Golgi
that reside
theoligosaccharide
D cleaves chains fromproteins --;
ER Golgitransport. [FromC J Beckersetal, 1987, 50t523
Cell ]
Endoglycosidase

O 583
SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y
T E C H N I Q U EF
 ClassA GlassB ClassC ClassD ClassE

Fate of Normal Accumulation Accumulation Accumulation Accumulation Accumulation

secreted secretion in the cytosol i n r o u g hE R in ER-to-Golgi in Golgi In secretory
proteins transoortvesicles vesicles
Defective Transport B u d d i n go f Fusionof Transportfrom Transportfrom
function i n t ot h e E R vesiclesfrom transportvesicles Golgi to secretory secretoryvesicles
t h e r o u g hE R with Golgi vesicles to cell surface

EXPERIMENTAL FTGURE l4-4 phenotypes of yeastsec highern , o n p e r m i s s i voen e . A n a l y s i o

s f d o u b l em u t a n t sp e r m i t t e d
mutantsidentifiedstagesin the secretorypathway.These the sequentialorder of the stepsto be determined.[Seep Novick
temperature-sensitive
mutantscanbe grouped intofiveclasses
based e t a | , 1 9 8 1C
, e l l 2 5 : 4 6a1n, d C A K a i s e r a nRdS c h e k m al g ng. O.Cett
on thesitewherenewlymadesecreted (reddots)accumulate
proteins 61:723l
whencellsareshiftedfromthe permissive
temperature to the

than the corresponding glycosylatedprotein, these proteins A Iarge number of yeast mutants initially were identified
can be readily distinguished(Figure14-3b). basedon their ability to secreteproteins at one temperature
This type of assaycan be usedto track movement of VSV and inability to do so at a higher, nonpermissivetempera-
G protein in virus-infectedcellspulse-labeledwith radioac- ture. Sfhen these temperature-sensitivesecretion (sec) mw-
tive amino acids. Immediately after labeling, all the ex- tants are transferred from the lower to the higher tempera-
tractedlabeledVSV G protein is still in the ER and is resist- ture, they accumulate secretedproteins at the point in the
ant to digestion by endoglycosidaseD, but with time an pathway blocked by the mutation. Analysis of such mutants
increasingfraction of the glycoprotein becomessensitivero identified five classes(A-E) characterizedby protern accu-
digestion. This conversion of VSV G protein from an endo- mulation in the cytosol, rough ER, small vesiclestaking pro-
glycosidase D-resistant form ro an endoglycosidase teins from the ER to the Golgi complex,
Golgi cisternae,or
D-sensitive form corresponds to vesicular transport of the constitutive secretory vesicles (Figure 14-4). Subsequent
protein from the ER to the cis-Golgi. Note that transport of characterization of sed mutants in the various classeshas
VSV G protein from the ER to the Golgi takes about 30 min- helped elucidate the fundamental components and molecu-
utes as measuredby either the assaybasedon oligosaccha- lar mechanismsof vesicletrafficking that
we discussin later
ride processing or fluorescencemicroscopy of VSVG-GFp sectlons.
(Figure 14-3c). A variety of assaysbased on specific carbo- To determine the order of the steps in the pathw ay, re-
hydrate modifications thar occur in later Golgi compart- searchersanalyzeddouble sec mutants. For instance,when
ments have been developedto measureprogression of VSV yeast cells contain mutarions in both class B and class D
G protein through each stageof the Golgi apparatus. functions, proteins accumulate in the rough ER, not in the
Golgi cisternae.Since proteins accumulate at the earliest
YeastMutants Define Major Stagesand Many blocked step; this finding shows that classB mutatrons must
Componentsin VesicularTransport act at an earlier point in the secretoryparhway than classD
mutations do. These studies confirmed that as a secreted
The generalorganization of the secretorypathway and many protein is synthesizedand processed,it moves sequentially
of the molecular componentsrequired for vesicletrafficking from the cytosol -+ rough ER --> ER-to-Golgi transport
are similar in all eukaryotic cells. Becauseof this .o.r.ruul vesicles-+_Golgi cisternae-+ secreroryvesiclesand finally is
exocytoseo.
The three methods outlined in this sectionhave delineated
the major steps of the secretory pathway and have con-
tributed to the identification of many of the proteins respon-
sible for vesiclebudding and fusion. Currently eachof the in-
dividual steps in the secretorypathway is being studied in
mechanisticdetail, and increasinglSbiochemical assaysand
moleculargeneticstudiesare usedto study eachof thesesteps
in terms of the function of individual protein molecules.

584 . c H A p r E R1 4 | vEstcuLAR
T R A F F t cs,E c R E T t o N
A,N D E N D o c y r o s t s
(a) (b)

Golgi isolated from G protein in Golgi from

cis-Golgi medial-Golgi trans-Golgi uninfected wild-tyPe cells infected mutant cells

N-Acetylglucosamine
transferaseI reaction lncubation
VSV-infected wild-type cells

Addition of
N-acetyl-
glucosamine
VSV-infected mutant cells to G protein
I = N - A c e t y l g l u c o s a m i n e@ = G a l a c t o s e
(no N-acetylglucosamine
O =Mannose a=N-Acetylneuraminicacid
transferasel)

FIGURE 14-5A cell-freeassaydemonstrates with a simpler high-mannose oligosaccharide containing onlytwo

EXPERIMENTAL
N-acetylglucosamine andfivemannose residues.(b)WhenGolgi
protein transpoftfrom one Golgi cisternato another.(a)A
fibroblasts
isessentialin thistypeof assayIn cisternae frominfected
isolated mutantcellsareincubated with Golgi
mutantlineof cultured
cisternae fromnormal, unrnfected the
cells, VSV G protein produced
thisexample, thecellslacktheenzyme N-acetylglucosamine
| (stepE in Figure14-14)ln wild-type cells,thisenzyme is in vitrocontains theadditional N-acetylglucosamine Thismodification
transferase
localizedto themedr,afGolgi andmodifies N-linked oligosaccharides iscarriedout by transferase enzymethat is movedby transport
of oneN-acetylglucosamine,InVSV-infected wild-type vesrcles fromthewild-typemedial-Golgi to the mutantcts-
cisternae
bytheaddition
on theviralG proteinismodified to a typical Golgicisternae in the reactionmixture[See W E Balch et al, 1984'Cell
theoligosaccharide
cells,
panel.In 39:405 and525;W A et
Braell al, 1984,
Cell 1;
39:51 and J E Rothman and
complex oligosaccharide,asshownin the trans-Golgi
infectedmutantcells,however, the G proteinreaches the cellsurface I S6llner, 1997,Science275:1212 l

Cell-FreeTransportAssaysAllow Dissection
o f I n d i v i d u a lS t e p si n V e s i c u l aTr r a n s p o r t
In vitro assaysfor intercompartmental transport are power-
ful complementary approachesto studieswith yeast sec mu'
tants for identifying and analyzingthe cellular components
responsible for vesicular trafficking. In one application of
this approach, cultured mutant cells lacking one of the en-
zymes that modify N-linked oligosaccharidechains in the
Golgi are infected with vesicular stomatitis virus (VSV). For
example, if infected cells lack N-acetylglucosamine trans-
feraseI, they produce abundant amounts of VSV G protein
but cannot add N-acetylglucosamine residues to the
oligosaccharidechains in the medial-Golgi as wild-type cells
do (Figure 14-5a). When Golgi membranes isolated from
such mutant cells are mixed with Golgi membranes from
wild-type, uninfected cells, the addition of N-acetylglu-
cosamineto VSV G protein is restored(Figure14-5b). This
modification is the consequenceof vesiculartransport of N-
acetylglucosaminetransferaseI from the wild-type medial-
Golgi to the cls-Golgi compartment from virally infected
mutant cells.Successfulintercompartmentaltransport in this
cell-freesystem dependson requirementsthat are typical of
a normal physiologicalprocess,including a cytosolic extract,
a sourceof chemicalenergyin the form of ATP and GTP, and
incubation at physiological temperatures'
In addition, under appropriate conditions a uniform
population of the transport vesiclesthat move N-acetylglu-
cosaminetransferaseI from the medial- to cls-Golgi can be
purified away from the donor wild-type Golgi membranes
ty centrifugation. By examining the proteins that are en-
riched in thise vesicles,scientistshave been able to identify
targeting and fusion of vesicleswith appropriate acceptor
-.-br".t.t. In vitro assayssimilar in general design to the
one shown in Figure 1,4-5 have been used to study various
transport stepsin the secretorypathway.
Techniquesfor Studyingthe SecretoryPathway
r AII assays for following t he trafficking of proteins
rn living cells require a way
through the secretorypathway ln
secretoryproteins and a way to identity
to label a cohort of
labeled proteins subsequentlyare
the compartments where
located.
r Pulse labeling with radioactive amino acids can specifi-
cally label a cohort of newly made proteins in the ER' AI-
ternatively, a temperature-sensitivemutant protein that is
retained in the ER at the nonpermissivetemperaturewill be
releasedas a cohort for transport when cells are shifted to
the permissivetemperature.

SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y
T E C H N I Q U EF 585
 r Transport of a fluorescentlylabeled protein along the se- ( a )C o a t e dv e s i c l eb u d d i n g
cretory pathway can be observed by microscopy (seeFig-
ure 14-2). Transport of a radiolabeledprotein commonly
is tracked by following comparrment-specificcovalent
m o d i f i c a t i o n sr o r h e p r o t e i n .
Many of the componentsrequired for intracellularprotein
afficking have been identified in yeast by analysisof tem- Soluble
perature-sensitivesec mutants defectivefor the secretionof
proteins at the nonpermissivetemperarure(seeFigure 14-4).
Membrane
r Cell-free assaysfor intercompartmentalprorein trans- cargo-receptor
port have allowed the biochemicaldissectionof individual prorern
stepsof the secretorypathway. Such in vitro reactronscan
be used to produce pure transport vesiclesand to rest the Coat proteins
Donor
biochemicalfunction of individual transportproteins. memorane Cytosol

(b) Uncoatedvesiclefusion
I!f,| Molecular
Mechanisms
of VesicularTraffic cwosor
ltfl.'T..""
Small membrane-boundedvesiclesthat transporr prorelns
from one organelle to another are common elemenisin the

type of vesicle, studies employing genetic and biochemical

techniqueshave revealedthat each of the different vesicular
transport stepsis simply a variation on a common theme. In protein complex
this sectionwe explore the basicmechanismsunderlying vesi- FIGURE 14-6 Overviewof vesiclebuddingand fusionwith a
cle budding and fusion, that all vesicletypeshave in common. target membrane.(a)Budding isinitiated
by recruitment
of a small
The budding of vesiclesfrom their parent membrane is GTP-binding proteinto a patchof donormembraneComplexes of
driven by the polymerization of soluble protein complexes coatproteins in thecytosolthenbindto thecytosolicdomainof
onto the membrane to form a proteinaceousvesiclecoat membrane cargoproteins, someof whichalsoactasreceptors that
(Figure 14-6a). Interactionsbetweenthe cytosolic portions bindsoluble proteinsin the lumen,therebyrecruiting
luminalcargo
of integral membraneproteins and the vesiclecoat gather the proteinsintothe buddingvesicle(b)Afterbeingreleased and
appropriate cargo proteins into the forming vesicle.Thus the shedding itscoat,a vesicle
fuseswith itstargetmemorane In a
coat not only gives curvature to the membrane to form a processthatinvolves interaction
of coqnate SNARE proteins.
vesiclebut also acts as the filter to determine which oroteins
are admitted into the vesicle.
reversiblepolymerization of a distinct set of protein subunits
The integral membrane proteins in a budding vesiclein-
(Table 14-1). Each rype of vesicle,named for its primary
clude v-SNAREs, which are crucial to eventual fusion of the
coat proterns,transports cargo proteins from particular par-
ent organellesto particular destination organelles:

r COPI vesiclestransport proteins from the rough ER to

the Golgi.

COPI vesiclesmainly transport proteins in the retrograde

irection betweenGolgi cisternaeand from the cls-Golei
ack to the rough ER.

r Clathrin vesiclestransport proteins from the plasma

Assemblyof a ProteinCoat DrivesVesicle membrane (cell surface)and the trans-GoIgi network to late
F o r m a t i o na n d S e l e c t i o no f C a r g oM o l e c u l e s endosomes.

Three types of coated vesicleshave been characterized,each Every vesicle-mediatedtrafficking step is thought to
with a different type of protein coat and each formed by utilize some kind of vesicle coat; however, a specific coat

586 . c H A p r E R1 4 I vEstcuLAR
T R A F F t cs,E c R E T t o N
A,N D E N D o c y r o s t s
 TYPI MEDIATEI)
STEP
IRANSPORT PROTEINS
C()AT GTPase
ASS0CIATED
VESICIE

ER to cls-Golgi 1
Sec23lSec24and Secl3/Sec3 Sarl
COPII
Sec16
complexes,

cls-Golgi to ER Coatomerscontalnrngseven ARF

COPI
Later to earlier Golgi cisternae differentCOP subunits

Clathrin* AP1 comPlexes ARF

Clathrinand adapterprotelns tr ans-G olgi to endosome

tr ans -Golgi to endosome Clathrin + GGA ARF

Plasmamembrane to endosome Clathrin * AP2 comPlexes ARF

Golgi to lysosome,melanosome, AP3 complexes ARF

or plateletvesicles

.Each vesiclescontains clathrin

t1,peof Ap complex consists of four different subunits. It is not known whether the coat of AP3

protein complex has not been identified for every type ol and clathrin vesicles,this GTP-binding protein is known as
vesicle.For example,researchers have not yet identifiedthe ARF protein A different but related GTP-binding protein
coat proteins surrounding the vesiclesthat move protelns known as Sarl protein is present in the coat of COPII vesi-
from the trans-Golgito the plasma membrane during either cles. Both ARF and Sarl are monomeric proteins with an
constitutive or regulated secretion. overail structuresimilar to that of Ras' a key intracellular
The general scheme of vesicle budding shown in Fig- signal-transducingprotein (seeFigure 16-24)' ARF and Sarl
ure 14-6a appliesto all three known types of coated vesicles. pr"ot.inr, like Ras, belong to the GTPase superfamily of
Experimentswith isolated or artificial membranesand puri- switch proteins that cycle between inactive GDP-bound and
fied coat proteins have shown that polymerizationof the coat activeGTP-boundforms (seeFigure 3-32)'
proteins onto the cytosolic face of the parent membrane is The cycle of GTP binding and hydrolysis by ARF and
necessaryto producethe high curvatureof the membranethat Sarl are tho,tght to control the initiation of coat assembly,
is typical of a transport vesicleabout 50 nm in diameter.Elec- as schematicafy depicted for the assemblyof COPII vesicles
tron micrographs of in vitro budding reactions often reveal
structufes that exhibit discrete regions of the parent mem-
brane bearing a dense coat accompanied by the curvature
characteristicof a completedvesicle(Figure14-7). Suchstruc-
tures, usually calleduesiclebwds, appearto be intermediates
that are visible after the coat has begunto polymerizebut be-
fore the completedvesiclepinchesoff from the parent mem-
brane. The polymerized coat proteins are thought to form
sometype of curved lattice that drivesthe formation of a vesi-
cle bud by adheringto the cytosolicface of the membrane'

A ConservedSet of GTPaseSwitch Proteins

ControlsAssemblyof Different VesicleCoats
Based on in vitro vesicle-buddingreactions with isolated r, EXPERIMENTAL FIGURE 14-7 Vesiclebudscan be visualized
membranesand purified coat proteins,scientistshave deter- during in vitro buddingreactions. WhenpurifiedCOPII coat
mined the minimum set of coat components required to components areincubated with isolatedERvesiclesor artificial
form each of the three maior types of vesicles.Although phospholipid (liposomes),
vesicles of the coatproteins
polymerization
most of the coat proteinsdiffer considerablyfrom one type on thevesicle surfaceinduces emergence of highlycurvedbuds ln
of an in vitrobuddingreaction, notethe
of vesicleto another,the coats of all three vesiclescontain a th electron micrograph
di inctmembrane coat,visible asa darkproteinlayer,present on the
small GTP-bindingprotein that acts as a regulatorysubunit
buds lFrom
vesicle K Matsuoka etal, 1988,Ceil93(2):263
]
to control coat assembly(seeFigure 1,4-6a).For both COPI
. 587
S F V E S I C U L ATRR A F F I C
M E C H A N I S MO
MOLECULAR
 E Sarl membranebinding,
GTP exchange
in Figure 14-8. First, an ER membrane protein known as
Sec1.2catalyzesrelease of GDp from cyrosolic Sarl.GDp
Hydrophobic
GTP GDP N-terminus and binding of GTP. The Sec12 guanine nucleotide-
/ \ exchangefactor apparently receivesand integratesmultiple
Sart
Cytosol as yet unknown signals,probably including the presenceof
Jec tz -
. cargo proteins in the ER membrane that are ready to be
transported. Binding of GTp causes a conformational
ER lumen change in Sarl that exposes its hydrophobic N-terminus,
which then becomesembeddedin the phospholipid bilayer
and tethers Sarl.GTP to the ER membrane. The membrane_
Sec23/Sec24
attached Sarl.GTP drives polymerization of cytosolic com-
plexes of COPII subunits on the membrane.eventually lead-
ing to formation of vesicle buds. Once COPII vesiciesare
releasedfrom the donor membrane, the Sarl GTpase activ_
ity hydrolyzes Sarl.GTP in the vesicle membrane to
E :3:'liT' Sarl.GDP with the assistanceof one of the coat subunits.
This hydrolysis triggers disassemblyof the COpII coat. Thus
Sarl couples a cycle of GTp binding and hydrolysis to the
formation and then dissociationof the COpII coat.
ARF protein undergoesa similar cycle of nucleotide ex_
change and hydrolysis coupled to the assembly of vesicle
coats composedeither of COpI or of clathrin and other coat
proteins (AP complexes),discussedlater. A covalent protein
modification known as a myristate anchor on the N-termi_

p cre hydrotysis

P;
ARF'GTP with the membrane servesas the foundation for
further coat assembly.
Drawing on the structural similarities of Sarl and ARF
to other small GTPaseswitch proteins, researchershave con_
structed genesencoding mutant versionsof the two proteins
I Coatdisassembly
that have predictable effectson vesiculartraffic when trans-

Uncoated
vesicle
FIGURE 14-8 Modelfor the role of Sarl in the assemblyand
disassembly of COPIIcoats.Step[: Interaction of soluble GDp_
boundSarlwith theexchange factorSecl2, an ERinregrar
membrane protein, with target membranes.Addition of a nonhydrolyzable GTp
catalyzes exchange of GTpfor GDpon Sarl In
the GTP-bound formof Sar1,itshydrophobic analog to in vitro vesicle-buddingreactions causesa similar
N-terminus extends
outwardfromthe protein's surface andanchors blocking of coat disassembly.The vesiclesthat form in such
Sarlto the ER
membrane. Stepf,l: Sarlattached to the membrane serves reactions have coats that never dissociate,allowing their
asa
bindingsitefor the Sec23/Sec24 coatproteincomplex. Membrane composition and structure to be more readily analyzed.The
cargoproteins arerecruited to theformingvesicle budby bindingof purified COPI vesiclesshown in Figure 14-9 were produced
specific shortsequences (sorting signals) in theircytosolic regions to in such a budding reacrion.
siteson the Sec23/Sec24 complex, Somemembrane cargoproteins
alsoactasreceptors that bindsoluble proteins in the lumen.The
coatiscompleted by assembly of a second typeof coatcomplex
TargetingSequenceson CargoproteinsMake
composed of Sec13 andSec31 (notshown)StepB: Afterthe SpecificMolecularContactswith Coat proteins
vesicle coatiscomplete, the Sec23coatsubunitpromotes GTp In order for transport vesiclesto move specificproteins from
h y d r o l y sbiysS a r l S t e p@ : R e l e a soef S a r l . G Dfpr o mt h ev e s i c l e
one compartment to the next, vesicle buds must be able to
membrane causes disassembly of thecoat.[See S Sprrngeret at, 1999,
Cell97:145I discriminate among potential membrane and soluble cargo
proteins, acceptingonly those cargo proteins that should J_
vance to the next compartment and excluding those that

588 . c H A p r E R1 4 | vEstcuLAR
T R A F F I cs,E c R E T t o N
A.N D E ND O C Y T O S I S
 should remain as residentsin the donor compartment' In ad-
dition to sculpting the curvature of a donor membrane' the
vesiclecoat functions in selectingspecificproteins as cargo'
The primary mechanism by which the vesicle coat selects
cargo moleculesis by directly binding to specific sequences,
or sorting signals, in the cytosolic portion of membrane
cargoproteins(seeFigure 14-6a).The polymerizedcoat thus
acts as an affinity matrix to cluster selectedmembrane cargo
proteins into forming vesicle buds. Since soluble proteins
within the lumen of parent organelles cannot contact the
coat directly, they require a different kind of sorting signal'
Soluble luminal proteins often contain what can be thought
of as luminal sorting signals,which bind to the luminal do-
mains of certain membrane cargo proteins that act as recep-
tors for luminal cargo proteins. The properties of several
A EXPERIMENTAL FIGURE 14-9 Coatedvesiclesaccumulate known sorting signalsin membrane and soluble proteins are
during in vitro buddingreactionsin the presence of a
summarizedin Table 14-2.We describethe role of thesesig-
nonhydrolyzable analogof GTP. WhenisolatedGolgimembranes
nals in more detail in later sectlons.
areincubated with a cytosolic COPIcoatproteins,
extractcontaining
formandbudoff fromthe membranes
vesicles of a
Inclusion
nonhydrolyzable analogof GTPin the buddingreactionprevents Rab GTPasesControl Dockingof Vesicles
disassembly of the coataftervesicle Thismicrograph
release shows
on TargetMembranes
COPIvesicles generated in sucha reactionandseparatedfrom
membranes by centrifugation prepared
Coatedvesicles in thisway A secondset of small GTP-binding proteins, known as Rab
canbe analyzed to determine theircomponentsandproperties proteins, participate in the targeting of vesiclesto the appro-
of L Orci]
[Courtesy priate target membrane' Like Sarl and ARF, Rab proteins

LUMENAL SORTING SIGNALS

(KDEL)
Lys-Asp-Glu-Leu ER-resident soluble protelns KDEL receptorin cls-Golgi
membrane

(M6P)
Mannose6-phosphate Soluble lysosomal enzymes M6P receptor in trans-Golgr
after processingin cls-Golgi membrane

Secretedlysosomal enzymes M6P recepror in plasma membrane Clathrin/AP2

CYTOPLASMICSORTING SIGNALS

ER-resident membrane protelns COPI ct and P subunits COPI

Lys-Lys-X-X(KKXX)

Cargo membrane proteins in ER COPIISec24subunit COPII

Di-acidic(e.g.,Asp-X-Glu)

AP2 complex ClathridAP2

Asn-Pro-X-Tyr(NPXY) LDL receptor in plasma
membrane

AP1 (p1 subunit) Clathrin/AP1

Tyr-X-X-<D(YXXO) Membrane proteins in
trans-Go|gi

Plasma membrane proteins AP2(p.2subunit) Clathrin/AP2

Plasma membrane proteins AP2 complexes Clathrin/AP2

Leu-Leu(LL)

in parentheses'
X : any amino acid; O - hydrophobic amino acid. Single-letteramino acid abbreviations are

S F V E S I C U L ATRR A F F I C
R E C H A N I S MO
MOLECULAM 589
 (a) Transport < FIGURE 14-10Modelfor dockingand fusionof transport
vesicle vesicleswith their target membranes. (a)Theproteins shownin
thisexample participatein f usionof secretory vesicleswith the
plasmamembrane, but similarproteins mediate allvesicle-fusion
events.Step[:A Rabproteintethered viaa lipidanchorto a
secretoryvesicle bindsto an effectorproteincomplex on the plasma
membrane, therebydockingthetransport vesicleon the appropriate
V e s i c l e d o cki n sl E Rab.GTP
targetmembrane. StepE: A v-SNARE protein(inthiscase,VAMp)
interactswith the cytosolicdomains of the cognatet-SNAREs (inthis
case,syntaxin andSNAP-25) Theverystablecoiled-coil SNARE
complexes thatareformedholdthe vesicle closeto the target
membraneStepB: Fusion of the two membranes immediately
followsformationof SNARE complexes, but precisely how this
occursrsnot known Step@: Following membrane fusion,NSFin
conjunction with ct-SNAP proteinbindsto the SNARE complexes.
TheNSF-catalyzed hydrolysis of ATpthendrivesdissociation of the
Target SNARE complexes, freeingthe SNARE proteins for anotherroundof
memDrane vesiclefusionAlsoat thistime,Rab.GTp ishydrolyzed to Rab.GDp
anddissociates fromthe Rabeffector(notshown)(b)TheSNARE
complexNumerous noncovalent interactions betweenfour longo
helices,two fromSNAP-25 andoneeachfromsyntaxin andVAMp,
stabilizethe coiled-coil
structure[See J E Rothman andT Sollner,1997,
Science2T6:1212, andW Weis andR Scheller,1gg1,Nature 395:328 part
(b)fromY A ChenandR H Scheller, 2001,Nat Rev. Mot Cett Biot2(2):98
I
SNARE
complex\

Rab'GTP is tetheredto the vesiclesurface,it is thought to in-

teract with one of a number of different large proteins,
Membrane tu.'on known as Rab effectors, attached to the target membrane.
JE Binding of Rab.GTP to a Rab effector docks the vesicleon
an appropriate target membrane (Figure 14-10, step [). Af-
ter vesiclefusion occurs, the GTp bound to the Rab protein
is hydrolyzed to GDP, triggering the releaseof Rab.GDp,
o-SNAP
which rhen can undergo another cycle of GDp-GTp ex-
change,binding, and hydrolysis.
cis-SNARE Severallines of evidencesupport the involvement of spe_
comptex cific Rab proteins in vesicle-fusionevents.For instance,ihe
yeast SEC4 gene encodesa Rab protein, and yeast cells ex-
D i s a s s e m b l yo f
pressingmutant Sec4proteins accumulate secretoryvesicles
SNAREcomplexes that are unable to fuse with the plasma membrane (classE
mutants in Figure 14-4).In mammalian cells,Rab5 protein
is localized to endocytic vesicles,also known as early endo-
somes. These uncoated vesiclesform from clathrin-coated
vesiclesjust after they bud from the plasma membrane dur-
ing endocytosis(seeFigure 14-1, step pt). The fusion of
( b ) S N A R Ec o m o l e x early endosomeswith eachother in cell-freesysremsrequrres
the presenceof Rab5, and addition of Rab5 and GTp to cell-
free extracts acceleratesthe rate at which thesevesiclesfuse
with each other.A long coiled protein known as EEA1 (early
endosome antigen 1), which resideson the membrane of the
Syntaxin early endosome,functions as the effector for Rab5. In this
case, RabS.GTP on one endocytic vesicle is thought to
specifically bind to EEA1 on the membrane of another
belong to the GTPasesuperfamily of switch proteins. Con_ endocytic vesicle, setting the stage for fusion of the two
version of cytosolic Rab.GDp to Rab.GTp, iatalyzed by a vesicles.
specific guanine nucleotide-exchangefactor, induces a con-
A different type of Rab effector appearsto function for
formational changein Rab that enablesit to interact with a
each vesicletype and at each step of the secretorypathway.
surface protein on a particular transport vesicle and insert
Many questions remain about how Rab proteins are rar_
its isoprenoid anchor into the vesicle membrane. Once
geted to the correct membrane and how specific complexes

590 . c H A p r E R1 4 | v E S t c u L ATRR A F F I sc E
, c R E T t oA
NN. DE N D O C Y T O S I S
form between the different Rab proteins and their corre-
sponding effector protetns.
PairedSetsof SNAREProteinsMediate Fusion
of Vesicleswith TargetMembranes
As noted previously,shortly after a vesiclebuds off from the
donor membrane, the vesiclecoat disassembles to uncover a
vesicle-specific membrane protein, a v-SNARE (seeFigure
14-6b1.Likewise, eachtype of target membranein a cell con-
tains I-SNARE membrane proteins. After Rab-mediated
docking of a vesicleon its target (destination)membrane,the
interaction of cognate SNAREs brings the two membranes
close enough together that they can fuse.
One of the best-understood examples of SNARE-
mediatedfusion occursduring exocytosisof secretedproteins
(Figure 14-10, steps f,l and B). In this case,the v-SNARE,
membrane protein), is
known as VAMP (zesicle-associated
incorporated into secretory vesiclesas they bud from the
trans-Golgi network. The I-SNAREs aresyntaxin, anintegtal
membrane protein in the plasma membrane, and SNAP-25,
which is attachedto the plasma membraneby a hydrophobic
lipid anchor in the middle of the protein. The cytosolic region
in each of thesethree SNARE proteins contains a repeating
heptad sequencethat allows four a helices-one from VAMP,
one from syntaxin, and two from SNAP-2S-Io coil around
one another to form a four-helix bundle. The unusual stabil-
ity of this bundled SNARE complex is conferred by the
arrangementof hydrophobic and charged amino residuesin
the heptad repeats.The hydrophobic amino acids are buried
in the central core of the bundle, and amino acidsof opposite
chargeare alignedto form favorableelectrostaticinteractions
between helices.As the four-helix bundles form, the vesicle
and target membranesare drawn into closeapposition by the
embeddedtransmembranedomains of VAMP and syntaxin.
In vitro experiments have shown that when liposomes
containingpurified VAMP are incubatedwith other liposomes
containing syntaxin and SNAP-25, the two classesof mem-
branesfuse,albeit slowly. This finding is strong evidencethat
the close apposition of membranesresulting from formation
of SNARE complexesis sufficient to bring about membrane
fusion. Fusion of a vesicleand target membraneoccurs more
rapidly and efficiently in the cell than it does in liposome ex-
periments in which fusion is catalyzed only by SNARE pro-
teins. The likely explanation for this differenceis that in the
cell, other proteinssuch as Rab proteinsand their effectorsare
involved in targetingvesiclesto the correct membrane.
Yeastcells,like all eukaryotic cells,expressmore than 20
different related v-SNARE and I-SNARE proteins. Analysesof
yeast mutants defectivein each of the SNARE geneshave iden-
tified specific membrane-fusion events in which each SNARE
protein participates.For all fusion eventsthat have been exam-
ined, the SNAREs form four-helix bundled complexes,similar
to the VAMP/syntaxin/SNAP-25 complexes that mediate fu-
sion of secretoryvesicleswith the plasma membrane.However,
in other fusion events(e.g.,fusion of COPII vesicleswith the
crs-Golgi network), each participating SNARE protein con-
tributesonly one ct helix to the bundle (unlike SNAP-25'which
contributes two helices);in thesecasesthe SNARE complexes
comprise one v-SNARE and three I-SNARE molecules.
Using the in vitro liposome fusion assay'researchershave
tested the ability of various combinations of individual
v-SNARE and I-SNARE proteins to mediate fusion of donor
and target membranes.Of the very large number of different
combinations tested,only a small number could efficiently me-
diate membrane fusion. To a remarkable degree,the functional
combinations of v-SNAREs and I-SNAREs revealedin thesein
vitro experimentscorrespond to the actual SNARE protein in-
teractions that mediate known membrane-fusioneventsin the
yeastcell. Thus the specificityof the interaction betweenSNARE
oroteins can account for much of the specificity of fusion be-
tween a particular vesicletype and its target membrane'
Dissociationof SNAREComplexesAfter
MembraneFusionls Driven by ATPHydrolysis
After a vesicle and its target membrane have fused, the
SNARE complexes must dissociateto make the individual
SNARE proteins available for additional fusion events' Be-
causeof the stability of SNARE complexes,which are held
together by numerous noncovalent intermolecular interac-
tiJrs, their dissociation dependson additional proteins and
the input of energY.
The first clue that dissociation of SNARE complexesre-
quired the assistanceof other proteins came from in vitro
tiansport reactions depleted of certain cytosolic proteins'
The observedaccumulation of vesiclesin these reactions in-
dicated that vesiclescould form but were unable to fuse with
a targetmembrane.Eventually two proteins, designatedNSI
o-SNaR were found to be required for ongoing vesicle
"nd
fusion in the in vitro transport reaction. The function of NSF
in vivo can be blocked selectively by N-ethylmaleimide
-SH group
(NEM), a chemical that reactswith an essential
on NSF (hencethe name, NEM-sensitive /actor)'
Among the classC yeastsecmutants are strains that lack
functionaf Sec18or Secl7, the yeast counterpartsof mam-
malian NSF and o-SNAP, respectively.\fhen these class C
mutants are placed at the nonpermissivetemperature, they
accumulate nR-to-Golgi transport vesicles;when the cells
are shifted to the lower, permissivetemperature'the accu-
mulated vesiclesare able to fuse with the cis-Golgi'
Subsequentto the initial biochemicaland geneticstudies
identifying NSF and ct-SNAP,more sophisticated in vitro
transport assayswere developed.Using thesenewer assays're-
searchershave shown that NSF and a-SNAP proteins are not
sion assaysand in the yeast mutants after a loss of Sec17or
Sec18were a consequence of free SNARE proteinsrapidly be-
coming sequestered in undissociated SNARE complexesand
thus b-ing unavailable to mediate membrane fusion'
TRAFFIC . 591
M O L E C U L A RM E C H A N I S M 5O F V E 5 I C U L A R
 Molecular Mechanismsof VesicularTraffic
r The three well-characterizedtransport vesicles-COpl, !,''ir*, $cotgi
j' network
COPII, and clathrin vesicles-aredistinguishedby the pro-
teins that form their coats and the transport routes they
mediate(seeTable 14-1).
r All types of coated vesiclesare formed by polymerization
of cytosoliccoat proteinsonto a donor (parent)membrane iF,,f-q'"',
to form vesicle buds that eventually pinch off from the
membrane to releasea complete vesicle.Shortly after vesi-
cle release,the coat is shed,exposingproteinsrequiredfor
fusion with the target membrane(seeFigure 14,6).
Small GTP-bindingproteins (ARF or Sarl) belongingto
e GTPase superfamily control polymerization of coat
proteins, the initial step in vesicle budding (see Fig_
ure 14-8).After vesiclesare releasedfrom the donor mem_
brane, hydrolysis of GTP bound to ARF or Sarl triggers
disassemblyof the vesiclecoats.
pecific sorting signalsin membrane and luminal pro-
s_ofdonor organellesinteracrwith coat proteinsd.riing
c l e b u d d i n g .r h e r e b yr e c r u i t i n gc a r g o p r o t e i n sr o v e s i -
(seeTable 14-2).
r A secondset of GTP-bindingproteins,the Rab proteins, Rough
regulate docking of vesicleswith the correct rarget mem- ER
brane. Each Rab appearsto bind to a specificRab effector
associatedwith the target membrane.
r Each v-SNARE in a vesicular membrane specifically
binds to a complex of cognateI-SNARE proteins in the tar- A FIGURE 14-11 Vesicle-mediated protein trafficking between
get membrane, inducing fusion of the two membranes. the ERand crs-Golgi. Stepsfi-f,t: Forward (anterograde) transport
After fusion is completed, the SNARE complex is disas- is mediated by COPII vesrcles,whichareformedby polymerization of
soluble COPII coatproteincomplexes (green) on the ERmembrane
sembledin an MP-dependent reacrionmediatedby other
v-SNAREs (orange) andothercargoproteins (blue)in the ER
cytosolic proteins (seeFigure 14-10).
membrane areincorporated intothe vesicle by interacting with coat
proteinsSoluble cargoproteins (magenta) arerecruited by binding
to appropriate receptors in the membrane of buddingvesicles
M EarlyStagesof the Secretory Dissociation of the coatrecycles freecoatcomplexes andexposes
Pathway v-SNARE proteins on the vesicle surfaceAfterthe uncoated vesicle
becomes tetheredto the crs-Golgi membrane in a Rab-mediated
In this section we take a closer look at vesicular traffic process, pairingbetweenthe exposed v-SNAREs andcognate
I-SNARE i nst h e G o l g m
i e m b r a nael l o w sv e s i c lfeu s i o nr.e l e a s i n o
the contentsintothe crs-Golgi compartment (seeFigure14-101.
Steps@-@: Reverse (retrograde) transport, mediated by vesicles
coatedwith COPIproteins (purple), recycles the membrane bilayer
andcertainproteins, suchasv-SNAREs and missorted ER-resident
tain newly synthesizedproteins destined for the Golgi, cell proteins (notshown),fromthe crs-Golgi to the ER All SNARE
p r o t e i nasr es h o w ni n o r a n g ea l t h o u g vh- S N A R aEnsdI - S N A R E a rse
surface,or lysosomesas well as vesiclecomponents such as
d i s t i n cpt r o t e i n s
v-SNAREs that arerequired ro targer vesiclesto the cls-Golei
membrane. Proper sorring of proteins between the ER anld correctly delivered to the Golgi advance through successive
Golgi also requires reverse retrograde transport from the compartmentsof the Golgi by cisternalmaturarion.
cls-Golgito the ER and is mediatedby COpI vesicles(Fig_
ure 14-11). This retrogradevesicletransport servesro rerrreve
v-SNARE proteins and rhe membraneitielf back to the ER to COPIIVesiclesMediate Transportfrom the ER
provide the necessarymaterial for additional rounds of vesi_ to the Golgi
cle budding from the ER. COpl-mediated retrograde trans, COPII vesicleswere first recognizedwhen cell-free extracts
port also retrieves missorted ER-resident proteins from the of yeast rough ER membraneswere incubated with cytosol
cis-Golgi to correct sorting mistakes.proteins that have been and a nonhydrolyzable analog of GTp. The vesicles that

592 . c H A p r E R1 4 I vEstcuLAR
T R A F F , .s, E c R E T t o N
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COPIIcoat
/"
COPIVesiclesMediate RetrogradeTransport
w i t h i n t h e G o l g i a n d f r o m t h e G o l g it o t h e E R
COPI vesicleswere first discoveredwhen isolated Golgi
fractions were incubated in a solution containing .ytorJl
and a nonhydrolyzable analog of GTp (seeFiguie 14-9).
Subsequentanalysisof thesevesiclesshowed that the coat
is formed from large cytosolic complexes, called
coatomers,composedof sevenpolypeptide subunits.yeast
cells containing temperature-sensitive
murations in COpI
proteins accumulateproteins in the rough ER at the non_
permissive temperature and thus are categorizedas class B
secmutants (seeFigure 14-4). Although discoveryof these
mutants initially suggestedthat COpI vesiclesmediateER_
to-Golgi transport, subsequentexperiments showed that
their main function is retrograde transport, both between
Golgi cisternaeand from the cis-Golgi to the rough ER
(seeFigure 14-11, right). BecauseCOpI mutanrs cannor
recyclekey membrane proteins back to the rough ER, the
ER gradually becomesdepleted of ER proteins such as v-
SNAREs necessaryfor COpII vesiclefunction. Eventually.
vesicleformation from the rough ER grinds to a halt; se_
cretory proteins continue to be synthesizedbut accumu_
late in the ER, the defining characteristicof classB secmu-
tants. The generalability of secmurants involved in either
COPI or COPII vesiclefunction to eventually block both
anterograde and retrograde transport illustrates the
594 o c H A p r E R1 4 | vEstcuLAR
T R A F F t cs,E c R E T t o N
A,N D E N D o c y r o s t s
Video:KDELReceptorTrafficking
< FIGURE 14-13Roleof the KDEtreceptorin retrievalof
ER-resident luminalproteinsfrom the Golgi.ERluminal
proteins,especially
thosepresent at highlevels, canbe passively
incorporated intoCOPII vesicles
andtransported to the Golgi
(stepsE and Z). Manysuchproteins beara C-terminal KDEL
(Lys-Asp-Glu-Leu)sequence (red)thatallowsthemto be retrieved
TheKDELreceptor, locatedmainlyin the crs-Golgi networkandin
bothCOPII andCOPIvesicles, bindsproteins bearing the KDEL
sortingsignalandreturnsthemto the ER(stepsEl and 4). This
retrieval
system prevents depletionof ERluminalproteins suchas
thoseneededfor properfoldingof newlymadesecretory
proteinsThebindingaffinityof the KDELreceptor isvery
to pH Thesmalldifference
sensitrve in the pHof the ERand
Golgifavorsbindingof KDEl-bearing proteins to the receptor in
Golgi-derived vesicles
andtheirrelease in the ER [Adapted fromJ
Semenza etal, 1990,
Cell61:13491
fundamental interdependence of these two rransporr
processes.
As discussedin Chapter 13, the ER contains severalsolu-
ble proteins dedicated to the folding and modification of
newly synthesizedsecretory proteins. These include the chap-
erone BiP and the enzymeprotein disulfide isomerase,which
are necessaryfor the ER to carry out its functions. Although
such ER-residentluminal proteins are not specificallyselected
by COPII vesicles,their sheer abundancecausesthem to be
continuouslyloadedpassivelyinto vesiclesdestinedfor the cls-
Golgi. The transport of thesesolubleproteins back to the ER,
mediated by COPI vesicles,preventstheir eventualdepletion.
Most soluble ER-residentproteins carry a Lys-Asp-GIu-
Leu (KDEL in the one-lettercode) sequenceat their C-termi-
nus (seeTable 14-2). Severalexperimentsdemonstratedthat
this KDEL sorting signal is both necessaryand sufficient to
causea protein bearingthis sequenceto be located in the ER.
For instance,when a mutant protein disulfide isomerase
lacking thesefour residuesis synthesizedin cultured fibrob-
lasts, the protein is secreted.Moreover, if a protein that nor-
mally is secretedis alteredso that it containsthe KDEL signal
at its C-terminus,rhe protein is locatedin the ER. The KDEL
sorting signalis recognizedand bound by the KDEL receptor,
a transmembraneprotein found primarily on small transport
vesiclesshuttling between the ER and the cls-Golgi and on
the cis-Golgi reticulum. In addition, soluble ER-residentpro-
teins that carry the KDEL signal have oligosaccharidechains
s6s V M H r _ V d A U O I _ 3 U ) l S3 H r l O S I 9 V J _ SA ' l U V 3
snqJ 'de.{\gfpd ,{rolorf,osaql ur lueulr€durof, rorlJpo ue ol ol -lorpaw aql ruorJ pue r.dlou-lotpaw aqt ot -sD aqr I'uort
tuoql SurtJodsuerlpue tuarutredruoc ralel E r.uoJtsaudz 'p.remro; suralo.rd .{rolarcas Surlrrer solJrsal lJodsueJl
-ua r31og Jo UE Sur,tar.rtar 'trodsue.rl ape.rSorlaraterpeu
IIErusqrr,r\ sluaurlrBdruol Io 1rs lrlprs lllprtuossa ue sE^\
.,{1a4r1
rsoru solrrse^asoqt lele^\oH '(SI-tI arnSrg)reqlo xaldruoc r31og eql reqr lq8noql sE^{tr sreoddueur roJ
-ue ot luerutJgdtuocr31o5 euo ruorJ suralord e^otu ]eql '(sqelsuorlEJ
xelduroc r31og eql qtr.&\petertosse self,rse^11erusdueu -lJlpour eJuanbasalrletuasa.rdore ro1 g1-71 a;n8lg aas)
Jo
slEa^er ddocsorcrru uortf elo 'paapul 'r?1o'j-suou aql lueruuedruof, lxau oql yo seudzue 3ur.{yporu oqt roJ saterls
-qns oqr se Surrr.ras
luatul.redruof auo ur surer{f ereJpdr{ogJel
(>pels r3log eqr
porlrpou oql qBno.rqref,uonbesur Sur,tour
ue e>lrlqrnur satBredoxelduoc
suratord qtr^ 'eurl .,(lgurasse
r31og eql 'a1oqmoqr uO '>1cers r3log oql lrsuel daql se sural
'pla]uro) -ord d.rolo:rasol paqrplte salerpdqog:el palur1-O ro pa>lurl
I t E 9 : S t w a e o t g ^ a A u u V ' S 8 61 Sp u e
plaluro>g ea5l r6;o9 aq1 ur sdeis 6ursse>otd ut sa)ualalltpLuol+ -51 Surd;rpou ur po^lo^ur ere leql saseroJsuenldsordppue
] l n s a lu e f s a p u e q l e s o O r l o p a ) u r l - N+ o a t n ] ) n l l sa q ] u t s u o t i p u p n saseprsordp a.re seurdzuaaqt ,o duey41'ureluoc deqr reqr
'urelordorI16uerlpLUuJeLu
;etrdril e to+ slua^o0urssatotdr61o9 sou.{zua oql ol Surproooe roqloup ouo ruog .roylp r31og
eql sluaserder{ennq}edsrql losol{r oqi LuoJ}peuodu.rrstostn>eld eql 'PuralsrJro 'sfEs
Jo sluaulreduof,gns aql PaU3DEIJ to
o p r l o a l ) n ur e b n su . r o t ; ' a L u et l ] p e u o ' a p u e q ) ) e s o 6 r 1eoq 1o 1 s t e 6 n s tos po>lJEtse ur peSue.rrEueuo orE r{rrq,{\ 'sluerulredurol
ppe sau,izueasero+suel] trltad5 (ll dels) sanptsalasol:e1e6 -qns rnoJ ro eerql olur pezrueSrosr xeydruocr3log eq1
aql ]o tl)ea ol anprsolpoe )ruruJelneull{late-ry ue 1o a6e1ur1 {q
{11eup r ,uLe ( g d a } s )s o n p t s aa. ts o l t e ; e 6o a r q t u o t l l p p el i q r 6 1 o 9
} o
uorlernlelNleurotsr) [q srnrrg
-sue4 aq] ur pelalduot sr 6urssatotd(E dols) pappe st aso)n+
!6loC aql qOnorql uodsuerl operboroluv
al6urse pue '(E dals) penouLatoJesanptsa.i osouupLuolorJ.t oMl
'(E pue
E sdels)poppeere sanprsol()VN)lg) euru.reso>n16;,i1e>e-X7 'de,tr.qredd.rolarcesaql
oorql 'oJaH't61ocl1erpaw aql ol uorle.rnleur leuralsr),iq senoLrL
uralordoqt '(ll dals) r61o9-sr:aql ut sanptsalasouueuloorql 1o se8elsrelEl ur rnrf,o oslEfeqt 8ur>llrlJerturolord rElnf,rse^
,to lenourorJaUV sluoulueduo> pale:rpuloql 01 paztle)ola.te Jo sarnleet letuaurepunJeJEsluJrulJedruorolrl oql uae.r\laq
dals qree 6urz,ile1et saul{zuaaqf 'slla) a}eJqayon u! oeuJa}s!) sureto.rdleurr.unlolqnlos 1o Surycdcaroql pue 'surelord pue
t6;o9-suerl pue '-Fpa.u'-sl) ulqllrvl suralo.rdo>I;6 uo suleq) sprdqoqdsoqdauerqurau ;o Surledcaraql (selJrso^uodsuerl
a p u e q > r e s o b t l op e l u r l - N ; o 6 u r s s e > o l 6t l - t l l u n g l l V pepunoq-euerqruou otur surolord 1o drrua e^uJeles oql
'salrrse^ (aperSo;rer)
plce clutuelnou;A1ecy-1y = I osocnl = V IdOl pu€ (aperSo.rarue)UaOf, qloq
aso]oele!=a O s o u u e l A =o olur SurpeoyoSrer Jo lrlcglcads aql dq pellonuot dlareurrr
euruesocnlb;Ilacv-N = I -1nssacordparrln8er pue o^uJelosdlq8H e sr xalduroc r31og
pue UE or{l ueo,trloqsuralord;o Suruortrued aqr 'd1.reo13
uf uloll 't.rodsue.rr
yodsuerl
e;crsen
gg-or-r31o5 aperSone.ralerpou solJrsolIdOl ler{l SunBc
r6roe -lpul 'UE or{t or >lleq
I leu8rs srqr Surreaqsurolord elerrlor
z1cy11c191s1uey11 or elgpun are osle rnq leu8rsXX)X eqt purq ot alqeun ore
_^ z(cyqc19)s(uey11 dluo rou dlaOC ro nIdOJ 3ur>1ce1 sluetntu lsea,(alrlrsues
-arnleradural 'UE ar{t ot tJodsueJtaperSo.uarJoJ sel3rse^
IdO) orur suratord JuEJqruoru elurod.rocur ol luarf,
-rttns pu€ dresseceu qroq sr '(.raruoleorIdOl aql ur slrun
T -gns aprrdad,(1od ua,rasaql yo ozvrr)slrunqns fl pue o y4g3
er{t Jo xaldtuor E ol spurg rlJlrl^a 'pu8rs Sutltos XXXX
slql '(Z-tI a1qe1 aas) yosoldr eqt sere; r{11{a\'luaur8es
lz( z(cVNctg)s(uen) Ieurrural-) rraql to pue dre,r oql tE of,uanbasy-y-sd1-sd1
z 1 c y y c 1 9 l s 1 u e t A ) (go)V N c t e urpluof, r3log aqr ruory UE aqt ot lleq pat.rodsue.rta.re
v leql surelord euerqrueu Jer{lopup roldarer lgq) or{J
v-dno 'ug eql
Jo reqt uerlt re^{ol dllqSas sr r31og aqr;o gd aqt esneroqUg
eql ur saprrdadasaql eseeloror rnq r31og-sll:eqt ur saprrdad
,11d
lC:D pulq ot elqe sr roldof,or eqr teqr rq8noql sr lr pue
,ry\ol
te pue8q slr o1 dlrqSri arotu spurg rotdarer -IAC) or{I
'G.t-Vt e.rn8rg) eqr ot ueql urntor pue >lro.ryueu
UA r31og-srr
aqr or padecsa o^pq leqr leu8rs Surl.rosIEC) aql Sururetuor
suralo.rdalqnlos elarrler o1 dlureur slce rotdacarlge) eql
leql alerrpur s8urpurl osJrJJ'>lro,ry\tau r81og-sr ar{l sp rEJ sE
<- ls?ol lE pelrodsuert uaeq pue UE ar{rr}ol e^eq tsnru suratord
]!x3
eseqleurl eruostE snqt !>lro,rtlaur81og-slr.ror31og-srraqr ur
.,{1uo
punoy seudzua Aqpazllvw ore teqt suonerrJrpou qlr,v\
 Video: 3-D Model of a Golgi Complex K.€l

< EXPERIMENTAL FIGURE 14-15 Electron

micrographof the Golgicomplexin an
Formingsecretory exocrinepancreatic cellrevealsboth
vesicle
anterogradeand retrogradetransport
trans-Golginetwork vesicles. A Iargesecretory vesicle canbe
seenformingfromthe trans-Golgi network
-l Elements of the rough ER are on the bottom
trans
andleftin thismicrograph Adjacent to the
,,,uaiut lG.olsi roughERaretransitional elements from
. I Crsrernae
cts ,) whichsmoothprotrusions appearto be
buddingThesebudsformthe smallvesicles
cis-Golginetwork thattransport secretory proteins fromthe
roughERto the Golgicomplexlnterspersed
ER-to-Golgi a m o n gt h eG o l gci i s t e r n aaer eo t h e rs m a l l
t r a n s p o r vt e s i c l e s vesicles now knownto functionin
Smooth protrusion retrograde, not anterograde, transport
[Courtesy G Palade ]
T r a n s i t i o n aell e m e n t s

the Golgi appearsto have a highly dynamic organization.
To see the effect this retrograde transport has on the or-
ganization of the Golgi, consider the net effect on the me-
dial-Golgi compartment as enzymesfrom the trans-Golgi
move to the medial-Golgi while enzymesfrom the medial-
G o l g i a r e r r a n s p o r t e dr o t h e c i s - G o l g i . A s t h i s p r o c e s s
continues, the medial-Golgi acquires enzymes from the
trans-Golgi while losing medial-Golgi enzymesand thus
g r a d u a l l y b e c o m e sa n e w t r a n s - G o I g i c o m p a r t m e n t . I n
this wa5 secretory cargo proteins can acquire carbohy-
drate modification in the proper sequentialorder without
being moved from one cisternato another vra antero,
grade vesicletransport.
The first evidencethat the forward transporr of cargo
proteins from the cis- to the trans-Goigi occurs by a non-
vesicular mechanism, called cisternal maturation, came
from careful microscopic analysisof the synthesisof algal
scales.These cell-wall glycoproteins are assembledin the
cls-Golgi into large complexes visible in the electron mi-
gen precursor often form in the lumen of the cls-Golgi
(see Figure 19-24). The procollagen aggregaresare roo
l a r g et o b e i n c o r p o r a t e di n t o s m a l l t r a n s p o r tv e s i c l e sa, n d
investigators could never find such aggregaresin trans-
port vesicles.These observationssuggestthat the forward
m o v e m e n t o f t h e s e a n d p e r h a p s a l l s e c r e t o r yp r o t e i n s
from one Golgi compartment to another does not occur
via smallvesicles.
A particularly elegant demonstration of cisrernal matu-
ration in yeast takes advantageof different-coloredversions
of GFP to image two different Golgi proteins simultane-
ously.Figure 14-16 shows how a cis-Golgiresidentprotein
labeled with a green fluorescent protein and a trans-Golgt
protein labeled with a red fluorescentprorein behavein the
same yeast cell. At any given moment individual Golgi cis-
terna appear to have a distinct compartmental identity, in
the sensethat they contain either the cls-Golgi protein or the
trans-Golgi protein but only rarely contain both proteins.
However, over time an individual cisterna labeled with the
cis-Golgi protein can be seento progressivelylose this pro-
tein and acquire the trans-Golgi protein. This behavior is ex-
actly that predicted for the cisternal maturation model, in
which the composition of an individual cisterna changesas
Golgi residentproteins move from later to earlier Golgi com-
partments.
Numerous controversial questions concerning mem-
brane flow within the Golgi stack remain unresolved. For
example, although most protein traffic appears to move
through the Golgi complex by a cisternal maturation mech-
anism,thereis evidencethat at leastsomeof the COpI trans-

s96 CHAPTER
14 | V E S T C U L ATR
R A F F t CS, E C R E T T OANN, D E N D O C Y T O S T S
L6S AVA HT_VdUOt_lU):StHr lo st9vl_s ulJ.v'l
'LI-il ernSr{ 'tpor
Idoc eql Jo slrun
ur PezrrErur.uns ar? rSloc-suErl oql uorJ pnq tEqr soltrsa^ 'aouenbes
-qns ol spurg r.{rrr.{,ry\ XX)) E sr selJrso^IdOC
'de^4.r{reddrolarces oql ur alEI rnf, olul surelord ouprgruoru stf,orrp leqt sleu8rs Surlros aql
Jo seddt snolrel ar{I
-lo tpql sluela Surssoro:dda1 pue 'uaqt Suorue surelord 'self,rse^
to ouo IdOJ lq r31og-slr eql uro{ pa^orrlor oq
o3.rer ele8arSasreqr susruer{f,aruagt '>lrozuteur31o5-suul7 upJ self,Ise^IIdOI ruroJ ol papoau suralold ouerqr.uory r
eql tuort pnq rPqt solJrse^to spur>lruaraJJrpeql ssnf,srpe.4 'G.t-Vt ern8rg
uorlf,ossrqr uI 'uorlpurlsop IpurJ rreqt o1 .(rorrrloproJ selr
aas) selorsea1493 eperSonor olur suralord UE pailos
-rsa^ sPur{ ruero}Jrp requrnu E
Jo Jo Jo euo otur palJos Orp -srru slruJal eueJquau r31og-slr eql ur uralord JoldeJ
deql 'ara11'tuoutredruoc r31o9 Ielsrp tsoru agl '4romrau -ar rt;tcadse ol acuanbas 'leu8rs3ur
Ie^orrtorsrqr;o Surpurg
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-dlrporu-arerpdqoqreoosoql ToldoJOJurelord oueJqr.uoru E ol
Jo slo^ol luerlrJJnssurelurpru
stuerulredruocr31og rerlrpo ol ratpl tuorJ sollrsol IdO:) Surpurqdq salcrsa,tIIdOf, ol pale8rel o.redlqeqo.rdsurelord
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1o 3ur1o11;e.rraperSo.uar oqI
dg rno perrrptr ere surer{t aprreqrceso8rlorraql ot suorl ur leu8rs Surl.rosJer{lo Jo f,rprf,E-rpe Surureluoc surelo.rd
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leurelsrJ dq xalduroo r31og aqr ;o
e)e! sua.q ar{l or elet sD eqr urort alotu suraloJd o8rec sy 'xaloruor
I€los/€Ilas
e pue 'xaldruoc VZ)eSrcZ)eS e '1re5 uralo.rd Surpurq
fennqle4 -dIC sleot IIdOJ r
IIBlusagt :sluouodruof,aargl asr.rduroc
A,ro1ane5
oql lo sobelstale'l @ 'fit-Vt arn8tg ees)uorlcarrp as.rerr
-or oqt ur sutalord uodsuel self,rsal1493 :r31og-sp aqt
or dH q8nor oql tuory suratord lrodsue.4 soltrsel IIdOf, r
'uorlf,eJrp
^e^ qled ftolalra5 oql lo sabel5{;.re3
ape.rSortare ur t.rodsuertrelnf,rsal IdOf, dq Surrrou saurdz
-ua r31og luaprsor uo spuadap teql lrodsuerl ep€r8orel
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ueqrraqrer)epe.r8
'>1cersr31og aql ur sluaruueduor rarlree ol rolpl -oJetue
uE ur oloru pue (seur.{zue
r31o9ueqr raqrer)
suralord
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l.rod
I Z00L:lttarn]eN'9OOZ suralotd
'te ]a^asotu.rorll r6;o9a1e;;o 161o9 euolsnlrrnoqssa6er-ur
,Losauosmouoqaql 1)oSlo y6t1 qlrnn
ute6puesuralotd 16;o9
{;tea ssol{q paztJollereql uotleln}eul iaq]ropolaqelareeurl auo{ue qltqM 'aeuialst)
161o9 uor})ollo)
1o }o }e ;o
ssa:orde nnollo]
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}o peJtpe sl aq1 AdorsoDrLllesde;-au11{q pabeulr alp(otualsolonl} poj)pauso
luauruadxe srql luauyeduro)stq]ul peztle)ol-o)atesuralotd qloq ol posn+1ta5urelotd161o9 a1e;oql pup(o)ua)salonl] ueat6)639 o1
q)rqMurpouad;auqe burnnollol'eulalsr) palelost aqi ut pate)ol pasn;y6r4urelordr6;o9^lreaoql 6urssaldxa sllarlseo1';1erpea{
stouolepausc-I)asuoq]pue eulalsr)paielostaql ul pele)ol sl dlg 6ugnr;e ur uorleJnleu leuJolst) 16;o9ale,rlsuourap surelold
-7614Iluo 1st13a6er-ur
aq1,Lo6urssa:otd {q palelosr 'eulo}st) uorsn;pa66et-a)ua)saronlJ
lelrOrp 9t-tt lUngll'lVINll,llUldx:l V
l--.'l
tun I
 "i &,,""""""""''"'' < FIGURE 14-17Vesicle-mediated protein
i-.t, F
.i,,,-,,,:r,
'i,,,-'-1.,,:' ::"
,, plaSma trafficking from the trans-golginetwork.
Plasma
/-
membrane COPI(purple) (ll) mediate
vesicles retrograde
/ A I transport withintheGolgi.Proteins that
I
l/
E _.r'
functionin the lumenor in the membrane
the lysosome aretransported
of
fromthe trans-
I _rr'!_
''"'l'l' Golginetworkviaclathrin coated(red)vesicles
fl
d ' \ ^
(B). Afteruncoating, thesevesicles fusewith
lateendosomes, whichdeliver theircontents
to the lysosome Thecoaton mostclathrin
vesicles contains additionalproteins(AP
complexes) not indicatedhereSomevesicles
,:.
fransGolgi rl,,;
e"H::',." fromthe trans-Golgi carrying cargodestined
network for the lysosome fusewith the lysosome
,_,,,,,.,
directly(E), bypassing theendosome These
,rdF,1e'"5,*ri&aq,ubl,1. vesicles arecoatedwith a typeof APcomplex
(blue);it is unknownwhetherthesevesicles
alsocontainclathrin. Thecoatproteins
FS q'q**"*6'*'' surrounding (4) andregulated
constitutive
, ...,..,,,,'.,;i il
Lvsosome ($) secretory vesicles
arenot yetcharacterized;
r-t. E thesevesicles carrysecreted proteinsand
\ plasma-membrane proteinsfromthe trans-
Golginetworkto thecellsurface

fransGolgi

/r,-r\
Video: Birth of a Clathrin Coat \\-//
VesiclesCoatedwith Clathrinand/or Adapter
ProteinsMediate SeveralTransportSteps
The best-characterizedvesicles that bud from the trans-
Golgi network (TGN) have a two-layered coat: an outer
layer composed of the fibrous protein clathrin and an inner
Iayer composedof adapter protein (AP) complexes.Purified
clathrin molecules,which have a three-limbed shape, are
calledtriskelions, from the Greek for "three-legged" (Figure
14-18a). Each limb contains one clathrin heavy chain
Binding site
for assembly (180,000MV) and one clathrinlight chain (=35,000-40,000
particles M'W). Triskelions polymerize to form a polygonal lattice
with an intrinsic curvature (Figure 14-18b). V/hen clathrin
polymerizeson a donor membrane, it does so in association
with AP complexes,which assemblebetweenthe clathrin lat-
FIGURE14-18 Structureof clathrin coats. (a)A clathrin tice and the membrane. Each AP complex (340,000 M\f)
molecule,calleda triskelion,is composedof threeheavyand three contains one copy eachof four different adapter subunit pro-
lightchains lt hasan intrinsiccurvaturedue to the bend in the teins. A specificassociationbetween the globular domain at
heavychains(b) Clathrincoatswere formed in vrtroby mlxrng the end of each clathrin heavy chain in a triskelion and one
purifiedclathrinheavyand Iightchainswith Ap2 complexes in the subunit of the AP complex both promotes the co-assemblyof
absenceof membranesCryoelectron mrcrographs of morethan clathrin triskelions with AP complexesand adds to the sta-
1000assembled hexagonalclathrrnbarrelparticles were analyzedby bility of the completed vesiclecoat.
digitalimageprocessing to generatean averagestructural
By binding to the cytosolic face of membraneprorelns,
representation. The processed imageshowsonlythe clathrinheavy
chainsin a structurecomposedof 36 triskelions.
adapter proteins determine which cargo proreins are
Threerepresenralve
triskelionsare highlightedin red,yellow,and green partof the Ap2 specificallyincluded in (or excludedfrom) a budding trans-
complexes packedinto the interrorof the clathrincagearealso port vesicle. Three different AP complexes are known
visiblein this the processed (4P1, AP2, AP3), each with four subunits of different,
representation [SeeB pishvaee andG
Payne,1998, Cell95:443 part(b)from Fotinet al , 2004,Nature432:573 though related,proteins.Recently,a secondgeneraltype of
I
adapterprotein known as GGA has beenshown to contain

598 C H A P T E R1 4 | V E S T C U L ATRR A F F | CS
, E C R E T T OA
NN, D ENDOCYTOStS
in a single 70,000 M\7 polypeptide both clathrin- and Exoplasmicface
cargo-bindingelementssimilar to those found in the much
larger hetero-tetramericAP complexes.Vesiclescontaining
each type of adapter complex (AP or GGA) have been
found to mediate specifictransport steps(seeTable 14-1).
All vesicleswhose coats contain one of these complexes
utilize ARF to initiate coat assemblyonto the donor mem-
brane. As discussedpreviously,ARF also initiatesassembly
of COPI coats.The additional featuresof the membraneor
protein factors that determine which type of coat will as-
semble after ARF attachment are not well understood at
this time. Integral
Vesicles that bud from the trans-GoIgi network en
route to the lysosome by way of the late endosomehave
clathrin coats associatedwith either AP1 or GGA. Both
AP1 and GGA bind to the cytosolic domain of cargo pro-
teins in the donor membrane. Recent studies have shown
that membrane proteins containing a Tyr-X-X-O se-
quence,where X is any amino acid and O is a bulky hy-
drophobic amino acid, are recruited into clathrin/AP1
vesicles budding from the trans-Golgi network. This
YXXQ sorting signal interacts with one of the AP1 sub-
units in the vesiclecoat. As we discussin the next section,
vesicles with clathrin/AP2 coats, which bud from the vesicle
Clathrin-coated
plasma membrane during endocytosis,also can recognize
a FIGURE 14-19Modelfor dynamin-mediated pinchingoff of
the YXXO sorting signal. Vesiclescoated with GGA pro- bud forms, dynamin
clathrin/AP-coated After
vesicles. a vesicle
teins and clathrin bind cargo molecules with a different thatisnotwellunderstood,
overthe neck.Bya mechanism
polymerizes
kind of sorting sequence.Cytosolic sorting signals that of GTPleadsto release
hydrolysis of thevesicle
dynamin-catalyzed
specificallybind to GGA adapter proteins include Asp-X- fromthe donormembraneNotethatmembrane proteins in the
Leu-Leu and Asp-Phe-Gly-X-Osequences(where X and O donormembrane are into
Incorporated by
vesicles interacting with
are defined as above). APcomplexes fromK Takel
in the coat lAdapted et al, 1995,Nature
Some vesiclesthat bud from the trans-Golgi network 374:186]l
have coats composed of the AP3 complex. Although the
AP3 complex does contain a binding site for clathrin sim-
ilar to the AP1 and AP2 complexes,it is not clear whether
clathrin is necessaryfor function of AP3-containing vesi- oinched off from the donor membrane. In the case of
cles since mutants of AP3 that lack the clathrin binding clathrin/AP-coated vesicles' a cytosolic protein called dy-
site appear to be fully functional. AP3-coatedvesiclesme- namin is essentialfor releaseof complete vesicles.At the
diate trafficking to the lysosome, but they appear to by- later stages of bud formation, dynamin polymerizes
pass the late endosomeand fuse directly with the lysoso- around the neck portion and then hydrolyzes GTP. The en-
mal membrane.In certain types of cells, such AP3 vesicles ergy derived from GTP hydrolysis is thought to drive a
mediate protein transport to specializedstoragecompart- conformational changein dynamin that stretchesthe vesi-
ments related to the lysosome. For example, AP3 is re- cle neck until the vesiclepinchesoff (Figure 14-1,9).Intet-
quired for delivery of proteins to melanosomes,which estingly, COPI and COPII vesicles appear to pinch off
contain the black pigment melanin in skin cells, and to from-donor membraneswithout the aid of a GTPasesuch
platelet storage vesiclesin megakaryocytes,a large cell as dynamin. At presentthis fundamental differencein the
that fragments into dozens of platelets.Mice with muta- processof pinching off among the different types of vesi-
tions in either of two different subunits of AP3 not only cles is not understood.
have abnormal skin pigmentation but also exhibit bleed- Incubation of cell extracts with a nonhydrolyzable detiv-
ing disorders.The latter occur becausetears in blood ves- ative of GTP provides dramatic evidencefor the importance
selscannot be repaired without plateletsthat contain nor- of dynamin in pinching off of clathrin/AP vesiclesduring en-
mal storagevesicles. docytosis.Such treatment leadsto accumulation of clathrin-
coated vesicle buds with excessively long necks that are
surrounded by polymeric dynamin but do not pinch off
D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f (Figure 14-20). Likewise, cells expressingmutant forms of
of ClathrinVesicles dynamin that cannot bind GTP do not form clathrin-coated
A fundamental step in the formation of a transport vesicle vesiclesand instead accumulate similar long-necked vesicle
that we have not yet considered is how a vesicle bud is buds encasedwith polymerized dynamin.

PATHWAY . 599
L A T E RS T A G E sO F T H E S E C R E T O R Y
 < EXPERIMENTAL FIGURE 14-20 cTp hydrolysisby dynaminis
requiredfor pinchingoff of clathrin-coated vesiclesin cell-free
extracts.A preparation of nerveterminals, whichundergoextensive
e n d o c y t o sw l sa, sl y s e db y t r e a t m e nwt i t h d i s t i l l ew
d a t e ra n d
incubated with GTP-1-S, a nonhydrolyzable derivative of GTpAfter
sectioning t h, e p r e p a r a t i owna st r e a t e dw i t h g o l d - t a g g eadn t i -
d y n a m ian n t i b o dayn dv i e w e di n t h e e l e c t r om n i c r o s c o pTeh i s
image,whichshowsa long-necked clathrin/AP-coated budwith
polymerizd e yd n a m i lni n i n gt h e n e c k r, e v e a tl sh a tb u d sc a nf o r mi n
the absence of GTPhydrolysis, but vesicles cannotpinchoff. The
extensive polymerization of dynaminthat occursin the presence of
G T P - 1 -p5r o b a b ldy o e sn o t o c c u rd u r i n gt h e n o r m abl u d d i n g
process[From K Takel et al , 1995,Nature 374:186; courtesy of pietro
D eC a m i l l i

As with COPI and COPII vesicles,clathrin/Ap vesicles endosomeis a carbohydrateresidue,mlnnose 5-phospbate

normally lose their coat soon after their formation. Cytoso-
lic Hsc70, a constitutive chaperoneprotein found in all eu-
karyotic cells, is thought to use energy derived from the hy-
drolysis of ATP to drive depolymerization of the clathrin
coat into triskelions.Uncoatingnot only releasestriskelions
for reusein the formation of additional vesiclesbut also ex-
poses v-SNAREs for use in fusion with targer membranes.
Conformational changes thar occur when ARF switches
from the GTP-bound to GDP-bound state are thousht to
regulatethe timing of clathrin coat depolymerization.How
the action of Hsc70 might be coupled to ARF switching is
not well understood.
M a n n o s e6 - P h o s p h a t R
e e s i d u eT
s a r g e tS o l u b l e
Proteinsto Lysosomes
Most of the sorting signals that function in vesicular traf-
ficking are short amino acid sequencesin the targeted pro-
tein. In contrast, the sorting signal that directs soluble lyso-
somal enzymes from the trans-Golgi network to the late
(M6P), which is formed in the cis-Golgi. The addition and
initial processing of one or more preformed N-linked
oligosaccharideprecursors in the rough ER is the same for
lysosomal enzymesas for membrane and secretedproteins,
yielding core Man6(GlcNAc)2chains (seeFigure 13-18). In
the cls-Golgi, the N-linked oligosaccharidespresenton most
lysosomal enzymesundergo a two-step reaction sequence
that generatesM6P residues(Figure 14-21.).The addition of
M6P residuesto the oligosaccharide chains of solublelyso-
somal enzymesprevents theseproteins from undergoing the
further processing reactions characteristic of secretedand
membrane proteins (seeFigure 14-14).
As shown in Figure 14-22, the segregationof M6p-
bearing lysosomal enzymesfrom secretedand membrane
proteins occurs inthe trans-Golgi network. Here, transmem-
brane mannose 6-phosphate receptors bind the M6p
residues on lysosome-destinedproreins very tightly and
specifically.Clathrin/AP1 vesiclescontaining the M6p recep-
tor and bound lysosomal enzymesthen bud from the trais-
Golgi network, lose their coats, and subsequentlyfuse with

Lysosomalenzyme

\
\
G l c N A cp h o s p h o t r a n s f e r a s e Catalyticsite Recognitionsite
FIGURE 14-21Formationof mannose6-phosphate (M6p) andboundby thisenzyme, phosphorylated GlcNAc groupsare
residuesthat target solubleenzymesto lysosomes. TheM6p addedspecifically to lysosomal enzymesStepA: Afterrelease of a
residues
thatdirectproteinsto lysosomesaregenerated in the crs- modifiedproteinfromthe phosphotransferase, a phosphodiesterase
Golgibytwo Golgi-resident
enzymes Step[: An N-acetylglucosamineremoves the GlcNAc Aroup,leaving a phosphorylated mannose
(GlcNAc)phosphotransferasetransfers
a phosphorylated GlcNAc r e s i d u e otnh el y s o s o meanl z y m e[ S
, e e AB c a n t o r eatl ,j 9 9 2 , JB i o l
groupto carbonatom6 of oneor moremannose residues Because Chem 267:23349,and S Kornfeld, 1987, FASEBI 1i4621
onlylysosomalenzymes containsequences (red)thatarerecoqnized

600 CHAPTER
14 | V E S T C U L ATR
R A F F t CS, E C R E T T OANN, D E N D O C Y T O S t S
 M6P
receptor
Exterior

Plasmamembrane

Cytosol

Clathrin-
Receptor- coated
mediated vesicle
endocytosis

Recyclingof
M6P receptor
Uncoated
endocytic
vesicle

El
v

{:l-,--$
Late
endosome
(low pH)

A ,#o.,"0
transport
vesicle
,ri'i"'r"''*b!i ,,

trans-
Clathrin-coated
Golgi
vesicle
network

FfGURE '14-22frallicking of solublelysosomalenzymes andaredephosphorylated, lateendosomes subsequently fusewith a

from the trans-Golginetwork and cell surfaceto lysosomes. lysosome (step4) Notethatcoatproteins andM6Preceptors are
Newlysynthesized lysosomal enzymes, produced in the ER,acquire recycled (steps EEI andEE), andsomereceptors aredeliveredto
mannose 6-phosphate (M6P)residues in thecrs-Golgi (seeFigure thecellsurface (stepE). Phosphorylated lysosomal enzymes
14-21)Forsimplicity, onlyonephosphorylated oligosaccharide chain occasionallyaresortedfromthe trans-Golgi and
to the cellsurface
isdepicted, althoughlysosomal enzymes typically havemanysuch secretedThesesecreted enzymes canbe retrieved by receptor-
c h a i n sI n t h e t r a n s - G o lngei t w o r kp, r o t e i ntsh a tb e a rt h e M 6 P mediated endocytosis (steps6-ts), a process thatcloselyparallels
sortingsignalinteract with M6Preceptors in the membrane and traffickingof lysosomalenzymes from the trans-Golgi networkto
thereby aredirected intoclathrin/AP1 vesicles (steptr) Thecoat lysosomes [See et al, 1988,Cell52:329,5Kornfeld,
G Griffiths 1992,Ann
surrounding released vesicles is rapidly depolymerized (stepE), and Rev.Biochem 61:307;andG Griffiths andJ,Gruenberg, 1991,Trends
Cell
the uncoated transport vesicles fuse with late endosomes (stepB) B i o1
l :51
Afterthe phosphorylated enzymes dissociate fromthe M6Preceptors

the late endosome by mechanismsdescribed previously. Furthermore, a phosphatasewithin late endosomesusually

BecauseM6P receptorscan bind M6P at the slightly acidic removesthe phosphatefrom M6P residueson lysosomal en-
pH (=6.5) of the trans-Golgi network but not at a pH less zymes, preventing any rebinding to the M6P receptor that
than 6, the bound lysosomal enzymes are releasedwithin might occur in spite of the low pH in endosomes.Vesicles
Iate endosomes,which have an internal pH of 5.0-5.5. budding from late endosomesrecyclethe M6P receptor back

L A T E RS T A G E SO F T H E S E C R E T O RPYA T H W A Y 601
to the trans-Golgi network or, on occasion, to the cell
surface. Eventually, mature late endosomes fuse with
lysosomes,delivering the lysosomal enzymes to their final
destination.
The sorting of soluble lysosomal enzymesin the trans-
Golgi network (Figure L4-22, steps[-4) sharesmany of
the features of trafficking between the ER and cls-Golgi
compartments mediated by COPII and COPI vesicles.First,
mannose 6-phosphateacrs as a sorring signal by interacting
with the luminal domain of a receptor protein in the donor
membrane.Second,the membrane-embedded receptorswith
their bound ligands are incorporated into the appropriate
vesicles-in this case, either GGA or AP1-containing
clathrin vesicles-by interacting with the vesiclecoat. Third,
thesetransport vesiclesfuse only with one specificorganelle,
here the late endosome,as the result of interactions between
specificv-SNAREs and I-SNAREs. And finally, intracellular
transport receptorsare recycledafter dissociatingfrom their
bound ligand.
Study of LysosomalStorageDiseases
RevealedKey Componentsof the Lysosomal
Sorting Pathway
FE A groupof geneticdisorders
termedlysosomal
stor-
3l age diseasesare causedby the absenceof one or more
lysosomalenzymes.As a result, undigestedglycolipids and
extracellularcomponentsthat would normally be degraded
by lysosomalenzymesaccumulatein lysosomesas large in-
clusions.I-cell diseaseis a particularly severetype of lyso-
somal storagediseasein which multiple enzymesare miss-
ing from the lysosomes.Cells from affected individuals
Iack the N-acetylglucosaminephosphotransferasethat is
required for formation of M6P residueson lysosomal en-
zymes in the cls-Golgi (see Figure 1,4-21). Biochemical
comparisonof lysosomalenzymesfrom normal individuals
with those from patientswith I-cell diseaseled to the initial
discoveryof mannose6-phosphateas rhe lysosomalsorting
signal. Lacking the M6P sorting signal, the lysosomalen-
zymes in I-cell patients are secreted rather than being
sorted to and sequestered in lysosomes.
lVhen fibroblasts from patients with I-cell disease
are
grown in a medium containing lysosomalenzymesbearing
M6P residues,the diseasedcellsacquirea nearly normal in-
tracellular content of lysosomal enzymes.This finding indi-
catesthat the plasmamembraneof thesecellscontainsM6p
receptors,which can internalize extracellular phosphory-
lated lysosomalenzymesby receptor-mediated endocytosis.
This process,used by many cell-surfacereceptorsto bring
bound proteins or particlesinto the cell, is discussedin de-
tail in the next section. It is now known that even in normal
cells, some M6P receptorsare transported to the plasma
membrane and some phosphorylated lysosomal enzymes
are secreted(seeFigure 14-22). The secretedenzymescan be
retrieved by receptor-mediatedendocytosisand directed to
lysosomes.This pathway thus scavenges any lysosomalen-
zymes that escapethe usual M6P sorting parhway.
602 C H A P T E R1 4 | VEStCULAR
T R A F F t CS, E C R E T T OA
NN, D ENDOCYTOStS
Hepatocytes from patients with l-cell diseasecontain
a normal complementof lysosomalenzymesand no inclu-
sions, even though these cells are defective in mannose
phosphorylation.This finding implies that hepatocytes(the
most abundant type of liver cell) employ an M6P-inde-
pendent pathway for sorting lysosomal enzymes.The na-
ture of this pathway, which also may operate in other cells
types,is unknown. I
ProteinAggregationin the trans-GolgiMay
Functionin Sorting Proteinsto Regulated
SecretoryVesicles
As noted in the chapter introduction, all eukaryotic cells
continuously secretecertain proteins, a process commonly
called constitwtiue secretion. Specialized secretory cells also
store other proteins in vesiclesand secretethem only when
triggered by a specific stimulus. One example of such regu-
lated secretioz occurs in pancreatic B cells, which store
newly made insulin in special secretoryvesiclesand secrete
insulin in responseto an elevation in blood glucose(seeFig-
ure 15-33). These and other secretorycells simultaneously
utilize two different types of vesiclesto move proteins from
the trans-Golgi network to the cell surface:regulated trans-
port vesicles,often simply called secretoryvesicles,and un-
regulated transport vesicles,also called constitutive secre-
tory vesicles.
A common mechanismappearsto sort regulatedproteins
as diverseas ACTH (adrenocorticotropichormone), insulin,
and trypsinogen into regulated secretory vesicles.Evidence
for a common mechanismcomesfrom experimentsin which
recombinant DNA techniquesare used to induce the synthe-
sis of insulin and trypsinogenin pituitary tumor cells already
synthesizingACTH. In these cells, which do not normally
express insulin or trypsinogen, all three proteins segregate
into the same regulated secretory vesiclesand are secreted
together when a hormone binds to a receptor on the pitu-
itary cellsand causesa rise in cytosolic Ca2*. Although these
three proteins share no identical amino acid sequencesrhat
might serveas a sorting sequence,they obviously have some
common feature that signals their incorporation into regu-
lated secretoryvesicles.
Morphologic evidencesuggeststhat sorting into the regu-
lated pathway is controlled by selectiveprorein aggregadon.
For instance,immature vesiclesin this pathway-those that
have just budded from the trans-Golginerwork-contain dif-
fuse aggregatesof secretedprotein that are visible in the elec-
tron microscope.These aggregatesalso are found in vesicles
that are in the processof budding, indicating that proteins
destinedfor regulated secretoryvesiclesselectivelyaggregate
together before their incorporation into the vesicles.
Other studies have shown that regulated secretoryvesi-
cles from mammalian secretorycells contain three proteins,
chromogranin A, chromogranin B, and secretogranin II,
that together form aggregateswhen incubated at the ionic
conditions (pH = 6.5 and 1 mM Ca2*; thought to occur in
the trans-Golgi network; such aggregatesdo not form at the
( a )P r o i n s u l i na n t i b o d y < EXPERIMENTAL FIGURE 14-23Proteolyticcleavageof
proinsulinoccursin secretoryvesiclesafter they have budded
from the trans-Golgi network.Serial sectionsof the Golgiregion
*,* cellwerestained
of an insulin-secreting with (a)a monoclonai
antibody that recognizes proinsulinbut not insulinor (b)a different
antibody that recognizes insulinbut not proinsulinTheantibodies,
whichwereboundto electron-opaque goldparticles,appearasdark
dotsin theseelectron micrographs (seeFigure 9-21).lmmature
*
secretoryvesicles(closed arrowheads) andvesiclesbuddingfromthe
b trans-Golgi(arrows) stainwiththe proinsulin antibody but notwith
insulinantibodyThese vesiclescontaindiffuseproteinaggregates
t that includeproinsulinandotherregulated secreted proteinsMature
vesicles(openarrowheads) stainwith insulinantibody but notwith
proinsulinantibody andhavea densecoreof almostcrystalline
insulinSincebuddingandimmature secretoryvesiclescontain
proinsulin(notinsulin),the proteolyticconversion of proinsulinto
,$fri insulinmusttakeplacernthesevesicles aftertheybudfromthe
..**Y
t Sr: trans-Golgi
vesicle
networkTheinsetin (a)showsa proinsulin-rich
surrounded by a proteincoat(dashed
secretory
ltne)lrromL orcietal,
.,'.,k:,. 1987, Cell 49:865;courtesyof L Orci l

( b ) I n s u l i na n t i b o d y
SomeProteinsUndergoProteolyticProcessing
After Leavingthe trans-Golgi
For somesecretoryproteins(e.g.,growth hormone) and cer-

w'
,:asi
tain viral membraneproteins (e.g.,the VSV glycoprotein),
removal of the N-terminal ER signal sequencefrom the nas-
cent chain is the only known proteolytic cleavagerequired to
convert the polypeptide to the mature, active species(see

t Figure 13-6). However, some membraneand many soluble

secretoryproteins initially are synthesizedas relatively long-
lived, inactive precursors, termed proproteins, that require
further proteolytic processingto generatethe mature, active
proteins. Examples of proteins that undergo such processing
are soluble lysosomal enzymes)many membrane proteins
such as influenza hemagglutinin (HA), and secretedproteins
such as serum albumin, insulin, glucagon, and the yeast c{.
mating factor. In general, the proteolytic conversion of a
G proprotein to the correspondingmature protein occurs after
the proprotein has been sorted in the trans-Golgi network to
appropriate vesicles.
I In the caseof soluble lysosomalenzymes,the proproteins
are called proenzymes,which are sorted by the M5P recep-
/ tor as catalytically inactive enzymes.In the late endosomeor
lysosomea proenzymeundergoesa proteolytic cleavagethat
generatesa smaller but enzymaticallyactive polypeptide.
0 . 5u m
Delaying the activation of lysosomal proenzymesuntil they
reach the lysosomepreventsthem from digestingmacromol-
eculesin earlier compartments of the secretorypathway.
neutral pH of the ER. The selectiveaggregationof regulated Normall5 mature vesiclescarrying secretedproteins to
secretedproteins together with chromogranin A, chromo- the cell surface are formed by fusion of several immature
granin B, or secretograninII could be the basisfor sorting of ones containing proprotein. Proteolytic cleavageof propro-
theseproteins into regulatedsecretoryvesicles.Secretedpro- teins, such as proinsulin, occurs in vesiclesafter they move
teins that do not associatewith theseproteins,and thus do away from the trans-Golgi network (Figure 14-23). The pro-
not form aggregates,would be sorted into unregulatedtrans- proteins of most constitutively secreted proteins (e.g.,
p o r t v e s i c l e bs y d e f a u l t . albumin) are cleaved only once at a site C-terminal to a

L A T E RS T A G E SO F T H E S E C R E T O RPYA T H W A Y 603
 (a) Constitutivesecretedproteins and thus were unable to mate with cells of the opposite mat-
ing type (seeFigure 21,-19).The wild-type KEX2 gene en-
Proalbumin
codesan endoproteasethat cleavesthe cr-factorprecursor at
NH3+mCOO-
a site C-terminal to Arg-Arg and Lys-Arg residues.Using the
| ,u,'nJooo.o,""r" KEX2 gene as a DNA probe, researcherswere able to clone
+ a famtly of mammalian endoproteases,all of which cleavea
protein chain on the C-terminal side of an Arg-Arg or Lys-
NH^*|J I
Aro Aro I
- Y I
Albumin coo-
Arg sequence.One, calledfurin, is found in all mammalian
cells; it processesproteins such as albumin that are secreted
(b) Begulatedsecretedproteins
by the continuous pathway. In contrast, the PC2 and PC3
Proinsulin endoproteasesare found only in cells that exhibit regulated
secretion;theseenzymesare localized to regulated secretory
vesiclesand proteolytically cleave the precursors of many
NH.t Arg Arg C Lys Arg hormones at specificsites.

enooprolease

C Lys Arg SeveralPathwaysSort MembraneProteins

to the Apical or BasolateralRegion
o f P o l a r i z e dC e l l s

N H- -. r-l- B
?ft?l A lcoo-
The plasma membrane of polarized epithelial cells is di-
- T -l -A-r4g-Arr-gl -l I vided into two domains, apical and basolateral; tight junc-
IIS-S tions located between the two domains prevent the move-
carbox'Pe'tidase
ment of plasma-membrane proteins between the domains
(seeFigure 19-9). Severalsorting mechanismsdirect newly
[ [^E-] t-^Er synthesizedmembrane proteins to either the apical or baso-
lateral domain of epithelial cells, and any one protein may
s__l__s I be sorted by more than one mechanism.Although these
.[' -l---l- sorting mechanisms are understood in general terms, the
IBI Insutin|ll
molecular signals underlying the vesicle-mediatedtransport
S-S of membrane proteins in polarized cells are not yet known.
A FIGURE 14-24 Proteolyticprocessingof proproteinsin the As a result of this sorting and the restriction on protein
constitutiveand regulatedsecretionpathways.Theprocessing of movement within the plasma membrane due to tight junc-
proalbumin andproinsulin istypical of theconstitutive andregulated tions, distinct setsof proteins are found in the apical or ba-
pathways, respectively. Theendoproteases that functionin such solateral domain. This preferential localization of certain
processing cleave at the C-terminal sideof two consecutive amino transport proteins is critical to a variety of important phys-
acids(a)Theendoprotease furinactson the precursors of constitutive iological functions, such as absorption of nutrients from the
secreted proteins(b)Twoendoproteases, PC2andPC3,acton the intestinal lumen and acidification of the stomach lumen (see
precursorsof regulated secreted proteinsThefinalprocessing of Figures11-29 and 11-30).
manysuchproteins iscatalyzed by a carboxypeptidase that Microscopic and cell-fractionation studies indicate that
sequentiallyremoves two basicaminoacidresidues at theC-terminus
proteins destined for either the apical or the basolateral
of a polypeptide[See D Steineretal,1992,] Biol.Chem261:234351
membranes are initially located together within the mem-
branes of the trans-Golgi network. In some cases,proteins
destined for the apical membrane are sorted into their own
transport vesiclesthat bud from the trans-Golgi network
dibasic recognition sequencesuch as Arg-Arg or Lys-Arg and then move to the apical region, whereas proteins des-
(Figure 14-24a). Proteolytic processingof proteins whose tined for the basolateral membrane are sorted into other
secretionis regulated generally entails additional cleavages. vesiclesthat move to the basolateral region. The different
In the case of proinsulin, multiple cleavagesof the single vesicle types can be distinguished by their protein con-
polypeptide chain yields the N-terminal B chain and the stituents, including distinct Rab and v-SNARE proteins,
C-terminal A chain of mature insulin, which are linked by which apparently target them to the appropriate plasma-
disulfide bonds, and the central C peptide, which is lost and membrane domain. In this mechanism, segregationof pro-
subsequentlydegraded(Figure14-24b). teins destined for either the apical or basolateral mem-
The breakthrough in identifying the proteasesresponsr- branes occurs as cargo proteins are incorporated into
ble for such processingof secretedproteins came from analy- particular types of vesicles budding from the trans-Golgi
sis of yeastwith a mutation inthe KEX2 gene.Thesemutant network.
cells synthesizedthe precursor of the cr mating factor but Such direct basolateral-apicalsorting has been investi-
could not proteolytically process it to the functional form gated in cultured Madin-Darby canine kidney (MDCK)

604 C H A P T E R1 4 | VEStCULAR
T R A F F | CS
, ECRETION
A,N D E N D O C Y T O S t S
(') video: regation of Apical and BasolateralCargo in the Golgi of Live Cells

Influenzavirus HA glycoprotein < FIGURE 14-25 Sortingof proteins

destinedfor the apicaland basolateral
VSV G glycoprotein plasmamembranesof polarizedcells.When
culturedMDCKcellsareinfectedsimultaneously
Direct apical w i t h V S Va n d i n fl u e n z av i r u st,h e V S VG
g l y c o p r o t e (i np u r p l ei)s f o u n do n l yo n t h e
basolateral membrane, whereas the influenza HA
glycoprotein (green)isfoundonlyon the apical
membrane S.o m ec e l l u l aprr o t e i n(so r a n g cei r c l e ) ,
especially thosewith a GPIanchor,arelikewise
sorteddirectly to the apicalmembrane and
othersto the basolateral membrane (notshown)
viaspecific transport vesicles that bud fromthe
trans-Golgi network.In certainpolarized cells,
someapicaland basolateral proteins are
Apical protein transported togetherto the basolateral surface;
Clathrin- the apicalproteins (yellowoval)then move
coatedpit selectively, by endocytosis andtranscytosis, to
Tight the apicalmembrane. [AfterK Simons andA
Basolateral A p i c a lp l a s m a
o l a s m am e m b r a n e junction membrane Wandinger-Ness, 1990,Cell62:201 , andK Mostov
et al , 1992, I CellBiol 116:577 l

cells, a line of cultured polarized epithelial cells (seeFig-
we 9-34).In MDCK cellsinfectedwith the influenzavirus,
progeny viruses bud only from the apical membrane,
whereas in cells infected with vesicular stomaritis virus
(VSV), progeny viruses bud only from the basolateralmem-
brane. This difference occurs becausethe HA glycoprotein
of influenza virus is transported from the Golgi complex ex-
clusively to the apical membrane and the VSV G protein is
transported only to the basolateral membrane (Figure
14-25). Furthermore, when the geneencoding HA protein is
introduced into uninfected cells by recombinant DNA tech-
niques, all the expressedHA accumulatesin the apical mem-
brane, indicating that the sorting signal resides in the HA
glycoprotein itself and not in other viral proteins produced
during viral infection.
Among the cellular proteins that undergo similar apical-
basolateral sorting in the Golgi are those with a glyco-
sylp h osph atidy lino sito I (G PI ) m embrane an chor. In MD CK
cells and most other types of epithelial cells, GPl-anchored
proteins are targeted to the apical membrane.In membranes
GPl-anchored proteins are clustered into lipid rafts, which
are rich in sphingolipids (seeChapter 10). This finding sug-
gests that lipid rafts are localized to the apical membrane
along with proteins that preferentially partition them in
many cells.However, the GPI anchor is not an apical sorting
signal in all polarized cells; in thyroid cells, for example,
GPl-anchored proteins are targeted to the basolateralmem-
brane. Other than GPI anchors, no unique sequenceshave
been identified that are both necessaryand sufficient to tar-
get proteins to either the apical or basolateral domain. In-
stead, each membrane protein may contain multiple sorting
signals, any one of which can target it to the appropriate
plasma-membranedomain. The identification of such com-
plex signals and of the vesicle coat proteins that recognize
them is currently being pursued for a number of different
proteins that arc sorted to specific plasma-membranedo-
mains of polarized epithelial cells.
Another mechanism for sorting apical and basolateral
proteins, also illustratedin Figure 14-25, operatesin hepa-
tocytes. The basolateral membranesof hepatocytesface the
blood (as in intestinalepithelialcells),and the apical mem-
branes line the small intercellular channels into which bile
is secreted.In hepatocytes,newly made apical and basolat-
eral proteins are first transported in vesiclesfromthe trans-
Golgi network to the basolateralregion and incorporated
into the plasma membraneby exocytosis(i.e.,fusion of the
vesiclemembranewith the plasma membrane)'From there,
both basolateral and apical proteins are endocytosedin the
same vesicles, but then their paths diverge. The endocy-
tosed basolateralproteins are sorted into transport vesicles
that recycle them to the basolateral membrane. In contrast,
the apically destined endocytosed proteins are sorted into
transport vesiclesthat move across the cell and fuse with
the apical membrane, a process called transcytosis. This
process also is used to move extracellular materials from
one side of an epithelium to another. Even in epithelial
cells,such as MDCK cells,in which apical-basolateral pro-
tein sorting occurs in the Golgi, transcytosismay provide a
"fail-safe" sorting mechanism.That is, an apical protein
sorted incorrectly to the basolateral membrane would be
subjectedto endocytosisand then correctly delivered to the
apical membrane.

PATHWAY
L A T E RS T A G E SO F T H E S E C R E T O R Y 605

Later Stages of the Secretory Pathway
t The trans-Golgi network (TGN) is a major branch point
in the secretory pathway where soluble secretedproteins,
Iysosomal proteins, and in some cells membrane proteins
destinedfor the basolateralor apical plasma membrane are
segregatedinto different transport vesicles.
r Many vesiclesthat bud from the trans-Golgi network as
well as endocytic vesicles bear a coat composed of AP
(adapterprotein) complexesand clathrin (seeFigure 1.4-1.8).
r Pinching off of clathrin-coatedvesiclesrequiresdynamin,
which forms a collar around the neck of the vesiclebud and
hydrolyzes GTP (seeFigure 14-191.
r Soluble enzymesdestined for lysosomesare modified in
the cls-Golgi, yielding multiple mannose 5-phosphate
(M6P) residueson their oligosaccharide
chains.
r M6P receptors in the membrane of the trans-Golgi net-
work bind proteins bearing M6P residuesand direct their
transfer to late endosomes,where receptors and their lig-
and proteins dissociate.The receptorsthen are recycledto
the Golgi or plasma membrane, and the lysosomalenzymes
are deliveredto lysosomes(seeFigure 14-22).
r Regulated secretedproteins are concentratedand stored
in secretory vesiclesto await a neural or hormonal signal
for exocytosis.Protein aggregationwithin the trans-GoIgi
network may play a role in sorting secretedproteins to the
regulatedpathway.
r Many proteins transported through the secretory path-
way undergo post-Golgi proteolytic cleavagesthat yield the
mature, active proteins. Generally,proteolytic maturation
can occur in vesiclescarrying proteins from the trans-Golgi
network to the cell surface,in the late endosome,or in the
lysosome.
r In polarized epithelial cells, membrane proteins destined
for the apical or basolateral domains of the plasma mem-
brane are sorted in the trans-Golgi network into different
transport vesicles(seeFigure 14-25). The GPI anchor is the
only apical-basolateralsorting signal identified so far.
r In hepatocytes and some other polarized cells, all
plasma-membraneproteins are directed first to the basolat-
eral membrane. Apically destined proteins then are endo-
cytosed and moved acrossthe cell to the apical membrane
(transcytosis).
flp Receptor-Mediated
Endocytosis
In previous sectionswe have explored the main pathways
whereby secretoryand membraneproteins synthesizedon the
rough ER are delivered to the cell surface or other destina-
tions. Cells also can internalize materials from their sur-
roundings and sort theseto particular destinations.A few cell
types (e.g., macrophages)can take up whole bacteria and
other large particles by phagocytosis,a nonselectiveacrin-
605 C H A P T E R1 4 | V E S T C U L ATRR A F F | CS
, E C R E T T OA
NN, D ENDOCYTOS|S
mediated processin which extensionsof the plasma mem-
brane envelop the ingested material, forming large vesicles
called phagosomes(seeFigure 9-2).ln contrast, all eukary-
otic cells continually engage in endocytosis, a process in
which a small region of the plasma membraneinvaginatesto
form a membranelimited vesicleabout 0.05-0.1 pm in di-
ameter. In one form of endocytosis, called pinocyrosls, small
droplets of extracellular fluid and any material dissolvedin it
are nonspecificallytaken up. Our focus in this section,however,
is on receptor-mediatedendocytosis,in which a specific recep-
tor on the cell surface binds tightly to an extracellular macro-
molecular ligand that it recognizes;the plasma-membrane
region containing the receptorJigand complex then buds in-
ward and pinches off, becoming a transport vesicle.
Among the common macromolecules that vertebrate
cellsinternalizeby receptor-mediatedendocytosisare choles-
terol-containing particles called low-density lipoprotein
(LDL), the iron-binding protein transferrin, many protein
hormones (e.g., insulin), and certain glycoproteins. Recep-
tor-mediated endocytosis of such ligands generally occurs
via clathrin/AP2-coatedpits and vesiclesin a processsimilar
to the packaging of lysosomal enzymesby mannose 6-phos-
phate (M6P) in the trans-Golgi network (seeFigure 1.4-22).
As noted earlier, some M6P receptors are found on the cell
surface,and theseparticipate in the receptor-mediatedendo-
cytosis of lysosomalenzymesthat are mistakenly secreted.In
general, the transmembranereceptor proteins that function
in the uptake of extracellular ligands are internalized from
the cell surface during endocytosisand are then sorted and
recycled back to the cell surface,much like the recycling of
M5P receptors to the plasma membrane and trans-Golgi.
The rate at which a ligand is internalized is limited by the
amount of its correspondingreceptor on the cell surface.
Clathrin/AP2 pits make up about 2 percent of the surface
of cells such as hepatocytesand fibroblasts. Many internal-
ized ligands have been observed in these pits and vesicles,
which are thought to function as intermediatesin the endo-
cytosis of most (though not all) ligands bound to cell-surface
receptors (Figure 1,4-26).Some receptors are clustered over
clathrin-coated pits even in the absenceof ligand. Other re-
ceptors diffuse freely in the plane of the plasma membrane
but undergo a conformational change when binding to lig-
and, so that when the receptor-ligandcomplex diffuses into
a clathrin-coatedpit, it is retained there. Two or more types
of receptor-boundligands, such as LDL and transferrin, can
be seenin the samecoated pit or vesicle.
CellsTakeUp Lipidsfrom the Blood in the Form
of Large,Well-DefinedLipoproteinComplexes
Lipids absorbed from the diet in the intestinesor stored in
adipose tissue can be distributed to cells throughout the
body. To facilitate the mass transfer of lipids between cells,
animals have evolved an efficient way to packagefrom hun-
dreds to thousands of lipid molecules into water-soluble,
macromolecular carriers, called lipoproteins, that cells can
take up from the circulation as an ensemble.A lipoprotein
particle has a shell composed of proteins (apolipoproteins)
 (a) (b)

s,,..: ".;
lvl

l'
rf
,..s

LDL-ferritin
pit
Clathrin-coated
(c) (d)

"e

EXPERIMENTAL FIGURE 14-26The initialstagesof receptor- microscopy at periodic

intervals (a)A coatedpit,showingthe
mediatedendocytosisof low-densitylipoprotein(LDL) clathrincoaton the inner(cytosolic) of the pit,soonafterthe
surface
particlesare revealedby electronmicroscopy. Culturedhuman temperature was raised.(b)A pit LDLapparently
containing closing
fibroblastswereincubated in a mediumcontarning LDLparticles on itselfto forma coatedvesicle(c)A coatedvesicle containing
covalently linkedto theelectron-dense, iron-containingprotein ferritin-tagged LDLparticles (d)Ferritin-taggedLDLparticles
in a
ferritin;eachsmallironparticle in ferritinisvisible
asa smalldot smooth-surfaced earlyendosome 6 minutesafterinternalization
underthe electron microscope. Cellsinitiallywereincubated at 4'C; began.[Photographs courtesyof R Anderson bypermission
Reprinted from
at thistemperature LDLcanbindto itsreceptor, but internalization J Goldsternetal, Nature279:619 Copyrightl979,MacmillanJournals
doesnot occur. Afterexcess LDLnot boundto the cellswaswashed LrmrtedSeealsoM 5 Brown andJ Goldstein,1986,Scrence232:341
away,thecellswerewarmedto 37 'C andthenprepared for

and a cholesterol-containingphospholipid monolayer. The
shell is amphipathic becauseits outer surfaceis hydrophilic,
making theseparticles water soluble, and its inner surfaceis
hydrophobic. Ad;acent to the hydrophobic inner surface of
the shell is a core of neutral lipids containing mostly choles-
teryl esters,triglycerides, or both. Mammalian lipoproteins
fall into different classes,defined by their differing buoyant
densities.The class we will consider here is low-density
lipoprotein (LDL). A typical LDL particle, depicted in Fig-
ve 1.4-27,is a sphere20-25 nm in diameter.The amphipathic
outer shell is composed of a phospholipid monolayer and a
single molecule of a large protein known as apoB-100; the
core of a particle is packed with cholesterol in the form of
cholesterylesters.
Two generalexperimental approacheshave been used to
study how LDL particles enter cells.The first method makes
use of LDL that has been labeledby the covalent attachment
12sIto the side chains of tyrosineresiduesin
of radioactive
apoB-100 on the surfacesof the LDL particles.After cul-
tured cells are incubated for severalhours with the labeled
LDL, it is possibleto determine how much LDL is bound to
the surfacesof cells. how much is internalized,and how
much of the apoB-100 component of the LDL is degradedby
enzymatic hydrolysis to individual amino acids. The drp:l-
125I-
d"tion of apoB-100 can be detectedby the releaseof
tyrosine into the culture medium. Figure 14-28 shows the
time course of eventsin receptor-mediatedcellular LDL pro-
cessingdetermined by pulse-chaseexperimentswith a fixed
12sI-labeledLDL. These experiments
concentration of
clearly demonstrate the order of events:surface binding of
LDL -+ internalization -+ degradation. The second ap-
proach involves tagging LDL particles with an electron-
dense label that can be detected by electron microscopy.
Such studiescan revealthe details of how LDL particles first
bind to the surface of cells at clathrin-coated endocytic pits
and then remain associatedwith the coated pits as they in-
vaginate and bud off to form coated vesiclesand finally are
transportedto endosomes(seeFigure 1'4-26).

R E C E P T O R - M E D I A TE
ENDD O C Y T O S I S 607
 Polar 12
su rface
10
Apolar \\
,yi core D e gr a d a t i o n
Td
F.n
II b
o
J Binding

ApolipoproteinB
x- @A

l l'--
/______)_
'
LDL 30 40 50 60 120
T i m ea t 3 7 ' C( m i n )
FIGURE 14-27Modelof low-densitylipoprotein(LDL).This
class andthe otherclasses of lipoproteins havethesamegeneral A EXPERIMENTAL FIGURE 14-28Pulse-chase experiment
structure: an amphipathic shell,composed of a phospholipid demonstratesprecursor-product relationsin cellularuptakeof
monolayer (notbilayer), cholesterol, andprotein, anda hydrophobic LDL.Cultured normalhumanskinfibroblasts wereincubated in a
mediumcontaining t"I-LDLfor 2 hoursat 4 "C(thepulse)After
core,composed mostlyof cholesteryl esters or triglycerides or both
excess 12sl-LDL
notboundto thecellswaswashed away,thecellswere
b u tw i t hm i n o a r m o u n tosf o t h e rn e u t r al il p i d (seg , ,s o m ev i t a m i n s )
Thismodelof LDLis basedon electron microscopy andotherlow- incubateda|37"Cfor theindicated amounts of timein theabsence of
r e s o l u t i obni o p h y s i cmael t h o d sL D Li su n i q u ei n t h a ti t c o n t a i nosn l y externalLDL(thechase)Theamounts of surface-bound,internalized.
anddegraded (hydrolyzed) 12sl-LDL
weremeasured. Bindingbut not
a single molecuo l ef o n et y p eo f a p o l i p o p r o t (eai np o Bw) ,h i c h
appears to wraparoundtheoutside of the particle asa bandof internalization
or hydrolysisof LDLapoB-100 occurs duringthe4'C
proteinTheotherlipoproteins containmultiple apolipoprotein pulse,Thedatashowtheveryrapiddisappearance of bound1251-LDL
molecules, oftenof different types[Adapted fromM Krieger, 1995, in fromthesurface it
as is internalized
afterthecellshavebeenwarmed
E Haber,
ed , Molecular
Cardiovascular
Medrcine.
Scientific
American to allowmembrane movements Aftera lagperiodof 15-20minutes,
lysosomaldegradation of theinternalized12sl-LDLcommences tV]
pp 31-47I
Medicrne, [See
S BrownandJ L Goldstein,1976, Ce//9:663
1

Receptorsfor Low-DensityLipoprotein and is now known to be causedby mutations in the LDLR

a n d O t h e r L i g a n d sC o n t a i nS o r t i n gS i g n a l s gene.In patientswho have one normal and one defectivecopy
That TargetThem for Endocytosis of the LDLR gene (heterozygotes),LDL cholesterol in the
blood is increasedabout twofold. Those with rwo defective
The key to understandinghow LDL particlesbind to the cell
LDLR genes(homozygotes)have LDL cholesterollevelsthat
surface and are then taken up into endocytic vesiclescame
are from fourfold to sixfold as high as normal. FH heterozy-
from discovery of the LDL receptor (LDLR). The LDL
gotes commonly develop cardiovasculardiseaseabout 10
receptor is an 839-residueglycoprotein with a single trans-
years earlier than normal people do, and FH homozygotes
membrane segment; it has a short C-terminal cytosolic
usually die of heart attacks beforereachingtheir late 20s.
segmentand a long N-terminal exoplasmic segmentthat
A variety of mutations in the geneencodingthe LDL re-
containsa B-propellerdomain and a ligand-bindingdomain.
ceptor can causefamilial hypercholesterolemia. Some mu-
Seven cysteine-rich imperfect repeats form the ligand-
tations prevent the synthesisof the LDLR protein; others
binding domain, which interacts with the apoB-100
prevent proper folding of the receptor protein in the ER,
moleculein a LDL particle. Figure 14-29 shows how LDL
l e a d i n g t o i t s p r e m a t u r e d e g r a d a t i o n( C h a p t e r 1 3 ) ; s t i l l
receptor proteins facilitate internalization of LDL particles
other mutations reduce the ability of the LDL recepror to
by receptor-mediatedendocytosis. After internalized LDL
bind LDL tightly. A particularly informative group of mu-
particles reach lysosomes,lysosomal proteaseshydrolyze
tant receptorsare expressedon the cell surface and bind
their surface apolipoproteins and lysosomal cholesteryl es-
LDL normally but cannot mediate the internalization of
teraseshydrolyze their core cholesterylesters.The unesteri-
bound LDL. In individualswith this type of defect,plasma-
fied cholesterolis then free to leavethe lysosomeand be used
membranereceptorsfor other ligands are internalizednor-
as necessaryby the cell in the synthesisof membranesor var-
mally, but the mutant LDL receptor is not recruited into
ious cholesterolderivatrves.
coated pits. Analysis of this mutant receptor and other
mutant LDL receptors generatedexperimentally and ex-
The discoveryof the LDL receptor and an understand-
pressedin fibroblastsidentified a four-residuemotif in the
cytosolic segmentof the receptor that is crucial for its in-
ternalization: Asn-Pro-X-Tyr,where X can be any amino
diseasethat is marked by elevated plasma LDL cholesterol
acid. This NPXY sorting signalbinds to the AP2 complex,

508 c H A P T E R1 4 | V E S T C U L ATR
R A F F t CS, E C R E T t O A
NN, D ENDOCYTOStS
 A t n e u t r a lp H , l i g a n d - b i n d i n g
arm is free to bind another
LDL oarticle

Coated Plasmamembrane
pit -+ ._f

Clathrin,/ E
AP2
complex

Coated
vesicle

Early
endosome

Lysosome

Fattyacids

FIGURE 14-29Endocyticpathwayfor internalizinglow- (earlyendosome) fuseswith the lateendosome.TheacidicpH in this

densitylipoprotein(tDL).Step[: Cell-surface LDLreceptorsbind compartment causesa conformational changein the LDLreceptor
to an apoBproteinembedded in the phospholipid
outerlayerof LDL of the boundLDLparticleStepZl: Thelate
that leadsto release
particles Interactionbetween the NPXYsortingsignalin thecytosolic endosome fuseswith the lysosome,andthe proteins andlipidsof the
tailof the LDLreceotor andthe AP2comolexincoroorates the freeLDLparticle arebrokendownto theirconstituent partsby
receptor-ligand complex intoformingendocytic vesiclesStepE: enzymes in the lysosome. Step[: TheLDLreceptor recycles
to the
Clathrin-coated pits(orbuds)containing receptor-LDL
complexes are cellsurface,whereat the neutralpHof the exterior mediumthe
pinched off bythe samedynamin-mediated mechanism usedto form receptorundergoes a conformational changesothatit canbind
clathrin/AP1 vesicles
on the trans-Golgr network(seeFigure14-19) anotherLDLparticle. [See M S Brown 1986,science
andJ L Goldstein,
StepB:After thevesicle coatisshed,the uncoated endocyticvesicle 232:34,andG Rudenko 298:2353
etal,2002,Sclence l

linking the clathrin/AP2 coat to the cytosolic segmentof
the LDL receptor in coated pits. A mutation in any of the
conservedresiduesof the NPXY signalwill abolishthe abil-
ity of the LDL receptor to be incorporated into coated pits.
A small number of individuals who exhibit the usual
symptoms associatedwith familial hypercholesterolemia
produce normal LDL receptors.In these individuals, the
geneencodingthe AP2 subunit protein that binds the NPXY
sorting signal is defective.As a result, LDL receptorsare not
incorporated into clathrin/AP2 vesiclesand endocytosisof
LDL particles is compromised.Analysis of patients with this
genetic disorder highlights the importance of adapter pro-
teins in protein trafficking mediated by clathrin vesicles.I
Mutational studieshave shown that other cell-surfacere-
ceptors can be directed into forming clathrin/AP2 pits by a
different sorting signal: Tyr-X-X-O, where X can be any
amino acid and Q is a bulky hydrophobic amino acid. This
Tyr-X-X-O sorting signal in the cytosolic segment of a te-
ceptor protein binds to a specific cleft in one of the protein
subunits of the AP2 complex. Becausethe tyrosine and O
residues mediate this binding' a mutation in either one
reducesor abolishesthe ability of the receptor to be incor-
porated into clathrin lAP2-coatedpits.Moreover, if influenza
HA protein, which is not normally endocytosed,is geneti-
cally engineeredto contain this four-residue sequencein its
cytosolic domain, the mutant HA is internalized' Recall

RECEPTORE
-MD I A T E DE N D O C Y T O S I S 609
from our earlier discussionthat this same sorting signal re-
cruits membraneproteins into clathrin/AP1 vesiclesthat bud
from the trans-Golgi network by binding to a subunit of AP1
(seeTable 14-2). All these observationsindicare rhat Tyr-X-
X-O is a widely used signal for sorting membraneproteins to
clathrin-coatedvesicles.
In some cell-surfaceproteins, however, other sequences
(e.g.,Leu-Leu)or covalentlylinked ubiquitin moleculessignal
endocytosis.Among the proteins associatedwith clathrin/AP2
vesicles,several contain domains that specifically bind to
ubiquitin, and it has been hypothesizedthat thesevesicle-as-
sociated proteins mediate the selectiveincorporation of ubiq-
uitinated membrane proteins into endocytic vesicles.As de-
scribed later, the ubiquitin tag on endocytosedmembrane
proteins is also recognized at a later stage in the endocytic
pathway and plays a role in delivering these proteins into the
interior of the lysosome,where they are degraded.
The Acidic pH of Late EndosomesCausesMost
Receptor-Ligand Complexesto Dissociate
The overall rate of endocytic internalization of the plasma
membrane is quite high: cultured fibroblasts regularly internal-
ize 50 percent of their cell-surfaceproteins and phospholipids
each hour. Most cell-surfacereceptorsthat undergo endocyto-
sis will repeatedlydeposit their ligands within the cell and then
recycle to the plasma membrane, once again to mediate inter-
nalization of ligand molecules.For instance,the LDL receptor
makes one round trip into and out of the cell every 10-20 min-
utes,for a total of severalhundred trips in its 20-hour life span.
Internalizedreceptor-ligandcomplexescommonly follow
the pathway depicted for the M5P receptor inFigure 14-22
and the LDL receptor in Figure L4-29. Endocytosedcell-sur-
face receptorstypically dissociatefrom their ligands within
late endosomes,which appearas sphericalvesicleswith tubu-
Iar branching membraneslocateda few micrometersfrom the
cell surface.The original experimentsthat definedthe late en-
dosome sorting vesicleutilized the asialoglycoproreinrecep-
tor. This liver-specific protein mediates the binding and inter-
nalization of abnormal glycoproteinswhose oligosaccharides
terminate in galactose rather than the normal sialic acid;
hence the name asialoglycoprotein. Electron microscopy of
liver cells perfusedwith asialoglycoproteinreveal that 5-10
minutes after internalization, ligand molecules are found in
the lumen of late endosomes,while the tubular membraneex-
tensionsare rich in receptor and rarely contain ligand. These
findings indicate that the late endosomeis the organelle in
which receptorsand ligands are uncoupled.
The dissociationof receptor-ligandcomplexesin late en-
dosomesoccurs not only in the endocytic pathway but also
in the delivery of soluble lysosomal enzymesvia the secre-
tory pathway (seeFigure 1.4-22).As discussedin Chapter 11,
the membranesof late endosomesand lysosomescontain V-
classproton pumps that act in concert with Cl channelsto
acidify the vesiclelumen (seeFigure 11-13). Most receptors,
including the M5P receptor and cell-surfacereceptors for
LDL particles and asialoglycoprotein, bind their ligands
tightly at neutral pH but releasetheir ligands if the pH is
Iowered to 6.0 or below. The late endosomeis the first vesi-
cle encounteredby receptor-ligandcomplexeswhose luminal

(a) (b)
Cell surface [pH =7.0] Ree l ased
Endosome tpH =51
Ligand-binding L D Lp a r t i c l e
arm (R1-R7
Cholesterolesters I
LDL LDL Surface of
receptor B-propeller pid
domain particle B-propeller
monolayer domain becomes
NPXY protein positivelycharged,
andthen binds
to the ligand-
b i n d i n ga r m

FIGURE 14-30Modelfor pH-dependent bindingof LDt

particlesby the LDLreceptor.Schematic depictionof LDLreceptor Ligand-
at neutralpHfoundat thecellsurface (a)andat theacidicpHfound b i n d i n ga r m
in the interiorof the lateendosome (b) (a)nt the cellsurface,
apoB- R6
100on thesurface of a LDLparticle bindstightlyto the receptor.
Of
thesevenrepeats (R1-R7) p-propeller
in the ligand-binding arm,R4andR5
domain
appearto be mostcritical for LDLbinding. (b,top)Withinthe
endosome, histidine residues in the B-propellerdomainof the LDL
receptorbecomeprotonatedThepositively chargedpropeller can
bindwith highaffinityto the ligand-binding arm,whichcontains
negatively charged residues, causing releaseof the LDLparticle(b,
bottom)Experimental electron density andCotracemodelof the
extracellularregionof the LDLreceptor at pH 5 3 basedon x-ray
crystallographicanalysis. Inthisconformation, extensivehydrophobic
andionicinteractions occurbetween the B propellerandthe R4and
R5repeats. lPart(b)fromG Rudenko et al, 2002,Science2gS:2353]

610 C H A P T E R1 4 | V E S T C U L ATRR A F F t CS, E C R E T T OA

NN, D ENDOCYTOS|S
pH is sufficiently acidic to promote dissociationof most en-ceptor-ligandcomplex doesnot dissociatein late endosomes.
docytosedreceptorsfrom their tightly bound ligands. Nonetheless,changesin pH also mediate the sorting of re-
The mechanism by which the LDL receptor releases ceptors and ligands in the transferrin pathway, which func-
bound LDL particles is now understood in detail (Figure tions to deliver iron to cells.
1,4-30).At the endosomalpH of 5.0-5.5, histidineresidues A major glycoprotein in the blood' transferrin transports
in the B-propeller domain of the receptor become proto- iron to all tissuecellsfrom the liver (the main site of iron stor-
age in the body) and from the intestine (the site of iron ab-
nated, forming a site that can bind with high affinity to the
negatively charged repeats in the ligand-binding domain. sJrption). The iron-free form, apotransferrin, binds two Fe3*
This intramolecular interaction sequestersthe repeats in a ions very tightly to form ferrotransferrin. Arllmammalian cells
conformation that cannot simultaneouslybind to apoB-100, contain cell-surfacetransferrin receptors that avidly bind fer-
thus causingreleaseof the bound LDL particle. rotransferrin at neutral pH, after which the receptor-bound
ferrotransferrinis subjectedto endocytosis.Like the compo-
The EndocyticPathway Deliverslron to Cells nentsof an LDL particle,the two bound Fe'* atoms remain in
the cell, but the apotransferrinpart of the ligand doesnot dis-
without Dissociationof the Receptor-Transferrin
sociate from the receptor, and within minutes after being en-
C o m p l e xi n E n d o s o m e s docytosed,apotransferrinis secretedfrom the cell.
The endocytic pathway involving the transferrin receptor As depicted in Figure 14-3'J',the explanation for the be-
and its ligand differs from the LDL pathway in that the re- havior of the transferrin receptor-ligand complex lies in the

E
Ferrotransferrin _ E
>^ Apotransferrin
Exterior
(pH7.01
Transferrin
receptor
<t dissociatesfrom
receptorat
neutralpH

Adapter complex
Clathrin

5t

E
Apotransferrin
is recycledto
cell surface

-r+
Low pH causesreleaseof Fe3' Fe' Aootransferrin
f r o m l i g a n d ;l i g a n dr e m a i n s
bound to receptor
Late endosome

A FIGURE 14-31Thetransferrincycle,which operatesin all endocyticvesiclesfusewith the membrane of the endosomeFe*3is

growing mammaliancells.StepE: Thetransferrin dimercarrying released complex
fromthe receptor-ferrotransferrin in the acidiclate
two boundatomsof Fe*3,calledferrotransferrin,bindsto the endosome compartment StepE: Theapotransferrin proteinremains
transferrinreceptorat thecellsurfaceStep[: Interactionbetween boundto itsreceptor at thispH,andtheyrecycleto thecellsurface
thetailof thetransferrinreceptorandtheAP2adapter complex together.Step@: TheneutralpHof the exterior mediumcauses
incorporates the receptor-ligand
complexintoendocyticclathrin- releaseof the iron-freeapotransferrin A
[See Ciechanoveretal, 1983,
coatedvesicles StepsB and4:The vesicle coatisshedandthe J BiolChem258:9681 l

R E C E P T O R - M E D I A T E D E N D O C Y T O .S I S 6 1 1
unique ability of apotransferrin to remain bound to the
transferrin receptor at the low pH (5.0-5.5) of late endo-
somes.At a pH of lessthan 5.0, the two bound Fe3* atoms
dissociatefrom ferrotransferrin, are reduced to Fe2* by an
unknown mechanism,and then are exported into the cytosol
by an endosomal transporter specific for divalent metal
ions. The receptor-apotransferrincomplex remaining after
dissociationof the iron atoms is recycledback to the cell sur-
face. Although apotransferrin binds tightly to its receptor
at a pH of 5.0 or 6.0, it doesnot bind at neutral pH. Hence
the bound apotransferrin dissociatesfrom the transferrin
receptor when the recycling vesiclesfuse with the plasma
membrane and the receptor-ligandcomplex encountersthe
neutral pH of the extracellular interstitial fluid or growth
medium. The recycled receptor is then free to bind another
molecule of ferrotransferrin, and the releasedapotransferrin
is carried in the bloodstream to the liver or intestineto be re-
loaded with iron.
Receptor-Mediated Endocytosis
r Some extracellular ligands that bind to specific cell-
surface receptors are internalized, along with their recep-
tors, in clathrin-coated vesicleswhose coats also contain
AP2 complexes.
r Sorting signalsin the cytosolic domain of cell-surfacere-
ceptors target them into clathrin/AP2-coatedpits for inter-
nalization. Known signals include the Asn-Pro-X-Tyr,
Tyr-X-X-O, and Leu-Leu sequences(seeTable 14-2).
r The endocytic pathway delivers some ligands (e.g., LDL
particles) to lysosomes,where they are degraded. Trans-
port vesiclesfrom the cell surfacefirst fuse with late endo-
somes,which subsequentlyfuse with the lysosome.
r Most receptor-ligandcomplexes dissociatein the acidic
milieu of the late endosomel the receptors are recycled to
the plasma membrane,while the ligands are sorted to lyso-
somes (seeFigurel4-29).
Iron is -imported into cells by an endocytic pathway in
hich Fe3+ ions are releasedfrom ferrotransferrin in the
late endosome.The receptor-apotransferrincomplex is re-
cycled to the cell surface, where the complex dissociates.
releasingboth rhe receptorand apotransfeirinfor reuse.
M DirectingMembraneproteins
and CytosolicMaterialsto the Lysosome
The major function of lysosomesis to degradeextracellu-
lar materials taken up by the cell and intracellular com-
ponents under certain conditions. Materials to be de-
graded must be delivered ro the lumen of the lysosome,
where the various degradativeenzymesreside.As just dis-
cussed,endocytosedligands (e.g.,LDL particles) that dis-
sociate from their receptors in the late endosome subse-
quently enter the lysosomal lumen when the membrane of
612 C H A P T E R1 4 | VESICULAR
T R A F F | CS, E C R E T | O N
A,N D E N D O C Y T O S T S
the late endosome fuses with the membrane of the lyso-
some (seeFigure 1,4-29).Likewise, phagosomescarrying
bacteria or other particulate matter can fuse with lyso-
somes,releasingtheir contents into the lumen for degra-
dation.
It is apparent how the general vesicular trafficking
mechanism discussedin this chapter can be used to de-
liver the luminal contents of an endosomal organelle to
the lumen of the lysosome for degradation. However,
vesicular trafficking cannot allow for delivery of mem-
brane proteins or cytosolic materials to the lysosomal lu-
men. As we will seein this section.the cell has two differ-
ent specializedpathways for delivery of these molecules
to the lysosome interior for degradation. The first path-
way is used to degrade endocytosedmembrane proteins
and utilizes an unusual type of vesiclethat buds into the
lumen of the endosometo produce a multivesicular endo-
some.The secondpathway, known as autophagS involves
the de novo formation of a double membrane organelle
known as an autophagosomethat envelopscytosolic ma-
terial, such as soluble cytosolic proteins or sometimesor-
ganellessuch as peroxisomesor mitochondria. Both path-
ways lead to fusion of either the multivesicular endosome
or autophagosomewith the lysosome,depositingthe con-
tents of these organelles into the lysosomal lumen for
degradation.
MultivesicularEndosomesSegregate
MembraneProteinsDestinedfor the
LysosomalMembranefrom Proteins
Destinedfor LysosomalDegradation
Residentlysosomalproteins, such as V-classproton pumps
and amino acid transporters, can carry out their functions
and remain in the lysosomal membrane, where they are
protected from degradation by the soluble hydrolytic en-
zymes in the lumen. Such proteins are delivered to the lyso-
somal membrane by transport vesiclesthat bud from the
trans-Golgi network by the same basic mechanisms de-
scribed in earlier sections.In contrast, endocytosedmem-
brane proteins such as receptor proteins that are to be de-
graded are transferred in their entirety to the interior of the
lysosome by a specializeddelivery mechanism. Lysosomal
degradation of cell-surface receptors for extracellular sig-
naling molecules is a common mechanism for controlling
the sensitivityof cells to such signals(Chapter 15). Recep-
tors that become damaged also are targeted for lysosomal
degradation.
Early evidencethat membranescan be delivered to the
lumen of compartments came from electron micrographs
showing membrane vesiclesand fragments of membranes
within endosomesand lysosomes(seeFigure 9-2c).parallel
experimentsin yeastrevealedthat endocytosedreceptor pro-
teins targeted to the vacuole (the yeast organelle equivalent
to the lysosome)were primarily associatedwith membrane
fragments and small vesicleswithin the interior of the vac-
uole rather than with the vacuole surfacemembrane.
 Plasmamembrane

Early endosome

*''*'..,"--.,"*l
41b|i':ee,{d,ert!}

I
i-",
rl
!4&"edctu4*{*rl

'\ Go|gi
".''*","-,"',.,"*".,'
Lysosome
J Lateendosome/
rr,r..,***,npaadt
body
multivesicular

FIGURE 14-32Deliveryof plasma-membrane proteinsto the internal (stepB) Fusion

vesicles endosome
of a multivesrcular
lysosomalinteriorfor degradation.Early endosomes carrying directlywith a lysosome vesicles
the internal
releases intothe lumen
endocytosed plasma-membrane proteins (blue)andvesicles carrying of the lysosome, where they canbe degraded (stepZl) Because
lysosomal membrane proteins (green) fromthe trans-Golgi network protonpumpsandotherlysosomal membrane proteinsnormally
are
fusewith the lateendosome, transferring theirmembrane proteins not incorporated intointernalendosomal theyaredelivered
vesicles,
a l e m b r a n(es t e p tsr a n d Z ) P r o t e i nt o
t o t h ee n d o s o m m s be to the lysosomal membrane andareprotected fromdegradation. [see
degraded, suchasthosefromtheearlyendosome, areincorporated F.Reggiori Eukaryot
andD J Klionsky,20Q2, 1:11,
Cell andD J Katzmann
intovesicles that bud into the interiorof the lateendosome, et al. 2002.Nature Mol CellBiol3:893l
Rev.
eventually forminga multivesicular endosome containing manysuch

These observations suggest that endocytosed mem-
brane proteins can be incorporatedinto specializedvesicles
that form at the endoso-ai -e-brane (Figure 1,4-32).Al-
though thesevesiclesare similar in size and appearancero
,r"nrlo., vesicles,they differ topologically.Transport vesi-
cles bud outward from the ,.rifu.. of a donor trganelle
into the cytosol, whereas vesicleswithin the endosomebud
inward from the surface into the lumen (away from the cy-
tosol). Mature endosomescontaining numerous vesiclesin
their interior are usually calledmultiuesicularendosomes
(or bodies). Eventually,the surfacemembrane of a multi-
vesicular endosome fuses with the membrane of a lyso-
some,thereby deliveringits internal vesiclesand the mem-
brane proteins they contain into the lysosomeinterior for
degradation.Thus the sorting of proteins in the endosomal
membrane determineswhich onei will remain on the lyso-
some surface (e.g., pumps and transporters) and *hi.h
ones will be incolpor"t.d i.rto internal vesicles and ulti-
mately degradedin lysosomes.
Vany of the proteins required for inward budding of
the endosomal membrane were first identified by muta-
tions in yeast that blocked delivery of membraneproteins
to the interior of the vacuole. More than 10 ,rr.h "bod-
ding" proteins have beenidentified in yeast,most with sig-
nificanl similaritiesto mammalian proteins that evidentiy
perform the same function in mammalian cells. The current
model of endosomalbuddine to form multivesicularendo-
somesin mammalian cells is basedprimarily on studiesin
yeast (Figure 1'4-33).Most cargo proteins that enter the
multivesicularendosomearetaggedwith ubiquitin. Cargo
proteins destined to enter the multivesicular endosome
usually receivetheir ubiquitintagat the plasma membrane'
the TGN, or the endosomal membrane. We have already
seenhow ubiquitin tagging can serve as a signal for degra-
dation of cytosolic or misfolded ER proteins by the protea-
some (seeChapters 3 and 13). When used as a signal for
proteasomaldegradation,the ubiquitin tag usualjy consists
of a chain of covalently linked ubiquitin molecules(polyu-
biquitin), whereasubiquitin used to tag proteins for entry
into the multivesicularendosomeusually takes the form of
a single(monoubiquitin) molecule.In the membraneof the
endosome a ubiquitin-tagged peripheral membrane pro-
tein, known as Hrs, facilitates loading of specific mo-
noubiquitinatedmembranecargo proteins into vesiclebuds
directed into the interior of the endosome. The ubiquiti-
nated Hrs protein then recruits a set of three different
protein complexesto the membrane.TheseESCRT (endo-
somal sorting complexes required for /ransport) proteins
include the ubiquitin-binding protein Tsg101..The mem-
brane-associatedESCRT proteins act to complete vesicle
budding, Ieading to releaseof a vesicle carrying specific
membranecargo into the interior of the endosome'Finally,
an ATPase, known as Vps4, usesthe energy from ATP hy-
drolysis to disassemblethe ESCRT' releasingthem into the

TO THE LYSOSOME
MATERIALS ' 613
SND CYTOSOLIC
M E M B R A N EP R O T E I N A
DIRECTtNG
 Cytosol
Ubiquitin
Hrs protein
Endosomar
Cargo proTer
ns vesicle
Lumen of endosome
cytosol for another round of budding. In the fusion event
that pinchesoff a completedendosomalvesicle,the ESCRT
proteins and Vps4 may function like SNAREs and NSF, re-
spectively, in the typical membrane-fusion process dis-
cussedpreviously (seeFigure 14-10).
RetrovirusesBud from the plasmaMembrane
by a ProcessSimilarto Formationof
M u l t i v e s i c u l aE
r ndosomes
The vesiclesthat bud into the interior of endosomeshave a
topology similar to that of envelopedvirus particles that bud
from the plasma membrane of virus-infected cells. More-
over, recent experimentsdemonstratethat a common set of
proteins is required for both types of membrane-budding
events. In fact, the two processesso closely parallel each
other in mechanistic detail as to suggestthat enveloped
viruses have evolved mechanismsto recruit the cellular
proteins used in inward endosomal budding for their own
purposes.
The human immunodeficiency virus (HIV) is an en-
veloped retrovirus that buds from the plasma membrane of
infected cells in a process driven by viral Gag protein, the
major structural component of completed virus particles.
Gag protein binds to the plasma membrane of an infected
cell and =4000 Gag molecules polymerize into a spherical
shell, producing a structure that looks like a vesicle bud
protruding outward from the plasma membrane. Muta-
tional studies with HIV have revealed that the N-terminal
segmentof Gag protein is required for associationwith the
plasma membrane, whereas the C-terminal segmentrs re-
quired for pinching off of complete HIV particles. For in-
stance, if the portion of the viral genome encoding the C-
terminus of Gag is removed, HIV buds will form in infected
614 C H A P T E R1 4 | V E S T C U L ATRR A F F t CS, E C R E T T OA
NN, D ENDOCYTOS|S
< FIGURE 14-33 Model of the
mechanismfor formation of
m u l t i v e s i c u l aern d o s o m e sI .n e n d o s o m a l
b u d d i n gu, b i q u i t i n a t eHdr so n t h e
endosomm a l e m b r a ndei r e c t lso a d i n g
of
c e m b r a ncea r g op r o t e i n(sb l u ei)n t o
s p e c i f im
vesicle budsandthenrecruits cytosolic
E S C Rt T o t h e m e m b r a n(es t e pE ) N o t et h a t
both Hrsandthe recruited cargoproteins are
taggedwith ubiquitinAfterthe setof
b o u n dE S C RcTo m p l e x emse d i a t m e embrane
f u s i o na n dp i n c h i nogf f o f t h ec o m p l e t e d
vesicle (stepZ), theyaredisasssembled by
the ATPase Vps4and returnedto the cytosol
(stepB) Seetextfor discussion [Adapted
fromO Pornillos et al , 2002,Trends CellBiol.
12:569,1
cells, but pinching off does not occur, and thus no free virus
particles are released.
The first indication that HIV budding employs the same
molecular machinery as vesicle budding into endosomes
came from the observation that Tsg101, an ESCRT pro-
tein, binds to the C-terminus of Gag protein. Subsequent
findings have clearly established the mechanistic parallels
between the two processes.For example, Gag is ubiquiti-
nated as part of the processof virus budding, and in cells
with mutations in Tsg101 or Vps4, HIV virus buds
accumulatebut cannot pinch off from the membrane (Fig-
ure 14-34). Moreover, when a segment from the cellular
Hrs protein is added to a truncated Gag protein, proper
budding and releaseof virus particles is restored. Taken
together, these results indicate that Gag protein mimics the
function of Hrs, redirecting ESCRT to the plasma mem-
brane, where they can function in the budding of virus
particles.
Other enveloped retroviruses such as murine leukemia
virus and Rous sarcomavirus also have beenshown to require
ESCRT complexesfor their budding, although eachvirus ap-
pearsto have evolveda somewhatdifferent mechanismto re-
cruit ESCRT complexesto the site of virus budding.
The Autophagic Pathway Delivers Cytosolic proteinr or
Entire Organelles to Lysosomes When cells are placed
under stresssuch as conditions of starvation, they have the
capacity to recycle macromoleculesfor use as nutrients in a
process of lysosomal degradation known as autophagy
("eating oneself"). The autophagicpathway involves the for-
mation of a flattened double-membrane cup-shapedstruc-
ture that envelops a region of the cytosol or an entrre or-
ganelle (e.g., mitochondrion), forming an autophagosome,
or autophagic uesicle (Figure 1,4-35).The outer membrane
 Membrane
ffi aod.urt:HIVBuddingfromthe Plasma
(a) HIV virus < FIGURE 14-34 Mechanismfor
HIV envelope Core particle budding of HIVfrom the Plasma
membrane.Proteins required for formation
of endosomes
multivesicular are exploited
by HIVfor virusbuddingfromthe plasma
membrane. (a)Budding of HIVparticles
Extracellular
space fromH|V-infected cellsoccursby a similar
mechanism asin Figure14-33,usingthe
Plasmamembrane encoded
virally Gagproteinandcellular
ESCRT andVps4(stepsn-B).
Cytosol UbiquitinatedGagneara buddingparticle
functionslikeHrs Seetextfor discussion.
H I VG a g Ubiquitin
protein (b)Inwild-type cellsinfectedwith HIVvirus
+Pi budfromthe plasma
particles membrane
andarerapidlyreleased intothe
disassembly
as space.
extracellular (c)In cellsthatlackthe
functionalESCRT protein Tsg101, theviral
Gagproteinformsdenseviruslike
structures,but buddingof thesestructures
fromthe plasma membrane cannotbe
(c) viral
completed andchains of incomplete
budsstillattached to the plasma membrane
accumulate. [WesSundquist, Universityof
UtahI

of an autophagic vesiclecan fuse with the lysosome,deliver- bounded organelle.Although the origin of this membrane is
ing a large vesicle,bounded by a singlemembrane bilayer,to not known, most studiessuggestthat the autophagic vesicle
the interior of the lysosome.Similar to the situation that oc-
curs when multivesicular endosomes are delivered to the
lysosome,lipasesand proteaseswithin the lysosomewill de-
grade the autophagic vesicleand its contents into their mo-
lecular components.Amino acid permeasesin the lysosomal
membrane then allow for transport of free amino acids back
into the cytosol for use in synthesisof new proteins.
The formation and fusion of autophagic vesicles are
thought to take place in three basic steps.Although the un-
derlying mechanismsfor each of these steps remain poorly
understood,they are thought to be related to the basicmech- autophagic vesicle.
anisms for vesiculartrafficking discussedin this chapter.
Autophagic Vesicle Growth and Completion New
Autophagic Vesicle Nucleation The autophagicvesicleis membrane must be delivered to the autophagosomemem-
thought to originate from a fragment of a membrane- brane in order for this cup-shapedorganelle to grow' This

M A T E R I A L ST O T H E L Y S O S O M E
SND CYTOSOLIC
M E M B R A N EP R O T E I N A
DIRECTING
515
 Autophagic pathways
A FIGURE 14-35 The autophagicpathway.Theautophagic
pathwayallowscytosolic proteinsandorganelles to be delivered
t h e l y s o s o mianlt e r i ofro r d e g r a d a t i oInn,t h e a u t o p h a g pi ca t h w a y ,
a cup-shaped structure formsaroundportionsof the cytosolor
a n o r g a n e l lseu c ha sa p e r o x i s o maes,s h o w nh e r eC o n t i n u e d
addition o f m e m b r a neev e n t u a llleya d st o t h ef o r m a t i o o nf a n
autophagosome vesicle that envelops itscontentsby two complete
m e m b r a n e( s t e pE ) F u s i o on f t h e o u t e rm e m b r a nwei t h t h e
growth is likely to occur by the fusion of some type of trans-
port vesiclewith the membrane of the autophagosome.Al-
though the origin of such vesiclesis not known, the endo-
some is a likely candidate. Some of the proteins that
participate in the formation of autophagosomeshave been
identified in geneticscreensin yeast for mutants that are de-
fective in autophagy. A subset of these proteins appear to
form a coat structure on the surfaceof the autophagoso-e.
One of theseproteins is Atg8, shown in Figure 14-35, which
is covalently linked to the lipid phosphatidylethanolamine
and thus becomesattached to the cytoplasmic leaflet of the
autophagic vesicle.This coat may give the autophagosome
its cup-shapedstructure.
Autophagic Vesicle Targeting and Fusion The outer
membrane of the completed autophagosomeis thought to
contain a set of proteins that target fusion with the mem_
brane of the lysosome. Two vesicle-tetheringproteins have
been found to be required for autophagosomefusion with
the lysosome, but the corresponding SNARE proteins have
not been identified. Fusion of the autophagosomewith the
lysosomeoccurs after Atg8 has beenreleasedfrom the mem-
brane by proteolytic cleavage,and this proteolysis step only
occurs once the autophagic vesiclehas completely formed a
sealeddouble,membranesystem.Thus AtgS protein appears
616 . c H A p r E R1 4 | vEstcuLAR
T R A F F t cs,E c R E T t o N
A,N D E N D o c y r o s t s
membrane of a lysosome
releasesa single-layervesicle
andits
to contentsintothe lysosome (stepfl) Afterdegradation
interior of
the protern
andlipidby hydrolasesin the lysosome interior,
the
releasedaminoacidsaretransported across the lysosomalmembrane
intothecytosolProteinsknownto participate in theautophagic
pathwayinclude Atg8,whichformsa coatstructure aroundthe
autophaqosome
to mask fusion proteins and to prevent premature fusion of
the autophagosomewith the lysosome.
Directing Membrane Proteins and Cytosolic Materials
to the Lysosome
r Endocytosed membrane proteins destined for degrada-
tion in the lysosomeare incorporated into vesiclesthat bud
into the interior of the endosome. Multivesicular endo-
somes,which contain many of these internal vesicles,can
fuse with the lysosometo deliver the vesiclesto the interior
of the lysosome(seeFigure 14-32).
r Some of the cellular components (e.g.,ESCRT) that me-
diate inward budding of endosomalmembranesare usedin
the budding and pinching off of envelopedviruses such as
HIV from the plasma membrane of virus-infectedcells (see
Figures 14-33 and 14-34).
r A portion of the cytoplasm or an entire organelle (e.g.,
peroxisome)can be envelopedin a flattened membrane and
eventually incorporated into a double-membrane au-
tophagic vesicle.Fusion of the outer vesiclemembranewith
the lysosomedeliversthe envelopedcontentsto the interior
of the lysosomefor degradation (seeFigure 14-35).
 ability to image vesicular transport of cargo proteins in live
cells gives hope that some of these more subtle aspectsof
vesiclefunction may be clarified in the near future.
The biochemical, genetic, and structural information pre-
sented in this chapter shows that we now have a basic un-
derstanding of how protein traffic flows from one mem- KeyTerms
brane-boundedcompartment to another.Our understanding
of theseprocesseshas come largely from experimentson the AP (adapter protein) multivesicular endosomes
function of various types of transport vesicles.Thesestudies complexes598 613
have led to the identification of many vesicle components anterogradetransPott 592 Rab proteins589
and the discovery of how these components work together
ARF protein 587 receptor-mediated
to drive vesicle budding, to incorporate the correct set of endocytosis 605
cargo moleculesfrom the donor organelle' and then to me- autophagy 51.4
cisternal maturation 596 regulatedsecretion602
diate fusion of a completed vesiclewith the membrane of a
target organelle. clathrin 585 retrogradetransPort592
Despite these advances,important stagesof the secre- constitutive sectetiol 602 secmutants584
tory and endocytic pathways remain about which we know COPI586 secretorypathwaY580
relatively little. For example,we do not yet know what types sortingsignals589
COPII585
of proteins form the coats of either the regulatedor the con- 505
dynamin 599 transcytosis
stitutive secretoryvesiclesthat bud from the trans-Golgi net- -
endocytic pathway 579 tr ans G olgi network (TGN)
work. Moreover, the types of signals on cargo proteins that
580
might target them for packaging into secretoryvesicleshave ESCRT proteins613
not yet been defined. Another baffling processis the forma- transportvesicles 580
late endosome580
tion of vesiclesthat bud away from the cytosol, such as the I-SNAREs586
low-density lipoprotein
vesiclesthat enter multivesicular endosomes.Although some (LDL) 607 v-SNAREs585
of the proteins that participate in formation of these "inter-
mannose6-phosphate
nal" endosome vesiclesare known, we do not know what
(M6P) 600
determinestheir shapeor what type of processcausesthem
to pinch off from the donor membrane. Similarly, the origin
and growth of the membrane of the autophagic vesicle is Reviewthe ConcePts
also poorly understood. In the future, it should be possible
for these and other poorly understood vesicle-trafficking 1.. The studies of Palade and colleaguesused pulse-chase
steps to be dissectedthrough the use of the same powerful labeling with radioactively labeled amino acids and autora-
combination of biochemical and genetic methods that have diography to visualizethe location of newly synthesizedpro-
delineated the working parts of COPI, COPII, and teins in pancreaticacinar cells.These early experimentspro-
clathrin/AP vesicles. vided invaluable information on protein synthesis and
Questions still remain about vesicletrafficking between intercompartmental transport. New methods have evolved,
the ER and cls-Golgi, betweenGolgi stacks,and betweenthe but two basic requirementsare still necessaryfor any assay
tr ans- G olgi and endosome, the best-char acterized transport to study this type of protein transport. What two require-
steps. In particular, our understanding of how proteins are ments must be met? Briefly describe the experimental ap-
actually sorted between these organelles is incomplete proachesneededto meet the criteria.
largely becauseof the highly dynamic nature of all the or- NSF. It is a class C
2. Sec18is a yeast gene that encodes'$fhat
ganelles along the secretory pathway. Although we know pathway. is the mecha-
mutant in the yeast secretory
many of the details of how particular vesicle components trafficking? As indicated by
nistic role of NSF in membrane
function, we cannot account for why their functions are re- does an NSF mutation produce
its class C phenotype, why
stricted to specific stagesin the overall flow of anterograde at what appears to be only one
accumulation of vesicles
and retrograde transport steps.For example' we cannot ex-
stageof the secretorYPathway?
plain why COPII vesiclesfuse with one another to form a IUfhatis
new cls-Golgi stack, whereas COPI vesiclesfuse with the 3. Vesiclebudding is associatedwith coat proteins'
the role of coat proteins in vesicle budding? How are coat
membrane of the ER, since both vesicletypes appear to con-
proteins recruited to membranes?!7hat kinds of molecules
tain similar sets of v-SNARE proteins. In the same vein, we
do not know what feature of the Golgi membrane actually are likely to be included or excluded from newly formed
distinguishesa COPl-coated vesiclebud from a clathrin/AP- vesicles?What is the best-known example of a protein likely
coated bud. In both cases,binding of ARF protein to the to be involved in vesiclepinching off?
Golgi membrane appearsto initiate vesiclebudding. The so- 4. Treatment of cells with the drug brefeldin A (BFA) has
lution to these problems will require a more integrated un- the effect of decoating Golgi apparatus membranes, result-
derstandingof the flow of vesicular traffic in the context of ing in a cell in which the vast majority of Golgi proteins are
the entire secretory pathway. Recent improvements in our found in the ER.'What inferencescan be made from this

R E V I E WT H E C O N C E P T S 617
 observation regarding roles of coat proteins other than
promoting vesicle formation? Predict what type of muta-
tion in Arfl might have the same effect as treating cells
with BFA.
5. An antibody to an exposed '.hinge" region of BCOpI
known as EAGE blocks the function of BCOpI when mi-
croinjected into HeLa cells. predict what the conse-
quencesof this functional block might be for anterograde
transport from the ER to the plasma membrane. propose
an experiment to test whether the effect of EAGE mi-
croinjection is initially on anterograde or retrograde
transport.
6. Specificity in fusion between vesiclesinvolves two dis-
crete and sequentialprocesses.Describe the first of the two
processesand its regulation by GTpase switch proteins.
What effect on the sizeof early endosomesmight reiult from
overexpressionof a mutant form of Rab5 that is stuck in the
GTP-bound state?
7. Sorting signalsthat causeretrograde transport of a pro-
tein in the secretory pathway are sometimesknown as re-
trieval sequences.List the two known examplesof retrieval
sequencesfor soluble and membrane prot;ins of the ER.
How does the presenceof a retrieval sequenceon a soluble
ER protein result in its retrieval from the cls-Golgi complex?
Describehow the concept of a retrieval sequenceis essential
to the cisternal-progressionmodel.
8.
_Clathrinadapter protein (Ap) complexesbind directly
to the cytosolic face of membrane proteins and also inter-
act with clathrin. Sfhat are the four known adapter pro_
tein complexes?rVhy may clathrin be consideredto be an
accessoryprotein to a core coat composedof adapter pro_
teins?
9, I-cell diseaseis a classicexample of an inherited human
defect in protein targeting that affects an enrire classof pro-
teins, soluble enzymesof the lysosome. !7hat is the molecu-
lar defectin I-cell disease?Why doesit affectthe targeting of
an entire class of proteins? lfhat other types of mutati,ons
might produce the samephenotype?
10. The TGN, trans-Golgi network, is the site of multiple
versus apical cell surfacesin MDCK cells versus heoato_
cytes.
12. \What mechanisticfeaturesare shared by (a) the forma_
tion of multivesicular endosomesby budding into the inte_
rior of the endosome and (b) the outward Uuaaing of HIV
virus at the cell surface?You wish to design a peptide in-
618 C H A P T E R1 4 | VES|CULAR
T R A F F | CS, E C R E T | O N
A,N D E N D O C Y T O S | S
hibitor/competitor of HIV budding and decideto mimic in a
synthetic peptide a portion of the HIV Gag protein. Which
portion of the HIV Gag protein would be a logical choice?
'Sfhat
normal cellular processmight this inhibitor block?
13. The phagocytic and autophagicpathways servetwo fun-
damental roles, but both deliver their vesiclesto the lyso-
some. !7hat are the fundamental differences between the
two pathways? Describe the three basic steps in the forma-
tion and fusion of autophagic vesicles.
Analyze the Data
In order to examine the specificity of membrane fusion con-
ferred by specificv-SNAREs and I-SNAREs (seeMacNew et
aI., 2000, Nature 407:15 3-1 59), liposomes (artificial lipid
membranes)were reconstitutedwith specificI-SNARE com-
plexes or with v-SNAREs. To measurefusion, the v-SNARE
liposomes also contained a fluorescent lipid at a relatively
high concentration such that its fluorescenceis quenched.
(Quenchingis reducedfluorescencerelative to that exoected.
In this case,quenching occurs becausethe fluoresceni lipid,
are too concentrated and interfere with each other's ability
to becomeexcited.) On fusion of theseliposomeswith those
lacking the fluorescent lipid, the fluorescent lipids are di-
luted, and quenching is alleviated. Three sets of liposomes
were prepared using yeast t-SNARE complexes:those con-
taining plasma membrane I-SNAREs, Golgi I-SNAREs, or
vacuolar I-SNAREs. Each of these was mixed with fluores-
cent liposomes containing one of three different yeast v-
SNAREs. The following data were obtained.
v-SNARE1 v-SNARE2 v-SNARE3
TSNARE=
plasma
membrane
q)
c)
= Golgi
TSNARE
6
o)
f
L
TSNARE=
vacuolar
Time after mixing
a. $fhat can be deduced from these data about the
specificity of membrane fusion events?
b. Ifhere might you expect to find v-SNAREs 1. 2.
and 3 in yeast?
c. \What kind of experiment could be designedto de-
termine where in the secretorypathway a given v-SNARE is
requiredin vivo?

d. The cytoplasmic domain of v-SNARE 2 has been
expressedand purified from E. coli. Yarious amounts of this
domain are incubated either with the Golgi I-SNARE lipo-
somes or with v-SNARE 2 liposomes' The liposomes are
then washed free of unbound protein. The various liposomes
are then mixed, as indicated below, and the fluorescenceof
each sample is measured t hour after mixing' How can the
data be explained? What would you predict the outcome to
be if yeastwere to overexpressthe cytoplasmic domain of v-
SNARE 2?
a = v - S N A R E2 l i p o s o m e si n c u b a t e dw i t h t h e
o \\ -.._- c y t o p l a s m i cd o m a i n o f v - S N A R E2 a n d
t h e n m i x e d w i t h G o l g i I - S N A R El i p o s o m e s
o -t-
o -\-
0) -r = G o l g i I - S N A R El i p o s o m e si n c u b a t e dw i t h
o
o) t h e c y t o p l a s m i cd o m a i n o f v - S N A R E2 a n d
t h e n m i x e d w i t h V - S N A R E2 l i p o s o m e s
II
Amount of v-SNARE2
cytoplasmicdomain added
References
Techniques for Studying the Secretory Pathway
Beckers,C. J., et al. 1.987.Semi-intactcellspermeableto macro-
molecules:use in reconstitutionof protein transport from the endo-
plasmic reticulum to the Golgi complex' Cell 5O:523-534.
Kaiser,C. A., and R. Schekman.1990. Distinct setsof SEC
genesgovern transport vesicleformation and fusion early in the
secretorypathway. Cell 6l:723-7 33.
Novick, P.,et al. 1981. Order of eventsin the yeastsecretory
pathway. Cell 25:461'469.
Lippincott-Schwartz,J.,et al. 2001. Studyingprotein dynamics
in living cells.Nature Reu.Mol. Cell Biol.2:444456.
Orci, L., et al. 1989. Dissectionof a singleround of vesicular
transport: sequentialintermediatesfor intercisternalmovementin
the Golgi stack.Cell 56:357-368.
Palade,G.1975. Intracellularaspectsof the processof protein
synthesis.Science789:347-3 5 8.
Molecular Mechanisms of Vesicular Traffic
Bonifacino,J. S., and B. S. Glick. 2004.The mechanismsof vesi-
cle budding and fusion. Cell 116:153-66.
Grosshans,B. L., D. Ortiz, and P. Novick. 2006. Rabs and their
effectors: achieving specificity in membrane traffic' Proc. Natl.
Acad. Sci.USA 103:11821-11'827.
Jahn,R., et al. 2003. Membranefusion.Cel/ 112:51'9-533'
Kirchhausen,T. 2000. Three ways to make a vesicle.Nature
Reu.Mol. Cell Biol.7:187-1'98.
McNew, J. A., et al. 2000. Compartmentalspecificityof
cellular membranefusion encodedin SNARE proteins.Nature
407 1.53-159.
Ostermann,J., et al. 1993. Stepwiseassemblyof functionally
activetransport vesicles.Cell 7 5 :1'015-1'025'
Schimmoller,F., I. Simon, and S. Pfeffer.1998. Rab GTPases,
directorsof vesicledocking'J. Biol' Cbem.273:22161-221'64'
'Weber,
T., er.al. 1998. SNAREpins:minimal machineryfor
membranefusion. Cell 92:759-772.
'Wickner,
\7., and A. Haas. 2000. Yeasthomotypic vacuole
fusion: a window on organelletrafficking mechanisms'Ann' Reu'
Biochem. 69:247-275.
Zerial,M., and H. McBride.2001. Rab proteins as membrane
organizers.Nature Reu.MoI. Cell Biol. 22L07-tI7 '
Early Stages of the Secretory Pathway
Barlowe, C. 2003. Signalsfor COPll-dependentexport from the
ER: what's tire ticket out? Trends CeII Biol' t3:295-300'
Lee, M. C., et al. 2004. Bi-directionalprotein-transportbetween
the ER and Golgr. Ann. Reu. Cell Deu. Biol' 20:87-123'
Letourneut,F, et al. 1994. Coatomeris essentialfor retrieval of
dilysine-tagged.proteins to the endoplasmicreticulum' Cell
79:1.L99-1207.
Losev, E., et al. 2006. Golgi maturation visualized in living
y east.N atur e 441:10 02-t00 6.
Pelham,H. R. 1995' Sorting and retrieval befiveenthe endo-
plasmic reticulum and Golgi apparat.ts'Curr' Opin' Cell Biol'
/:)JU-)J).
Later Stages of the Secretory Pathway
Bonifacino,J. S. 2004. The GGA proteins:adaptorson the
move. Na/. Reu.MoI. Cell Biol. 5:23-32'
Bonifacino,J. S., and E. C. Dell'Angelica' 1'999'Molecular
basesfor the recbgnition of tyrosine-baied sorting signals'J' Cell
Biol. 745:923-926.
Edeling,M. A., C. Smith, and D. Owen' 20-0-6'I'ife of a clathrin
..";i;tGi; from clathrin and AP structures' Nat' Reu' MoL Cell
Biol.7:3244.
Fotin, A., et aI.2004. Molecular model for a complete-clathrin
lattice from .i..tro.t cryomicroscopy'Nature 432:573-579'
Ghosh,P.,et al. 2003' Mannose 6-phosphate-receptors: new
twists in the tale. Nature Reu.Mol. CeIl Bio' 4:202-213'
Mostov, K. E., M. Verges,and Y. Altschuler'2.000'Membrane
traffic in polarizeiepithelial ceIls.Curr' Opin' Cell Biol'
122483490.
Schmid,S. t997. Clathrin-coatedvesicleformation and protein
sorting: an integratedprocess'Ann. Reu' Biochem' 66:511-548'
Simons.K.. and E. Ikonen. 1997. Functional rafts in cell mem-
branes.Nature 387:569-572.
Song,B. D., and S. L. Schmid.2003' A molecular motor or a
,.gulatoii Dynamin's in a classof its own' Biochemistry
42:1369-1376.
Steiner,D. F.' et al. 1'996.The role of prohormone convertases
in insulin biosynihesis:evidencefor inherited defectsin their action
i., -"n ,nd experimentalanimals.DiabetesMetab' 22294-104'
Tooze.S. A.. et al. 2001. Secretorygranule biogenesis:rafting to
the SNARE. Trends CeIl Biol' ll:11'6-122'
Receptor-Mediated EndocYtosis
Brown, M. S., and J' L. Goldstein' 1986' Receptor-mediated
pathway for cholesterolhomeostasis'Nobel Prize Lecture' Science
232:34-47.
Kaksonen,M., C. P.Toret' and D' G' Drubin' 2006' Harnessing
Jy""-i.s for clathrin-mediatedendocytosis'Nat' Reu'Mol'
".ri"
Cell Biol.7A044l4.
Rudenko, G., et al. 2002. Structureof the LDL receptorextra-
cellular domain at endosomalpH. Science298:2353-2358'
REFERENCES 619
 DirectingMembraneProteinsand cytosolicMaterials Lemmon, s. K.,andL. M. Traub.2000.sortingin theendoso-
to the Lysosome malrystemin yeasiandanimalcells.c"* oil". iri-ai.t.
t2:457466'
. HurleSJ. H., andS.D. Emr.TheESCRT
complexes:
structure
Ain.ii,;rii.- RNAvirus
;ti:;;If:lgi:,:;#;):;-rrarricking ""iiiu' b"dl,:;"#;;3,';:;t;1ii.'?;,H;1;;"-'orenveroped
Shintani,J..,an{ D..J. Klionsky.2004.A_u_toplragy
.Katzmann,D._J.,et al. 2002.Receptordownregulationand ,.
disease:
in healthand
multivesicular-bodysorting.Nature Riu.Mot. CatTiii.i,SiigOs. a double-edged sword.Science30699C-l995'.
Khalfan,W. A., and D..J.Klionsky.2002.Molecularmachinery
requiredfor autophagyan_d the cytopias- to vacuoletargeting
pathwayin S. cereuisiae. - (Cvi)
Curr. Opin. Cell Biol. t+469475.

I
620 o cHAprER
14 | vEsrcuLAR
TRAFFT.,
sEcRETroN,
ANDENDocyrosrs
cLASSIC EXPERIMENT
OUT OF THECELL
A PROTEIN
FOLLOWING
J. Jamieson and G. Palade, 1965, Proc. Natl. Acad. Sci.IJSA 55(2):424-431
The advent of electron microscopy al-
lowed researchersto seethe cell and its
structuresat an unprecedentedlevel of
detail. George Palade utilized this tool
not only to look at the fine details of
the cell but also to analyze the process
of secretion. By combining electron
microscopy with pulse-chaseexperi-
ments, Paladeuncoveredthe path pro-
teins follow to leave the cell.
Background
In addition to synthesizingproteinsto
carry out cellular functions, many cells
must also produce and secreteaddi-
tional proteins that perform their du-
ties outside the cell. Cell biologists,
including Palade, wondered how se-
creted proteins make their passage
from the inside to the outside of the
cell. Early experimentssuggestingthat
proteins destinedfor secretionare syn-
thesizedin a particular intracellular lo-
cation and then follow a pathway to
the cell surface employed methods to
disrupt cells synthesizing a particular
secreted protein and to separate their
various organelles by centrifugation.
Thesecell-fractionationstudiesshowed
that secretedproteins can be found in
membrane-bounded vesicles derived
from the endoplasmic reticulum (ER),
where they are synthesized,and with
zymogen granules, from which they
are eventually released from the cell.
Unfortunately, results from thesestud-
ies were hard to interpret due to diffi-
culties in obtaining clean separationof
all of the different organellesthat con-
tain secretoryproteins. To further clar-
ify the pathway, Palade turned to a
newly developedtechnique, high-reso-
lution autoradiography, that allowed
him to detect the position of radioac-
tively labeled proteins in thin cell sec-
tions that had been prepared for elec-
14
tron microscopy of intracellular or-
ganelles.His work led to the seminal
finding that secreted proteins travel
within vesicles from the ER to the
Golgi complex and then to the plasma
membrane.
TheExperiment
Palade wanted to identify which cell
structuresand organellesparticipate in
protein secretion. To studY such a
complex process,he carefully chosean
appropriate model systemfor his stud-
ies, the pancreaticexocrine cell, which
is responsible for producing and se-
creting large amounts of digestive en-
zymes. Becausethese cells have the
unusual property of expressing only
secretory proteins, a general label for
newly synthesized protein' such as
radioactively labeled leucine,will only
be incorporated into protein molecules
that are following the secretory
pathway.
Palade first examined the Protein
secretionpathway in vivo by injecting
live guinea pigs with ['H]-leucine,
which was incorporated into newlY
made proteins, thereby radioactively
labeling them. At time points from
4 minutes to 15 hours. the animals
were sacrificed,and the pancreatictis-
sue was fixed. By subjectingthe speci
mens to autoradiography and viewing
them in an electronmicroscope,Palade
could trace where the labeled proteins
were in cells at various times. As ex-
pected, the radioactivity localized in
vesiclesat the ER at tim^epoints imme-
diately following the [rH]-leucine in-
iection and at the plasma membrane at
the later time points. The surprise
came in the middle time points. Rather
than traveling straight from the ER to
the plasma membrane, the radioac-
t i v e l y l a b e l e d p r o t e i n s a p p e a r e dt o
F O L L O W I N GA P R O T E I NO U T O F T H E C E L L
stop off at the Golgi comPlex in the
middle of their iourney. In addition,
there never was a time point where the
radioactively labeledproteins were not
confined to vesicles.
The observation that the Golgi
complex was involved in protein secre-
tion was both surprising and intrigu-
ing. To thoroughly addressthe role of
this organelle in protein secretion,
Palade turned to in vitro pulse-chase
experiments, which Permitted more
precise monitoring of the fate of
labeled proteins. In this labeling
technique,cells are exposedto radiola-
beled precursor' in this case ['H]-
leucine, for a short Period known as
the pulse. The radioactive precursor is
then replacedwith its nonlabeled form
for a subsequentchaseperiod. Proteins
synthesized during the pulse period
will be labeled and detectedby autora-
diography, whereas those synthesized
during the chase period, which are
nonlabeled, will not be detected.
Palade began by cutting guinea Pig
pancreasinto thick slices,which were
then incubated for 3 minutes in media
containing [3H]-leucine.At the end of
the pulse, he added excess unlabeled
leucine. The tissue sliceswere then ei-
ther fixed for autoradiography or used
for cell fractionation. To ensure that
his results were an accurate reflection
of protein secretion in vivo' Palade
meticulously charucterized the system.
Once convinced that his in vitro sys-
tem accurately mimicked protein se-
cretion in vivo, he Proceeded to the
critical experiment. He pulse-labeled
tissue slicis with [3Hl-leucine for 3
minutes, then chased the label for 7,
1,7, 37 , 57 , and 1 17 minutes with unla-
beled leucine. Radioactivity, again
confined in vesicles'began at the ER,
then traveled in vesiclesto the Golgi
complex and remained in the vesicles
621
 < FIGURE 1 The synthesisand movementof guineapig
pancreaticse€retoryproteinsas revealedby electronmicroscope
autoradiography. Aftera periodof labelingwith [3H]-leucine,the
tissueisfixed,sectionedfor electronmicroscopy, andsubjected to
autoradiography. Theradioactive decayof [3tt]in newlysynthesized
proteinsproduces autoradiographic in an emulsion
Arains placedover
thecellsection (whichappear inthemicrograph asdense, wormlike
granules) thatmarkthe position of newlysynthesized proteins(a)At the
endof a 3-minute periodautoradiographic
labeling Arainsareoverthe
roughER(b)Following a 7-minute chaseperiod withunlabeled leucine,
mostof the labeled proteinshavemovedto the Golgivesicles (c)Aftera
37-minute chase,mostof the proteins areoverimmature secrerory
vesicles(d)Aftera 117-minute chase,the majority of theproteinsare
overmaturezymogen granulesfCourtesyof J Jamieson andG palade]
(c) (d)

as they passedthrough the Golgi and
onto the plasma membrane (seeFig-
ure 1). As the vesiclestraveledfarther
along the pathway, they becamemore
densely packed with radioactive pro-
t e i n . F r o m h i s r e m a r k a b l es e r i e so f a u -
toradiograms at different chasetimes,
Palade concluded that secretedpro-
teins rravel in vesiclesfrom rhe Ei{ ro
the Golgi and onto the plasma mem-
brane and that throughout this
process,they remain in vesiclesand do
not mix with the rest of the cell.
Discussion
Palade'sexperimenrs gave biologists
the first clear look at the stagesof the
secretorypathway. His studieson pan-
creatic exocrine cells yielded two fun-
damental observations.First, that se-
creted proteins pass through the Golgi
complex on their way out of the cell.
This was the first function assignedto
the Golgi complex. Second, secrered
proteins never mix with cellular pro-
teins in the cytosol; they are segregated
into vesiclesthroughout the pathway.
These findings were predicated from
two rmportant aspects of the experi-
mental design. Palade'scareful use of
electronmicroscopy and autoradiogra-
phy allowed him to look at the fine de-
tails of the pathway. Of equal impor-
tance was the choice of a cell tyoe
devoted to secretion, the pancreatic
exocrine cell, as a model system. In a
different cell type, significant amounts
of nonsecretedproteins would have
also been produced during the label-
ing, obscuring the fate of secretory
proteins in particular.
Palade's work set the stage for
more detailed studies. Once the secre-
tory pathway was clearly described,
entire fields of research were opened
u p t o i n v e s t i g a t i o inn t h e s y n t h e s ias n d
movement of both secretedand mem-
brane proteins. For this groundbreak-
ing work, Palade was awarded the
Nobel Prize for Physiology and Medi-
cine in 1974.

622 C H A P T E R1 4 | v E s t c u L A RT R A F F | CS, E C R E T T O A
NN, D ENDOCYTOSTS
 CHA PTER

I:
CELLSIGNALING
SIGNAL
TRANSDUCTION
AND SHORT-TERM
Thestorageand metabolismof triglycerides is regulatedby many CELLULARRESPONSES
importanthormones. This3T3-11adipocyte isstainedfor three
dropletsat differentstagesof their
proteinsthat Iinetriglyceride
maturation-perilipin in green,andTlP47in
in blue,adipophilin
red.Courtesv PerrvBickeland NathanWolins

o cell lives in isolation; cellular communication is a receptors; signaling from such intracellular receptors is dis-
fundamental property of all cellsand shapesthe func- cussedin detail Chapter 7. Somesmall signaling moleculesare
tion and abilities of every living organism. Even hydrophilic and are transported by membrane proteins into
single-celledorganismshave the ability to communicatewith the cell cytoplasm in order to influence cell behavior'
each other or other organisms.Eukaryotic microorganisms, Most signalingmolecules,however,are too large and too
such as yeasts, slime molds, and protozoans, use secreted hydrophilic to penetrate through the plasma membrane'
moleculescalled pheromones to coordinate the aggregation These bind to cell-surfacereceptors that are integral proteins
of free-living cells for sexual mating or differentiation under
certain environmental conditions. Yeastmating-type factors OUTLINE
are a well-understood example of pheromone-mediatedcell-
to-cell signaling (Chapter 21). More important in plants and 15.1 F r o mE x t r a c e l l u l aSri g n a tl o C e l l u l a r
animals are extracellular signaling molecules that function ResPonse 62s
within an organism to control metabolism of sugars, fats'
and amino acids, the growth and differentiation of tissues, 15.2 Receptors
StudyingCell-Surface 627
the synthesisand secretionof proteins, and the composition
15.3 Highly ConservedComponentsof
of intracellular and extracellular fluids. Animals also re- 632
Pathways
IntracellularSignal-Transduction
spond to many signals from their environment, including
light, oxygen, odorants, and tastantsin food. 15.4 GeneralElementsof G Protein-Coupled
Many extracellular signaling molecules are synthesized ReceptorSystems
and releasedby signaling cells within the organism. In all cases
signaling molecules produce a specific response only in target 15.5 G Protein-CoupledReceptorsThat
cells that have receptors for the signaling molecules' Many R e g u l a t el o n C h a n n e l s
types of chemicalsare used as signals:small molecules(e.g.,
15.6 G Protein-CoupledReceptorsThat Activate
amino acid or lipid derivatives,acetylcholine),peptides(e.g', 646
or Inhibit AdenYlYlCYclase
ACTH and vasopressin),soluble proteins (e.g., insulin and
growth hormone) and many proteinstetheredto the surfaceof 15.7 G Protein-Coupled ReceptorsThat Activate
a cell or bound to the extracellular matrix. Most receptors PhospholipaseC 6s3
bind a singlemoleculeor a group of closelyrelatedmolecules.
Some signaling molecules,especiallyhydrophobic mole- 15.8 IntegratingResponses of Cellsto
culessuchas steroids,retinoids,and thyroxine' spontaneously E n v i r o n m e n t aI ln fl u e n c e s
diffuse through the plasma membrane and bind to intracellular

623
 OverviewAnimation:Extracellular tlltl
Signaling
< FIGURE 15-1 Generalprinciplesof signalingby cell-surface
receptors. Communication by extracellular signals usually involves
t h ef o l l o w i n g
s t e p sS: y n t h e soi sf t h e s i g n a l i nmg o l e c u lbey t h e
signaling cellanditsrncorporation intosmallintracellular vesicles fil),
itsrelease intothe extracellular spaceby exocytosis (Z), andtransport
o f t h e s i g n atlo t h et a r g e ct e l l( B ) w h e r et h e s i g n a l i nm g olecule
o bindsto a specific cell-surface receptor proteinleading to activation
o of the receptor (4). Theactivated receptor theninitiates one or
m o r ei n t r a c e l l u lsai g
r n a l - t r a n s d u cpt iaotnh w a y(sE t )l e a d i n tgo
specif ic changes, usually short-term, in cellular function,metabolism.
or movement (EE)or to long-term changes in geneexpresston or
Cell surface development ((p). Termination of the cellular response iscaused by
receptor intracellular signaling molecules thatinhibitreceptor function(Z)
andby removal of the extracellular signal(S)

the samepathway may be referred to by different names. For-

,a uo
/l\@ @/\
Modificationof Modificationof
cellularmetabolism, geneexpression,
function,movement develooment
on the plasma membranes.Cell-surfacereceptors generally
consist of three discretesegments:a segmenton the extracel-
Iular surface, a segment that spans the plasma membrane,
and a segmentfacing the cytosol. The signalingmoleculeacts
as a ligand, which binds to a structurally complementarysite
on the extracellular or membrane-spanningdomains of the
receptor. Binding of the ligand induces a conformational
change in the receptor that is transmitted through the
membrane-spanning domain to the cytosolicdomain, resulting
in binding to and subsequentactivation(or inhibition) of othei
proteins in the cytosol or attached to the plasma membrane.
The overall process of converring extracellular signals into
intracellular responses,as well as the individual stepsin this
process,is termed signal transduction (Figure 15-1).
In all eukaryotesthere are only about a dozen classesof
bined with biochemical analyseshave enabledresearchersto
trace many entire signalingpathways from binding of ligand
to receptorsto the final cellular resDonses.
tunatelS as researchershave discoveredthe molecular details
of more and more receptorsand pathways, some overarching
principles and mechanismsare beginning to emerge.These
shared featurescan help us make senseof the wealth of new
information concerningcell-to-cellsignaling.
Perhapsthe most numerous class of receptors-found
in organismsfrom yeast to human-are commonly called
G protein-coupled receptors (GPCRs). The human genome
encodes about 900 G protein-coupled receptors including
receptorsin the visual, olfactory (smell),and gustatory (taste)
systems,many neurotransmitter receptors, and most of the
receptorsfor hormones that control carbohydrate, amino acid,
and fat metabolism. Essentiallyrhe same signaling pathway is
usedin yeastfor signalingby mating factors(Chapter21). This
chapter focusesmainly on these receptors, which usually in-
duce short-term changesin cell function. Activation of many
cell-surfacereceptors alters the pattern of gene expression by
the cell, leading to cell differentiation and other long-term
consequences. Thesereceptorsand the intracellular signaling
pathways they activate are explored in Chapter 16. Certain
other cell-surfacereceptorsare normally closed ion channels
that open in responseto ligand binding, allowing a particular
type of ion to cross the plasma membrane.These receptors,
called ligand-gatedion channels,are especiallyimportant in
nerve cells;they are discussedin Chapter 23.
In this chapter we first review the generalprinciples of cell
signaling and describehow cell-surfacereceptorsare identified
and characterized.Next we discussseveral features of many
signal-transduction pathways and their regulation that have
been conservedthroughout evolution and are found in a wide
variety of organisms.We then describethe common elementsin
G protein-<oupled signalingpathways and examine in molecu-
lar detail the mechanismsby which diverse cellular functions
are controlled by a relatively small set of G protein--coupledre-
ceptorsand their associatedsignalingpathways. Finally, we will
seethat a signal-transductionpathway activated by one recep-
tor can affect the signaling pathway downstream of a second
kind of receptor-sometimes positivelg at other times in a neg-
21iysrn2nnsl-in order to integrate cellular responsesto multi-
ple extracellular signals.This kind of signal integration plays a
key role in the body's responseto differing needsfor glucose.

624 ' C H A P T Et R
5 | c E L Ls T G N A L TrN: sGr c N A LT R A N S D U c T oANN Ds H o R T - T E RCME L L U L ARRE s p o N s E s
 FromExtracellular
Signal
to CellularResponse
Communicationby extracellularsignalswithin an organism
usually involves the following steps (seeFigure 15-1): tr
synthesis and Z release of the signaling molecule by the
signalingcell; E transport of the signalto the targetcell; 4
binding of the signal by a specific receptor protein Ieading
to a conformationalchange;E initiation of one or more ln-
tracellular signal-transductionpathways by the activated
receptor; 6 specific changes in cellular function, metabo-
lism, or development;and feedbackregulation usually in-
volving Z deactivation of the receptor and B removal of
the signaling molecule, which together terminate the cellu-
lar response.
S i g n a l i n gM o l e c u l e sC a nA c t L o c a l l y
or at a Distance
Releasedsignaling moleculestravel to their target cells (see
Figure 15-1, step B). Some are transportedlong distances
by the blood; others have more local effects.In animals, sig-
naling by extracellular moleculescan be classifiedinto three
types-endocrine, paracrine,or autocrine-based on the dis-
tance over which the signalacts (Figure 15-2a-c).In addition,
certain membrane-bound proteins on one cell can directly
signalan adiacentcell.
( a ) E n d o c r i nsei g n a l i n g
ooo

. o^ o
S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e ':'
,.1

S i g n a l i n gM o l e c u l e s Hormonesecretion
i n t o b l o o d b y e n d o c r i n eg l a n d Distanttarget cells
In humans rapid responsesto changesin the environment
are primarily mediated by the nervous system and by hor-
monesincluding small peptides(e.g.,insulin and adrenocor- ( b ) P a r a c r i n se i g n a l i n g
ticotropic hormone,ACTH) and small (nonpeptide)molecules
such as the catecbolamines(e.g., epinephrine, norepineph-
o
rine, and dopamine).The cellsthat make thesesignalingmol-
eculesare found in the pancreas(insulin), the pituitary gland n nau
lO, o
(ACTH), the adrenal glands (epinephrine and norepineph-
rine), neurons (norepinephrine)and the part of the brain
termed the hypothalamus (dopamine).Small signalingmole-
SecretorYcell Adjacenttarget cell
cules,including catecholamineneurotransmitters,are synthe-
sizedin the cytosol and then transported into secretoryvesi-
cles (Chapter 23), whereaspeptide and protein hormones are ( c ) A u t o c r i n es i g n a l i n g
synthesizedand processedby the secretorypathway detailed Key:
in Chapter 14. In both casesvesiclescontaining thesesignal-
E x t r a c e l l u l asri g n a l
ing moleculesaccumulatejust under the plasma membrane
where they are held, awaiting a releasesignal (seeFigure 15-1,
s t e p[ ) . @ Receptor

Stimulation of signalingcells is almost always coupled ? Mem brane-attached

to a rise in the local concentration of Ca"- near the vesi- s i gn a l
cles, causing immediate fusion of the membranes of the
v e s i c l e sa n d t h e p l a s m a m e m b r a n e ,l e a d i n gt o e x o c y t o s i s Targetsites on same cell
of the storedpeptideor protein hormone or small molecule
i n t o t h e s u r r o u n d i n gm e d i u m o r b l o o d ( s e eF i g u r e 1 5 - 1 ,
( d ) S i g n a l i n gb y p l a s m am e m b r a n e - a t t a c h epdr o t e i n s
s t e pZ ) .
Releasedpeptidehormonespersistin the blood for only
secondsto minutes before being degraded by blood and
tissue proteases.Releasedsmall molecules such as cate-
cholaminesare rapidly inactivatedby enzymesor taken up
via transporters into specific cells. The initial actions of
these signalingmoleculeson target cells (the activation or \"'_-.'**--,-,,-,.."*'-.'"_-
inhibition of specificenzymes)usually only lastssecondsor S i g n a l i n cg e l l
minutes. Thus the catecholaminesand some peptide hor- a FIGURE 15-2 Generalschemesof intracellular signaling'
mones can mediate short-term responsethat are terminated (a-c)Cell-to-cell by
signaling extracellular occurs
chemicals over
by their own degradation. For sustainedor longer-term re- distancesfrom a few in
micrometers and
autocrine paracrine
sponses,such as cell division or cell differentiation,the cells signalingto severalmetersin endocrine (d)Proteins
signaling.
must be exposedto signalingmoleculesfor extendedperi- attached to the plasmamembrane directly
of onecellcaninteract
ods. usually hours. with cell-surface on adjacent
receptors cells

RESPONSE
F R O M E X T R A C E L L U L ASRI G N A LT O C E L L U L A R 625
 In endocrine signaling, the signaling molecules are syn-
thesized and secrered by signaling cells (endocrine cells),
transported through the circulatory systemof the organism,
and finally act on target cells distant from their site of
synthesis.The term hormone generally refers to signaling
moleculesthat mediate endocrine signaling.
In paracrine signaling, the signaling molecules releasedby
a cell affect only those target cells in close proximity. The con-
duction by a neurotransmitter of a signal from one nerve cell
to another or from a nerve cell to a muscle cell (inducing or
inhibiting muscle contraction) occurs via paracrine signaling.
Many growth factors regulating development in multicellular
organisms also act at short range. Some of these molecules
bind tightly to the extracellular matrix, unable to signal, but
subsequentlycan be releasedin an active form. Many devel-
opmentally important signals diffuse away from the signaling
cell, forming a concentration gradient and inducing differeni
cellular responsesdepending on the distance of a particular
target cell from the site of signal release(Chapter 22).
In autocrine signaling cells respond to substancesthat
they themselvesrelease.Somegrowth factors act in this fash-
ion, and cultured cellsoften secretegrowth factors that stim-
ulate their own growth and proliferation. This type of sig-
naling is particularly characteristicof tumor cells, many of
which overproduceand releasegrowth factors that stimulate
inappropriate, unregulatedself-proliferation as well as influ-
(a) Residuesessentialto
tight binding with receptor
Growth
hormone
Residuesessential
to tight binding with
hormone
Growth
normone
receptor
-ooc
EXPERIMENTAL FTGURE 15-3 Smallpatchesof aminoacids
are important for specificbinding between growth hormone
and its receptor.Theoutersurfaceof the plasmamembrane is
towardthe bottomof the figure,andeachreceptormolecule is
anchored to the membrane by a hydrophobic membrane-spanning
cthelix(notshown)thatisa continuation of the carboxyl
terminus
depicted in thefigureAs determined fromthethree-dimensional
structureof the groMh hormone-growth hormonerecepror comptex,
28 aminoacidsin the hormone areat the bindinginterfacewith one
receptorEachof theseaminoacidswasmutated, oneat a ttme.to
alanine,andthe effecton receptor bindingwasdetermined (a)From
thisstudyit wasfoundthatonlyeightaminoacidson growthhormone
(pink)contribute 85 percent
of the bindingenergy; theseaminoacids
626 C H A P T E R1 5 I C E L LS I G N A L I N GI : S I G N A LT R A N S D U C T I OA
encing adjacentnontumor cells;this processmay lead to for-
mation of a tumor mass.
Signalingmoleculesthat are integral membrane proteins
located on the cell surfacealso play an important role in de-
velopment (Figure 15-2d).In some cases,such membrane-
bound signalson one cell bind receptorson the surfaceof an
adjacent target cell to trigger its differentiation. In other
cases,proteolytic cleavageof a membrane-bound signaling
protein releasesthe extracellular segment,which functions
as a soluble signaling molecule.
Some signaling molecules can act at both short and long
ranges. Epinephrine, for example, functions as a neurotrans-
mitter (paracrine signaling) and as a systemichormone (en-
docrine signaling). Another example is epidermal growrh
factor (EGF), which is synthesizedas an integral plasma
membrane protein. Membrane-bound EGF can bind to
and signal an adjacentcell by direct contact. Cleavageby an
extracellularmatrix proteasereleasesa soluble form of EGF,
which can signalin either an autocrineor a paracrinemanner.
Bindingof SignalingMoleculesActivates
Receptorson TargetCells
When a signaling molecule arrives at atargetcell, it binds to a
receptor(seeFigure 15-1, step 4). The vast majority of recep-
tors are activated by binding of secretedor membrane-bound
(b) (c)
H3'
aredistantfromeachotherin the primarysequence but adjacent
in the foldedproteinSimilar studiesshowedthat two tryptophan
residues (blue)in the receptor contributemostof the energyfor
bindinggrowthhormone, although otheraminoacidsat the interface
with the hormone (yellow)arealsoimportant. (b)Bindingof growth
hormone to onereceptor molecule isfollowedby (c)bindingof a
second receptor (purple)to theopposing sideof the hormone; this
involves thesamesetof yellowandblueaminoacidson the receptor
but differentresidues on the hormone.As we seein the nextchapter,
suchhormone-induced receptordimerization isa commonmechanism
for activationof receptorsfor proteinhormones[AfterB Cunningham
andJ,Wells, 1993,L Mol Biol234:554, andT.Clackson andJ Wells.
1995.
Sclence267:383 l
NN D S H o R T . T E R Mc E L L U L A RR E s P o N s E s
molecules(e.g.,hormones,growth factors,neurotransmitters,
and pheromones).Somereceptors,however,are activatedby
changesin the concentrationof a metabolite (e.g.,oxygen or
nutrients) or by physicalstimuli (e.g.,light, touch, heat).
As noted earlier,the specificreceptor protein for any hy-
drophilic extracellular signaling molecule is almost always
located on the surfaceof the target cell. The signaling mole-
cule, or ligand, binds to a site on the extracellular domain of
the receptor as a consequenceof their molecular comple-
mentarity (Figure15-3; seealso Chapter2). Binding of a lig-
and causes a conformational change in the membrane-
bound receptor that initiates a sequenceof reactionsleading
to a specific cellular responseinside the cell. Different cell
types may have different sets of receptors for the same lig-
and, each of which can induce a different response.Or the
samereceptor may be found on various cell types, and bind-
ing of a particular ligand to a particular receptor may trigger
a different responsein each type of cell. In theseways, one
ligand can induce different cells to respond in a variety of
ways.
Ligand binding to G protein-coupled receptors triggers
an intracellular protein (a trimeric G protein) to exchange
one bound GDP nucleotide for a GTP. The conformational
change induced by GTP binding, in turn, affects interaction
of the G protein with downstream signal-transductionpro-
teins. The human receptors for the hormones epinephrine,
serotonin, and glucagon are examplesof G protein-coupled
receptors. As we discuss in other chapters, the conforma-
tional change resulting from ligand binding to some other
classesof receptors activates (or occasionally inhibits) the
phosphorylating, or kinase, activity of the receptor's intra-
cellular domain, or of an associatedcytosolic kinase en-
zyme. Subsequentphosphorylation of substrate proteins in
the cytosol modifies their activities. Activation of still other
receptors involves proteolysis or disassemblyon intracellu-
lar multiprotein complexes, leading to releaseof signal-
transductionproteins.
From ExtracellularSignal to Cellular Response
r Extracellular signalingmoleculesregulate interactions
between unicellular organisms and are critical regula-
tors of physiology and development in multicellular
organlsms.
r Extracellular signaling molecules bind to receptors, in-
ducing a conformational change in the receptor (activa-
tion) and consequent alteration in intracellular activities
and functions.
r Signalsfrom one cell act on nearby cells in paracrine
signaling, on distant cells in endocrine signaling, or on
the signaling cell itself in autocrine signaling (see Fig-
ure 15-2).
r External signals include membrane-anchoredand se-
creted proteins and peptides (both dissolved in the extra-
cellular fluid or embedded in extracellular matrix), small
lipophilic molecules (e.g., steroid hormones, thyroxine),
small hydrophilic molecules(e.g.,epinephrine),gases(e.g.,
nitric oxide), and physicalstimuli (e.g.,light).
r Binding of extracellularsignalingmoleculesto cell-surface
receptorstriggers a conformational changein the receptor,
which in turn leads to activation of intracellular signal-
transduction pathways that ultimately modulate cellular me-
tabolism, function, or geneexpression(seeFigure 15-1).
Receptors
StudyingCell-Surface
The responseof a cell or tissueto specificexternal signalsis
dictated (a) by the cell's complement of receptors that can
recognize the signals, (b) the signal-transductionpathways
activated by those receptors, and (c) the intracellular
processesaffected by those pathways. Recall that the inter-
action betweenligand and receptor causesa conformational
changein the receptor protein that enablesit to interact with
other proteins, thereby initiating a signalingcascade(seeFig-
ure 15-1, steps4 and E). In this sectionwe explorethe bio-
chemical basisfor the specificity of receptor-ligand binding,
as well as the ability of different concentrationsof ligand to
activate a pathway. $(/e also examine experimental tech-
niques used to purify and characterize receptor proteins.
Many of thesemethods are applicable to receptorsthat me-
diate endocytosis(Chapter 1'4\or cell adhesion(Chapter 19)
as well as those receptorsthat mediate signaling.
Typical cell-surface receptors are present in 1000 to
50,000 copies per cell. This may seem like a-large number,
but a "typical" mammalian cell contains -10tu total protein
moleculesand:106 proteins on the plasma membrane.Thus
the receptor for a particular signaling molecule commonly
constitutes only 0.1 to 5 percent of plasma membrane pro-
teins. This low abundance complicates the isolation and
purification of cell-surface receptors. The purification of
receptors is also difficult becausethese integral membrane
proteins first must be solubilized from the membrane with a
nonionic detergent (see Figure 1'0-23) and then separated
from other cellularproteins.
ReceptorProteinsBind LigandsSpecifically
Each receptor generallybinds only a singlesignalingmole-
cule (ligand) or a group of very closely structurally related
molecules. The binding specificity of a receptor refers to
its ability to distinguishclosely related substances.Ligand
binding depends on weak, multiple noncovalent forces
(i.e., ionic, van der \faals, and hydrophobic interactions)
and molecular complementarity between the interacting
surfacesof a receptor and ligand (seeFigure 2-1,2).The
insulin receptor, for example, binds insulin and related
hormones called insulin-like growth factors 1' and2 (IGF-1
and IGF-2), but no other hormones.SimilarlS the receptor
that binds growth hormone does not bind other hormones
of similar structure, and acetylcholinereceptorsbind only
this small molecule and not others that differ only slightly
S T U D Y I N GC E L L - S U R F A CREE C E P T O R S 627
 in chemical structure. As these examplesillustrate, all re-
ceptors are highly specificfor their ligands.
Not only is each receptor protein characterized by its
binding specificityfor a particular ligand, the resulting recep-
tor-ligand complex exhibits effector specificity becausethe
complex mediatesa specificcellular response.Many signal-
ing molecules bind to multiple types of receprors,each of
which can activate different intracellular signalingpathways
and thus induce different cellular responses.For instance,the
surfacesof skeletal muscle cells, heart muscle cells, and the
pancreatic acinar cells that produce hydrolytic digestiveen-
zymes each have different types of receptors for acetyl-
choline. In a skeletalmusclecell, releaseof acetylcholinefrom
an adjacent neuron triggers contraction by activating an
acetylcholine-gatedion channel.In the autoimmune paralytic
diseasemyasthenia gravis, the body makes antibodies that
block the activity of its own acetylcholinereceptors.In heart
muscle,the releaseof acetylcholineby different neurons acti-
vatesa G protein-coupled receptorand slows the rate ofcon-
traction, and thus the heart rate. Releaseof acetylcholine
near pancreaticacinar cells triggers a rise in cytosolic [C"t*]
that induces exocytosis of the digestive enzymes stored in
secretorygranules to facilitate digestion of a meal. Thus
formation of different acetylcholine-receptorcomplexes in
different cell types leadsto different cellular responses.
On the other hand, different receptorsof the same class
that bind different ligands often induce the same cellular re-
sponsesin a cell; thus different extracellularsignalscan cause
a common changein the behaviorof a cell. In liver cells,for in-
stance,the hormonesepinephrine,glucagon,and ACTH bind
to different membersof the G protein{oupled receptor familS
but all these receptorsactivate the same signal-transduction
pathway, one that promotes synthesisof a small intracellular
signalingmolecule(cyclicAMP) that in turn regulatesvarious
metabolic functions, including glycogenbreakdown. As a re-
sult, all three hormones have the same effect on liver-cell me-
tabolism, as further detailedin Sections15.6 and 15.8.
The DissociationConstantls a Measureof the
Affinity of a Receptorfor tts Ligand
Ligand binding to a receptor usually can be viewed as a sim-
ple reversiblereaction, where the receptor is representedas
R, the ligand as L, and the receptor-ligand complex as RL:
koff
R+L = RL
kot
Ao66is the rate constant for dissociation of a ligand from its
receptor,and Ao, is the rate constant for formation of a re-
ceptor-ligandcomplex from free ligand and receptor.
At equilibrium the rate of formation of the receptor-ligand
complex is equal to the rate of its dissociation,and can be
describedby the simple equilibrium-binding equation
f R l fL l
ro :
lnl_l
628 ' c H A P T E R1 5 C E L LS T G N A L T NrG
: s T G N A LT R A N S D U C T o NA N D s H o R T - T E R MC E L L U L A R
| REspoNSEs
where [R] and [L] are the concentrations of free receptor
(that is, receptorwithout bound ligand) and ligand, respec-
tively, at equilibrium, and [RL] is the concentration of the
receptor-ligand complex. Ka, the dissociation constant, is a
measure of the affinity of the receptor for its ligand. For a
simple binding reaction, Ka : ko,;.lko,.The lower ftos is rela-
tive to &o., the more stable the RL complex-the tighter
the binding-and thus the lotuer the value of K6. Another
way of seeingthis key point is that K6 equals the concen-
tration of ligand at which half of the receptorshave a lig-
and bound; at this ligand concentration [R] : [RL] and
thus, from Equation 1,5-2,Kd: [L]. The lower the K6, the
lower the ligand concentration required to bind 50 percent
of the cell-surfacereceptors. The K6 for a binding reaction
here is essentiallyequivalentto the Michaelis constant K-,
which reflects the affinity of an enzyme for its substrate
( C h a p t e r3 ) .
Like all equilibrium constants,however, the value of K6
does not depend on the absolute values of &o6and Ao,, only
on their ratio. In the next sectionwe learn how K6 valuesare
experimentally determined.
BindingAssaysAre Usedto DetectReceptors
a n d D e t e r m i n eT h e i rA f f i n i t i e sf o r L i g a n d s
Usually receptorsare detectedand measuredby their ability
to bind radioactive ligands to intact cells or to cell frag-
ments. Figure 15-4 illustrates such a binding assayfor in-
teraction of insulin with insulin receptors on liver cells.
The amounts of radioactive insulin bound to its receotor
on cells growing in Petri dishes(vertical axis) were mias-
ured as a function of increasinginsulin added to the ex-
tracellular fluid (horizontal axis). Both the number of
ligand-binding sitesper cell and the K6 value are easilyde-
termined from the specificbinding curve (curve B), usually
by applying readily available computer curve-fitting pro-
grams to the experimentalvalues.Assuming each receptor
generally binds just one ligand molecule, the number of
ligand-binding sitesequals the number of active receptors
per cell. In the example shown in Figure 1.5-4,the value of
Ka is 1.4 x 10-10 M. In other words. an insulin concen-
tration of 1.4 x 10-10 M in the extracellular fluid is re-
quired for 50 percent of a cell'sinsulin receptorsto have a
bound insulin. A receptor usually has a different affinity
for each of the ligands that it can bind. For instance,sim-
ilar binding assaysshowed that the Ka for binding of in-
(1s-1) sulin-like growth factor 1(IGF-1) to the insulin receptor is
3 x 10-o M. Thus an -200-foId higher concentration of
IGF-1 than insulin is required to bind 50 percent of the in-
sulin receptors.
Direct binding assayslike the one in Figure 1.5-4 are
feasible with receptors that have a strong affinity for their
ligands, such as the erythropoietin receptor (K6 : 1 x
10-10 M) and the insulin receptoron liveriells (Ka : 1.4 x
10-10 M). However,many ligands such as epinephrineand
other catecholaminesbind to their receptors with much
( 1s- 21 lower affinity. If the K6 for binding is greater than -1 X
10-7 M, a casewhen the rate .orrrt".rt &"s is relativelylarge
 manipulations required for the measurement(relatively low
kor).
c)

6 40,000 Competitive binding is often used to study synthetic

th Specificbinding analogsof natural hormones that activate or inhibit
0)

receptors.Theseanalogs,which are widely usedin research

o 30,000
on cell-surfacereceptorsand as drugs, fall into two classes:
E agonists, which mimic the function of a natural hormone
c
= 20,000 by binding to its receptor and inducing the normal re-
o sponse,and antagonists,which bind to the receptor but in-
duce no response.By occupying ligand-binding sites on a
Nonspecificbinding
o
10,000 receptor, an antagonist can block binding of the natural
hormone (or agonist)and thus reducethe usual physiolog-
N
= ical activity of the hormone. In other words' antagonists
inhibit receptorsignaling.
0 0.2 0.4 0.6 0.8 1.0 Consider for instance the drug isoproterenol used to
[ 1 2 5i1n]s u l i n( n M ) treat asthma.Isoproterenolis made by the addition of two
A EXPERIMENTAL FIGURE 15-4 Fot high-affinityligands, methyl groups to epinephrine(seeFigure 1'5-5,right).lso-
bindingassayscandeterminethe K6 and the numberof proterenol, an agonist of the epinephrine-responsiveG
receptorsper cell.Shownherearedatafor insulin-specific receptors protein-coupled receptors on bronchial smooth muscle
o n t h e s u r f a c oe f l i v e rc e l l sA. s u s p e n s i o nf c e l l si s i n c u b a t efdo r cells, binds about tenfold more strongly (lO-fold lower
t hourat 4'C with increasing concentrations of 12sl-labeled insulin; K6) than does epinephrine(seeFigure 15-5, left). Because
the low temperature is usedto preventendocytosis of the cell-surface activation of these receptors promotes relaxation of
receptors. Thecellsareseparated f rom unboundinsulin,usually by bronchial smooth muscle and thus opening of the air pas-
c e n t r i f u g a t i oann,dt h e a m o u not f r a d i o a c t i v b i toy u n dt o t h e mi s
sagesin the lungs, isoproterenol is used in treating bronchial
m e a s u r eT d h et o t a lb i n d i n gc u r v eA r e p r e s e ni tnss u l i sn p e c i f i c a l l y
asthma, chronic bronchitis, and emphysema.In contrast'
b o u n dt o h i g h - a f f i n irtey c e p t o a r ssw e l la si n s u l i n o n s p e c i f i c a l l y
activation of a different type of epinephrine-responsive
boundwith low affinityto othermolecules on the cellsurfaceThe
contribution of nonspecific bindingto totalbindingis determined G protein-coupled receptors on cardiac muscle cells in-
by repeating the bindingassay in the presence of a 10O-fold excess creasesthe heart contraction rate. Antagonists of this recep-
of unlabeled insulin, whichsaturates allthespecific high-affinity sites tor, such as alprenolol and related compounds' are referred
In thiscase,allthe labeled insulinbindsto nonspecific sites,yielding to as beta-blockers; such antagonists are used to slow
curveC Thespecific bindingcurveB iscalculated asthedifference heart contractionsin the treatment of cardiac arrhythmias
betweencurvesA and C As determined by the maximumof the and angina. I
specif ic bindingcurveB,the numberof specif ic insulin-binding sites
(surface receptors) percellis33,000TheK6istheconcentration of
M a x i m a lC e l l u l a rR e s p o n s teo a S i g n a l i n g
insulinrequired to bindto 50 percent of the surface insulinreceptors
(inthiscaseabout16,500receptors/cell) Thus,the K6isabout1 4 MoleculeUsuallyDoesNot RequireActivation
x 1 0 - 1 0M , o r O 1 4 n M [ A d a p t ef rdo mA C i e c h a n oevtea rl, 1 9 8 3 , of AII Receptors
Cell32:267l All signalingsystemsevolvedsuch that a rise in the level of
extracellularsignalingmoleculesinducesa proportional re-
sponsein the respondingcell. For this to happen the bind-
compared to fro,, then it is likely that during the seconds ing affinity (Ka value) of a cell-surfacereceptor for a cir-
to minutes required to measurethe amount of bound lig- culating hormone must be greater than the normal
and, some of the receptor-boundligand will dissociateand (unstimulated) Ievel of that hormone in the extracellular
'We
thus the observedvalueswill be systematicallytoo low. fluids or blood. can see this principle in practice by
One way to detect weak binding of a ligand to its recep- comparing the levelsof insulin presentin the body and the
tor is in a competition assaywith another ligand that binds K6 for binding of insulin to its receptoron liver cells' 1.4 x
to the samereceptor with high affinity (low K6 value). In this 10-10 M. Suppose,for instance,that the normal concen-
type of assay,increasingamounts of an unlabeled,low-affin- tration of insulin in the blood is 5 x 10-12 M. By substi-
ity ligand (the competitor) are added to a cell sample with a tuting this value and the insulin K6 into Equation 1'5-2,we
constant amount of the radiolabeled,high-affinity ligand can calculatethe fraction of insulin receptorswith bound
(Figure15-5). Binding of unlabeledcompetitor blocks bind- insulin-[Rl-]/(lRLl +[R])-at equilibrium as 0.0344; that
ing of the radioactive ligand to the receptor; the concentra- is, about 3 percent of the total insulin receptors will be
tion of competitor required to inhibit binding of half the ra- bound with insulin. If the insulin concentration rises five-
dioactive ligand approximates the K6 value for binding of fold to 2.5 x L0-11 M, the number of receptor-hormone
the competitor to the receptor. It is possible to accurately complexes will rise proportionately, almost fivefold, so
measurethe amount of the high-affinity ligand bound in this that about 15 percent of the total receptors will have
assay because little dissociatesduring the experimental bound insulin. If the extent of the inducedcellular response

S T U D Y I N GC E L L - S U R F A CREE C E P T O R S 629
 100 OH
l*
cH -cH2 -
I NH2- CH3

P80 (EP)
Epinephrine

EOU
c
o
?'
cH-cH2-NH2-CH
. ?'.
(lPl
lsoproterenol CHs
E40
c
CH,
il- oH gH.
E20 l*l
cH2-cH -cH2 -NH2-CH
I
CHs
0-
10-8 10-6 1O-4 Alprenolol(AP)
Competitor (M)
concentration
A EXPERIMENTAL FTGURE 15-5 Forlow-affinityligands, inhibition
of ['H]alprenolol
binding versus epinephrine
or isoproterenol
bindingcan be detectedin competitionassays.In thisexample, concentration,suchasshownhere,theconcentration of the competitor
the synthetic ligandalprenolol, whichbindswith highaffinityto the thatinhibits
alprenololbindingby 50 percent approximates theK6
e p i n e p h r i nr e c e p t oorn l i v e rc e l l s( K a= 3 x 1 O - eM ) ,i s u s e dt o valuefor competitor binding.Notethat the concentrations of
detectthe bindingof two low-affinity ligands,the naturalhormone competitorsareplottedon a logarithmic scaleTheK6for bindingof
epinephrine (EP) anda synthetic ligandcalledisoproterenol (lp)Assays epinephrine on livercellsisonly-5 x 1O-sM and
to itsreceptor
areperformed asdescribed in Figure 15-4butwith a constant amount wouldnot be measurable by a directbindingassay with
of [3H]alprenolol to whichincreasing amounts of unlabeled epinephrine [3H]epinephrine. TheK6for bindingof isoproterenol,whichinduces
or isoproterenol areadded.At eachcompetitor concentration, the thenormalcellular response,ismorethantenfoldlower
amountof boundlabeled alprenolol isdetermined In a plotof the

parallels the number of hormone-receptor complexes, imal binding versus ligand concentration differs from a
[RL], as is often the case,then the cellular responsesalso plot of the percentageof maximal cellular responseversus
will increaseby about fivefold. ligand concentration.
On the other hand, supposethat the normal concentra-
tion of insulin in the blood is the same as the K6 value of
1.0 P h y s i o l o g i c ar el s p o n s e
1.4 x 10-10 M; in this case,50 percent of the toial recep-
!L"X,
tors would have a bound insulin. A fivefold increasein the
insulin concentrationto 7 x 10-10 M would result only in 0.8
-=
G6

a 66 percent increase in the fraction of receptors with xL

0.6 Fractionof surfacereceptors
bound insulin (to 83 percent bound). Thus, in order for a OL
with boundligand
rise in hormone concentrationto causea proportional in- bf
h=

crease in the fraction of receptors with bound ligand, the 0.4 Ligandconcentration
for 50Yophysiologicalresponse
normal concentration of the hormone must be well below
the Ka value. LL 0.2
K6for ligand binding
In generalthe maximal cellular responseto a particular
ll
ligand is induced when much less than 100 percent of its
01234
receptorsare bound to the ligand. This phenomenoncan be
Relative
concentration
of ligand
revealed by determining the extent of the responseand of
receptor-ligand binding at different concenrrations of lig- EXPERIMENTAL FIGURE 15-6 The maximalphysiological
and (Figure 15-6). For example, a typical red blood (ery- responseto an externalsignaloccurswhen only a fraction of
throid) progenitor cell has -1000 surface receptors for the receptorsare occupiedby ligand.Forsignaling pathways
erythropoietin, the protein hormone that induces these t h a te x h i b itth i sb e h a v i opr ,l o t so f t h e e x t e not f l i g a n db i n d i n gt o
cells to proliferate and differentiate into red blood cells. the receptor and of physiological response at differentligand
concentrations differ.In the exampleshownhere,50 percentof
Becauseonly 100 of these receptors need to bind erythro-
the maximal physiological response isinduced at a ligandconcentration
poietin to induce division of a progenitor cell, the ligand
at whichonly 18 percentof the receptors areoccupiedLikewise,
concentration neededto induce 50 percent of the maximal
8 0 p e r c e not f t h e m a x i m ar le s p o n si sei n d u c e w d h e nt h e l i g a n d
cellular responseis proportionally lower than the K6 value concentration equalsthe K6value,at which50 percentof the
for binding. In such cases,a plot of the percentageof max- receptors areoccupied

630 C H A P T E R1 5 | C E L LS I G N A L I N Gl : S I G N A LT R A N S D U C T I O A
NN D S H O R T - T E R M
CELLULAR
RESpONSEs
Sensitivityof a Cellto ExternalSignals
ls Determinedby the Numberof Surface
Receptorsand Their Affinity for Ligand
Becausethe cellular responseto a particular signaling mole-
cule dependson the number of receptor-ligand complexes,
the fewer receptors present on the surface of a cell, the less
sensitiuethe cell is to that ligand. As a consequence,a
higher ligand concentration is necessaryto induce the
physiological response than would be the case if more
receptorswere present
To illustrate the important relationship betweenreceptor
number and hormone sensitivity,let's extend our example of
a typical erythroid progenitor cell. The K6 for binding of ery-
10 M. As we
thropoietin (Epo) to its receptor is about 10
noted above, only 10 percent of the :1000 erythropoietin
receptorson the surfaceof a cell must be bound to ligand to
induce the maximal cellular response.'Wecan determine the
ligand concentration, [L], needed to induce the maximal
responseby rewriting Equation 15-2 as follows:
(15-3)
where R1 : [R] + [RL], the total number of receptorsper
cell. If the total number of Epo receptorsper cell, R1, is 1000,
K6 is 10-10 M, and tRLl is 100 (the number of Epo-occupied
receptorsneededto induce the maximal response),then an
11M
Epo concentration([L]) of 1.1 x 10 will elicit the max-
imal response.If the total number of Epo receptors (R1) is
reducedto 200/cell,then a ninefold higher Epo concentration
(10-to M) is required to occupy 100 receptorsand induce the
maximal response.Clearly, therefore, a cell's sensitivity to a
hormone is heavily influencedby the number of receptorsfor
that hormone that are presentas well as the Kd.
Epithelial growth factor (EGF), as its name implies,
stimulates the proliferation of many types of epithe-
lial cells, including those that line the ducts of the mam-
mary gland. In about 25 percent of breast cancers the
tumor cells produce elevated levels of one particular EGF
receptor called HERZ. The overproduction of HER2
makes the cells hypersensitiveto ambient levels of EGF
that normally are too low to stimulate cell proliferation; as
a consequencegrowth of these tumor cells is inappropri-
ately stimulated by EGF. Understanding of the role of
HER2 in certain breast cancersled to development of mon-
oclonal antibodies that bind HER2, and thereby block
binding of EGF; these antibodies have proven useful in
treatment of thesebreastcancerpatients.I
The HER2-breast cancer connection vividly demon-
stratesthat regulation of the number of receptorsfor a given
signaling molecule expressedby a cell plays a key role in di-
recting physiological and developmentalevents.Suchregula-
tion can occur at the levelsof transcription, translation, and
post-translational processing,or by controlling the rate of
receptor degradation.Alternatively, endocytosisof receptors
on the cell surfacecan sufficiently reducethe number present
to terminate the usual cellular response at the prevailing
signal concentration. As we discussin later sections,other
mechanismscan reduce a receptor's affinity for ligand, and
thus reduce the cell's responseto a given concentration of
ligand. Thus reduction in a cell's sensitivity to a particular
ligand, calleddesensitization,can result from various mech-
anisms and is critical to the ability of cells to respond appro-
priately to external signals.
ReceptorsCan Be Purifiedby Affinity
Techniques
Becauseof their low abundancespecialtechniquesare neces-
sary to isolate and purify receptors. Cell-surfacereceptors
can be identified and followed through isolation procedures
by affinity labeling.In this technique,cellsare mixed with an
excessof a radiolabeled ligand for the receptor of interest.
After unbound ligand is washed away the cells are treated
with a chemical agent that covalently crosslinks the bound
labeled ligand molecules to receptors on the cell surface.
Once a radiolabeled ligand is covalently cross-linked to its
receptor,it remains bound even in the presenceof detergents
and other denaturing agents that are used to solubilize re-
ceptor proteins from the cell membrane. The labeled ligand
provides a means for detectingthe receptor during purifica-
tion procedures.
Another technique often used in purifying cell-surface
receptorsthat retain their ligand-binding ability when solu-
bilized by detergentsis similar to affinity chromatography
using antibodies (seeFigure 3-37c). To purify a receptor by
this technique, a ligand for the receptor of interest, rather
than an antibody, is chemically linked to the beads used to
form a column. A crude, detergent-solubilizedpreparation
of membrane proteins is passedthrough the column; only
the receptor binds, while other proteins are washed away.
Passageof an excessof the soluble ligand through the col-
umn causesthe bound receptor to be displaced from the
beadsand eluted from the column. In some cases'a receptor
can be purified as much as 100,000-fold in a single affinity
chromatographic step.
ReceptorsAre Frequently Expressed
from ClonedGenes
Once the amino acid sequenceof a purified receptor has
been determined its gene can be cloned. A functional ex-
pression assay of the cloned cDNA in a mammalian cell
that normally lacks the encoded receptor can provide
definitive proof that the proper protein indeed has been
obtained (Figure 15-7). Suchexpressionassaysalso permit
investigators to study the effects of mutating specific
amino acids on ligand binding or on "downstream" signal
transduction, thereby pinpointing the receptor amino
acids responsiblefor interacting with the ligand or with
c r i t i c a l s i g n a l - t r a n s d u c t i opnr o t e l n s .
S T U D Y I N GC E L L - S U R F A CREE C E P T O R S 631
 clone encoding the receptor is identified, the sequenceof the
$ Receotorfor cDNA can be determinedand that of the receptorprotein de-
Tf_ ..
|( ilgano orner duced from the cDNA sequence.Cells overexpressingthe re-
: than X
ii
ceptor protein can be usedto purify large amounts of the pro-
,l tein, which can be used to determine its three-dimensional
.t,
{ iI structure.This structural information can provide additional
! insightsinto the mechanismsby which the receptorfunctions,
as well as suggestinghow new types of drugs might interact
L i g a n dX
with the receptor to treat diseases.
Genomic studies coupled with functional expressionas-
No binding of X; no cellular response
saysare now being used to identify genesfor previously un-
known receptors. In this approach, stored DNA sequences
are analyzed for similarities with sequencesknown to en-
code receptor proteins (Chapter 6). Any putative receptor
genesthat are identified in such a searchthen can be tested
for their ability to bind a signaling molecule or induce a re-
sponsein cultured cells by a functional expressionassay.

Binding of X; normal cellular response
A EXPERIMENTAL FIGURE 15-7 Functionalexpression assay
can identify a cDNAencodinga cell-surfacereceptor.Targetcells
lackingreceptors for a particular
ligand(X)arestablytransfectedwith
a cDNAexpression vectorencoding the receptor
Thedesignof the
expressionvectorpermits selection
of transformedcellsfromthose
thatdo not incorporate thevectorintotheirgenome(seeFigure
5-32b)Providing
thatthesecellsalready express
allthe relevant
signal-transductionproteins,
thetransfected
cellsexhibitthe normal
cellular
response to X if the cDNAin factencodes thef unctional
receptor.
Cell-surfacereceptors for many signaling molecules are
presentin such small amounts that they cannot be purified by
affinity chromatographyand other conventional biochemical
techniques.Theselow-abundancereceptor proreins can now
be identified and cloned by various recombinant DNA tech-
niques, eliminating the need to isolate and purify them from
cell extracts.In one technique,a library of cloned cDNAs pre-
pared from the entire nRNA extracted from cells that produce
the receptor is inserted into expression vectors by techniques
describedin Chapter 5. The recombinanr vectors then are
transfected into cells that normally do not synthesizethe
receptorof interest(seeFigure 15-7). Only the very few trans-
fected cells that contain the cDNA encoding the desiredre-
ceptor synthesizeit; other transfectedcellsproduce irrelevant
proteins. The rare cellsexpressingthe desiredreceptorcan be
detectedand purified by various techniquessuch as fluores-
cence-activatedcell sorting using a fluorescent-labeledligand
for the receptor of interest (seeFigure 9-28). Once a cDNA
CharacterizingCell-SurfaceReceptors
r Receptorsbind ligandswith considerablespecificity,which is
determinedby noncovalent interactions betweena ligand and
specificamino acids in the receptorprotein (seeFigure 15-3).
r The concentration of ligand at which half its receprors
are occupied, the K4, can be determined experimentally
and is a measureof the affinity of the receptor for the lig-
and (seeFigure 15-4).
r The maximal responseof a cell to a particular ligand
generally occurs at ligand concentrationsat which most of
its receptorsare still not occupied(seeFigure 15-6).
r Becausethe amount of a particular receptor expressedis
generallyquite low (rangingfrom -1000 to 50,000 mole-
cules per cell), biochemical purification may not be feasi-
ble. Genes encoding low-abundance receptors for specific
ligands often can be isolated from cDNA libraries rrans-
fected into cultured cells.
r Functional expression assayscan determine if a cDNA
encodesa particular receptor and are useful in studying the
effects on receptor function of specific mutarions in irs
sequence(seeFigure 15-7).
HighlyConserved Components
of Intracellular
Signal-Transduction
Pathways
External signals induce two major types of cellular re-
sponses:( 1 ) changesin the activity or function of specificen-
zymes and other proteins that pre-exist in the cell, and (2)
changesin the amounts of specific proteins produced by a
cell, most commonly by modification of transcription factors
that stimulate or repressgene expression (seeFigure 15-1,
step Et). In general, the first type of responseoccurs more
rapidly than the second. Signaling from G protein-coupled
receptors, described in detail later in this chapter. often

632 CHAPTER
15 I C E L LS I G N A L I N G
I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A RR E s P o N s E s
results in changesin the activity of pre-existingproteins, al-
though activation of these receptors on some cells also in-
duceschangesin geneexpression.Other classesof receptors
operate primarily but not exclusively to modulate gene ex-
pression. Transcription factors activated in the cytosol by
thesepathways move into the nucleus,where they stimulate
(or occasionally repress) transcription of specific target
genes.'Weconsider thesesignalingpathways, which regulate
transcription of many genesessentialfor cell division and for
many cell differentiation processes,in the following chapter.
Several intracellular proteins or small molecules are em-
ployed in a variety of signal-transductionpathways. These in-
clude cytosolic enzymesthat add or remove phosphate groups
from specifictarget proteins. Ligand binding to a receptor acti-
vates or inhibits these enzymes,whose action in turn activates
or inhibits the function of their target proteins. G proteins,
another component of many signal-transductionpathways,
shuttle betweena statewith a bound GTP that is capableof ac-
tivating other proteins and a statewith a bound GDP that is in-
active. A number of small molecules (e.g., Ca2* and cyclic
AMP) are also frequently used in intracellular signal-transduc-
tion pathways: A rise in the concentration of one of thesemol-
eculesresults in its binding to an intracellular target protein,
causing a conformational changein the protein that modulates
(a) GTP-bound"on" state
Gly-60 Thr-35
Switch ll
Gly-60
Switch I
A FIGURE 15-8 Switchingmechanism for G proteins.Theability
with otherproteins
of a G proteinto interact andthustransduce a conformation,
signaldiffersin the GTP-bound "on" stateandGDP-bound "off"
state(a)Inthe active"on" state.two domains, termedswitchI
(green)andswitchll (blue), areboundto theterminal1 phosphate
of GTP through interactions
with the backboneamide groupsof a
conserved threonine andglycineresidue(b)Releaseof the1
D O MP O NE N T SO F I N T R A C E L L U L ASRI G N A L - T R A N S D U C T I O
H I G HL Y C O N S E R V E C
its function. Here we review the basicproperties of theseintra-
cellular signal-transducingmolecules.The rules and exceptions
that govern how they are usedin particular signaling pathways
are developedfurther in subsequentsectionsof this chapter.
GTP-BindingProteinsAre FrequentlyUsed
As On/Off Switches
We introduced the large group of intracellular switch pro-
teins that form the GTPase superfamily in Chapter 3' These
guanine nucleotide-binding proteins are turned "on" when
they bind GTP and turned "off" when the GTP is hy-
drolyzed to GDP (seeFigure 3-32). Signal-inducedconver-
sion of the inactive to active state is mediated by a guanine
nucleotide-exchangefactor (GEF), which causesreleaseof
GDP from the switch protein. Subsequentbinding of GTP' fa-
vored by its high intracellular concentration relative to its
binding affinity, induces a conformational change in at least
two highly conserved segmentsof the protein' termed switch
I and switch II, allowing the protein to bind to and activate
other downstream signaling proteins (Figure 15-8). The in-
trinsic GTPase activity of the protein then hydrolyzes the
bound GTP to GDP and P1,thus changingthe conformation
of switch I and switch II from the active form back to the
(b) GDP-bound"off" state
GDP
phosphate by GTPase-catalyzed causes
hydrolysis switchI andswitch
ll to relaxintoa different the inactive"off" stateThe
ribbonmodels shownhererepresent both conformations of Ras,a
monomeric G protein A similarspring-loadedmechanism switches
thectsubunitin trimeric G proteinsbetween the activeandinactive
conformations by movement of threeswitchsegmentsfAdapted from
I Vetter andA Wittinghofel2001,Scrence29411299 |
PA
NT H W A Y S 633
inactiveform. The rate of GTP hydrolysisregulatesthe length
of time the switch protein remainsin the activeconformation
and able to signal downstream: The slower the rate of GTP
hydrolysis, the longer the protein remains in the active state.
The rate of GTP hydrolysis is often modulated by other pro-
teins. For instance,both GTPase-actiuatingproteins (GAP)
and regulator of G protein signaling (RGS) proteins acceler-
ate GTP hydrolysis.Many regulatorsof G protein activity are
themselvescontrolled by extracellular signals.
Two large classesof GTPaseswitch proteins are used in
signaling. Trimeric (large) G proteins, ai noted already, di-
rectly bind to and are activated by certain cell-surfacerecep-
tors. The activated receptor functions as a GEF and triggers
releaseof GDP and binding of GTP. Monomeric (small) G
proteins, such as Ras and various Ras-likeproteins,play cru-
cial roles in many parhwaysthat regulatecell division and cell
motility; theseG proteinsfrequentlyundergo activaringmuta-
tions in cancers. Ras is linked indirectly to receptors via
adapterproteins and GEF proteins discussedin the next chap-
ter. All G switch proteins contain regions like switch I and
switch II that modulate the activity of specificeffector proteins
by direct protein-protein interactions when the G protein is
bound to GTP. Despite these similarities, the nvo classesof
GTP-binding proteins are regulatedin very different ways.
ProteinKinasesand Phosphatases
are Employed
i n V i r t u a l l yA l l S i g n a l i n gP a t h w a y s
Activation of virtually all cell-surfacereceptorsleads directly
or indirectly to changesin protein phosphorylation through
the activation of protein kinases,which add phosphategroups
to specific residues,or protein phosphatases,which remove
phosphategroups. Animal cells contain two types of protein
kinases:those that add phospharero rhe hydroxyl grorrp ort
tyrosine residues and those that add phosphate to the hy-
droxyl group on serineor threonine (or both) residues.phos-
phatasescan act in concertwith kinasesto switch the function
of various proteins on or off (seeFigure 3-33). At last count
the human genome encodesabout 500 protein kinases and
100 different phosphatases.In some signalingpathways, rhe
receptor itself possesses intrinsic kinase or phosphataseactiv-
ity; in other pathways, the receptor interacts with cytosolic or
membrane-associated kinases.Importantly the activity of all
kinasesis highly regulated.Commonly the catalytic activity of
a protein kinase itself is modulated by phosphorylation by
other kinases, by direct binding to other proteins or by
changesin the levels of various small intracellular signaling
molecules.The resultingcascadesof kinaseactiviry .o--
mon feature of many signalingpathways. "r. "
In general, each protein kinase phosphorylates specific
residuesin a set of target proteins whose patterns of expres-
sion generally differ in different cell types.Many proteins are
substratesfor multiple kinaseseachof which phosphorylates
differentamino acids.Each phosphorylationevenican mod-
ify the activity of a particular target protein in different
ways, some activating its function, others inhibiting it. An
example we encounter later is glycogen phosphorylase
kinase, a key regulatory enzymein glucosemetabolism.
634 CHAPTER
15 | C E L LS I G N A L I N G
l : 5 I G N A LT R A N S D U C T I OANN D S H O R T - T E RC
The activity of all protein kinasesis opposed by the ac-
tivity of protein phosphatases,some of which are themselves
regulated by extracellular signals.Thus the activity of a pro,
tein in a cell can be a complex function of the activitiesof the
usually multiple kinasesand phosphatasesthat act on it. Sev-
eral examplesof this phenomenon occur in regulation of the
cell cycle and are describedin Chapter 20.
S e c o n dM e s s e n g e rC
s a r r ya n d A m p l i f y S i g n a l s
from Many Receptors
The binding of ligands ("first messengers")to many cell-
surfacereceptorsleads to a short-lived increase(or decrease)
in the concentration of certain low-molecular-weight intra-
cellular signaling molecules termed second messengers.
These,in turn, bind to other proteins modifying their activity.
One secondmessengerusedin virtually all metazoancells
is Ca2* ions. I(e noted in Chapter 11 that the concentration
of Ca2* free in the cytosol is kept very low (-10-7 M) by
ATP-poweredpumps that continually transport Ca2* out of
the cell or into the endoplasmicreticulum (ER). The cytosolic
Ca2* level can increasi from l0- ro 100-fold by a signal-
induced releaseof Ca2* from ER srores or by its import
through calcium channelsfrom the extracellularenvironment.
In muscle,a signal-inducedrise in cytosolicCa2* triggerscon-
traction (see Figure 17-33). In endocrine cells, a similar
increasein Ca"* inducesexocytosisof secretoryvesiclescon-
taining hormones.In nerve cells,a Ca2* increaseleadsto the
exocytosis of neurotransmitter-containing vesicles (see
Chapt^er23).ln all cells this rise in cytosolic Ca2* is sensed
by Ca'*-binding proteins, particularly those of the EF hand
family, such as calmodulin, all of which contain the helix-
loop-helix motif (seeFigure 3-9a). The binding of Caz* to
calmodulin and other EF hand proteins causesa conforma-
tional changethat permits the protein to bind various target
proteins, thereby switching their activities on or off (seeFig-
u r e3 - 3 1 ) .
Another nearly universally used second messengeris
cyclic AMP (cAMP). In many eukaryoric cells, a rise in
cAMP triggers activation of a particular protein kinase that
in turn inducesvarious changesin cell metabolism in differ-
ent types of cells. In other cells, cAMP regulatesthe activity
of certain ion channels.The structures of cAMP and three
other common secondmessengersare shown in Figure 15-9.
In later sectionsof this chapter,we examine the specificroles
of secondmessengers in signalingpathways activated by var-
ious G protein-coupled receptors.
Becausesecondmessengerssuch as Ca2* and cAMP dif-
fuse through the cytosol much faster than do proteins, they
are employed in pathways where the downstream target is
located in an intracellular particle or organelle (such as a se-
cretory vesicle)distant from the plasma membrane receptor.
Another advantageof secondmessengersis that they facili-
tate amplification of an extracellular signal. Activation of a
single cell-surfacereceptor moleculecan result in an increase
in perhapsthousandsof cAMP moleculesor Ca2* ions in the
cytosol. Each ofthese, in turn, by activating its target protein
affects the activity of multiple downstream proteins.
ME L L U L A R E S p O N S E s
llll+
FocusAnimation:secondMessengers
in signal Pathways
NH, o

(*
N -A*
ill
-2\N'/" 5
o-cH2 c H 3 - ( c H 2 ) , - ?o - 4 ,
ol
cH3-(cH2)"-q-o-cH
tl
o
3
o:P-o oH Fatty acyl groups
cH2oH
I I
o- o Glycerol
lnositol
3',5'-CyclicAMP 3',5'-CyclicGMP 1,2-Diacylglycerol 1,4,5-trisphosphate
(cAMP) (cGMPl (DAG} (tP3)

A c t i v a t e s p r o t e i n k i n a s eA ( P K A ) Activates protein kinaseG (PKG) proteinkinase

Activates C OpensCa2+ in
channels
and ooens cation channelsin (PKC) reticulum
theendoPlasmic
rod cells

A FIGURE 15-9 Fourcommonintracellular secondmessengers. phosphatidylinositol

membrane-bound alsoact
derivatives
Themajordirecteffector effectsof eachcompoundareindicated messenqers.
assecond
belowitsstructural
formula.Calcium ion(Ca2*)andseveral

pathways. GPCR pathways usually have short-term effects in

Highly Conserved Components of Intracellular
Signal-TransductionPathways
r Protein kinasesand phosphatasesare employed in virtu-
ally all signaling pathways; their activities are highly regu-
lated by receptors.
r Other conserved proteins that act in many signal-
transduction pathways include monomeric and trimeric G
switch proteins(seeFigure 15-8).
r The cytosolic concentrationsof secondmessengers, such
as Ca2* and cAMP, increase or occasionally decreasein
responseto binding of ligand to cell-surfacereceptors(see
Figure 15-9).Thesenonprotein, low-molecular-weightintra-
cellular signalingmolecules,in turn, regulatethe activitiesof
enzymesand nonenzymaticproteins in signalingpathways.
GeneralElementsof
G Protein-CoupledReceptorSystems
As noted above, perhaps the most numerous class of
receptors-found in organisms from yeast to man-are the G
proteircoupled receptors (GPCRs). Receptor activation by
ligand binding triggers activation of the coupled trimeric G
protein, which interacts with downstream signal-transduction
proteins. All GPCR signaling pathways share the following
common elements: (1) a receptor that contains seven
membrane-spanningdomains; (2) a coupled trimeric G pro-
tein, which functions as a switch by cycling between actrve
and inactive forms; (3) a membrane-boundeffector protein;
and (4) feedbackregulation and desensitizationof the signal-
ing pathway. A secondmessengeralso occursin many GPCR
pathways.Thesecomponentsare modular and can be "mixed
and matched" to achievean astonishinqnumber of different
the cell by quickly modifying existing proteins' either enzymes
or ion channels.Thus thesepathways allow cells to respond
rapidly to a variety of signals,whether they be environmental
stimuli such as light or hormonal stimuli such as epinephrine.
In this section, we first consider the general features of
GPCR signaltransductionand then discusseach of the mem-
brane-bound components in turn: the receptor,the trimeric G
protein, and the effectorproteins.In Section15.5-15'7 we de-
scribe GPCR pathways that involve several different effector
proteins. The short-term signalingdescribedin thesesections
of this chapter often can be turned into long-term signaling in-
volving changesin transcription and, as a consequence,cell
differentiation,as describedin Chapter 16.
G Protein-CoupledReceptorsAre a Large
and DiverseFamilywith a CommonStructure
and Function
All G protein-<oupled receptors have the same orientation in
the membrane and contain seventransmembranect-helicalre-
gions (H1-H7), four extracellular segments,and four cytosolic
segments(Figure 15-10). Invariably the N-terminus is on the
exoplasmic face and the C-terminus is on the cytosolic face of
the plasma membrane.The carboxyl-terminalsegment(C4)'
the C3 loop, and, in some receptors'also the C2 loop are in-
volved in interactions with a coupled trimeric G protein. There
are many subfamilies of G protein-coupled receptors that are
conservedthrough evolution; membersof thesesubfamiliesare
especiallysimilar in amino acid sequenceand structure.
The exterior surface of all G protein-coupled receptors
mainly consistsof hydrophobic amino acidsthat allow the pro-
tein to be stably anchored in the hydrophobic core of the
plasma membrane. One G protein{oupled receptorswhose
structure is known in molecular detail is rhodopsin, which
is composed of the protein opsin covalently bound to the

O F G P R O T E I N - C O U P L ERDE C E P T O S
G E N E R A LE L E M E N T S RY S T E M S 635
 epinephrineor hydrophobic like many odorants and retinal.
Figure 15-11 depictsa model of the complex formed betvveen
Exterior the B2-adrenergicreceptor and the hormone epinephrine. As
with retinal, epinephrineis thought to bind (in this case,non-
covalently)in the middle of the plane of the membrane,inter-
actingwith amino acidsin the interior-facingside of severalof
the membrane-spanning a helices.
Cytosol As an exampleof the diversity and functionality of GPCR
coo-
proteins, we will considerthe different G protein-coupled re-
G protein ceptors for epinephrine that are found in different types of
i nteraction mammalian cells. The hormone epinephrine is particularly
FIGURE 15-10Generalstructureof G protein-coupled important in mediating the body's responseto stress(fight-
receptors. All receptors of thistypehavethe sameorientation in or-flight response),such as fear or heavy exercise,when tis-
the membrane andcontainseventransmembrane o-helical regions suesmay have an increasedneed to catabolizeglucose and
( H 1 - H 7 f)o, u re x t r a c e l l usl a
e rg m e n(t E
s 1 - E 4a) n
, df o u rc y t o s o l i c fatty acids to produce ATP. These principal metabolic fuels
segments (C1-C4)Thecarboxyl-terminal segment (C4),the C3 loop, can be supplied to the blood in secondsby the rapid break-
and,in somereceptors, alsothe C2 loopareinvolved in interactions down of glycogen to glucosein the liver and of triacylglyc-
with a coupled trimeric G protein erols to fatty acids in adipose(fat) cells.
In mammals, the liberation of glucoseand fatty acids
can be triggered by binding of epinephrine (or its derivative
Iight-absorbing visual pigment 11-cls-retinal.In rhodopsin, norepinephrine) to B-adrenergicreceptors on the surface of
the sevenmembrane-embedded o helicesof opsin completely hepatic (liver) and adiposecells. Epinephrine bound to B-
surround a central segment to which retinal is covalently adrenergicreceptorson heart musclecellsincreasesthe con-
bound. In this case, binding of the ligand, retinal, does not traction rate, which increasesthe blood supply to the tis-
trigger a conformational change in the receptor; rather, ab- sues.In contrast, epinephrinestimulation of B-adrenergic
sorption of a quantum of light by the bound retinal inducesa receptors on smooth muscle cells of the intestrne causes
changein opsin conformation that activatesit, as we discuss them to relax. Another type of epinephrine receptor,the cv-
in Section15.5. adrenergic receptor, is found on smooth muscle cells lining
The amino acids that form the interior of different G pro- the blood vesselsin the intestinal tract, skin, and kidneys.
tein-coupledreceptorsare diverse,allowing differenrreceprors Binding of epinephrineto thesereceptorscausesthe arteries
to bind very different small molecules,be they hydrophilic like to constrict,cutting off circulation to theseperipheral organs.

View from external surfaceface

Exterior

Membrane

Gytosol

H e l i x5 H e l i x6

A FIGURE 15-11 Structuralmodelof complexformed between groupforms an ionicbond with the carboxylate sidechainof
epinephrineand the B2-adrenergic '1
receptor.(left)sideview.The a s p a r t a t e 1 3 ( D 1 1 3i n
) H 3 ; t h e c a t e c h orli n ge n g a g e si n h y d r o p h o b i c
approximate location
of the membrane phospholipidbilayer is interactions with phenylalanine 290 (F2e0) in H6; and two hydroxyl
indicatedThethreect-heltcesthatparticipate
in epinephrine binding groupson the catecholring hydrogen-bond to the hydroxylgroupsin
arecoloredred(Helix5),green(Helix3),andpurple(Helix6) (right) threeserineresidues (5203, 5204and S2o7) in H5 iseep L Freddolino et
viewfromexternal face.Epinephrineatomsarecolored grey(C), al ,2004,Proc Nat'lAcad SciUSA101:2736, adaptedfrom modelprovided
red(O)andpurple(N) Epinephrine interacts
with several residuesin byW A Goddardl
the receptor
thatarewithinthe planeof the membraneltsamino

636 CHAPTER
15 | C E L LS I G N A L t N G AN
t : S T G N A TL R A N S D U C T T O N DS H O R T - T E C
REML L U L ARRE S P O N S E S
 Effect on Although all ct- and B-adrenergicreceptorsbind epineph-
adenylyl cyclase rine, different receptors are coupled to different G proteins
that induce different downstream signaling pathways, Iead-
I n h i b i t s( b i n d sG " i ) ing to different cellular responses.Studies with chimeric
adrenergicreceptors,like those outlined in Figure 15-12' sug-
gest that the long C3 loop between ct helices5 and 5 is im-
portant for interactionsbetweena receptorand its coupled G
o,2-Adrenergicreceptor (wild typel
protein. PresumablS ligand binding causesthese helicesto
move relative to each other in a way that allows the loop to
bind and activatethe transducing G protein. Other evidence
Activates(binds G"") indicatesthat the C2 loop, joining helices3 and4, also con-
tributes to the interaction of somereceptorswith a G protein.

B2-Adrenergicreceptor (wild type)

G Protein-CoupledReceptorsActivate Exchange
of GTPfor GDPon the q.Subunit of a Trimeric
G Protein
Activates(bindsG"r) Trimeric G proteins contain three subunits designatedct, B, and
1. Both the G. and G, subunits are linked to the membrane by
covalently attached lipids. During intracellular signaling, the B
and 1 subunits remain bound together and are usually referred
Chimeric receptor 1
to as the Gs, subunit. In the resting state' when no ligand is
bound to the receptor,the Go subunit has a bound GDP and is
complexed with Gpr. Binding of a normal hormonal ligand
I n h i b i t s( b i n d sG " i ) (e.g.,epinephrine)or an agonist (e.g.,isoproterenol)to a G
protein{oupled receptor changesthe conformation of its cy-
tosol-facing loops and enablesthe receptor to bind to the G.
subunit (Figure15-13, stepsI and Z). This binding releases
Chimeric receptor 2
the bound GDP; thus the activatedligand-bound receptor func-
tions as a guanine nucleotide-exchangefactor (GEF) for the
G* subunit (stepB). Next GTP rapidly binds to the "empty"
guanine nucleotide site in the Go subunit, causing a change in
CONCLUSION
the conformation of its switch segments(seeFigure 15-8). These
changesweaken the binding of G* with both the receptor and
the Gs, subunit (step4).
y f G p r o t e i nb i n d i n g
R e g i o nd e t e r m i n i n gs p e c i f i c i t o In most cases,Go'GTl which remains anchored in the
( c o m p a r ec h i m e r a s1 a n d 2 )
membrane, then interacts with and activates an associated
effector protein, as depictedin Figure 15-13 (step E). In
A EXPERIMENTAL FIGURE 15-12Studieswith chimeric
some cases,G.'GTP inhibits the effector.Moreover, depend-
adrenergicreceptorsidentifythe long C3loop as criticalto
ing on the type of cell and G protein, the G9" subunit, freed
interactionwith G proteins.Xenopus oocytes weremicroinjected
from its a subunit, will sometimestransduce a signal by in-
withmRNAencoding a wild-type o2-adrenergic, B2-adrenergic, or
teractingwith an effectorprotein.
chimeric o-B receptors AlthoughXenopus oocytes do not normally
express adrenergic receptors, theydo express G proteins thatcancouple In any case,the active G.'GTP state is short-lived be-
to theforeignreceptors expressed on thesurface of microinjected cause the bound GTP is hydrolyzed to GDP in minutes,
oocytesTheadenylyl cyclase activity of the injected cellsin the presence catalyzed by the intrinsic GTPase activity of the G' sub-
of epinephrine agonists wasdetermined andindicated whether the unit (see Figure 15-13, step El). The conformation of the
expressed adrenergic receptor boundto thestimulatory (G*,)or Go then switches back to the inactive G*'GDP state,
inhibitory (G")typeof oocyteG proteinComparison of chimeric blocking any further activation of effector proteins' The
receptor 1,whichinteracts withG..,andchimeric receptor 2, which rate of GTP hydrolysis is further enhancedby binding of
interacts with G", showsthatthe G proteinspecificity isdetermined the G*'GTP complex to the effector; the effector thus
primarily bythesourceof thecytosol-facing C3 loop(yellow) between functions as a GTPase-activating protein (GAP). This
cthelices 5 and6. [See B Kobilka etal,1988, Sclence240:1310] mechanism significantly reducesthe duration of effector
activation and avoids a cellular overreaction. In many
These diverse effects of epinephrine help orchestrate inte- cases,a noneffector RGS protein also acceleratesGTP hy-
grated responsesthroughout the body all directed to a drolysis by the Go subunit, further reducing the time dur-
common end: supplying energy that can be used for the ing which the effector remains activated' The resulting
rapid movementof major locomotor musclesin responseto G..GDP quickly reassociateswith Gp" and the complex
bodily stress. becomesready to interact with an activated receptor and

O F G P R O T E I N - C O U P L ERDE C E P T O S
G E N E R A LE L E M E N T S RY S T E M S 637
 Signaling{ltlt
OverviewAnimation:Extracellular

I e i n d i n go f h o r m o n ei n d u c
a c o n f o r m a t i o n aclh a n g e
In receptor Hormone

p Activated receptor |
b i n d st o G " s u b u n i t
I
,+m
I
(

'

Bindingof GTPto G"

triggersdissociationof G,
boitr from the receptorant
from Gsn

FIGURE 15-13Generalmechanismof the activationof bindsto andactivatesan effectorprotein(stepS) Hydrolysis of GTP

effector proteinsassociatedwith G protein-<oupledreceptors. terminatessignaling
andleadsto reassembly of thetrimeric
G protein,
TheG* andG9,subunits of trimericG proteins
aretetheredto the returningthesystem state(step6) Binding
to the resting of another
membrane bycovalentlyattachedlipidmolecules(wigglyblacklines) ligandmoleculecauses repetitionof thecycleInsomepathways, the
Following ligandbinding, exchange of GDpwithGTBand proteinisactivated
effector bythefreeGs,subunit[After W Oldham
dissociationof the G proteinsubunits,
(stepsn-E), thefreeG* GTp andH Hamm, 2006,(QuaftRev. Biophys40:(tnpress)l

start the process all over again. Thus the GPCR signal-
transduction system contains a built-in feedback
mechanism that ensures the effector protein becomes
activated only for a few seconds or minutes following
receptor activation; continual activation of receptors via
Iigand binding is essentialfor prolonged activation of the
effector.
Early evidencesupporting the model shown in Figure
15-13 came from studieswith compounds that can bind to
Go subunits as well as GTP does, but cannot be
hydrolyzed by the intrinsic GTPase. In some of these
compounds, the P-O-P phosphodiesterlinrcageconnecr-
ing the B and 1 phosphatesof GTP is replaced by a non-
hydrolyzable P-CH2-P or P-NH-P linkage. Addition of
such a GTP analog to a plasma membrane prepararion in
the presence of the natural ligand or an agonist for a
particular receptor results in a much longer-lived activa-
tion of the associatedeffector protein than occurs with
GTP. In this experiment, once the nonhydrolyzable GTP
analog is exchangedfor GDP bound to Go, it remainsper-
manently bound to Go. Becausethe Go.analogcomplex is
as functional as the normal G*.GTP complex in activating
the effector protein, the effector remains permanently
actrve.

538 CHAPTER
15 I C E L LS I G N A L I N G
I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RC
ME L L U L A R E S P o N s E S
r=
ffi eodcast: by Fluorescence
Activationof G ProteinsMeasured (FRET)
EnergyTransfer
Resonance
(b)
cAMP
e

440nm 490nm light

Excitation I r r ' ,
(cyan) 440nm 15 30 45 60
O
T i m e( s )
A EXPERIMENTAL FIGURE 15-14Activationof G proteinsoccurs oI 527-nm(yellow) li9ht,characteristicof YFPHowever, if ligand
within seconds of ligandbindingin amoebacells.Intheamoeba bindingleads to dissociationof theG" andGu"subunits, then
Dictyostelium
discoideumcell,cAMPactsasan extracellular signaling fluorescence energytransfercannotoccur.In thiscase,irradiation of
moleculeandbindsto a G protein-coupled receptor;it isnota second cellsat 440nmcauses emission of 490-nmlight(cyan) characteristic
messenger Amoebacellsweretransfected with genesencoding two oI CFP(right)(b)Plotof the emission of yellowlight(527nm)from
fusionproteins:
a G* fusedto cyanfluorescentprotein (CFP),a mutant a single transfected amoeba cell before andafteraddition of
formof greenfluorescentprotein(GFp), anda Gsfusedto another extracellular AMP
cyclic (anows), the ligand for the G protein-<oupled
GFpvariant,yellowfluorescent (YFP)CFPnormally
protein fluoresces receptor in thesecells.Thedropin yellowfluorescence, whichresults
490-nmlight;yFB521-nm light (a)WhenCFpandYFParenearby, as fromthedissociation of theG*-CFP fusionprotein fromtheGpy-YFP
in theresting
G" Gp"complex, fluorescence
energytransfer canoccur fusionprotein, occurs withinseconds of cAMPaddition. [Adapted
betweenCFPandYFP(eft) Asaresult,irradiationofrestingcellswith fromCJanetopoulosetal ,2001,Science291:2408ll
440-nmlight(whichdirectly excitesCFPbutnotYFP) causes emission

GPCR-mediated dissociation of trimeric G proteins
recently has been detectedin living cells. These studieshave
exploited the phenomenon of fluorescenceenergy transfer,
which changesthe wavelength of emitted fluorescencewhen
two fluorescent proteins interact. Figure 15-14 shows how
this experimental approach has demonstrated the dissocia-
tion of the Go'Gs" complex within a few secondsof ligand
addition, providing further evidencefor the model of G protein
cycling.This generalexperimentalprotocol can be usedto fol-
low the formation and dissociationof other protein-protein
complexesin living cells.
Different G ProteinsAre Activated
by Different GPCRs and ln Turn Regulate
Different Effector Proteins
All effector proteins in GPCR pathways arc either
membrane-boundion channelsor enzymesthat catalyzefor-
mation of the secondmessengersshown in Figure 15-9. The
variations on the theme of GPCR signaling that we examine
in Sections1,5.5-1.5.7arise becausemultiple G proteins are
encoded eukaryotic genomes.At last count humans have
in
2L different Go subunits encoded by 16 genes, several of
which undergo alternative splicing; 6 Gp subunits; and 12
G" subunits. So far as is known, the different GB, subunits
function similarly.
Table 15-1 summarizesthe functions of the maior classes
of G proteins with different Go subunits. For example, the
different typesof epinephrinereceptorsmentionedpreviously
are coupled to different G proteins that influence effectors
differently,and thus have distinct effectson cell behavior in a
target cell. Both subtypesof B-adrenergicreceptors,termed
B1 and 82, arecoupled to a stimulatory G protein (G,) whose
alpha subunit (G.,) activatesa membrane-boundeffector en-
zyme called adenylyl cyclase. Once activated' this enzyme
catalyzessynthesisof the second messengercAMP. In con-
trast, the cr2-adrenergicreceptor is coupled to a Go; protein
that inhibits adenylyl cyclase,the sameeffector enzymeasso-
ciated with B-adrenergicreceptors.The Gooprotein, which is
coupled to the ct1-adrenergicreceptor' activates a different
effector enzyme)phospholipase C, that generatestwo other
secondmessengers(DAG and IP3)' Examples of signaling
pathways that use each of the G. proteins listed in Table 15-
1 are describedin the following three sections.
Some bacterial toxins contain a subunit that pene-
trates the plasma membrane of target mammalian
cellsand in the cytosol catalyzesa chemicalmodification of
Go proteins that prevents hydrolysis of bound GTP to
GDP. For example, toxins produced by the bacterium Vib-
rio cholera, which causes cholera' or certain strains of
E. coli modify the Go, protein in intestinal epithelialcells.

O F G P R O T E I N - C O U P L ERDE C E P T O S
G E N E R A LE L E M E N T S RY S T E M S 639
 G.CIASS ASS0CTATED
IFFECT0R 2NO
MESSTNGEB RTCEPT()R
EXAMPIIS

Go. Adenylyl cyclase cAMP (increased) (epinephrine)

B-Adrenergic receptor;
receptors for glucagon, serotonin, vasopressin

Goi Adenylyl cyclase cAMP (decreased) ct2-Adrenergic receptor

K* channel (Gp" Change in membrane potential Muscarinic acetylcholine receptor
activates effector)

Goolf Adenylyl cyclase cAMP (increased) Odorant receptors in nose

Goq Phospholipasec IP3, DAG (increased) cr1-Adrenergicrecepror

G*o PhospholipaseC IP3, DAG (increased) Acetylcholine receptor in endothelial cells

Go. cGMP phosphodiesterase cGMP (decreased) Rhodopsin (light receptor) in rod cells

"A given G. subclassmay be associatedwith more than one effector protein. To date, only one major G.. has been identified, but multiple G.o and
G"; proteins have been described.Effector proteins commonly are regulated by G* but in some casesby Gu" or the combined action of G* a.td ip".
IP3 : lnor',ot 1,4,5-trisphosphate;DAG : 1,2-diacylglycerol.
souRcEs: SeeL. Birnbaum eg 1992, Cell 77:1069; Z. Farfel et al., 1999, New Eng. Med. 340:1,012;and K. Pierce er al., 2002, Nature Reu. Mol. Cell
J.
Biol. 32639.

As a result, Go, remains in the active state, continuously
activating the effector adenylyl cyclase in the absence of
hormonal stimulation. The resultingexcessiverise in intra-
cellular CAMP leads to the loss of electrolytesand water
into the intestinal lumen, producing the watery diarrhea
characteristicof infection by thesebacteria.The toxin pro-
duced by Bordetella pertwssis,a bacterium that commonly
infects the respiratory tract and causeswhooping cough,
catalyzesa modification of Goi that prevents releaseof
bound GDP. As a result, G.; is locked in the inactive state,
reducing the inhibition of adenylyl cyclase.The resulting
increase in cAMP in epithelial cells of the arrways pro-
motes loss of fluids and electrolytesand mucus secretionI
General Elements of G Protein-Coupled
Receptor Systems
r G protein-coupled receptors are a large and diverse
family with a common structure of sevenmembrane-span-
ning cr helices.
r Trimeric G proteins transducesignalsfrom coupled cell-
surface receptors to associatedmembrane-bound effector
proteins, which are either enzymesthat form second mes-
sengers(e.g.,adenylylcyclase)or ion channelproteins (see
T a b l e1 5 - 1 ) .
r Signalsmost commonly are transducedby G., a GTpase
switch protein that alternatesbetween an active (,,on")
state with bound GTP and inacive ("off',) state with GDp.
The B and 1 subunits, which remain bound togethe! occa-
sionally transducesignals.
r Hormone-occupied receptors act as guanine
nucleotide-exchangefactors (GEFs) for Go proreins, car-
alyzingdissociationof GDP and enabling GTP to bind. The
resulting change in conformation of switch regions in Go
causesit to dissociatefrom the GB" subunit and the recep-
tor and interactwith an effectorprotein (seeFigure 15-13).
r Fluorescenceenergy transfer experiments demonstrate
receptor-mediateddissociationof coupled Go and Gs, sub-
units in living cells(seeFigure 15-14).
G Protein-Coupled
Receptors
ThatRegulatelon Channels
One of the simplest cellular responsesto a signal is the
opening of ion channels essentialfor transmission of nerve
impulses.Nerve impulsesare essentialto the sensoryper-
ception of environmental stimuli such as light and odors, to
transmission of information to and from the brain, and to
the stimulation of muscle movement. During transmission
of nerve impulses, the rapid opening and closing of ion
channelscauseschangesin the membranepotential. Many
neurotransmitter receptors are simply ligand-gated ion
channels,which open in responseto binding of a ligand.
Such receptorsinclude some types of glutamate,seroronin,
and acetylcholine receptors, including the acetylcholine re-
ceptor found at nerve-muscle synapses.Ligand-gated ion
channels that function as neurotransmitter receDrorsare
c o v e r e di n C h a p r e r2 3 .
Some neurotransmitter receptors, however, are G pro-
tein-coupled receptorswhose effector proteins are a Na* or
K* channel. Neurotransmitter bindins to these receDrors

640 CHAPTER
15 I C E L LS I G N A L I N G
I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A RR E s P o N S E S
causesthe associatedion channel to open or close,leading to
changesin the membrane potential. Still other neurotrans-
mitter receptors,as well as odorant receptorsin the noseand
photoreceptors in the eye, are G protein-coupled receptors
that indirectly modulate the activity of ion channelsvia the
action of secondmessengers. In this section,we considertwo
G protein-coupled receptorsthat illustrate the direct and in-
direct mechanismsfor regulating ion channels:the mus-
carinic acetylcholinereceptor of the heart and the light-
activatedrhodopsin of the eye.
AcetylcholineReceptorsin the Heart Muscle
Activate a G ProteinThat OpensK* Channels
Activation of muscarinic acetylcholine receptors in cardiac
muscle slows the rate of heart muscle contraction. (Because
muscarine,an acetylcholineanalog,also activatestheserecep-
tors, they are termed "muscarinic.") This type of acetylcholine
receptor is coupled to a Go; protein, and ligand binding leads
to opening of associatedK* channels(the effectorprotein) in
the plasmamembrane(seeTable 15-1). The subsequentefflux
of K* ions from the cytosol causesan increasein the magni-
tude of the usual inside-negativepotential acrossthe plasma
membrane that lasts for several seconds.This state of the
membrane, called hyperpolarization, reducesthe frequency
of muscle contraction. This effect can be determinedexperi-
mentally by direct addition of acetylcholineto isolatedheart
musclecells and measurementof the potential using a micro-
electrodeinsertedinto the cell (seeFigure 11-18).
Acetylcholine
Exterior
Cytosol
Active muscarinic
acetylcholinereceptor
A FIGURE15-15 Activation of the muscarinicacetylcholine
receptor and its effector K* channel in heart muscle.Bindingof
acetylcholine triggersactivationof the G. subunitand its dissociation (-60
l a y ( s e eF i g u r e1 5 - 1 3 )I.n t h i s
f r o m t h e G u , s u b u n i ti n t h e u s u a w
case,the releasedGu" subunit(ratherthan G"i GTP)bindsto and
opensthe associated effectorprotein,a K* channel The increasein
the membrane,which reducesthe
K* permeability hyperpolarizes
frequencyof heart musclecontractionThoughnot shown here,
activatronis terminatedwhen the GTPbound to G*| is hydrolyzed
GDPand G"i GDPrecombines with Gsn [SeeK Hoet al , 1993,Nafure
362:31. andY Kuboet al . 1993,Nature 362:127
As depicted in Figure 15-15, the signal from activated
muscarinic acetylcholine receptors is transduced to the
effectorprotein by the releasedGp" subunit rather than by
G..GTP. That GB" directly activates the K* channel was
demonstratedby patch-clampingexperiments,which can
measureion flow through a single ion channel in a small
patch of membrane (seeFigure 1'1-21'1. \fhen purified GB"
protein was added to the cytosolic face of a patch of heart
muscle plasma membrane, K* channels opened immedi-
ately, even in the absenceof acetylcholine or other neuro-
transmitters-clearly indicating that it is the Gg" protein
that is responsiblefor opening the effector K- channels
and not G.'GTP.
Rhodopsins
Light ActivatesGo.-Coupled
The human retina contains two types of photoreceptor cells,
rods and cones, which are the primary recipients of visual
stimulation. Cones are involved in color vision, while rods
are stimulated by weak light like moonlight over a range of
wavelengths.The photoreceptorssynapseon layer upon
Iayer of interneuronsthat are innervated by different combi-
nations of photoreceptor cells. All these signals are
processedand interpreted by the part of the brain called the
uisual cortex.
As noted aheady,rhodopsin consistsof the protein opsin'
which has the usual GPCR structure,covalently linked to the
light-absorbing pigment 11-cis-retinal.Rhodopsin is local-
ized to the thousand or so flattened membrane disks that
make up the outer segmentof rod cglls (Figure 15-16). A hu-
man rod cell contains about 4 x 10/ moleculesof rhodopsin.
Kt channel
The trimeric G protein coupled to rhodopsin, called trans-
++
ducin (Gr), contains the Go, subunit (seeTable 15-1); like
rhodopsin, G*. is found only in rod cells.
Upon absorption of a photon, the retinal moiety of
rhodopsin is immediatelyconvertedfrom the cis to the all-
trans isomeq causinga conformational changein the opsin
portion that activatesit (Figure 1'5-1'7).This is equivalent
to the conformational change that occurs upon Iigand
binding by other G protein-coupled receptors.Analogous
K* to other G protein-coupled receptors' the light-activated
form of rhodopsin interacts with and activatesa Go pro-
tein. in this caseGo,. Activated opsin is unstableand spon-
taneously dissociatesinto its component parts, releasing
oosin and all-trans-retinal,thereby terminating visual sig-
n"ling. In the dark, free all-trans-retinal is converted back
to LI-cis-retinal,which can then rebind to opsin' re-form-
ing rhodopsin.
In the dark, the membrane potential of a rod cell is
about -30 mV, considerablylessthan the resting potential
to -90 mV) typical of neurons and other electrically
active cells.This state of the membrane,called depolariza-
tion, causesrod cells in the dark to constantly secreteneu-
rotransmitters, and thus the neurons with which they
synapse are continually being stimulated. The depolarized
to stateof the plasmamembraneof restingrod cellsis due to the
presenceof a large number of open nonselectiueion chan-
| nels that admit Na+ and Ca2*, as well as K*. Absorption
ION CHANNELS
G P R O T E I N - C O U P L ERDE C E P T O RTSH A T R E G U L A T E 641
 (a)

Outer
segment

',4:--
-----
1"'"11,","n
rhodopsin

Mitocho

Rough
endoplasmic
reticulum

Inner Cilium
segment

Nucleus

S y n aptic
body
Humanrod cell o5Pm
' '
F I G U R1E5 - 1 6H u m a nr o d c e l l .( a )S c h e m a tdi ica g r a m
of an rodcellindicated by the bracket in (a),Thisregionincludes the
entirerodcell.At thesynaptic body,the rodcellformssynapses with j u n c t i o on f t h ei n n e r a n o
d u t e r s e g m e nltpsa r t ( b ) f r oRmG K e s s e l
oneor morebipolarinterneurons Rhodopsin, a light-sensitive G and R H Kardon,1979, Tissuesand Organs.A Text-Atlas
of ScanninqElectron
protein-coupled receptor,is located in theflattened membrane disks Microscopy,W H Freemanand Company.p 91 l
of the outersegment. (b)Electron micrograph of the regionof the

of light by rhodopsin leads to closing of these channels, A c t i v a t i o no f R h o d o p s i nI n d u c e sC l o s i n g

causing the membrane potential to become more jnsjde
negatrve.
The more photons absorbed by rhodopsin, rhe more
c h a n n e l sa r e c l o s e d ,t h e f e w e r N a * a n d C a 2 * i o n s c r o s s
the membrane from the outside, the more negative the
membrane potential becomes,and the lessneurotransmit-
ter is released.This change is transmitted to the brain
where it is perceivedas light. Remarkably,a singlephoton
absorbed by a resting rod cell produces a measurablere-
sponse,a more inside negative change in the membrane
potential of about 1 mV which in amphibians lasrsa sec-
ond or two. Humans are able to derecia flash of as few as
five photons.
o f c G M P - G a t eC d a t i o nC h a n n e l s
Opening of GPCR-stimulatedK* channelsin the heart re-
quiresonly an acrivatedG protein (seeFigure 15-15).In con-
trast, the closing of cation channelsin the rod-cell plasma
membranerequireschangesin the concentrationof the sec-
ond messengercyclic GMP, or cGMP (seeFigure 15-9). Rod
outer segmentscontain an unusually high concentration
(:0.07 mM) of cGMP, which is continuouslyformed from
GTP in a reaction catalyzedby guanylyl cyclase,which ap-
pears to be unaffectedby light. However, light absorption by
rhodopsin inducesactivation of a cGMP phosphodiesterase,
which hydrolyzescGMP to 5'-GMP. As a result,the cGMp

642 CHAPTER
1s I C E L LS I G N A L I N G
l : S I G N A LT R A N S D U C T T O
A N D S H O R T - T E RcME L L U L A R
RESpONSEs
 11-cis-Retinal
moietv < F I G U R 1E5 - 1 7T h e l i g h t - t r i g g e r esdt e p i n v i s i o n .T h el i g h t -
'1
a b s o r b i npgi g m e n1t - c i s - r e t i ni a
scl o v a l e n tbl yo u n dt o t h e a m i n o
r e s i d uien o p s i nt,h e p r o t e i np o r t i o no f
g r o u po f a l y s i n e
rhodopsinAbsorption of lightcauses rapidphotoisomerrzation of
the boundcrs-retinal to the all-trans isomer, formingthe unstable
intermediate meta-rhodopsin ll, or activated opsin,whichactivates
Lysineside chain
G, proteinsWithin seconds, all-trans-retinal dissociates fromopsin
+ and is converted by an enzymebackto the ctsisomer, whichthen
:N-(CH?)d- Opsin rebinds to anotheropsinmolecule. [See J Nathans, 1992, Biochemistry
I t- 31:4923 I
H H
Rhodopsin

As depictedin Figure 15-18, cGMP phosphodiesterase is

L i gh t - in d u c e d
isomerization
the effector protein for Go.. The free G*,'GTP complex that
( < 1 02 s ) is generatedafter light absorption by rhodopsin binds to the
two inhibitory 1 subunits of cGMP phosphodiesterase're-
leasingthe active catalytic ct and B subunits,which then con-
moietv
all-trans-Retinal vert cGMP to GMP. This is a cleat example of how signal-
tra N S induced removal of an inhibitor can quickly activate an
enzyme,a common mechanismin signalingpathways. A sin-
'11 gle moleculeof activatedopsin in the disk membranecan ac-
c:N-(cHz)o-E@ tivate 500 Go, molecules,each of which in turn activatesone
H cGMP phosphodiesterase,thereby amplifying the original
light signal early in this pathway.
Mefa-rhodopsin ll Direct support for the role of cGMP in rod-cell activity
(activatedopsin) has been obtained in patch-clamping studies using isolated
patches of rod outer-segmentplasma membrane, which
concentration decreasesupon illumination. The high level of contains abundant cGMP-gated cation channels. When
cGMP present in the dark acts to keep cGMP- gated cation cGMP is added to the cytosolic surface of these patches,
channelsopen; the light-induced decreasein cGMP leads to there is a rapid increasein the number of open ion channels;
channel closing, membrane hyperpolarization, and reduced cGMP binds directly to a site on the channel protein to keep
neurotransmitterrelease. them open,indicatingthat theseare nucleotide-gated channels.

Lig
Cytosol

Disk membrane

Disk lumen

E Inactive q TSI" Rod

PDE
plasma
membrane
l
/p\
GG
TD cGMP GMP
PP

\tr
\
\*o,
- )vo-
:;
cGMP L-------J
A FIGURE 15-18 Light-activated rhodopsinpathwayand the closingof cation Closed cGMP-gated
channels in rod cells.In dark-adapted a highlevelof cGMPkeeps
rodcells, nucleotide-gated ion channel

nonselectivecationchannels open Lightabsorptiongeneratesactivatedopsin,O* (steptr),

of GDPwithGTP(step High Dark,.li&pted
whichbindsinactive GDP-bound G*,protein
andmediates replacement
cytosolic ti€{f.
Z) ThefreeG*,GTPgenerated cGMPphosphodiesterase
thenactivates (PDE) by bindingto its cGMP : .'::"'
(step them fromthe cr
catalytic and subunits(step41 (a)
1 subunits E) anddissociating
inhibitory B Na*
Relievedof theirinhibition, of PDEconvert
thectandB subunits cGMPto GMP(stepE) The Ca2'
decrease
resulting of cGMPfromthe nucleotide-gated
cGMPleadsto dissociation
in cytosolic Open cGMP-gated
channelsintheplasma membrane andclosingof thechannels(step51 Themembrane then ion channel
becomes transiently hyperpolarized fromV Arshavsky E Pugh,
lAdapted and 1998, Neuron20|11 1

RD
G PROTEIN_COUPLE E C E P T O RTSH A T R E G U L A T EI O N C H A N N E L S 643
 state for only a fraction of a second.Thus cGMP phospho-
diesteraserapidly becomesinactivated, and the cGMP level
gradually risesto its original level when the light stimulus is
Rhodopsin
removed. This allows rapid responsesof the eye roward
Exterior moving or changing objects.
Recentx-ray crystallographicstudiesrevealhow the sub-
ligand units of G, protein interact with each other and with light-
activatedrhodopsin and provide clues about how binding of
Membrane GTP leadsto dissociationof Go from GB". As revealedin the
structuralmodel in Figure 15-19,two surfacesof Go. inter-
act with GB: an N-terminal region near the membrane sur-
face and the two adjacent switch I and switch II regions,
Cytosol
which are found in all G, proteins. G" directly contacts Gp
but not Gro. These models also suggestthat the nucleotide-
binding domain of Go., together with the lipid anchors at the
C-terminus of G" and the N-terminus of Got, form a surface
that binds to light-activatedrhodopsin(O', in Figure15-18),
promoting the releaseof GDP from Go. and the subsequent
binding of GTP. The subsequentconformational changesin
G.o, particularly those within switches I and II, disrupt the
molecular interactions between Go. and Gpr, leading to their
dissociation.The structural studieswith rhodopsin and Go,
are consistentwith data concerningother G protein-coupled
GDP receptors and are thought to be generally applicable to all
receptorsof this type.
Gq N_
GIl,
A FIGURE 15-19 Structuralmodelof rhodopsinand its Rod CellsAdapt to Varying Levelsof Ambient
associated trimericG protein,transducin(GJ. Thestructures of LightBecause o f O p s i nP h o s p h o r y l a t i o n
r h o d o p s iann dt h e G o .a n dG B "s u b u n i tws e r eo b t a i n ebdyx - r a y
crystallography
a n d B i n d i n go f A r r e s t i n
TheC-terminal segment of rhodopsin that follows
t r a n s m e m b r ahneel i xH 7a n de x t e n disn t ot h ec y t o s oi sl n o ts h o w n Cone cellsare insensitiveto low levelsof illumination, and
in thismodelTheorientation of G*,withrespect to rhodopsin and the activity of rod cells is inhibited at high light levels.
t h e m e m b r a niesh y p o t h e t i c iat li;sb a s e d o n t h ec h a r g e ano Thus when we move from bright daylight into a dimly
hydrophobicity of the proteinsurfaces andthe knownrhodopsin- lighted room, we are initially blinded. As the rod cells
bindingsiteson G.,.As in othertrimericG proteins, the Go,andG-, slowly becomesensitiveto the dim light, we gradually are
s u b u n i tcso n t a i nc o v a l e n tal yt t a c h eldi p i d s( r e da n db l u ej a g g e d able to seeand distinguish objects.This processof uisual
l i n e st)h a ta r et h o u g h to b e i n s e r t eidn t ot h e m e m b r a n Ien t h e
adaptation permits a rod cell to perceivecontrast over a
G D P - b o u nf odr ms h o w nh e r et,h eo s u b u n i(tb l u ea) n dt h e B
100,000-fold range of ambient light levels.This wide
s u b u n i(tg r e e ni n) t e r a cwt i t he a c ho t h e ra, sd o t h e s u b u n iat n d
B 1 range of sensitivity is possible becausedifferencesin light
s u b u n r( tr e d )b, u tt h es m a l^l ys u b u n i tw, h i c hc o n t a i nl u s s tt w o c t
helices, levels in the visual field, rather than the absolute amount
doesnot contactthe ctsubunitSeveral segments of the a
subunitarethoughtto interact of absorbed light, are used to form visual images. Light-
with an activated rhodopsin,
c a u s i nag c o n f o r m a t i o ncahla n g teh a tp r o m o t erse l e a soef G D pa n d dependentregulation of the rhodopsin signaling pathway
binding o f G T PB i n d i nogf G T pi,n t u r n ,i n d u c elsa r g e (seeFigure 15-18) is responsiblefor this extraordinarily
conformational changes in the switchregions of G.. leadingto its wide sensitivityrange.
dissociation from GBr.[Adapted fromH Hamm, 2OO1 , procNat'lAcad One process contributing to visual adaptation involves
SciUSA98:48'19, andW Oldham andH Hamm2006euart Rev. Biophvs phosphorylation of opsin in its active conformarion (O',) but
40:(lnpress) l not in its inactive, or dark form (O) by rhodopsin kinase
(Figure 15-20), a member of a classof GPCR kinases.Each
Like the K+ channelsdiscussedin Chapter 11, the cGMp- opsin molecule has three principal serine phosphorylation
gated channel protein contains four subunits [see Figure sites on its cytosol-facing surface; the more sites that are
7I-191. In this case each of the subunits is able to bind a phosphorylated,the less able O', is to activate Go. and thus
CGMP molecule. Three or four cGMp moleculesmust bind induce closing of cGMP-gated cation channels.Becausethe
per channel in order to open it; this allosteric interaction extent of opsin phosphorylation by rhodopsin kinase is pro-
makes channel opening very sensitive to small changes in portional to the amount of time each opsin molecule spends
cGMP levels. in the light-activatedform, it is a measureof the background
Conversion of active G.t.GTp back to inactive G...GDP (ambient) level of light. Under bright-light conditions, opsin
is acceleratedby a specificGTPase-activatingprorein (GAp). phosphorylation is increased,and consequentlyits ability to ac-
In mammals Go, normally remains in the active GTp-bound tivate Go, is reduced.In other words, rhodopsinis desensitized

644 t c H A P T E R1 s c E L L s T G N A L T Nr G
: sTGNAL
T R A N S D U c I o NA N D s H o R T - T E R M
I c E L L U L A RR E S p o N s E s
llll+ overviewAnimation:Extracellular
Signaling
Rhodopsin Activated
(dark adapted) opsrn
Rhodopsin
k in a s e
Activation of Slightly reduced
uot G o ,a c t i v a t i o n
A FIGURE 15-20Rod-cell adaptationto ambientlight level
changesand opsinphosphorylation. Light-activatedopsin(O*),
rhodopsin,
but notdark-adapted for rhodopsin
isa substrate kinase.
Theextentof opsinphosphorylation proportional
isdirectly to the
amountof timeeachopsinmolecule spendsin the light-activated
formandthusto theaverage ambientlightleveloverthe previous
few minutesTheabilityof O* to activateG*,isinverselyproportional
by bright light, and thus a greater increasein light inten-
sity is necessaryto generatea change in cGMP levels and a
visual signal. When the level of ambient light is reduced' the
opsins become dephosphorylatedand the ability to activate
Go, increases;in this case,relatively fewer additional photons
are necessaryto generatea visual signal. The importance of
opsin phosphorylation in visual adaptation is supported by
studies with rod cells from mice with mutant rhodopsins
bearing zero or only one of the target serineresidues.These
rod cells show a much slower than normal rate of deactiva-
tion in bright light.
The light-dependentdesensitizationof rod cellsis further
increasedby binding of the cytosolic protein B-arrestin. At
high ambient light (such as noontime outdoors), B-arrestin
binds to the phosphorylatedserineresidueson the C-terminal
opsin segment.Bound B-arrestincompletelypreventsinterac-
tion of Go. with phosphorylatedO*, totally blocking forma-
tion of the active G*.'GTP complex and causinga shutdown
of all rod-cell activity.
The negative-feedbackregulation of rod-cell activity by
rhodopsinkinaseand arrestinis similarto adaptation(or de-
sensitization)of other G protein-coupled receptors to high
ligand levels.Another mechanism,which appearsunique to
rod cells, also contributes to visual adaptation. In dark-
adapted cellsvirtually all the G., and GB" subunits are in the
outer segments.But exposure for 10 minutes to moderate
daytime intensitiesof light causesover 80 percent of the Go,
and Gp., subunits to move out of the outer segmentsinto
other cellular compartments. Although the mechanism by
which theseproteins move is not yet known, the result is that
Go, proteins are physically unable to bind activated opsin.
As occurs in other signalingpathways, multiple mechanisms
are thus used to inactivate signaling during visual adapta-
tion, presumably to allow strict control of activation of the
signaling pathway over broad rangesof illumination'
G P R O T E I N - C O U P L ERDE C E P T O RTSH A T R E G U L A T EI O N C H A N N E L S
High Very high
light l i gh t
^-*9 ^'_-.r-,
->
Arrestin
Greatlyreduced No
Gd activation Go, activation
to the numberof phosphorylated residueson O* Thusthe higher
the ambient lightlevel,
the greaterthe extentof opsin
phosphorylation andthe largerthe increasein lightlevelneededto
activatethe samenumberof G*,molecules At veryhighlightlevels,
anestinbindsto the completely phosphorylated opsin,forminga
complex thatcannot activateG*.at all. L
[See Lagnado andD Baylor,
1992,Neuron8:995, andA Mendez etal,2OOO, Neuron 28:153l
G Protein-Coupled Receptors That Regulate lon
Channels
r The cardiac muscarinic acetylcholine receptor is a
GPCR whose effector protein is a K* channel. Receptor
activation causesreleaseof the GB" subunit, which opens
K* channels(seeFigure 15-15). The resultinghyperpolar-
ization of the cell membrane slows the tate of heart mus-
cle contraction.
dopsin, the photosensitiveGPCR in rod cells' com-
thi protein opsin linked to 11-cis-tetinal. Light-
d isomerization of the ll-cis-retinal moiety pro-
duces activated opsin, which then activates the coupled
trimeric G protein transducin (Gr) by catalyzingexchange
of free GTP for bound GDP on the Go. subunit.
r The effector protein in the rhodopsin pathway is cGMP
phosphodiesterase'which is activated by the G..'GTP-
mediated release of inhibitory subunits. Reduction in
the cGMP level by this enzyme leads to closing of
cGMP-gated Na+/Ca2+ channels, hyperpolarization of
the membrane, and decreasedreleaseof neurotransmitter
( s e eF i g u r e1 5 - 1 8 ) .
r As with other Go proteins, binding of GTP to Go. causes
conformational changesin the protein that disrupt its mo-
lecular interactions with GB" and enable G.I'GTP to bind
to its downstream effector.
r Phosphorylation of light-activated opsin by rhodopsin
kinase interfereswith its ability to activate Gorl subsequent
binding of arrestin to phosphorylatedopsin further inhibits
its ability to activate Go, (seeFigure 15-20)' This general
mechaniim of adaptation' or desensitization,is utilized by
other GPCRs at high ligand levels.
645
 G Protein-Coupled ReceptorsThat
Activateor InhibitAdenylylCyclase
GPCR pathways that utilize adenylyl cyclaseas an effector
protein and cAMP as the secondmessengerare found in most
mammalian cells. These pathways follow the general GPCR
mechanismoutlined in Figure 15-13: Ligand binding to the
receptor activatesa coupled trimeric G protein that activates
adenylyl cyclase,which synthesizesthe diffusible secondmes-
sengercAMP. cAMP, in turn, activates a cAMp-dependent
protein kinase that phosphorylatesspecifictarger proteins.
To explore this GPCR/cAMP pathway we focus on the
first such pathway discovered-the hormone-stimulated
generation of glucose from glycogen, a srorage polymer of
glucose.The breakdown of glycogen (glycogenolysls/occurs
Positive (activation) and negative (inhibition) regulation
of adenylyl cyclaseactivity occurs in many cell types, pro-
viding fine-tuned conrrol of the cAMP level (Figure 75-21).
For example,the breakdown of triacylglycerolsto fatty acids
in adipose cells (lipolysls) is stimulated by binding of epi-
nephrine, glucagon, or ACTH to receptors that activate
adenylyl cyclase.On the other hand, binding of two other
hormones, prostaglandin PGEI or adenosine,to their re-
spective G protein-coupled receptors inhibits adenylyl cy-
clase.The prostaglandin and adenosinereceptorsactivarean
inhibitory G1protein that contains rhe sameB and ^ysubunits
as the stimulatory G. protein but a different ct subunit (G.1).
After the active G*;.GTP complex dissociatesfrom Gs,, it
binds to but inhibits (rather than stimulates) adenylyl cy-
clase,resulting in lower cAMP levels.

A d e n y l y lC y c l a s et s S t i m u l a t e da n d I n h i b i t e d
by Different Receptor-LigandComplexes
G protein-coupled receptors,but both receptorsinteract with
and activate the same Go, that activates adenylyl cyclase.
Hence, both hormones induce the samemetabolic responses.
Activation of adenylyl cyclase,and thus the cAMp level, is
proportional ro the total concentrationof Go, .GTp resulting
from binding of both hormones to their respectivereceptorsl
StructuralStudiesEstablishedHow G.r.GTp
Bindsto and ActivatesAdenylyl Cyclase
X-ray crystallographic analysis has pinpointed the regions in
G.,.GTP that interact with adenylyl cyclase.This enzyme is a
multipass transmembraneprotein with two large cltosolic seg-
mentscontaining the catalytic domains (Figure l5-22a). Because
suchtransmembraneproteins are notoriously difficult to crystal-
lize, scientistspreparedtlvo protein fragmentsencompassingthe
fwo catalytic domains of adenylyl cyclasethat tightly associate
with one another in a heterodimer.When these catalytic frag-
ments are allowed to associatein the presenceof G*..GTp and
forskolin, they are stabilizedin their activeconformations.
The resulting water-soluble complex (two adenylyl cy-
clasedomain fragments/G.,.GTP/forskolin) was catalytically
active and showedpharmacologicaland biochemicalproper-
ties similar to those of intact full-length adenylyl cyclase.In
this complex, two regionsof G*,.GTP,the switch II helix and
the o3-B5 loop, contact the adenylyl cyclasefragments (Fig-
ure 15-22b). Thesecontactsare thought to be responsiblefor
the activation of the enzymeby G*,.GTP.Recallthat switch II

S t i m u l a t o r yI E p i n e p h r i n e
hormone , { Glucagon
| [ AcrH
I
@
Exterior
Adenytyl ;
cycrase l
/tr\ :
Cytosol
Goi

Receptorfor
stimulatory
normone
\___-_____\aJ

Stimulatory
G protein
P
It
cAMP
P
\_______Y-

Inhibitory
G protein
Receptorfor
inhibitory
normone

complex complex
A FIGURE 15-21Hormone-induced activationand inhibitionof andtheircorresponding receptors
differ.Ligand-stimulated
formationof
adenylylcyclasein adiposecells.Ligandbindingto G*,-coupled activeGoGTPcomplexes occursbythesamemechanism in bothG*,
receptorscauses
activation
of adenylyl
cyclase,whereasligandbindingto andG* proteins (seeFrgure 15-13) However; G*,GTpandG.,.GTp
G" -coupledreceptors
causesinhibition
of theenzyme.TheGp,,subunit interact
differently
with adenylylcyclase,
sothatonestimulates andthe
in bothstimulatory
andinhibitory
G proteins isidenticar;
theG^subunits otherinhibits
itscatalytic
activity. A G Gilman,
[See 1984,
Cett36:57j
]

646 o CHAPTER
15 C E L Ls t c N A L I N Gr : s T G N A L
| TRANSDUCToN
AND SHoRT-TERM
c E L L U L A RR E S p o N s E s
 (a) lnactive PKA Active P](A
Adenylyl cyclase

Cytosol coo
RegulatorY
subunits

( b ) R e g u l a t o r (yR )s u b u n i ts t r u c t u r e
(b)
AKAP g
, Dimerization/dockin
a3,- tsS binding / domain
^i+^
7

Catalytic
subunit
// \' b i n d i n g
I site
Flexlble/
, linkers ,/
in
Forskol
Catalytic
subunit#
""w binding
Adenylyl cyclase site
catalytic fragments cNB_A-
A FIGURE 15-22Structureof mammalianadenylylcyclases
and their interactionwith G"'.GTP. (a)Schematic diagram of o
d-,
mammalian adenylyl cyclases Themembrane-bound enzyme
contains two simrlar catalytic domains on thecytosolic faceof the
m e m b r a naen dt w o i n t e g r aml e m b r a ndeo m a i n e
s a
, c ho f w h i c hi s CNB-B (c) Conformational
thoughtto containsixtransmembrane o helices(b)Modelof the c h a n g e sf r o m
three-dimensional structure of G., GTPcomplexed with two c A M Pb i n d i n g
fragments encompassing thecatalytic domainof adenylyl cyclase
cAMP
determined byx-raycrystallography Thect3-B5loopandthe helixin bound
theswitchll region(blue)of G"sGTPinteract simultaneously wttha
specific regionof adenylyl cyclase Thedarker-colored portionof G.,
Catalytic
isthe GTPase domain, whichissimilar in structure to Ras(seeFigure s ub u n i t
1 5 - 8 )t;h el i g h t epr o r t i o nr sa h e l i c adlo m a i nT h et w o a d e n y l y l bound
cyclase fragments areshownin orangeandyellowForskolin (green)
locksthecyclase fragments in theiractiveconformations [Part (a)see
W - 1T a n g a n dG A G i l m a n , 1 9C 9e2 l, l T O : 8P6a9r t ( b ) a d a p t e d f Jr oGm J
Tesmer et al, 1997,Science 278:1907 )

is one of the segmentsof a Go protein whose conformation is A FIGURE 15-23Structureof the regulatory(R)subunitsof
different in the GTP-bound and GDP-bound states (see proteinkinaseA and its activationby cAMP'(a)Protein kinase A
(PKA)consists of two regulatory (R)subunits (green)andtwo catalytic
Figure 15-8). The GTP-inducedconformation of Go. that
(C)subunits WhencAMP(redtriangle) bindsto theregulatory subunit,
favors its dissociationfrom Gs, is preciselythe conformation (b)
thecatalytic subunitisreleased, thusactivatingPKA The two
essentialfor binding of Go, to adenylyl cyclase.Other studies linkeranda dimerization/
regulatory subunitsarejorned bya flexible
indicate that Go1binds to a different region of adenylyl cy- activatingprotein(AKABFigure 15-28)
Oocting domain where A-kinase
clase,accountingfor its different effect. hastwo cAMP-binding domains, CNB-A and
canbind.EachRsubunit
CNB-8, anda binding subunit
sitefor a catalytic (arrow)(c)Binding of
cAMPActivatesProteinKinaseA by Releasing cAMPtotheCNB-Adomaindisp|acesthecata|yticsubunit|eading
activation WithoutboundcAMBoneloopof theCNB-A domain
C a t a l y t i cS u b u n i t s (purple)istna conformation thatcanbindthecatalytic (C)subunitA
In multicellular animals, virtually all the diverse effects of glutamate (E200) andarginine (R209)residue participatein binding of
cAMP are mediated through protein kinase A (PKA), also cAMP(red), whichcauses a conformational change (green) in the loop
called cAMP-dependent protein kinase. Inactive PKA is a thatprevents bindingof the loopto the C subunitlPart (b)after5 S
Biochim Biophys Acta1754i25 (c)
Part afterC Kim,N-H
tetramerconsistingof two regulatory(R) subunitsand two cat- et
Taylor ,al 2005,
alytic (C) subunits(Figure t5-23a). Each R subunit contains a andS 5 Taylor,
Xuong, 2005, science307:690l

. 647
G P R o T E I N _ C O U P L ERDE C E P T O RTSH A T A C T I V A T EO R I N H I B I TA D E N Y L Y LC Y C L A 5 E
 o
il tl
o-i-o-r-o-@ o
tl
oHoo OH OH
UDP-glucose Glycogen (n residues)

I u.oo"n
J "L"."

o o T
ilil
o-i-o-?-o-@
tl
oo OH
UDP Glycogen (n + 1 residues)

P'--rl n en
nn rorylasr:
J

HOCH2

OH
o-',
?+o
(
oHo
Glucose 1-phosphate Glycogen (n residuesl
FIGURE15-24Synthesis and degradationof glycogen. by glycogen
catalyzed phosphorylase
Becausetwo different
enzymes
Incorporation
of glucose
fromUDp-glucoseintoglycogen
iscatalyzed catalyze
theformation
anddegradation
of glycogen,
thetwo
by glycogen
synthase.
Removalof glucose
unitsfromgrycogen
ts reactions
canbe independently
regulated

pseudosubstratesequencethat binds to the active site in a
catalytic domain. By blocking substratebinding, the R sub-
units inhibit the activity of the catalytic subunits.Inactive
PKA is turned on by binding of cAMp. Each R subunit has
two distinct cAMP-binding sites,called CNB-A and CNB_B
(Figure I5-23b). Binding of cAMp ro an R subunit causesa
conformational change in the pseudosubstratedomain that
leadsto releaseof the associatedC subunit, unmaskingits cat-
alytic site and activatingits kinase activity (Figure 15-23c).
Binding of cAMP by an R subunit of protein kinase A
activity. Rapid activation of enzymesby hormone-triggered
dissociation of an inhibitor is a common feature of-manv
signaling pathways.
G l y c o g e nM e t a b o l i s ml s R e g u l a t e d
by Hormone-lnduced Activation of protein
K i n a s eA
Glycogen, alarge glucosepolymer, is the major storageform
of glucosein animals. Like all biopolymers,glycogenls syn-
thesizedby one set of enzymesand degradedby another (Fig,
ure 15-24). Degradation of glycogen, or glycogenolysis,in-
volvesthe stepwiseremoval of glucoseresiduesfrom one end
of the polymer by a phosphorolysisreaction, catalyzedby
glycogen p h osph oryla.se,yielding glucose1-phosphate.
In both muscle and liver cells, glucose 1-phosphatepro-
duced from glycogenis convertedto glucose6-phosphate.In
musclecells,this metaboliteentersthe glycolyticpathway and
is metabolizedto generateATP for usein powering musclecon-
traction (Chapter 12). Unlike musclecells,liver cellscontain a
phosphatasethat hydrolyzesglucose6-phosphateto glucose,
which is exported from these cells in parr by a glucose trans-
porter (GLUT2) in the plasma membrane(Chapter 11). Thus
glycogenstoresin the liver are primarily broken down to glu-
cose,which is immediatelyreleasedinto the blood and trans-
ported to other tissues,particularly the musclesand brain.
The epinephrine-stimulated activation of adenvlvl
cyclase,resulting increasein cAMp, and subsequentactiva-
tion of protein kinase A (PKA), enhancesthe conversion of
glycogen to glucose 1-phosphatein two ways: by inhibiting
glycogen synthesisand by stimulating glycogen degradation
(Figure 15-25a).PKA phosphorylatesand in so doing inac-
tivates glycogensynthase,the enzymethat synthesizesglyco-
gen. PKA promotes glycogen degradation indirectly by
phosphorylating and thus activating an intermediate kinase,

648 ' C H A P T E1Rs I c E L Ls t c N A L t N rG: s T G N ATLR A N S D U c T oANN D s H o R T - T E RCM

E L L U L ARRE s p o N s E s
(a) Increased
cAMP

GSO

G l u c o s e1 - p h o s p h a t e

(b) DecreasedcAMP

-
:;;;;;",t,*,
i

; e K A e r o t " i n k i n a s eA 1
GPK I P P P h o s p h o P r o t e iP n hosphatase
I G P K G l y c o g e np h o s p h o r y l a s ek i n a s e i
I G P G l y c o g e np h o s P h o r Y l a s e I
G S G l y c o g e ns y n t h a s e
i lP I n h i b i t o ro f P h o s p h o p r o t e l n l
________-_> G P phosphatase
UDP-glucose----------> Glycogen + UDP .

of phosphoprotein phosphatase (PP).

Binding of the phosphorylated
A FIGURE 15-25Regulationof glycogenmetabolismby cAMP
to
inhibitor PP preventsthis phosphatasefrom dephosphorylatingthe
in liver and musclecells.Activeenzymes arehighlightedin darker
activatedenzymes in the kinasecascade glycogen
or the inactive
shades; forms,in lightershades(a)An increase
inactive in cytosolic
synthase (b)A decrease in cAMPinactivatesPKA,leading to release
cAMPactivates proteinkinase A (PKA),
whichinhibitsglycogen
of the activeformof PPTheaction of thisenzyme promotes
synthesis and
directly promotes glycogen viaa protein
degradation
an inhibitor glycogen synthesisandinhibits glycogen degradation
kinase cascade At highcAMBPKAalsophosphorylates

glycogen phosphorylase kinase (GPK), that in turn phos- achieving a particular cellular responseand is a common
phorylates and activatesglycogen phosphorylase,the en- phenomenonin regulatory biology.
zyme that degradesglycogen.
The entire processis reversedwhen epinephrineis re- cAMP-MediatedActivation of Protein
moved and the level of cAMP drops, inactivating proteln KinaseA ProducesDiverseResponses
kinaseA (PKA). This reversalis mediatedby phosphoprotein
in Different CellTYPes
phosphatase,which removes the phosphateresiduesfrom
the inactiveform of glycogensynthase,therebyactivatingit'
and from the activeforms of glycogenphosphorylasekinase
and glycogen phosphorylase, thereby inactivating them
(Figure15-25b). Phosphoproteinphosphataseitself is regu-
lated by PKA. An inhibitor of phosphoproteinphosphatase
is normally inactive. When activatedPKA phosphorylates
this inhibitory protein, it can bind to phosphoproteinphos-
phatase,inhibiting its activity (seeFigure 1'5-25a).At low
cAMP levels, when PKA is inactive, the inhibitor is not
phosphorylatedand phosphoproteinphosphataseis active. array of hormone-induced cellular responsesin multiple
As a result, in the absence of cAMP the synthesis of b o d y c e l l s ( T a b l e1 5 - 2 ) '
glycogen by glycogen synthase is enhanced and the degra- Although protein kinase A acts on different substratesin
dation of glycogenby glycogenphosphorylaseis inhibited' different typei of cells, it always phosphorylatesa serine or
Epinephrine-inducedglycogenolysisthus exhibits dual threonine residuethat occurs within the same sequencemo-
regulation: activation of the enzymescatalyzing glycogen tif: X-Arg-(Arg/Lys)-X-(Ser/Thr)-O' where X denotes any
degradation and inhibition of enzymespromoting glyco- amino O denotesa hydrophobic amino acid' Other
".id "ttd kinases phosphorylate target residues
gen synthesis.Such coordinate regulation of syntheticand serine/threonine
degradativepathways provides an efficient mechanismfor within other sequencemotifs.

CYCLASE . 649
G P R O T E I N - C O U P L ERDE C E P T O RTSH A T A C T I V A T EO R I N H I B I TA D E N Y L Y L
 TlsslJE H0BM0NE
tNDuctNG
RtsE
tNcAMp cELtutAR
RESP0NSE
Adipose Epinephrine; ACTH; glucagon Increasein hydrolysis of triglyceride; decreasein amino acid uptake

Liver
grucagon lH,?Tr'1":1ffi:'J::;:,1'f:ff:trf:T:,T'"'J,:,li:',::,."
"3,iil,i.oiii,'e; rn gluconeogenesis(synthesisof glucose from amino acids)

ovarian follicle FSH; LH Increasein synthesisof estrogen,progesterone

Adrenal cortex ACTH Increasein synthesisof aldosterone, cortisol

Cardiac muscle Epinephrine Increasein contractron rate

Thyroid gland TSH Secretionof thyroxine

Bone Parathyroid hormone Increasein resorption of calcium from bone

Skeletal muscle Epinephrine Conversion of glycogen to glucose

Intestine Epinephrine Fluid secretion

Kidney Vasopressin Resorption of water

Blood platelets Prostaglandin I Inhibition of aggregation and secretion

''Nearly
all the effects of cAMP are mediated through protein kinase A (PKA), which
is activated by binding of cAMp.
s o u R C E :E . W . S u t h e r l a n d ,1 , 9 7 2 ,S c i e n c e1 7 7 : 4 0 1 .

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y
S i g n a l i n gP a t h w a y s _.- Epinephrine (10-10
M)
AmPlificatiorr -------.-"
Receptors are low-abundance proteins, typically present in r
only a few thousand copies per cell. yet the'..llul", .._ AAA Adenylyl
sponsesinduced by binding of a relatively small number of cyctase
hormone molecules to the available receptors mav require Amprification
production of tens of thousandsor even millions of second . r-l.l.\. c A M P ( 1 0 - 6M )
messenger or activatedenzymemoleculesper cell. Thus sub_
stantial signal amplification often -urt oi.u, in order for a
hormone signal to induce a significant cellular response.
ttttt Protein
k i n a s eA
In the caseof G protein--coupledreceptors,signal amplifi_
cation is possiblein part becauseboth recepto.sand G pro_ Am plification
teins can diffuse rapidly in the plasma membrane. A sinele Activated
@
epinephrine-GPcR complex causesconversionof up to 1-00 enzyme
inactive Go, moleculesto the active form before epinephrine A m p l i fi c a t i o n
dissociatesfrom the receptor.Each active G.,.GTq in turn, lz

activatesa single adenylyl cyclasemolecule. which then cat_

a Product
alyzes synthesisof many cAMp molecules during the time FIGURE 15-26Amplificationof an externalsignaldownstream
G.,.GTP is bound to it. from a cell-surfacereceptor.Inthisexample, bindlngof a single
The amplification that occurs in such an amplification cas_ epinephrinemolecule to oneGosprotein-coupled receptormolecule
cade dependson the number of steps in it and the relative induces synthesis
of a largenumber of cAMpmolecules, thefirstlevelof
concentrationsof the various components.In the epinephrine_ amplification.
Fourmolecules of cAMpactivate two molecules of
proteinkinaseA (PKA), buteachactivated pKAphosphorvlates and
induced cascadeshown
^in Figure 15-26, blood lwels of epi_ activatesmultiple
product molecules.
Thissecond levelof amplification
nephrineas low as 10-10 M can stimulateliver glycogenolysis
mayinvolve severalsequentialreactionsin whichthe productof one
and releaseof glucose.An epinephrine stimulus of thii maeni_
reactionactivates
the enzyme catalyzingthe nextreactionThemore
tude generatesan intracellularcAMp concentrationof 10-5"M.
stepsin sucha cascade, thegreater
thesignal amplificationposslble

650 . c H A p r E R1 s I c E L LS I G N A L I N G
l : S t G N A LT R A N S D U c I o NA N D s H o R T - T E R M
c E L L U L A RR E s p o N s E s
an amplificationof 104fold. Becausethreemore catalyticsteps
precedethe releaseof glucose,another 104 amplification can
occur,resultingin a 106amplificationof the epinephrinesignal.
In striated muscle the amplification is less dramatic, because
the concentrations of the three successiveenzymes in the
glycogenolyticcascade-protein kinase A, glycogen phos-
phorylase kinase, and glycogen phosphorylase-are tn a
1:10:240 ratio (a potential 240-foldmaximal amplification).
The epinephrine-inducedGPCR pathway leadingto glycogenol-
ysis,whether in liver or striatedmusclecells,dramaticallyillus-
trates how the effectsof an external signal can be amplified.
of the B-adrenergicreceptor, not those phosphorylated by
PKA, can be phosphorylatedby the enzymeB-adrenergicre-
ceptor kinase (BARK/, but only when epinephrineor an ago-
nist is bound to the receptor and the receptor is in its active
conformation. This processis called homologous desensitiza-
tion,becauseonly those receptorsthat are in their activecon-
formations are subject to deactivation by phosphorylation'
Another exampleof this regulatory mechanismis the desensi-
tization of rhodopsin by rhodopsin kinase.
Recall from our discussionof the rhodopsin pathway that

s o w n ' R e g u l a t eS i g n a l i n g
l e c h a n i s mD
S e v e r aM
from G Protein-CoupledRecePtors
For cellsto respond effectivelyto changesin their environment,
mechanismsmust exist to terminate the activation of signaling
pathways. Severalmechanismscontribute to termination of cel-
lular responsesto hormonesmediatedby B-adrenergicreceptors
and other G protein-coupled receptorscoupled to Go,. First, the
affinity of the receptor for its ligand decreaseswhen the GDP
bound to Go, is replacedwith GTP.This increasein the K6 of the
receptor-hormone complex enhancesdissociationof the ligand
from the receptor and thereby limits the number of Go, proteins sis. These interactions promote the formation of coated pits
that are activated. Second,the intrinsic GTPaseactivity of G.s and endocytosisof the associatedreceptors'thereby decreas-
convertsthe bound GTP to GDP, resulting in inactivation of the ing the number of receptorsexposedon the cell surface(Fig-
protein and decreasedadenylyl cyclaseactivity' Importantly' the ure 15-27).Eventually some of the internalizedreceptorsare
rate of hydrolysis of GTP bound to Go, is enhanced when Go,
binds to adenylyl cyclase,lesseningthe duration of cAMP pro-
duction; thus adenylyl cyclasefunctions as a GAP for Go,. More
generally,binding of most if not ail G*'GTP complexesto their Exterior
respectiveeffector proteins acceleratesthe rate of GTP hydroly-
sis.Finally, cAMP phosphodiesterase actsto hydrolyze cAMP to
5'-AMB terminatingthe cellular response. Thus the continuous
presenceof hormone at a high enough concentration is required
for continuous activation of adenylyl cyclaseand maintenance of
the hormone concentration falls CYtosol
an elevatedcAMP level. Once
sufficiently,the cellular responsequickly terminates'
Receptors can also be down-regulated by feedback re-
pression, in which the end product of a pathway blocks an Activationof l*rnoo.u,or,.
early step in the pathway. For instance, when a Go, pro- c - J u nk i n a s e
cascade J
tein-coupled receptor is exposedto hormonal stimulation
for severalhours, severalserineand threonineresiduesin the
cytosolicdomain of the receptorbecomephosphorylatedby Activation of MAP
protein kinaseA (PKA), the end product of the Go, pathway. kinasecascade
The phosphorylatedreceptorcan bind its ligand but cannot desensitization and
FIGURE 15-27Roleof p-arrestinin GPCR
efficiently activate Go,; thus ligand binding to the phospho- binds to phosphorylated serineand
signaltransduction' P-Arrestin
rylated receptorleadsto reducedactivation of adenylyl cy- residues in theC-terminal segment of G protein-coupled
threonine
clasecomparedwith ligand binding to a nonphosphorylated receptors (GPCRt. Clathrin andAP2,two otherproteins boundby
receptor.Becausethe activity of PKA is enhancedby the high promoteendocytosis of the receptor' B-Arrestin also
B-arrestin,
cAMP levelinducedby any hormone that activatesGo,,pro- functions in transducing signals from activated receptors by binding to
longed exposure to one such hormone, say, epinephrine, andactivating several cytosolic protein kinases. c-src activatesthe MAP
desensitizes not only B-adrenergicreceptorsbut also other kinase pathway, leading to phosphorylation of keytranscrrption factors
Go, protein-coupled receptorsthat bind different ligands (Chapter 16).Interaction of B-arrestin withthree other protelns,
(e.g., glucagon receptor in liver). This cross-regulationis including JNK-3 (aJunN-terminal results
kinase), in phosphorylation
andactivation of another transcription factor, c-Jun fAdaptedfromW
calledh eterologows desensitization.
forms Curr' Opin CellBiol 13:139, and K Pierce
Exposure of cells to epinephrinealso leadsto other Miller and R J Lefkowitz,2OO1 ,
domain Nature Rev.Mol Cell Biol 3:6391
of desensitization. Particularresiduesin the cytosolic et al , 2002,

CYCLASE . 651
G P R o T E I N _ C O U P L ERDE C E P T O RTSH A T A C T I V A T EO R I N H I B I TA D E N Y L Y L
 degradedintracellularlS and some are dephosphorylatedin
endosomes.Following dissociationof B-arrestin,the resensi-
tized (dephosphorylated)receptorsrecycleto the cell surface,
similar to recyclingof the LDL receptor (Chapter 14).
Desensitizationof many GpCRs and other classesof
r e c e p t o r so c c u r s b y r e c e p t o r p h o s p h o r y l a t i o n , a r r e s t i n
b i n d i n g , a n d e n d o c y t o s i so f l i g a n d - o c c u p i e dr e c e p t o r s ,
leading to their sequestrationinside the cell. In addition
to its role in regulating receptor activity, B-arrestin also
functions as an adapter protein in transducingsignals
frgm Gprotein-coupled receptorsto the nucleurlCh"pt.,
16). The multiple functions of p-arrestinillustrate the im-
portance of adapter proteins in both regulating signalingand
transducingsignalsfrom cell-surfacereceptors.
A n c h o r i n gP r o t e i n sL o c a l i z eE f f e c t so f c A M p
t o S p e c i f i cR e g i o n so f t h e C e l l
In many cell types,a rise in the cAMp level may produce a
responsethat is required in one part of the cell but is un_
One such anchoring protein (AKAP15) is tethered to the
cytosolic face of the plasma membranenear a particular type
of gated Ca2* channel in certain heart muscle cells. In the
heart, activarion of B-adrenergicreceptorsby epinephrine(as
part of the fight-or-flight response)leads to pKA-catalyzed
phosphorylation of these Ca2+ channels, causing them to
open; the resulting influx of Ca2* increasesthe rate of heart
A different AKAP in heart muscle anchors both protein
kinase A and cAMP phosphodiesterase(pDE) to the outer
nuclear membrane.Becauseof the closeproximity of pDE to
protein kinase A, negativefeedbackprovides tight local con-
trol of the cAMP concentration and hencelocal pKA activity
(Figure15-28).The localizationof protein kinaseA near the
nuclear membrane also facilitates entry of its catalytic sub-
units into the nucleus, where they phosphorylate and acti-
vatecertain transcriptionfactors (seeChapter 16).

G Protein-Coupled
Receptors
That Activateor Inhibit
AdenylylCyclase
r Ligand activationof G protein-coupledreceptorsthat
activateGo, resultsin the activationof the membrane-

E
B a s a lP D Ea c t i v i t y=
a
I n c r e a s e dc A M P :
E
P D Ep h o s p h o r y l a t i o n
resting state PKA activation a n d a c t i v a t i o nr ;e d u c t i o n
cAMP i n c A M Pl e v e l
>a
t
mAKAP
I

Cytosol
Outer
nuclear
memDrane

!f Returnto restingstate
FIGURE 15-28 Localization of proteinkinaseA (pKA)to the t h a tw h i c hc a nb e d e g r a d ebdy p D ET h er e s u l t i nbgi n d i n o
gf cAMp
nuclearmembranein heartmuscleby an A kinase-associated to the regulatory (R)subunits of pKAreleases theactivecatalytic (C)
protein.Thismemberof theAKAPfamily,designated mAKAB subunits intothecytosolSomeC subunits enterjntothe nucleus,
anchors bothcAMPphosphodiesterase (pDE) andthe regulatory wheretheyphosphorylate andthusactivate certaintranscrrptron
s u b u no
i tf P K At o t h en u c l e amr e m b r a nm
e ,a i n t a i n i n
t hge mi n a factors(Chapter 16),Concomitant phosphorylation of pDEby active
negative feedback loopthatprovides closelocalcontrolof the cAMp PKAcatalytic subunits stimulates itscatalyticactivity, therebyhydrolyzing
levelandPKAactivity. Step[:The basallevelof pDEactivity in the cAMPanddriving cAMPlevels backto basal andcausing reformation of
absence of hormone (resting state)keepscAMplevels belowthose theinactive PKAStep@: Subsequent dephosphorylation of pDE
necessary for PKAactivation. StepsA and E: Activation of B_ returns the complex to the resting state,[Adapted fromK L Dodqe
adrenergic receptors causes an increase in cAMplevelin excess of et al , 200 1, EMBOT 20:1921 )

652 CHAPTER
15 I C E L LS I G N A L I N G
I : S I G N A LT R A N S D U C T I OANN D S H o R T - T E R M
cELLULAR
REsPoNsEs
bound enzymeadenylyl cyclase,which converts ATP to the
cyclic AMP (cAMP).
secondmessenger
r Ligand activation of G protein-coupled receptors that
activate Goi results in the inhibition of adenylyl cyclaseand
lower levelsof cAMP.
r The switch regions in the activated forms of G.,'GTP
and Go1'GTP bind to the heterodimericactive site domains
in adenylyl cyclaseto activate or inhibit the enzyme, re-
spectively.
r cAMP binds cooperativelyto a regulatory subunit of
protein kinase A (PKA) releasingthe active kinase catalytic
subunit (seeFigure 15-23).
r PKA mediatesthe diverse effects of cAMP in most cells
(seeTable 15-2). The substratesfor PKA and thus the cel-
lular responseto hormone-inducedactivation of PKA vary
among cell types.
r In liver and muscle cells, activation of PKA induced by
epinephrine and other hormones exerts a dual effect, in-
hibiting glycogen synthesis and stimulating glycogen
breakdownvia a kinasecascade(seeFigure 15-25)'
r Signalingpathwaysinvolving secondmessengers and ki-
nasecascades amplify an externalsignaltremendously(see
Figure 75-26).
r BARK phosphorylatesligand-boundB-adrenergicrecep-
tors, leadingto the binding of B-arrestinand endocytosisof
the receptors.The consequentreduction in the number of
cell-surfacereceptorsrendersthe cell lesssensitiveto addi-
tional hormone.
r Localization of PKA to specificregions of the cell by an-
choring proteins restricts the effectsof cAMP to particular
subcellularlocations.
G Protein-CouPled That
RecePtors
C
ActivatePhosPholiPase
Calcium ions play an essentialrole in regulating cellular re-
sponsesto external signals and internal metabolic changes'
A, *. r"* in Chapter 11 the level of Ca2* in the cytosol is
maintainedat a submicromolarlevel (<0.2 ptM) by the con-
tinuous action of AlP-powere d Ca2* pumps' which trans-
port Ca2+ ions acrossthe plasma membrane to the cell exte-
iio, o, into the lumens of the endoplasmic reticulum and
other vesicles.Much intracellular Ca2* is also sequesteredin
the mitochondria.
A small rise in cytosolicCa2* inducesa variety of cellular
responsesincluding hormone secretion by endocrine cells,
secietionof digestiveenzymesby pancreaticexocrinecells,and
contractionof muscle(Table15-3).For example,acetylcholine
stimulationof G protein--coupledreceptorsin secretorycellsof

RISIINCAz+
INDUCI]I|G
ll()RMt)NE RESPONSE
CETLULAR

Pancreas(acinar cells) Acetylcholine Secretion of digestive enzymes' such as amylase and trypsinogen

Parotid (salivary) gland Secretion of amylase

Vascular or stomach Acetylcholine Contractton

smooth muscle

Liver Vasopressin Conversion of glycogen to glucose

Blood platelets Thrombin Aggregation, shape change, secretion of hormones

Mast cells Antigen Histamine secretion

Fibroblasts Peptide growth factors DNA synthesis,cell divisron

(e.g., bombesin and PDGF)

',Hormone stimulation leads to production of inositol 1,4,5-trisphosphate (lP:), a second messengerthat promotes releaseof Ca2* stored in the endo-
p l a s m i cr e t i c u l u m .
31,2231.5.
iou*.o, M. J. Berridge, 7987, Ann. Reu.Biochem. 5621.59;M. J. Berridge and R. F. hvine, 1984, Nature

RD
G PROTEIN_COUPLE E C E P T O RTSH A T A C T I V A T EP H O S P H O L I P A SCE 653
 I I
o o
A
v tUt 2
_,
'e6l< 4
=L
9l OH
Phosphatidylinositol
A FIGURE 15-29 Synthesis of secondmessengers DAGand lp3
from phosphatidylinositol (pl).Eachmembrane-bound pl kinase
placesa phosphate (yellowcircles)
on a specific groupon
hydroxyl
the inositol
ring,producingthe phosphorylated plpand
derivatives
the pancreasand parotid (salivary)gland inducesa risein Ca2*
that triggers the fusion of secretoryvesicleswith the plasma
membraneand releaseof their protein contentsinto the extra-
cellular space.In blood platelers,the rise in Ca2* induced by
thrombin stimulation triggers a conformational change in
thesecell fragmentsleadingto their aggregation,an lmporranr
step in blood clotting to preventleakageout of blood vessels.
In this secrion,we discussan important GpCR-triggered
signal-transducdonpathway that resultsin an elevarionof cy_
tosolic Ca'* ions. Binding of many hormonesto their G pro-
tein-<oupledreceptorson liver, fat, and other cellsactivatesG
PhosphorylatedDerivativesof InositolAre
l m p o r t a n tS e c o n dM e s s e n g e r s
A number of important second messengers,used in several
signal-transduction pathways, are derived from tire mem_
brane lipid_phosphatidylinositol (pI). The inositol group in
this phospholipid, which always faces the cytosoll .u., b.
reversiblyphosphorylatedat one or more positionsby the com_
bined actionsof variouskinasesand phosphatases discussedin
654 ' CHAPTER
ts c E L L s t c N A L ' N Gr : s T G N A L
I T R A N s D U c t o N A N D ' H o R T - T E R MC E L L U L A R E s p o N S E S
I
.)I o
I ATP ADP
QHH- a; -LH- .H
-> \n aH-a!
t- I'
o o
ID _ I
OH
I lr
o
Pl 4-phosphate Pl 4,5-bisphosphate
(PIP} (ptpzl
,lIl3iil3lh.;l;
(lP3)
PlP2Cleavageof PlP2by phospholipase C yieldsthe two important
secondmessengers DAG and lP3 [SeeA Toker andL C Cantley, 1997,
N a t u r e 3 8 7 i 6 7a3n,d C L C a r p e n t e r a n dCLC a n t l e y , 1 9 9C6u,r r . O p i C
n ell
Biol8:153I
Chapter 16. One derivarive of PI, the lipid phosphatidyl
inositol 4,5-bisphosphate (PIP2), is cleaved by activated
phospholipase C into two important second messengers: 1,2-
diacylglycerol (DAG), a lipophilic molecule that remains as-
sociated with the membrane, and inositol 1,4,5-trisphosphate
(IP3), which can freely diffuse in the cytosol (Figure 15-29).
'We
refer to downstream events involving these two second
messengers collectively as the IP j/DAG pathtuay.
C a l c i u ml o n R e l e a s e from the Endoplasmic
R e t i c u l u ml s T r i g g e r e db y l p 3
G protein-coupled receptors that activare phospholipaseC
inducean elevationin cytosolicCa2* evenwhen Ca2* ions are
absentfrom the surrounding extracellularfluid. In this situa-
tion, Ca2+ is releasedinto the cytosol from the ER lumen
through operation of the lPj-gated Ca2+ channel in the ER
membrane,as depicted in Figure 15-30 (step @). This large
channelprotein is composedof four identicalsubunits,eachof
which containsan IP3-bindingsite in the N-terminal cytosolic
domain. IPj binding inducesopening of the channel,allowing
Ca'* to flow down its concentrationgradientfrom the ER into
the cytosol. rX/henvarious phosphorylatedinositols found in
cellsare added to preparationsof ER vesicles,only Ip3 causes
releaseof Ca2* ions from the vesicles.This simpleexperiment
demonstratesthe specificityof the IP3effect.
The IP3-mediatedrise in the cytosolic Ca2* level is tran-
sient becauseCa2* pumps locatedin the plasmamembrane
and ER membrane actively transport Ca2l from the cytosol
to the cell exterior and ER lumen, respectively.Furthermore,
within a secondof its generation,the phosphatelinked to the
 llll+ FocusAnimation:SecondMessengers
in SignalingPathwa
Store-operated o 6
Ca2* - 9o^oo
@
o
c^ hha nan na ln e l . o "Oov o oao o
a

G coupledproteinreceptor(GPCR) PhospholipaC
se

Exterior
Plasma
membrane
Cytosol
\o ooo
PlP2 lPa

> FIGURE 15-30|P3/DAG pathwayand the ,/ (;)

elevation of cytosolic Ca2*. Thispathway canbe
triggered by ligandbindingto GPCRs
eitherthe Gooor G*oalphasubunitleading
thatactivate
to
r"'Vil
'^
kinaseC
"o
oo
activation of phospholipase C (stepIl). Cleavage of
PlP2 by phospholipase C yieldslP3andDAG(stepZ).
Afterdiffusing throughthecytosol, lP3interacts with lP3-gated
Ca2* channel
a n do p e n sC a 2 *c h a n n eilnst h em e m b r a noef t h e
endoplasmic reticulum (stepB), causing release of
storedCa2*ionsintothecytosol (stepZl) Oneof
several cellular responses induced by a risein cytosolic
Ca2*is recruitment of proteinkinase C (PKC) to the ooo A
- oo
plasma membrane (stepE), whereit isactivated by o
OO
o-
DAG(stepEl) Theactivated membrane-associated oO
o
k i n a scea np h o s p h o r y l vaat er i o ucse l l u l aern z y m easn d i^)+
reticulum
Endoplasmic
receptors, therebyaltering theiractivity (stepZ) As
endoplasmic reticulum Ca2-stores aredepleted, a
proteinassociated with the lP3-gated Ca2*channels
bindsto andopensstore-operated Ca'* channels in
the plasma membrane, allowinginfluxof extracellular
Ca2*(stepEl) [Adapted fromJ W Putney, 1999,Proc
Nat'lAcadSciUSA96z14669 l

carbon-S of IP3 (seeFigure 15-29\ is hydrolyzed,yielding tion of the cell-surface G protein-coupled receptor' How-
inositol 1,4-bisphosphate. This compoundcannot bind to the
IP3-gatedCa2* channel protein and thus does not stimulate
Ca2* releasefrom the ER.
\ilithout some means for replenishingdepletedstores of
intracellular Ca2*, a cell would soon be unableto increasethe
cytosolicCa2* levelin responseto hormone-inducedIP3.Patch-
clampingstudies(seeFigure 11-21)haverevealedthat a plasma
membrane Ca2* channel, called the store-operatedchannel,
opensin responseto depletionof ER Ca2* stores.In a way that
is not fully understood,depletionof Ca2* in the ER lumen leads
to a conformationalchangein a protein associated with the IP3-
gatedCa2* channelthat allows it to bind to the store-operated
Ca2* channel in the plasma membrane,causingthe latter to
open (seeFigure15-30,step E).
Continuous activation of certain G protein-coupled re-
cytosolicCa2*, is not understood.

potentiatesopening of thesechannelsby IP3, thus facilitating
ihe rapid rise in cytosolicCa2* following hormone stimula-
T h e C a 2 + / C a l m o d u lC i no m p l e xM e d i a t e sM a n y
C e l l u l a rR e s p o n s etso E x t e r n a S
l ignals
The small ubiquitous cytosolic protein calmodulin functions
as a multipurptse switch protein that mediatesmany cellular

RD
G PROTEIN_COUPLE E C E P T O RTSH A T A C T I V A T EP H O S P H O L I P A SCE 65s
 effects of Ca2* ions. Binding of Ca2* to four sites on
calmodulin yields a complex that interacts with and modu-
lates the activity of many enzymesand other protelns (see
Figure 3-31). Becausefour Ca2* bind to calmoJulin in a co-
operative fashion, a small change in the level of cytosolic
Ca2* leadsto a large changein the level of activecalmodulin.
One well-studied enzyme activated by the Ca2+/calmodulin
complex is myosin light-chain kinase,which regulatesthe ac-
tivity of myosin in muscle cells (Chapter 17). Another is
cAMP phosphodiesrerase,the enzyme that degradescAMp
to 5'-AMP and terminates its effects.This reaction thus
links Ca2+ and cAMP, one of many examples in which two
second messenger-mediated pathways interact to fine-tune
certain aspectsof cell regulation.
In many cells, the rise in cytosolic Ca2* following recep_
tor signaling via phospholipaseC-generated Ip3 leads io
phosphategroups from a transcription factor. An important
example of this mechanism involves T cells of the immune
systemin which Ca2* ions enhancethe activity of an essen-
tial transcription factor called NFAT (nuclear factor of acti-
sequencethat allows NFAT to move into the nucleus and
stimulate expressionof genesessentialfor the function of
T cells.
Diacylglycerol(DAG)Activatesprotein KinaseC,
W h i c h R e g u l a t e sM a n y O t h e r p r o t e i n s
After rts formation by phospholipaseC-catalyzed hydrolysis
of PIP2, the secondary messengerDAG remains associated
IP3/DAG pathway.
The activation of protein kinase C in different cells re_
556 ' CHAPTER
1s c E L L s t c N A L r N Gr : s T G N A L
I TRANSDUCToN
phosphorylatesvarious transcription factors; depending on
the cell type, these induce synthesisof mRNAs that trigger
cell division.
S i g n a l - l n d u c eR
d e l a x a t i o no f V a s c u l a S
r mooth
Musclels Mediated by cGMp-Activatedprotein
K i n a s eG
ffi Nitroglycerinhasbeenusedfor overa centuryas a
Ill treatment lor the intense chest pain of angina. It was
known to slowly decompose in the body to nitric oxide
(NO/, which causesrelaxation of the smooth muscle cells
surrounding the blood vesselsthat "feed" the heart muscle
itself, thereby increasingthe diameter of the blood vessels
and increasing the flow of oxygen-bearingblood to the
heart muscle. One of the mosr intriguing discoveriesin
modern medicine is that NO, a toxic gas found in car ex-
haust, is in fact a natural signalingmolecule.I
Definitive evidencefor the role of NO in inducins relax-
ation of smooth muscle came from a set of experirnentsin
which acetylcholinewas added to experimentalpreparations
of the smoorh muscle cells that surround blood vessels.
Direct application of acetylcholineto thesecellscausedthem
to contract, the expected effect of acetylcholine on these
muscle cells. But addition of acetylcholine to the lumen of
small isolated blood vesselscaused the underlying smooth
muscles to relax, not contract. Subsequentstudies showed
that in responseto acetylcholinethe endothelialcellsthat line
the lumen of blood vesselswere releasingsome substancethat
in turn triggeredmusclecell relaxation.That substanceturned
out to be NO.
\7e now know that endothelial cells contain a Go
protein-coupled receptor that binds acetylcholine and acti-
vates phospholipaseC, leading to an increasein the level of
cytosolic Ca2*. After Ca2* binds to calmodulin, the result-
ing complex stimulates the activity of NO synthase,an en-
zyme that catalyzes formation of NO from 02 and the
amino acid arginine. BecauseNO has a short half-life (2-30
seconds),it can diffuse only locally in tissuesfrom its site of
synthesis.In particular NO diffusesfrom the endothelial cell
into neighboring smooth muscle cells,where ir tnggers mus-
cle relaxation (Figure15-31).
The effect of NO on smooth muscle is mediated by the
secondmessengercGMP, which is formed by an intracellular
NO receptor expressedby smooth muscle cells. Binding of
NO to the heme group in this receptor leads to a conforma-
tional change that increasesits intrinsic guanylyl cyclase
activity, leading to a rise in the cytosolic cGMp level. Most
blood vessel.In this case,cGMP acts indirectly via protein
kinase G, whereasin rod cellscGMp acts directiy by bindlng
to and thus opening cation channels in the plasma mem-
brane (seeFigure 15-18).
AND sHoRT-TERM
CELLULARESpoNsES
Lumen of Acetylcholine < FIGURE 15-31The nitricoxide
blood vessel (NO)/cGMP pathwayand the relaxationof
arterialsmoothmuscle.Nitricoxideis
synthesized in endothelial cellsin response to
acetylcholine andthesubsequent elevation in
Endothelial cytosolicca'* (n-E). No diffuses locally
cells andactivates an intracellular
{
9F,.il;l throughtissues
NOreceptor with guanylyl cyclase in
activity
nearby smoothmuscle cells(E) Theresulting
risein cGMPactivates proteinkinase G (6 and
Z), leading to relaxation of the muscle and
thusvasodilation (ts) Thecell-surface receptor
factor(ANF)
for atrialnatriuretic alsohas
intrinsicguanylyl cyclaseactivity(notshown);
stimulation of thisreceptor on smooth
muscle cellsalso leadsto increased cGMP
andsubsequent muscle relaxation PP;:
pyrophosphate [SeeC S Lowenstein etal,
1994,Ann lnternMed 120:227 )
Smooth musclecells

Relaxation of vascular smooth muscle also is triggered
by binding of atrial natriureticfactor (ANF) and someother
peptidehormonesto their receptorson smooth musclecells.
The cytosolicdomain of thesecell-surfacereceptors,like the
intracellular NO receptor,possesses intrinsic guanylyl cy-
'$fhen
clase activity. an increasedblood volume stretches
cardiac muscle cells in the heart atrium, they releaseANF.
Circulating ANF binds to ANF receptorson the surfaceof
smooth musclecellssurroundingblood vessels,inducing ac-
tivation of their guanylyl cyclaseactivity and formation of
cGMP. Subsequentactivationof protein kinaseG causesdi-
lation of the vesselby the mechanismdescribedabove.This
vasodilationreducesblood pressureand countersthe stim-
ulus that provoked the initial releaseof ANF.
nitric oxide synthase,and protein kinasesor phosphatases
that control the activity of various transcription factors.
r Stimulation of acetylcholine G protein-coupled recep-
tors on endothelial cells induces an increase in cytosolic
Ca2* and subsequentsynthesisof NO. After diffusing into
surrounding smooth musclecells,NO activatesan intracel-
lular guanylate cyclaseto synthesizecGMP. The resulting
increasein cGMP leads to activation of protein kinase G,
which triggers a pathway resulting to muscle relaxation
and vasodilation(seeFigure 15-31).
r cGMP is also produced in vascular smooth muscle cells
by stimulation of cell-surfacereceptors that have intrinsic
guanylatecyclaseactivity. Theseinclude receptorsfor atrial
natriureticfactor (ANF).

G Protein-Coupled ReceptorsThat Activate IntegratingResponsesof Cells

PhospholipaseC
to EnvironmentalInfluences
r Simulation of someG protein-<oupledreceptorsleadsto ac-
no intracel-
tivation of G proteinscontaining the Gooor G.o alpha subunit' Just as no cell lives in isolation from other cells'
iular signaling pathway functions alone. All cells constantly
r TheseG proteins activatephospholipaseC, which gener- receive multiple signals from their environment including
ates two secondmessengers:diffusible IP3 and membrane- changesin hormone levels, metabolites, and gases such as
bound DAG (seeFigure 1,5-29). o*yg..t. All body cells constantly respond to behavioral de-
r IP3 triggers opening of IP3-gatedCa2* channels in the mands as well as to injury or infection. In this section, we
endoplasmic reticulum and elevation of cytosolic free consider the cellular responses to variations in the demand
Ca2*. In responseto elevatedcytosolic Ca2+, protein ki- for the key metabolite glucose.Cellular responsesto changes
nase C is recruited to the plasma membrane' where it is in other nutrients and to oxygen' which are largely reflected
activatedby DAG (seeFigure 15-30). in alterations in geneexpression'are covered in Chapter 7'
r A small rise in cytosolic Ca2* inducesa variety of cellu-
lar responsesincluding hormone secretion,contraction of I n t e g r a t i o no f M u l t i p l e S e c o n dM e s s e n g e r s
muscle,and plateletaggregation(seeTable 15-3). R e g u l a t e sG l Y c o g e n o l Y s i s
. The Ca2*/calmodulin complex regulatesthe activity of One way for cells to respond appropriately to a complex
to more
many different proteins,including cAMP phosphodiesterase, environment is to senseand integrate its responses

T O E N V I R O N M E N T AI N
OSF C E L L S
I N T E G R A T I NRGE S P O N S E LFLUENCES 657
 than one signal. Again, the breakdown of glycogen to glu-
cose (glycogenolysis)provides an excellent example. As de-
scribed in Section 15.6, epinephrine stimulation of muscle
and liver cells leads to a rise in the secondmessengercAMp,
which promotes glycogenbreakdown (seeFigure 15-25a). In
both muscle and liver cells, other second messengersalso
produce the same cellular response.
In muscle cells, stimulation by nerve impulses causesthe
releaseof Ca2* ions from the sarcoplasmicieticulum and an
increasein the cytosolic Ca2* concentration,which triggersmus-
cle contraction. The risein cytosolic Ca2* also activatesglycogen
phosphorylasekinase (GPK), thereby stimulating the degrada-
tion of glycogento glucose1-phosphate,which fuels prolonged
contraction. Recall that phosphorylation by cAMp-dependent
protein kinase A also activatesglycogenphosphorylasekinase.
Thus this key regulatory enzymein glycogenolysisis subject to
both neural and hormonal regulation in muscle(Figure 15-32a).
In liver cells, hormone-induced activation of the effector
protein phospholipaseC also regulatesglycogenbreakdown by
generating two second messengers,DAG and IP3. As we saw
Section15.7,1P3inducesan increasein cytosolicCa2*, which
activatesglycogenphosphorylasekinase as in musclecells,lead-
ing to glycogen degradation. Moreover, the combined effect of
.The dual regulation of glycogenphosphorylasekinase by
Ca'- and protein kinase A in both muscle and liver results
from its multimeric subunit structure (*gfE)+. The "ysubunit
is the catalytic enzyme; the regulatory c and
B subunits,
which are similar in structure,are phosphorylatedby protein
> FIGURE 15-32Integratedregulationof
glycogenolysis. (a)Neuronal stimulation of striated
muscle cellsor epinephrine
bindingto B-adrenergic
receptors on theirsurfaces
cytosolicconcentration
leadsto increased
of the secondmessenqers
Ca2*er cAMP,respectively. Thekeyregulatori
enzyme glycogen phosphorylase kinase(GpK)is
activatedby Ca2"ionsandby phosphorylation by
cAMP-dependent proteinkinase A (pKA)(b)In liver
cells,hormonal stimulationof B-adrenergic receptors
leadsto increased cytosolic
concentrations of cAMp
andtwo othersecondmessengers,
Enzymes aremarkedby whiteboxes.
diacylglycerol
(DAG)andinositol1,4,5-trisphosphate (tp3)
(+) :
activationof enzyme (-) : inhibition
activity;
658 ' C H A P T E R1 5 C E L Ls T G N A L T NrG: s T G N A LT R A N s D U C T o NA N D S H o R T - T E R M
| CELLULAR
kinase A; and the 6 subunit is calmodulin. Glycogen phos-
phorylase kinase is maximally active when Ca2* ions are
bound to the calmodulin subunit and at least the o. subunit
has beenphosphorylatedby protein kinaseA. In fact, binding
of Ca't to the calmodulin subunit may be essentialto the en-
zymattc activity of glycogen phosphorylase kinase. Phospho-
rylation of the cr and B subunirs increasesthe affinity of the
calmodulin subunit for Ca2*, enabling Ca2* ions to bind to
the enzymeat the submicromolar Ca2* concentrationsfound
in cells not stimulated by nerves. Thus increasesin the cy-
tosolic concentration of Ca2* or of cAMP or of both induce
incremental increasesin the activity of glycogenphosphory-
lasekinase.As a result of the elevatedlevel of cytosolic Ca2*
after neuronal stimulation of musclecells,glycogenphospho-
rylase kinase will be active even if it is unphosphorylated;
thus glycogen can be hydrolyzed to fuel conrinued muscle
contraction in the absenceof hormone stimulation.
Insulinand GlucagonWork Together
to Maintain a StableBlood GlucoseLevel
During normal daily living the maintenanceof normal blood
glucoseconcentrationsdependson the balance betweentwo
peptide hormones, insulin and glucagon, which are made in
distinct pancreatic islet cells and elicit different cellular
responses.Insulin, which contains two polypeptide chains
linked by disulfide bonds, is synthesizedby the p cells in the
islets; glucagon, a monomeric peptide, is produced by the o
islet cells.Insulin reducesthe level of blood glucose,whereas
glucagon increasesblood glucose.The availability of blood
glucoseis regulated during periods of abundance(following
a meal) or scarcity (following fasting) by the adiustmenr of
insulin and glucagon concentrationsin the blood.
(a) Musclecells (b) Livercells
Neural Hormonal Hormonal
stimulation stimulation stimulation
tt
Ca2+ cAMP
-'1'\
,i. i
**
lGprl++-@
I
+
*
Er
E -
l=r GT
@
Abbreviations:
PKA ProteinkinaseA Gp Glycogenphosphorylase
G P K G l y c o g e np h o s p h o r y l a skei n a s e GS G l y c o g e ns y n t h a s e
REspoNsEs
 Glucose I n s u l i n - c o n t aiinng After a meal, when blood glucose rises above its normal
secretoryvesicle level of 5 mM, the pancreatic B cells respond to the rise in glu-
cose(and amino acids)by releasinginsulin into the blood (Fig-
ure 15-33). The releasedinsulin circulatesin the blood and
binds to insulin receptors present on many different kinds of
rl;lli i I cells,including muscleand adipocltes (fat-storing cells).The in-
t, sulin receptor belongsto the classof receptorstermed receptor
rr fyrosine kinases (RTKs), which we describein Chapter 16' It
I can transducesignalsthrough an intracellular pathway leading
to the activation of protein kinase B. By an unknown mecha-
-70mV
nism, protein kinaseB triggersthe fusion of intracellular vesicles
?lg*^i containing the glucose transporter GLUT4 with the plasma
membrane(Figure15-34).The resultingtenfold increasein the
number of GLUT4 moleculeson the cell surfaceincreasesglu-
K+
coseinflux proportionallg thus lowering blood glucose'
As the blood glucose level drops, insulin secretion and
ATP-sensitive
Kt channels blood levelsdrop, and insulin receptorsare no longer being
activated as strongly. In response,cell-surfaceGLUT4 is in-
A FIGURE 15-33Secretion of insulinin responsetoarisein ternalized by endocytosis,lowering the level of cell-surface
blood glucose.Theentryof glucose intopancreatic p cellsis
GLUT4 and thus glucoseimport. Insulin stimulation of mus-
mediated bythe GLUT2 glucosetranspofter([) Because the K.
cle cells also promotes the conversion of glucoseinto glyco-
for glucose of GLUT2 is-20 mM,a risein extracellular glucose from5
gen, and it enhancesthe degradation of glucoseto pyruvate.
mM,characteristic of thefastingstate,causes a proportionate increase
Insulin also acts on hepatocytes(liver cells)to inhibit glucose
in the rateof glucose entry(seeFigure11-4) Theconversion of
synthesis from smaller molecules' such as lactate and ac-
glucose intopyruvate isthusaccelerated,resultingin an increase in the
concentration of ATPin the cytosol(El) Thebindingof ATPto ATP- etate, and to enhanceglycogen synthesisfrom glucose.The
sensitive K* channels closesthesechannels (B), thusreducing the net effect of all these actions is to lower blood glucoseback
effluxof K+ ionsfromthecellTheresulting smalldepolarization of the to the fasting concentration of about 5 mM while storing the
plasma membrane (4) triggerstheopening of voltage-sensitive Ca2* excessglucoseintracellularly as glycogenfor future use.
channels (Et) Theinfluxof Ca2*ionsraises thecytosolic Ca2- If the blood glucoselevel falls below about 5 mM, for ex-
concentration, triggering thefusionof insulin-containing secretory ample due to suddenmuscular activity, reducedinsulin secre-
vesicleswiththeplasma membrane andthesecretion of insulin(6) tion from pancreatic B cells induces pancreatic ct cells to
[Adapted fromJ Q Henquin, 2000,Diabetes49:1751 ] increasetheir secretionof glucagon into the blood. Like the

llll+ Technique
Animation:ReporterConstructs
( a ) R e s t i n gc e l l 2.5min (c) 5 min (d) 10 min

5lrt
| I

a EXPERIMENTAL FIGURE 15-34Insulinstimulationof fat membraneSuccessive images of the samecellaftertreatment with

cellsinducestranslocation of GLUT4from intracellular i n s u l ifno r 2 5 , 5 , a n d 1 0 m i n u t essh o w t h a t w i t t
h i m e i
, n c r e a sing
vesicles to the plasmamembrane.Inthisexperiment, fat cells numbers of theseGLUT4-containing membranes fusewith the
wereengineered to express
a chimeric proteinwhoseN-terminal plasma membrane, therebymovingGLUT4 to thecellsurface
endcorresponded to the GLUT4 sequence, followedbythe (arrows) andenabling it to transport glucose fromthe bloodintothe
entirety of the GFPsequence Whena cellisexposed to lightof cell Muscle cellsalsocontain insulin-responsive GLUT4 transporters
theexciting wavelength, GFPfluoresces yellow-green, indicating [Courtesy of J Bogan; see J Bogan et al , 2001, Mol Cell Biol21:47851
the position of GLUT4 cells(a),mostGLUT4
withincellsIn resting
is in internalmembranes thatarenot connected to the plasma

T O E N V I R O N M E N T AI N
OSF C E L L S
I N T E G R A T I NRGE S P O N S E LFLUENCES 659
epinephrinereceptor,the glucagon receptor,found primarily
on liver cells,is coupled to the Go, protein, whose effector pro-
tein is adenylyl cyclase.Glucagon stimulation of liver cells in-
ducesa rise in cAMP, leading to activation of protein kinase A,
which inhibits glycogen synthesisand promotes glycogenoly-
sis, yielding glucose 1-phosphate(seeFigures 1S-25a and
15-32b). Liver cellsconvert glucose1-phosphateinto glucose,
which is releasedinto the blood, thus raising blood glucose
back toward its normal fastinelevel.
Unfortunatelg these intricate and powerful control sys-
temssometimesfail, causingserious,evenlife-threatening
disease.Diabetes mellitus results from a deficiency in the
amount of insulin releasedfrom the pancreasin responseto
rising blood glucose(type I) or from a decreasein the ability
of muscleand fat cellsto respond to insulin (type II). In both
types, the regulation of blood glucoseis impaired, leading to
persistent elevated blood glucose concentrations (hyper-
glycemia) and other possiblecomplications if left untreated.
Type I diabetes is caused by an autoimmune process that
destroys the insulin-producing B cells in the pancreas.Also
called insulin-dependentdiabetes,this form of the diseaseis
generallyresponsiveto insulin therapy.Most Americanswith
diabetes mellitus have type II, or insulin-independent
diabetes,but the underlying causeof this form of the disease
is not well understood.Further identificat
pathways that control energymetabolism
vide insight into the pathophysiology of
leading to new methods for its prevention
lntegrating Responsesof Cellsto Environmental
Influences
r Glycogen breakdown and synthesisis regulated by mul-
tiple second messengersinduced by neural or hormonal
stimulation (seeFigure 1,5-32).
r A rise in blood glucose stimulates the releaseof insulin
from pancreatic B cells (see Figure 1S-33). Subsequent
binding of insulin to its receptor on muscle cells and
adipocytes leads to the activation of protein kinase B.
which promotesglucoseuptake and glycogensynrhesis,re-
sulting in a decreasein blood glucose.
A lowering of blood glucose stimulates glucagon re-
asefrom pancreaticct cells.Binding of glucagonto its G
protein-coupled receptor on liver cellspromotes glycogenoly-
sis by the cAMP-triggered kinase cascade(similar to epi-
nephrine stimulation under stress conditions) and an
increasein blood glucose.
In this chapter we focused primarily on signal-transduction
pathways activated by individual G protein-coupled recep-
tors. However, even these relatively simple pathways
presage the more complex situation within living cells.
650 C H A P T E R1 5 I C E L LS I G N A L I N GI : S I G N A LT R A N S D U C T I OA
Many G protein-coupled receptors form homodimers or
heterodimers with other G protein-coupled receptors that
bind ligands with different specificitiesand affinities. Much
current researchis focused on determining the functions of
thesedimeric receptorsin the body.
With :720 membersin total, the G protein-coupled re-
ceptors represent the largest protein family in the human
genome. Approximately half of these genes are thought to
encode sensory receptors; of these the majority are in the
olfactory systemand bind odorants. Of the remaining 360 G
protein-receptors,the natural ligand has been identified for
approximately 210 receptors,Ieaving 150 so-called orphan
GPCRs, that is putative GPCRs without known cognatelig-
ands. Many of these orphan receptors are likely to bind
heretofore unidentified signaling molecules, including new
peptide hormones. G protein-coupled receptorsalready rep-
resentthe largestclassof target moleculesfor drugs available
in the clinic, and thereforeorphan GPCRsrepresenta fruitful
resourcefor drug discoveryby the pharmaceuticalindustry.
One approach that has proven fruitful in identifying lig-
ands of orphan GPCRs involves expressingthe receptor genes
in transfectedcellsand using them as a reporter systemto detect
substancesin tissue extracts that activate signal-transduction
pathways in thesecells.This approach has already led to stun-
ning insights into human behavior. One example is two novel
peptides,termed orexin-A and orexin-B (from the Greekorexis,
meaning appetite), that were identified as the ligands for two
orphan GPCRs.Further researchshowedthat the orexin geneis
expressedonly in the hypothalamus, the part of the brain that
regulatesfeeding. Injection of orexin into the brain ventricles
causedanimals to eat more, and expressionof the orexin gene
increasedmarkedly during fasting. Both of these findings are
consistent with orexin's role in increasing appetite. Strikingly
mice deficient for orexins suffer from narcolepsy, a disorder
characterizedin humans by excessivedaytime sleepiness(for
mice, nighttime sleepiness).Moreover, very recenr repofts sug-
gest that the orexin system is dysfunctional in a majority of
human narcolepsypatients: Orexin peptidescannot be detected
in their cerebrospinal fluid (although there is no evidence of
mutation in their orexin genes). These findings firmly link
orexin neuropeptidesand their receptorsto both feedingbehav-
ior and sleepin both animals and humans.
One can only wonder about what other peptides and
small-moleculehormonesremain to be discovered,and the in-
sightsthat study of thesewill provide for our understanding of
human metabolism,growth, and behavior.
KeyTerms
adenylyl cyclase639 cAMP 634
adrenergicreceptors competition assay629
636 desensitization531
agonist 629 endocrine 525
arrcstin 645 functional expressionassay
attocrine 626 631
calmodulin 555 glucagon 659
NN D S H O R T - T E R M
c E L L U L A RR E S P o N s E S
G protein-coupled rhodopsin 635 channelsvia G proteins. Describethe similarities and differ-
rcceptors 624 secondmessengers634 encesbetweenthesetwo systems'
insulin 659 signal amplif ication 649 10. In liver and muscle cells, epinephrine stimulates the
IP3/DAG pathway 554 releaseof glucosefrom glycogenby inhibiting glycogensyn-
signal transdtction 624
thesisand stimulating glycogenbreakdown. Outline the mo-
muscarinic acetylcholine stimulatory G protein 539
leculareventsthat occur after epinephrinebinds to its receptor
receptors 641 transducin641 and the resultantincreasein the concentrationof intracellular
paracrine 626 trimeric G proteins 534 cAMP. How are the cAMP levels returned to normal? De-
phospholipaseC 539 visual adaptation 544 scribe the eventsthat occur after cAMP levels decline.
protein kinaseA 647 11. Continuous exposure of a G. protein-coupled receptor
protein kinase C 657 to its ligand leads to a phenomenon known as desensitiza-
tion. Describe several molecular mechanisms for receptor
Reviewthe Concepts desensitization.How can a receptor be reset to its original
'Sfhat
sensitizedstate? effect would a mutant receptor lack-
ing serineor threonine phosphorylation siteshave on a cell?
1. What common featuresare sharedby most of the differ-
ent cell signaling systems? 12. Visual adaptation and receptor desensitizationinvolve
2. Signalingby soluble extracellular moleculescan be clas- similar phosphorylation mechanisms.Describe how the
sified as endocrine,paracrine,or autocrine.Describehow B-adrenergicreceptorkinase(BARK) and rhodopsin kinase
play important roles in theseprocesses.!7hat role doesde-
these three types of cellular signaling differ. Growth hor-
phosphorylation play in thesereactions?
mone is secretedfrom the pituitary, which is located at the
base of the brain, and acts through growth hormone recep- 13. Sometimescells need to localize the effects of signaling
tors located on the liver. Is this an example of endocrine, systemsto specificsubcellularregions. One example is local-
'$7hat
ization of cAMP signals in heart muscle. proteins are
paracrine, or autocrine signaling?\7hy?
involved? How does this sYstemwork?
3, A ligand binds two different receptorswith a K6 value
of 1.0-' M for receptor 1 and a K6 value of 1,0 " M for re- 14. Inositol 1,4,5-trisphosphate(IP3) and diacylglycerol
ceptor 2. For which receptor does the ligand show the (DAG) are second messengermoleculesderived from the
greater affinity? Calculate the fraction of receptors that cleavageof the phosphatidylinositol 4,5-bisphosphate(PIP2)
have a bound ligand ([RL]/Rr) in the caseof receptor 1 and by activated phospholipaseC. Describethe role of IP3 in the
receptor2, if the concentrationof free ligand is 10-" M. releaseof Ca2* from the endoplasmic reticulum' How do
cells replenishthe endoplasmicreticulum stores of Caz*?
4.