Practice Exam III Week 9 (04/08-04/09) Take a deep breath. We know you can do it.

Be patient and read every question carefully before you answer any question. Answer what we ask you to answer. This practice exam is not entirely comprehensive. So remember to go back and review your earlier worksheets. Part I: True/False 1^)__F__ In mammalian cells the import of proteins into the mitochondria begins before the polypeptide chain is completely synthesized - in other words it occurs co-translationally. 2^)_T___ To function effectively, each transport vesicle must selectively take up only the appropriate proteins and must fuse only with the correct target membrane. 3^)_F___ After a protein enters the nucleus, its signal sequence is removed by the activity of a signal peptidase, which ensure that it will remain in the nucleus. 4^)__F__ Complementary Rab proteins on transport vesicles and target membranes bind to one another to allow transport vesicles to dock selectively at their appropriate target membranes. 5^)__T__ Diacylglycerol is an example of a second messenger used in cell signaling. 6^)___F_ The receptors involved in paracrine and synaptic signaling have the highest affinity for their respective receptors. 7^)_T___ The activity of any protein regulated by phosphorylation depends on the balance at any instant between the activities of the kinases that phosphorylate it and the phosphatases that dephosphorylate it. 8)^_F___ Binding of extracellular ligands to receptor tyrosine kinases (RTKs) activates the intracellular catalytic domain by propagating a conformational change across the lipid bilayer through a single transmembrane region. 9) T The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins. The fate of a protein with no sorting signal is to remain in the cytosol.

10) T

Part II: Multiple Choice 1^) If you were to remove the ER retention signal from protein disulfide isomerase (PDI),, which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be found? a. the ER b. the Cytosol c. extracellular fluid d. a lysosome

2^) Proteins that do not fold properly in the ER lumen are degraded in the cytosol by: a. ribosomes b. proteosomes c. signal peptidases d. ubiquitin 3^) The sorting of proteins to mitochondria and chloroplasts is: a. co-translational b. post-translational c. pre-translational d. gated transport 4^) Imagine a protein engineered with an ER signal sequence at its N-terminus and a nuclear localization signal near the C-terminus. The most likely final location for this protein is: a. the ER b. the nucleus c. the cytosol d. secreted 5^) Which of the following would be expected to happen in a mutated G protein that results in a decreased rate at which the signal causes the receptor to release GDP, but no affect on its affinity thereafter for GTP? a. a G-protein that was constantly activated b. a G-protein that was constantly inactivated c. a G-protein with a delayed activation response d. a G-protein with a delayed inactivation response 6) Receptor tyrosine kinases a. are able to function as monomers b. dimerize to bring the kinase domains of their receptor tails together c. able to phosphorylate G-proteins d. structured to have cytoplasmic domains that are enzymatically active 7) Which of the following is FALSE a. IP3 is produced directly by cleavage of an inositol phospholipid without the incorporation of an additional phosphate group b. The extracellular signal molecule acetylcholine has different effects on different cell types in an animal and often binds to different cell surface molecules on different cell types c. Both the GTP bound alpha subunits and nucleotide free beta gamma complexes--but not GDP bound fully assembled G-proteins -- can activate other molecules downstream of GPCRS. d. Calmodulin modulates the intracellular Ca2+ concentration.

8!) Actin filaments and microtubules share all of the following properties EXCEPT: a. They are involved in cell motility b. They are intrinsically polar structures c. They can associate with motor proteins d. They are assembled from subunits that are heterodimers e. They can be cross-linked into bundles 9!) Which of the following is NOT a characteristic of intermediate filaments a. They form the nuclear lamina b. They provide a mechanical stability to animal cells

c. Their protein composition is tissue specific d. They are composed of globular monomers that polymerize to form fibers e. They include the keratin filaments of epithelial cells 10!) Dynamic instability of microtubules stems from a. The complexity of the microtubule plus end b. The difference in critical concentration of GTP -tubulin and GDP-tubulin at the microtubule plus end c. The fact that one of the GTPs on the tubulin dimer is never hydrolyzed d. The intrinsic ability of the tubulin subunit to hydrolyze GTP to GDP e. More than one of the answers above is correct %11.) Which of the following is NOT true of chromosome segregation during mitosis? a. It requires motor-driven microtubule sliding. b. It requires polymerization and depolymerization of microtubules. c. It requires polymerization of intermediate filament fibers. d. It uses plus end- and minus end- directed molecular motors. %12.) Myosin motors require ATP hydrolysis to move along filaments because ATP hydrolysis a. is important to promote actin polymerization b. is required to generate the force necessary for the power stroke c. is required to initiate cytokinesis d. is driven by an energetically favorable process %13.) Dyneins and kinesins a. have sequence and structural homology b. are important during cytokinesis c. always transport cargo within the cell. d. alternate between high affinity and low affinity for microtubules. %14.) Which of the following statement(s) are FALSE? a. Long actin filaments work best for pushing the plasma membrane during cell crawling. b. Actin filaments underlie the plasma membrane in animal cells. c. Actin can generate force without the aid of motors. d. Some actin binding proteins can promote the formation of branched actin filaments. %15.) Two properties of microtubule polymerization are dynamic instability and treadmilling. Which of the following is NOT a true statement about these processes? a. Both are dependent on rates of polymerization and depolymerization of subunits. b. In one case the filament changes in size dramatically while in the other the size appears to remain the same. c. Both are influenced by the concentration of subunits in the surrounding environment. d. Both can be influenced by various classes of actin-binding proteins. Part III: Protein Localization ^1. Imagine a mutant cell in which the Ran protein involved in nuclear transport can no longer bind to GTP. What is the phenotype or expected outcome for this mutant cell? Please be specific but be brief! Explain what part of nuclear transport will be affected, if at all, by this mutation. Proteins will be imported into the nucleus normally. However, w/o RAN-GTP in the nucleus, the receptor will not release the cargo. Soon all the receptors full of cargo will be in the nucleus, leaving no receptors to transport new proteins into the nucleus from the cytosol.

#2. Imagine that you engineered a set of genes, each encoding a protein with a pair of conflicting signal sequences specifying different compartments. If the genes were expressed in a cell, predict which signal would win out for each of the following combinations (please circle one signal). a. A signal for import into the nucleus and a signal for import into the ER. b. A signal for import into the nucleus and a signal for the import into mitochondria. c. A signal for import into mitochondria and a signal for retention in the ER. d. A signal for import into the nucleus and a signal for export from the nucleus. Part IV: Cell Signaling 1.Briefly describe what they are and where in the GPCR signaling pathway the following components come into play: DAG

cAMP

GEF

AC (adenylyl cyclase)

GPCR

IP3

alpha-subunit

PKA

2) You are interested in cell size regulation and discover that signaling through a GPCR is important in controlling cell size in rat white blood cells. The G protein downstream of this receptor activates adenylyl cyclase, which ultimately leads to the activation of PKA. You find that cells that lack the GPCR are 15% smaller than normal cells, whereas cells that express a mutant, constitutively activated version of PKA are 15% larger than normal cells. Furthermore, the normal blood cells become smaller when treated with cholera toxin, which has been shown to inhibit certain subclasses of the a subunit of the G protein. Given these results, explain what you predict would happen to the cells size (bigger, smaller, or no change) if cells were treated in the following fashion.

a. You add pertussis toxin. Smaller. It would inhibit subclasses of the subunit of the G Protein.

b. You inhibit the RGS protein that normally works on the a subunit of the G protein involved in this pathway. Smaller.

c. You add a drug that increases the activity of cyclic AMP phosphodiesterase. Bigger.

d.You add a drug that inhibits adenylyl cyclase. No change.

e.You mutate the cAMP-binding sites in the regulatory subunits of PKA, so that the PKA binds more tightly to cAMP. No change.

3) Outline a general signaling pathway that would allow a yeast to move in response to yeast mating factor. This is a form of chemotaxis, or cell movement in a direction controlled by a chemical gradient. You do not know the steps in the middle, but you can predict generally how such a pathway would begin and end. (Hint, how would the cell receive the mating factor signal and how would the cell be able to move towards it?)

Part V: Cytoskeleton & Motor Proteins !1. Fill in the blanks: A. dynein and kinesin are motor proteins that use the energy of to move unidirectionally along microtubules. The direction of movement is determined by the concentration of the microtubules. B. Eukaryotic cilia and flagella contain bundles of stable intermediate filaments that originate from structures called . Microtubules in cilia and flagella have a unique _________ configuration and interact with a special motor protein called . C. A concentrated network of actin filaments underneath the plasma membrane forms the and is responsible for cell shape and movement. motor proteins use as energy to move along actin filaments, causing adjacent filaments to slide past each other in structure known as .

!2. About kinesin and dynein. Does dynein move things towards the nucleus of a cell and therefore towards a cell if it is coming from the outside? And does kinesin move things to the plasma membrane of a cell and therefore away? Both only move things inside cells. But dynein moves things toward the - ends of MT's and kinesin moves things toward the + ends.

%3. The figure below represents a typical time course of polymerization of filamentous actin from actin monomers.

% of actin subunits in filaments

Time A. Complete the figure by 1) labeling the axes, 2) naming each of the three phases represented by numerals I, II and III. B. Explain (in 1-2 sentences) the behavior of monomers in each of the three phases above. I. Nucleation (Lag Phase)

II. Elongation (Growth Phase)

III. Steady State (Equilibrium)

C. Indicate on the figure what the graph would look like if a large amount of a nucleating protein was added at the start? Label this curve C. D. What would happen to the curve if you added nibblerin, a protein that sequestered (bound up) actin monomers and prevented polymerization? Assume enough of this protein is added to bind up half of /

%4. In your spare time while preparing for your exam, you set up multiple microtubule motility assays to get a truly accurate measure of kinetic parameters for kinesin. To do this, you adhered kinesin to a glass slide and added fluorescent microtubules so that they could glide along, propelled by the adhered kinesins. A. President Pasquerella, wanting to encourage your quest for knowledge and willingness to do what no one else will do, offers to help you. The first time she does, however, she forgets to add taxol (a drug that binds tightly to microtubule, stabilizing the polymer). Explain briefly why you will be unable to obtain reliable measurements of microtubule velocity without taxol.

B. In a cell, kinesin can carry vesicles for long distances along microtubule tracks. Do you think the two head domains of a kinesin molecule are essential to accomplish this task or could a one headed kinesin function just as well? Explain your answer.

!5. A solution of alpha beta tubulin dimer (free of other proteins) is thought to nucleate microtubules by forming a linear protofilament about 7 dimers in length. At that point, the probabilities that the next dimer will bind laterally or to the end of the protofilament are about the same. The critical event for tubule formation is thought to be the first lateral association. How does lateral association promote the subsequent formation of a microtubule?

%- taken from Omar Quintero, BIO 220 Exam 3, 2008. ^- taken from Sharon Stranford, Cell BioExam III, 2010. !- taken from Cell Bio PLUMS, 2011