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Biochemistry of Lipids, Lipoproteins and Membranes

Biochemistry of Lipids, Lipoproteins and Membranes

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Biochemistry of Lipids, Lipoproteins and Membranes

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Jul 24, 2015


Biochemistry of Lipids: Lipoproteins and Membranes, Volume Six, contains concise chapters that cover a wide spectrum of topics in the field of lipid biochemistry and cell biology. It provides an important bridge between broad-based biochemistry textbooks and more technical research publications, offering cohesive, foundational information.

It is a valuable tool for advanced graduate students and researchers who are interested in exploring lipid biology in more detail, and includes overviews of lipid biology in both prokaryotes and eukaryotes, while also providing fundamental background on the subsequent descriptions of fatty acid synthesis, desaturation and elongation, and the pathways that lead the synthesis of complex phospholipids, sphingolipids, and their structural variants. Also covered are sections on how bioactive lipids are involved in cell signaling with an emphasis on disease implications and pathological consequences.

  • Serves as a general reference book for scientists studying lipids, lipoproteins and membranes and as an advanced and up-to-date textbook for teachers and students who are familiar with the basic concepts of lipid biochemistry
  • References from current literature will be included in each chapter to facilitate more in-depth study
  • Key concepts are supported by figures and models to improve reader understanding
  • Chapters provide historical perspective and current analysis of each topic
Jul 24, 2015

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Biochemistry of Lipids, Lipoproteins and Membranes - Elsevier Science

Biochemistry of Lipids, Lipoproteins and Membranes

Sixth Edition


Neale D. Ridgway, Ph.D.

Departments of Pediatrics and Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Roger S. McLeod, Ph.D.

Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Table of Contents

Cover image

Title page




Chapter 1. Functional Roles of Lipids in Membranes

1. Introduction and Overview

2. Diversity in Lipid Structure

3. Properties of Lipids in Solution

4. Engineering of Membrane Lipid Composition

5. Role of Lipids in Cell Function

6. Summary and Future Directions

Chapter 2. Approaches to Lipid Analysis

1. Introduction and Overview

2. Lipid Diversity

3. Chromatographic-Based Analysis of Lipids

4. Basic Concepts of Analytical Biochemistry

5. Lipid Mass Spectrometry

6. Future Directions

Chapter 3. Fatty Acid and Phospholipid Biosynthesis in Prokaryotes

1. Overview of Bacterial Lipid Metabolism

2. Membrane Systems of Bacteria

3. The Initiation Module

4. The Elongation Module

5. The Acyltransfer Module

6. The Phospholipid Module

7. Genetic Regulation of Lipid Metabolism

8. Future Directions

Chapter 4. Lipid Metabolism in Plants

1. Introduction

2. Plant Lipid Geography

3. Acyl-Acyl Carrier Protein Synthesis in Plants

4. Acetyl-Coenzyme A Carboxylase and Control of Fatty Acid Synthesis

5. Phosphatidic Acid Synthesis Occurs via Prokaryotic and Eukaryotic Acyltransferases

6. Membrane Glycerolipid Synthesis

7. Lipid Storage in Plants

8. Protective Lipids: Cutin, Waxes, Suberin and Sporopollenin

9. Sphingolipid Biosynthesis

10. Oxylipins as Plant Hormones

11. Sterol and Isoprenoid Biosynthesis

12. Future Prospects

Chapter 5. Fatty Acid Handling in Mammalian Cells

1. Introduction

2. Fatty Acid Biosynthesis

3. Fatty Acid Uptake, Activation and Trafficking

4. Fatty Acid Storage as Triacylglycerol in Lipid Droplets

5. Fatty Acid Use for Energy

6. Fatty Acids and Signalling

7. Fatty Acids and Disease Pathogenesis

8. Future Directions

Chapter 6. Fatty Acid Desaturation and Elongation in Mammals

1. Introduction

2. Elongation Reactions of Long-Chain Fatty Acids

3. Desaturation of Long-Chain Fatty Acid in Mammals

4. Transcriptional Regulation of Desaturases and Elongases

5. Summary and Future Directions

Chapter 7. Phospholipid Synthesis in Mammalian Cells

1. Introduction

2. Biosynthesis of Phosphatidic Acid and Diacylglycerol

3. Phosphatidylcholine Biosynthesis and Regulation

4. Phosphatidylethanolamine Biosynthesis and Regulation

5. Phosphatidylserine Biosynthesis and Regulation

6. Phosphatidylinositol and Polyphosphorylated Phosphatidylinositol

7. Biosynthesis of Phosphatidylglycerol and Cardiolipin

8. Fatty Acid Remodelling of Phospholipids

9. Future Directions

Chapter 8. Phospholipid Catabolism

1. Introduction

2. The Phospholipase A Family

3. Phospholipase C

4. Phospholipase D

5. Future Directions

Chapter 9. The Eicosanoids: Cyclooxygenase, Lipoxygenase and Epoxygenase Pathways

1. Introduction

2. Prostanoids

3. Prostanoid Biosynthesis

4. Prostanoid Catabolism and Mechanisms of Action

5. Leukotrienes and Lipoxygenase Products

6. Cytochrome P450S and Epoxygenase Pathways

7. Future Directions

Chapter 10. Sphingolipids

1. Introduction

2. Nomenclature and Structure

3. Sphingolipids Biosynthesis

4. Sphingolipid Degradation

5. Sphingolipid Signalling and Roles in Cell Regulation

6. Sphingolipid Biophysics

7. Sphingolipids in Disease Pathology

8. Perspectives

Chapter 11. Cholesterol Synthesis

1. Introduction

2. Cholesterol Synthesis – An Historical Overview

3. Targeting Cholesterol Synthesis Therapeutically

4. Sterol Pathway Intermediates

5. Enzymes of Cholesterol Biosynthesis

6. Oxysterols

7. Regulation of Cholesterol Synthesis

8. Summary

Chapter 12. Bile Acid Metabolism

1. Introduction

2. Bile Acid Structure and Physical Properties

3. Biosynthesis of Bile Acids

4. Enterohepatic Circulation of Bile Acids

5. Bile Acids as Signalling Molecules

6. Future Directions

Chapter 13. Lipid Modification of Proteins

1. Introduction

2. Attachment of Fatty Acids to Proteins

3. Attachment of Cholesterol to Hedgehog Proteins

4. Attachment of Isoprenoids to Proteins

5. Attachment of Phospholipids and Diacylglycerol Lipids to Proteins

6. Spotlight on Inhibitors of Lipid-Modifying Enzymes and Their Roles in Disease

7. Future Directions and Challenges

Chapter 14. Intramembrane and Intermembrane Lipid Transport

1. Introduction

2. Vesicular Trafficking of Lipids

3. Nonvesicular Transport of Lipids

4. Transbilayer Movement of Lipids

5. Specific Examples of Intracellular Lipid Transport

6. Future Directions

Chapter 15. High-Density Lipoproteins: Metabolism and Protective Roles Against Atherosclerosis

1. Introduction

2. High-Density Lipoprotein Formation

3. High-Density Lipoprotein Remodelling and Lipid Transfer

4. Extremes of High-Density Lipoprotein Cholesterol Levels and Relationship to Atherosclerosis

5. Protective Actions of High-Density Lipoproteins

6. High-Density Lipoprotein-Raising Therapies

7. Summary and Future Directions

Chapter 16. Assembly and Secretion of Triglyceride-Rich Lipoproteins

1. Overview of Apolipoprotein B-Containing Lipoproteins

2. Structure and Regulation of the Apolipoprotein B Gene

3. Structural Features of Apolipoprotein B

4. Assembly of Hepatic Very Low Density Lipoproteins

5. Regulation of Hepatic Very Low Density Lipoprotein Assembly and Secretion

6. Intracellular Degradation of Apolipoprotein B

7. Dysregulation of Very Low Density Lipoprotein assembly and Secretion

8. Assembly and Secretion of Chylomicrons

9. Hepatocyte and Enterocyte Models – Strengths and Limitations

10. Future Directions

Chapter 17. Lipoprotein Receptors

1. Introduction: Receptor-Mediated Lipoprotein Metabolism

2. Removal of Low-Density Lipoprotein from the Circulation

3. Post-translational Modulators of Low-Density Lipoprotein Receptor Activity

4. Receptor-Mediated Removal of Triacylglycerol-Rich Lipoproteins from the Plasma

5. Other Relatives of the Low-Density Lipoprotein Receptor Family

6. Roles of Lipoprotein Receptors in Signal Transduction

7. Scavenger Receptors: Lipid Uptake and Beyond

8. Outlook

Chapter 18. Atherosclerosis

1. Atherosclerosis

2. Lipoprotein Transport in Atherosclerosis

3. Lipoprotein Receptors and Lipid Transporters

4. Contributions of Lipoprotein-Mediated Inflammation to Atherosclerosis

5. New Emerging Mechanisms of Lipid Metabolism Influencing Atherosclerosis

6. Traditional and Evolving Lipid-Lowering Therapies for the Treatment of Atherosclerosis

7. Future Directions

Chapter 19. Diabetic Dyslipidaemia

1. Introduction to the Typical Dyslipidaemia of Insulin-Resistant States

2. Dyslipidaemia of Insulin-Resistant States: Key Factors and Mechanisms, with a Focus on Hepatic Lipoprotein Overproduction

3. Postprandial Dyslipidaemia and Intestinal Chylomicron Hypersecretion in Insulin-Resistant States

4. Low High-Density Lipoprotein in Insulin Resistance and Type 2 Diabetes

5. Treatment of the Dyslipidaemia of Insulin-Resistant States

6. Conclusions




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Khosrow Adeli,     The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Mikhail Bogdanov,     Department of Biochemistry and Molecular Biology, University of Texas–Houston, Medical School, Houston, TX, USA

Laura M. Bond,     Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

Andrew J. Brown,     School of Biotechnology and Biomolecular Sciences, The University of New South Wales (UNSW Australia), Sydney, NSW, Australia

H. Alex Brown

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA

Department of Biochemistry and The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

Alan Daugherty,     Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA

Paul A. Dawson,     Division of Pediatric Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA

Fang Ding,     Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Wenjiang, Sichuan, PR, China

William Dowhan,     Department of Biochemistry and Molecular Biology, University of Texas–Houston, Medical School, Houston, TX, USA

Sarah Farr,     The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Gordon A. Francis,     Department of Medicine, Centre for Heart Lung Innovation, University of British Columbia, Vancouver, BC, Canada

Anthony H. Futerman,     Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Murray W. Huff,     Departments of Medicine and Biochemistry, Robarts Research Institute, The University of Western Ontario, London, ON, Canada

Pavlina T. Ivanova,     Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA

Richard Lehner

Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, AB, Canada

Departments of Pediatrics and Cell Biology, University of Alberta, Edmonton, AB, Canada

Gary F. Lewis,     University Health Network, University of Toronto, Toronto, ON, Canada

Hong Lu,     Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA

Frederick R. Maxfield,     Department of Biochemistry, Weill Cornell Medical College, New York, NY, USA

Jeff G. McDonald,     Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, USA

Roger S. McLeod,     Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada

Anant K. Menon,     Department of Biochemistry, Weill Cornell Medical College, New York, NY, USA

Eugenia Mileykovskaya,     Department of Biochemistry and Molecular Biology, University of Texas–Houston, Medical School, Houston, TX, USA

Makoto Miyazaki,     Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA

Robert C. Murphy,     Department of Pharmacology, University of Colorado-Denver, Aurora, CO, USA

James M. Ntambi

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

Department of Nutritional Science, University of Wisconsin-Madison, Madison, WI, USA

Lucas M. O’Neill,     Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

Ariel D. Quiroga

Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, AB, Canada

Institute of Experimental Physiology (IFISE-CONICET), Faculty of Biochemical and Pharmacological Sciences, National University of Rosario, Suipacha, Rosario, Argentina

Marilyn D. Resh,     Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Neale D. Ridgway,     Departments of Pediatrics and Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada

Charles O. Rock,     Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN, USA

Katherine M. Schmid,     Department of Biology, Butler University, Indianapolis, IN, USA

Wolfgang J. Schneider,     Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria

Laura J. Sharpe,     School of Biotechnology and Biomolecular Sciences, The University of New South Wales (UNSW Australia), Sydney, NSW, Australia

William L. Smith,     Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA

Robert V. Stahelin

Department of Biochemistry & Molecular Biology, Indiana University School of Medicine-South Bend, South Bend, IN, USA

Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN, USA

Jennifer Taher,     The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Changting Xiao,     University Health Network, University of Toronto, Toronto, ON, Canada

Zemin Yao,     Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

Yong-Mei Zhang,     Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA


This represents the sixth edition of the textbook first edited by Drs. Dennis and Jean Vance in 1985. In taking on the editorship, we are revising the textbook that we used as an essential resource early in our academic careers. With this in mind we have strived to assemble a text that will inspire students to embrace the challenges of the future in lipid, lipoprotein and membrane biology.

Since the last edition that appeared in 2008 there have been remarkable advances in lipid and membrane biology in terms of identification of fundamental metabolic processes and their relationship to a broad spectrum of human diseases. The authors of this edition are at the forefront of these discoveries, and have provided chapters with a basic knowledge component coupled with unique insights into their respective fields of research. This edition also has a more defined focus on the impact on new technologies and relevance to chronic disease with a view to future studies of lipid metabolism. As with previous editions, the content is easily accessible to a broad spectrum of learners with a basic understanding of biochemistry and metabolism. The text also serves as a gateway to further exploration of topics, and provides a bridge between basic concepts and the current research literature. Thus undergraduate and graduate students will find this book to be an essential resource for course and research-related studies, while experienced researchers can use it as a reference guide to the lipid field.

All of the chapters have been revised from the fifth edition and new authors have taken on the task for many. We asked the authors to resist the temptation to be comprehensive; that we were not seeking to assemble a compendium of reviews. In addition, we limited the number of citations, attempting to glean classic and exceptional recent studies in each area. We are grateful that all the authors have complied. This edition of the book has full-color figures embedded in the text and standardized tables, which add visual appeal and clarity.

The contributors and editors assume full responsibility for the content and would appreciate any and all feedback for refinement of future editions.

Neale D. Ridgway,  and Roger S. McLeod,     Halifax, Nova Scotia, Canada, June 2015

Chapter 1

Functional Roles of Lipids in Membranes

William Dowhan, Mikhail Bogdanov,  and Eugenia Mileykovskaya     Department of Biochemistry and Molecular Biology, University of Texas–Houston, Medical School, Houston, TX, USA


Biological membranes define the outer limits of cells and organelles and are composed of phospholipids, glycolipids, sphingolipids, sterols and proteins. Each lipid class is composed of numerous variants within their respective polar and apolar domains. The apolar and polar nature of these amphipathic lipids is the basis for forming biological membranes with which membrane proteins associate either as integral proteins that span the membrane bilayer or as peripheral proteins that associate with the membrane surface. Individual lipids, once thought mainly to provide cell barrier function and a solvent for membrane proteins, are now recognised as critical components that directly influence an array of cellular functions. Physical and chemical properties of lipids that determine the properties of biological membranes, genetic approaches used to alter cellular lipid composition and examples of how lipid–protein interactions define specific roles for lipids in cell function are discussed.


Charge balance rule; Hydrophobic effect; Lipid diversity; Lipid–protein interactions; Membrane structure







   Extramembrane domain


   Monoglucosyl diacylglycerol


   DIglucosyl diacylglycerol


   10-N-nonyl acridine orange


   Phosphatidic acid












   Midpoint temperature


   Transmembrane domain

1. Introduction and Overview

Lipids as a class of molecules display a wide diversity in structure and biological function. A primary role of lipids is to form the membrane bilayer permeability barrier of cells and organelles (Figure 1). Glycerophospholipids (termed phospholipids hereafter) make up about 75% of total membrane lipids of prokaryotic and eukaryotic cells, but other lipids are important components. Table 1 shows the major lipids found in the membranes of various cells and organelles but does not include the minor lipids, many of which are functionally important. Sterols are present in all eukaryotic cells and in a few bacterial membranes. The major sterol of mammalian cells is cholesterol whereas yeast contain ergosterol. Bacteria do not make sterols but some species incorporate sterols from the growth medium. Interestingly Drosophila also must acquire cholesterol from exogenous sources. The ceramide-based sphingolipids are present in the membranes of all eukaryotes. Neutral diacylglycerol (DAG) glycans are major membrane-forming components in many Gram-positive bacteria and in the membranes of plants, while Gram-negative bacteria utilise a saccharolipid (Lipid A) as a major structural component of the outer leaflet of the outer membrane. The variety of hydrophobic domains of lipids results in additional diversity. In eukaryotes and eubacteria these domains are saturated and unsaturated fatty acids or lesser amounts of fatty alcohols; many Gram-positive bacteria also contain branched chain fatty acids. Instead of esterified fatty acids, Archaea contain long chain reduced polyisoprene moieties in ether linkage to glycerol. Such hydrophobic domains are highly resistant to the harsh environment of these organisms. Further stability of the lipid bilayer of Archaea comes from many of the hydrocarbon chains spanning the membrane with covalently linked head groups at each end. If one considers a simple organism such as Escherichia coli with three major phospholipids and several different fatty acids along with many minor precursors and modified lipid products, the number of individual phospholipid species ranges in the hundreds. In more complex eukaryotic organisms with greater diversity in both phospholipids and fatty acids, the number of individual species is in the thousands. Sphingolipids also show a similar degree of diversity and when added to the steroids the size of the eukaryotic lipidome dwarfs that of the proteome.

Figure 1  Model for membrane structure. This model of the plasma membrane of a eukaryotic cell is an adaptation of the original model proposed by Singer and Nicholson (1972) . The phospholipid bilayer is shown with integral membrane proteins largely containing α-helical transmembrane domains (TMDs). Peripheral membrane proteins associate either with the lipid surface or with other membrane proteins. Lipid rafts (dark green head groups) are enriched in cholesterol and contain a PI glycan-linked (GPI) protein. The purple head groups depict lipids in close association with protein. The irregular surface and wavy acyl chains denote the fluid nature of the bilayer.

Table 1

Lipid Composition of Various Biological Membranes

The data are expressed as mol% of total lipid. N.D. indicates not detected and blank indicates not analysed.

a Human (Tanford, 1980).

b Chinese hamster cells (Ohtsuka et al., 1993).

Saccharomyces cerevisiae inner and outer mitochondrial membrane (Zinser et al., 1991).

d Murine L cells (Murphy et al., 2000).

e Inner and outer membrane excluding Lipid A (Raetz, 1990).

Lipids provide the solvent within which integral membrane proteins (those whose transmembrane domains (TMDs) span the bilayer) are integrated. Peripheral proteins also interact with the membrane surface and are even found partially inserted into the lipid bilayer. These amphitropic proteins are found in the aqueous compartments of cells and interact with the membrane surface in a reversible manner. The lipid bilayer provides a rich and varied environment for proteins, which includes a highly hydrophobic interior bounded by the hydrophilic and/or charged lipid head groups. The latter organises water and counterions in a manner significantly differently from that of the cell aqueous phase, which imparts distinct properties to the aqueous layer in close contact with the membrane surface. Each lipid molecular class is made up of a wide spectrum of chemical and structural variants, which as an ensemble determine membrane fluidity, lateral pressure, permeability and surface charge. The lipid and protein components of the membrane are not held together by covalent interactions and therefore are in dynamic equilibrium undergoing transient interactions organised into the supermolecular structure of the lipid bilayer.

In this chapter, the diversity in structure, chemical properties and physical properties of lipids will be outlined. The various genetic approaches available for studying lipid function in vivo will be summarised. Finally, how the physical and chemical properties of lipids relate to their multiple functions in living systems will be reviewed to provide a molecular basis for the diversity of lipid structures in natural membranes. Due to space limitations, recent review articles and research articles, which contain the primary background references supporting the summaries in the text, are cited.

2. Diversity in Lipid Structure

Lipids are defined as those biological molecules readily soluble in organic solvents such as chloroform, ether or toluene. However, many peptides and some very hydrophobic proteins are soluble in organic solvents, and lipids with large hydrophilic domains such as saccharolipids are not soluble in these solvents. Here we will consider those lipids that contribute significantly to membrane structure or have a role in determining protein structure or function. The LIPID MAPS consortium (http://www.lipidmaps.org) in the United States, Lipid Bank (http://www.lipidbank.jp) in Japan and the LipidomicNet (http://www.lipidomicnet.org) in Europe have cooperated to devise classification systems, methodology and forums for the benefit of researchers.

2.1. Glycerolipids

The DAG backbone in eubacteria and eukaryotes is sn-3-glycerol (L-glycerol) esterified at the 1- and 2-position with long chain fatty acids (Figure 2) (Chapters 3 and 7). In Archae (Figure 3) sn-1-glycerol (D-glycerol) forms the backbone and the hydrophobic domain is composed of phytanyl (saturated isoprenyl) groups in ether linkage at the 2- and 3-positions (an archaeol) (Koga and Morii, 2007). In addition, two sn-1-glycerol groups are connected in ether linkage by two biphytanyl groups (dibiphytanyldiglycerophosphatetetraether) to form a covalently linked bilayer. Some eubacteria (mainly hyperthermophiles) have dialkyl (long chain alcohols in ether linkage) phospholipids and similar ether linkages are found in the plasmalogens of eukaryotes. The head groups of the phospholipids (boxed area of Figure 2) extend the diversity of lipids defining phosphatidic acid (PA, with OH), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI) and cardiolipin (CL). Archae analogues exist with head groups of glycerol and glyceromethylphosphate as well as all of the above except PC. Archae also have neutral glycan lipid derivatives in which mono- and disaccharides (glucose or galactose) are directly linked to the sn-1 position of archaeol (Figure 3). Plants (mainly in the thylakoid membrane) and many Gram-positive bacteria also have high levels of neutral DAG glycans with mono- or disaccharides linked to the 3-carbon of sn-3-glycerol (Chapter 4). In addition to head group diversity, a range of alkyl chains are attached to the glycerol moiety. In eubacteria, fatty acid chain lengths vary from 12 to 18 carbons and can be fully saturated or contain double bonds. Some Gram-positive bacteria contain odd-numbered, branched chain fatty acids rather than unsaturated fatty acids. Eukaryotic lipids contain fatty acid chains up to 26 carbons in length with multiple or no double bonds. Therefore, the diversity of glycerol-based lipids in a single organism is significant, but the diversity throughout nature is enormous.

Figure 2  Structure of glycerophosphate-based lipids. The lipid structure shown is 1,2 distearoyl- sn -glycerol-3-phosphocholine or phosphatidylcholine (PC). Substitution of choline in the box with the head groups shown on the right forms the other phospholipid structures. Cardiolipin (CL) is also referred to as diphosphatidylglycerol since it contains two PAs joined by a glycerol.

The majority of information on the chemical and physical properties of lipids comes from studies on the major phospholipid classes of eubacteria and eukaryotes with only limited information on the lipids from Archae. The biosynthetic pathways and the genetics of lipid metabolism have also been extensively studied in eubacteria (Chapter 3) and eukaryotes (Chapter 7). Clearly the archaeol lipids confer some advantage with respect to the environment of archaebacteria. Interestingly, the pathways for phospholipid biosynthesis in eubacteria and Archaea are very similar even though their lipids differ in chirality of the glycerol backbone. How the physical properties of the more commonly studied lipids change with environment will be discussed later.

Figure 3  Structure of dialkylglycerols in Archae . Archae have phytanyl chains in ether linkage to the 2- and 3-positions of sn -1-glycerol (archaeol). The 1-position can be derivatised with phosphodiesters. (A) Diphytanylglycerol (C20–C20 diether) with the stereochemistry of glycerol indicated. (B) Cyclic biphytanyl (C40) diether. (C) Biphytanyl diglycerol diether. (D) A glyceroglycan with either a mono or a disaccharide (glucose or galactose) at the 1-position of sn -1-glycerol. The R groups are ether-linked phytanyl chains. Similar glyceroglycans are found in eubacteria and plants with a sn -3-glycerol backbone and ester-linked fatty acid chains at the 1- and 2-positions.

2.2. Saccharolipids

The outer membrane of Gram-negative bacteria (Figure 4) contains lipopolysaccharide or endotoxin, which is a lipid made up of a backbone derived from glucosamine phosphate (Whitfield and Trent, 2014) rather than glycerophosphate. The core lipid (Lipid A, see Figure 5) in E. coli contains two glucosamine groups in β 1–6 linkage that are decorated at positions 2, 3, 2′ and 3′ with R-3-hydroxymyristic acid (C14) and at positions 1 and 4′ with phosphates. Further modification at position 6′ with a KDO disaccharide (two 3-deoxy-D-manno-octulosonic acids in a 1–3 linkage) results in KDO2–Lipid A that is further modified by an inner core, an outer core and the O antigen (Figure 4). Laboratory strains of E. coli such as K-12 and Salmonella typhimurium lack the O antigen found in the wild-type and clinically important strains.

Figure 4   Escherichia coli cell envelope. The complete cell envelope of Gram-negative bacteria contains an inner phospholipid bilayer and is the permeability barrier of the cell. The outer membrane is composed of an inner monolayer of phospholipid and an outer monolayer of the Lipid A portion of lipopolysaccharide (LPS). The structure of KDO 2 –Lipid A is shown in Figure 5 and is connected to a polysaccharide to build up the inner core, outer core and the O antigen repeat of LPS. The outer membrane is a permeability barrier allowing molecules of less than 750–1000   Da to pass through various pores in the outer membrane. The periplasmic space contains many proteins and the membrane-derived oligosaccharide (MDO), which is one component of the osmolarity regulatory system. MDO is decorated with sn -glycerol-1-phosphate and ethanolamine phosphate derived from PG and PE, respectively. The amino acid–sugar cross-linked peptidoglycan gives structural rigidity to the cell envelope. One-third of the lipoprotein ( lpp gene product) pool is covalently linked via its carboxyl terminus to the peptidoglycan and, in complex with the remaining lipoproteins as trimers, associates with the outer membrane via covalently linked fatty acids at its amino terminus. The amino terminal cysteine is blocked with a fatty acid, derived from membrane phospholipids, in amide linkage and is derivatised with diacylglycerol, derived from PG, in thioether linkage. Figure provided by and reproduced with permission from C.R. Raetz.

The core Lipid A forms the outer monolayer of the outer membrane bilayer of Gram-negative bacteria with the inner monolayer (Figure 4), which is made up of phospholipids (about 90% phosphatidylethanolamine (PE)). Lipopolysaccharide is modified postassembly in response to the environment including growth media, temperature, ionic properties and antimicrobial agents and displays additional diversity among enteric and nonenteric Gram-negative bacteria. Studies on Lipid A are of clinical importance because it is the primary antigen responsible for toxic shock syndrome.

Figure 5  Structure of KDO 2 –Lipid A. Lipid A is a disaccharide of glucosamine phosphate that is multiply acylated with both amide and ester linkages to fatty acids of the chain lengths indicated. As illustrated in Figure 4 , Lipid A is attached to KDO 2 that is then elongated with the remainder of the lipopolysaccharide structure. Figure provided by and reproduced with permission from C.R. Raetz.

2.3. Sphingolipids

All eukaryotic cells contain sphingolipids derived from the condensation of palmitoyl-coenzyme A and serine followed by slightly different species-specific conversion to the core ceramide molecule (Chapter 10). In higher eukaryotes there is additional diversity of the long chain base derived from palmitate with additional double bonds and hydroxyl groups as well as considerable diversity in the fatty acid in amide linkage, which can range up to 26 carbons in length. Yeast contain mainly derivatives of phytoceramide (4-hydroxy ceramide) and C26 fatty acid chains in amide linkage. The major classes of sphingolipids are grouped according to what is esterified at the primary hydroxyl β to the amide carbon of ceramide. Sphingomyelin has choline phosphate at this position whereas the glycosphingolipids have various lengths of oligosaccharides. The acidic glycosphingolipids, found primarily in mammalian cells, contain either sulphated sugars (sulphatide) or sialic acid (gangliosides) in the terminal sugar position. Yeast sphingolipids contain inositol phosphate and mannose inositol phosphate linked at this hydroxyl. Although the synthesis of sphingolipids occurs in the endoplasmic reticulum and the Golgi, they are primarily found in the outer leaflet of the plasma membrane.

3. Properties of Lipids in Solution

The matrix that defines a biological membrane is a lipid bilayer composed of a hydrophobic core excluded from water and an ionic surface that interacts with water and defines the hydrophobic–hydrophilic interface (Figure 1). Much of our understanding of the physical properties of lipids in solution and the driving force for the formation of lipid bilayers comes from the concept of the ‘hydrophobic bond’ as described by Walter Kauzmann (Kauzmann, 1959) in the context of the forces driving protein folding and later extended as the ‘hydrophobic effect’ by Charles Tanford (Tanford, 1980) to explain self-association of lipids within biological systems. The Tanford book is a must read for anyone wishing to work with membrane components. The ‘fluid mosaic’ model for membrane structure further popularised these concepts (Singer and Nicolson, 1972). This model envisioned membrane proteins as undefined globular structures freely moving in a homogeneous sea of lipids. Although this model stimulated research in the area of membrane proteins, it relegated lipids to a monolithic role as a fluid matrix within which membrane proteins reside and function. As will be become apparent, our current understanding of the role of lipids in cell function is as specific and dynamic as that of proteins, which are now more precisely defined with respect to their structure and interaction with lipids.

3.1. Why Do Polar Lipids Self-Associate?

Polar lipids are amphipathic in nature containing both hydrophobic domains that do not interact with water and hydrophilic domains that readily interact with water. The basic premise of the hydrophobic effect (Tanford, 1980) is that the hydrocarbon domains of polar lipids distort the stable hydrogen bonded structure of water by inducing ordered cage-like structures around the apolar domains. Self-association of the hydrophobic domains minimises the total surface area in contact with water, resulting in entropy-driven relaxation of the water structure and an energy minimum for the final self-associated molecular organisation. The polar domains of lipids interact through either hydrogen bonding or ionic interaction with water or other lipid head groups and therefore are energetically stable in an aqueous environment. The structural organisation that a polar lipid assumes in water is determined by its concentration and the law of opposing forces, that is, hydrophobic forces driving self-association of hydrophobic domains versus steric and ionic repulsive forces of the closely associated polar domains opposing self-association. At low concentrations, amphipathic molecules exist as monomers in solution. As the concentration of the molecule increases, its stability in solution as a monomer decreases until the unfavourable repulsive forces of closely packed polar domains are outweighed by the favourable self-association of the hydrophobic domains. At this point, a further increase in concentration results in the formation of increasing amounts of self-associated monomers in equilibrium with a constant amount of free monomer. This point of self-association and the remaining constant free monomer concentration is the critical micelle concentration. Due to the increased hydrophobic effect, a larger hydrophobic domain results in a lower critical micelle concentration. However, the larger the polar domain, because of either the size of neutral domains or the charge repulsion for like-charged ionic domains, the higher the critical micelle concentration due to the unfavourable steric hindrance or charge repulsion in bringing these domains into close proximity. The critical micelle concentration of amphipathic molecules with a net charge is lowered by increasing ionic strength of the medium due to dampening of the charge repulsion effect. Addition of chaotropic agents, such as urea, that disrupt water structure or organic solvents that lower the dielectric constant of water raises the critical micelle concentration by stabilising the hydrophobic domain in an aqueous environment. Therefore, the critical micelle concentration of the detergent sodium dodecyl sulphate is reduced 10-fold when the NaCl concentration is raised from 0 to 0.5  M but is increased on addition of urea or ethanol.

These physical properties and the shape of amphipathic molecules define three supermolecular structural organisations of polar lipids and detergents in solution (Figure 6). Detergents, lysophospholipids (containing only one alkyl chain) and phospholipids with short alkyl chains (eight or fewer carbons) have an inverted cone shape (large head group relative to a small hydrophobic domain) and self-associate above the critical micelle concentration with a small radius of curvature to form micellar structures with a hydrophobic core, excluding water. The micelle surface, rather than being a smooth spherical or elliptical structure with the hydrophobic domains completely sequestered inside a shell of polar residues that interact with water, is a very rough surface with many of the hydrophobic domains exposed to water. The overall structure reflects the optimal packing of amphipathic molecules at an energy minimum by balancing the attractive force of the hydrophobic effect and the repulsive force of close head group association. The critical micelle concentration for most detergents ranges from micromolar to millimolar. Lysophospholipids also form micelles with critical micelle concentrations in the micromolar range. However, phospholipids with chain lengths of 16 self-associate at a concentration around 10−¹⁰  M due to the hydrophobic driving force contributed by two alkyl chains. Phospholipids with long alkyl chains do not form micelles but organise into bilayer structures, which allow tight packing of adjacent side chains with the maximum exclusion of water from the hydrophobic domain. Due to repulsive forces of the head groups, significant lengths of the hydrocarbon chain near the glycerol backbone are exposed to water. In living cells phospholipids are not found free as monomers in solution but are organised into membrane bilayers or found complexed with proteins.

When long chain phospholipids are first dried to a solid from organic solvent and then hydrated, they spontaneously form large multilamellar bilayer sheets separated by water. Sonication disperses these sheets into smaller unilamellar bilayer closed structures that satisfy the hydrophobic nature of the ends of the bilayer sheets by forming sealed vesicles (also termed liposomes) defined by a continuous single bilayer and an aqueous core much like the membrane surrounding cells. Liposomes can also be made by physical extrusion of multilamellar structures through a small orifice or by dilution of a detergent–lipid mixture below the critical micelle concentration of the detergent (Patil and Jadhav, 2014).

Figure 6  Polymorphic phases and molecular shapes exhibited by lipids. The space-filling model for the micellar phase is of the β- D -octyl glucoside micelle (50 monomers). The polar portions of the detergent molecules (oxygen atoms are black ) do not completely cover the micelle surface (hydrocarbons in grey ), leaving substantial portions of the core exposed to bulk solvent. Inverted cone-shaped molecules form micelles. Polar lipids with two long alkyl chains adopt a bilayer or nonbilayer (H II ) structure, depending on the geometry of the molecule (cylindrical or cone shaped, respectively) and environmental conditions. The L β (ordered gel) and L α (liquid crystalline) bilayer phases differ in the order within the hydrophobic domain and in mobility of the individual molecules.

Cylindrical-shaped lipids (head group and hydrophobic domains of similar diameter), such as PC, form lipid bilayers. Cone-shaped lipids (small head groups relative to a large hydrophobic domain) such as PE containing at least one unsaturated fatty acid or CL in the presence of divalent cations favour an inverted micellar structure where the head groups sequester an internal aqueous core and the hydrophobic domains are oriented outwards and self-associate in nonbilayer structures. These are denoted as the hexagonal II (HII) and cubic phases (a more complex organisation similar to the HII phase). The ability of lipids to form multiple structural associations is referred to as lipid polymorphism. Lipids such as PE, PA, CL and monosaccharide derivatives of DAG can exist in either a bilayer or a nonbilayer phase depending on solvent conditions, alkyl chain composition and temperature. These phases are governed by the packing geometry of the hydrophilic and hydrophobic domains on self-association as discussed below.

Both cone-shaped and inverted cone-shaped lipids are considered nonbilayer-forming lipids and when mixed with bilayer-forming lipids change the physical properties of the bilayer by introducing lateral stress or strain within the bilayer structure. When bilayer-forming lipids are spread as a monolayer at an aqueous–air interface they orient with the hydrophobic domain facing air, and they have no tendency to bend away from or towards the aqueous phase due to their cylindrical symmetry. Monolayers of the asymmetric cone-shaped lipids (HII forming) tend to bend towards the aqueous interface (negative radius of curvature) while monolayers of asymmetric inverted cone-shaped lipids (micelle-forming) tend to bend away from the aqueous phase (positive radius of curvature). The distribution of lipids with different geometrical shapes between monolayers may determine the overall curvature of a bilayer membrane. The significance of shape mismatch in lipid mixtures is covered below.

3.2. Physical Properties of Membrane Bilayers

The organisation of DAG-containing polar lipids in solution is dependent on the nature of the alkyl chains, the head groups and the solvent conditions (i.e. ion content, pH and temperature). The transition between these phases for pure lipids in solution can be measured by various physical techniques such as ³¹P NMR and microcalorimetry. The difference between the ordered gel (Lβ) and the liquid crystalline (Lα) phases (Figure 6) is the viscosity or fluidity of the hydrophobic domains of the lipids, which is a function of temperature and the alkyl chain structure. At any given temperature, the ‘fluidity’ (the inverse of the viscosity) of the hydrocarbon core of the bilayer increases with increasing content of unsaturated or branched alkyl chain or with decreasing alkyl chain length. Due to the increased mobility of the fatty acid chains with increasing temperature, the fluidity and space occupied by the hydrophobic domain of lipids increases, which also tends to move the head groups apart. A bilayer-forming lipid such as PC assumes a cylindrical shape over a broad temperature range and with different alkyl chain compositions. When analysed in pure form, PC exists in either the Lβ or the Lα phase, mainly dependent on its alkyl chain composition and the temperature (Figure 6). Nonbilayer-forming lipids such as PE exist at low temperatures in the Lβ phase, at intermediate temperatures in the Lα phase and at elevated temperatures in the HII or cubic phase (Figure 7). The last transition is temperature dependent but also dependent on the shape of the lipid. The supermolecular organisation of lipids with relatively small head groups can change from cylindrical to conical (HII phase) with increasing unsaturation or length of the alkyl chains or with increasing temperature. As can be seen from Figure 7, the midpoint temperature (Tm) of the transition from the Lβ to Lα phase increases with an increase in the length of the fatty acids, but the midpoint of the transition temperature (TLH) between the Lα and the HII phases decreases with increasing chain length (or increasing unsaturation, not shown).

Figure 7  Phase behaviour of PE as a function of temperature and chain length. As hydrated lipids pass through a phase transition heat is absorbed as indicated by the peaks. The large peaks at the lower temperatures are due to the L β to L α transition and the smaller peaks at higher temperatures are due to the L α to H II transition. (A) Even numbered diacyl-PEs ranging from C12 to C20 (top to bottom). (B) Even numbered dialkyl-PEs in ether linkage ranging from C12 to C18 (top to bottom). The insets indicate an expanded scale for the transition to H II . Reprinted with permission from Seddon et al. (1983). Copyright 1983 American Chemical Society.

Similar transition plots as well as complex phase diagrams have been generated for mixtures of lipids. The physical property of a lipid mixture is collectively determined by each of the component lipids. Tm of biological membranes depends on its lipid composition. Liposomes composed of a single lipid exhibit a sharp phase transition while complex biological membranes display a broader transition with lipid composition finely tuned to reduce the sharpness of the transition. A large number of studies indicate that the Lα state of the membrane bilayer is required for cell viability, and cells adjust their lipid composition in response to many environmental factors so that the collective property of the membrane exhibits the Lα state. Cells regulate either their lipid fatty acid composition and/or their hydrophilic domain composition to maintain overall constant bilayer properties. Addition of nonbilayer-forming lipids to bilayer-forming lipids can result in nonbilayer formation but at a higher temperature than that for the pure nonbilayer-forming lipid. Addition of nonbilayer-forming lipids also adds other parameters of tension between the two monolayers and lateral stress within each monolayer. These lipids in each leaflet of the bilayer tend to reduce the radius of curvature of each monolayer that results in a tendency to pull the bilayer apart by curving the monolayers away from each other. This process results in potential energy residing in the bilayer that is a function of the presence of nonbilayer lipids. Forcing nonbilayer-forming lipids into a bilayer structure also exposes the hydrophobic core of the nonbilayer-forming lipids to the aqueous phase due to increasing lateral stress, which when relieved by insertion of proteins into the bilayer results in a release of free energy. Mixtures of lipids with dissimilar phase properties can also generate phase separations with local domain formation. Such discontinuities in the bilayer structure may be required for many structural organisations and cellular processes such as accommodation of proteins into the bilayer, movement of macromolecules across the bilayer, cell division and membrane fusion and fission events. The need for bilayer discontinuity may be the reason that all natural membranes contain a significant proportion of nonbilayer-forming lipids even though the membrane under physiological conditions is in the Lα phase.

Addition of cholesterol to lipid mixtures has a profound effect on the physical properties of a bilayer. Increasing amounts of cholesterol inhibit the organisation of lipids into the Lβ phase and favour a less fluid but more ordered structure than that of the Lα phase resulting in the lack of a phase transition normally observed in the absence of cholesterol. The solvent surrounding the lipid bilayer also influences these transitions, primarily by affecting the size of the head group relative to the hydrophobic domain (Figure 6). Ca²+ and other divalent cations (Mg²+, Sr²+ but not Ba²+) reduce the effective size of the negatively charged head groups of CL and PA, thus endowing nonbilayer properties. Low pH has a similar effect on the head group of PS. Since Ca²+ is an important signalling molecule that elicits many cellular responses, it is possible that part of its effect is transmitted through changes in the physical properties of membranes. In eukaryotes CL is found exclusively in the inner membrane (and to lesser extent in the outer membrane) of the mitochondria where Ca²+ fluxes play important regulatory roles.

3.3. What Does the Membrane Bilayer Look Like?

A primary role of biological membranes is to define the limits of a cell or organelle by maintaining a controlled permeability barrier to small polar and charged molecules (O2, CO2, H2O, H+, K+, HCO3−, Mg²+, Ca²+, etc.) as well as macromolecules. A second role is to provide the solvent and surface in which many essential biological processes are organised. Amphipathic lipid molecules are organised into a flexible noncovalently associated supermolecular structure that optimally satisfies these requirements. The functional properties of natural fluid bilayers are not only influenced by the hydrophobic core and the hydrophilic surface but by also the interface region containing bound water and ions. Figure 8(A) shows the distribution of the component parts of dioleoyl-PC across the bilayer (White et al., 2001) and illustrates the dynamic rather than static nature of the membrane. The length of the fatty acid chains defines the bilayer thickness of 30  Å for the above phospholipid. However, the thickness is not a static number as indicated by the probability of finding CH2 residues randomly distributed over a range of distances. Bilayer thickness can vary over the surface of a membrane if microdomains of lipids are formed with different alkyl chain lengths. The width (15  Å on either side of the bilayer) of the interface region between the hydrocarbon core and the free water phase (Figure 8(B)) is generally not appreciated. This region contains a complex mixture of chemical species defined by the ester linked glycerophosphate moiety, the variable head groups, bound water and ions. The many biological processes that occur within this interface region are dependent on its unique properties, including the steep polarity gradient within which surface-bound cellular processes occur.

Figure 8  The probability distribution for chemical constituents across a bilayer of PC. (A) The diagram was generated from X-ray and neutron diffraction data. The interface region between the hydrocarbon core and the free solvent region extends for approximately 15   Å on either side of the 30-Å-thick bilayer. The width of each peak defines the mobility of each constituent of PC. (B) As an α-helical peptide moves from either side of the bilayer towards the centre, the charge density of the environment steeply declines as indicated by the line. Figure adapted from White et al. (2001). Copyright 2001 The American Society for Biochemistry and Molecular Biology.

4. Engineering of Membrane Lipid Composition

Given the diversity in both lipid structure and function, how can the role of a given lipid be defined at the molecular level? Unlike proteins, lipids have neither inherent catalytic activity nor obvious functions in isolation (except for ligand–receptor interactions). Many potential functions of lipids have been uncovered serendipitously based on their effect on catalytic processes or biological functions studied in vitro. Although considerable information has accumulated with this approach, such studies are prone to artefacts. In addition, many of these functions have not been verified in living cells. The physical properties of lipids are as important as their chemical properties in determining function. Yet there is little understanding of how the physical properties of lipids measured in vitro relate to their in vivo function. Genetic approaches are generally the most useful for identifying in vivo function, but this approach has considerable limitations when applied to lipids. First, genes do not encode lipids, and in order to make mutants with altered lipid composition, the genes encoding enzymes along a biosynthetic pathway must be targeted. Therefore, the results of genetic mutation are indirect and many times far removed from the primary lesion. Second, a primary function of major membrane lipids is to provide the permeability barrier of the cell. Therefore, alterations in lipid composition may compromise cell permeability before other functions of a particular lipid are uncovered. Genetic approaches might strongly suggest that a lipid is essential for cell viability, but due to compromising cell viability the precise molecular basis for the requirement might be difficult to assess. The challenge is to use genetic information to manipulate the lipid composition of cells without severely compromising cell viability. In cases where this has been possible, the combination of the genetic approach to uncover phenotypes of cells with altered lipid composition, and the dissection in vitro of the molecular basis for the phenotype, has proven to be a powerful approach for defining lipid function. The most useful information to date has come from genetic manipulation of prokaryotic cells and eukaryotic microorganisms. However, the basic molecular principles underlying lipid function are generally applicable to more complex mammalian systems.

4.1. Alteration of Lipid Composition in Bacteria

The pathways for formation of the major phospholipids (PE, PG and CL) of E. coli (Chapter 3) were biochemically established mainly by Eugene Kennedy and coworkers (Figure 9) and subsequently verified using genetic approaches. The design of strains in which lipid composition can be genetically altered in a systematic manner has been very important in defining new roles for lipids in cell function (see Dowhan (2013) for a summary).

Surprisingly, E. coli mutants completely lacking either PE and PS or PG and CL are viable. Null mutants in the pgsA gene that cannot synthesise PG and CL are lethal, but suppressors of this mutation have been identified. In such mutants the major outer membrane lipoprotein precursor (Figure 4), which depends on PG for its lipid modification, accumulates in the inner membrane and apparently kills the cell. Mutants lacking this lipoprotein are viable without PG and CL but are temperature sensitive for growth at 42  °C, indicating that PG and CL are not absolutely required for viability, only for optimal growth. However, the anionic nature of these lipids (apparently substituted by increased levels of PA) is necessary for the proper membrane association and function of peripheral membrane proteins as discussed later. Mutants of E. coli lacking the amine containing lipids PE (psd null) or PS and PE (pssA null) are viable when grown in the presence of millimolar concentrations of Ca²+, Mg²+ and Sr²+ but have a complex mixture of defects in cell division, growth rate, outer membrane barrier function, energy metabolism and assembly of some membrane proteins (mainly including sugar and amino acid transporters).

Figure 9  Synthesis of native and foreign lipids in Escherichia coli . The native pathways ( blue arrows ), native lipids ( blue and grey ) and the respective gene names by the numbers are shown. 1) CDP-diacylglycerol synthase; 2) phosphatidylserine synthase; 3) phosphatidylserine decarboxylase; 4) phosphatidylglycerophosphate synthase; 5) phosphatidylglycerophosphate phosphatase encoded by three genes; 6) cardiolipin synthase (also encoded by clsB and clsC , which utilise PE and an unknown second substrate, respectively, rather than a second PG); 7) phosphatidylglycerol:premembrane-derived oligosaccharide (MDO) sn -glycerol-1- P transferase; 8) diacylglycerol kinase. The following enzymes and their respective gene names and sources noted have been expressed in E. coli and synthesise the indicated products ( red arrows ); 9) phosphatidylcholine synthase ( Legionella pneumophila ); 10) phosphatidylinositol synthase ( Saccharomyces cerevisiae ); 11) monoglucosyl diacylglycerol synthase ( Acholeplasma laidlawii ); 12) diglucosyl diacylglycerol synthase ( Acholeplasma laidlawii ); 13) lysyl t-RNA:phosphatidylglyerceol lysine transferase ( Staphococcus aureus ). Figure adapted from Dowhan (2013). Copyright 2013 Elsevier Press.

The plasticity in specific lipid requirements for viability of E. coli has been utilised to design strains in which phospholipid composition can be varied at steady state as well as temporally over the growth cycle of a culture. Such variability is attained through a combination of null mutants and placing specific genes under the control of regulated promoters. In addition, incorporation of genes from other organisms has made possible the expression of foreign lipids in E. coli in place of the naturally occurring phospholipids. This collection of mutants (Figure 9) has been instrumental in defining specific roles for some of the phospholipids of E. coli. Since the foreign lipids display an array of physical and chemical properties (Figure 10), their ability to suppress the phenotype of mutants lacking specific natural lipids has been used to establish which property of a given lipid is necessary to support a particular cellular function (Dowhan, 2013).

Figure 10  Structure and physical properties of lipids. The glycerol backbone ( red ) shown in ester linkage to fatty acids (aliphatic chains R 1 –R 4 ) at the sn -1 and sn -2 positions and either in phosphodiester linkage for phospholipids or in a glycosidic linkage for glycolipids at the sn -3 position. Head groups are colour coded to indicate the charge nature of each head group. Figure and legend taken from Dowhan (2013). Copyright 2013 Elsevier Press.

4.2. Alteration of Lipid Composition in Yeast

The pathways of phospholipid synthesis and the genetics of lipid metabolism in the yeast Saccharomyces cerevisiae (Henry et al., 2012) are as well understood as those in E. coli. S. cerevisiae has pathways similar to those of E. coli for CDP-DAG-dependent synthesis of PE and PG. However, CL synthesis in all eukaryotes involves transfer of a phosphatidyl moiety from CDP-DAG to PG rather than from one PG to another PG as in bacteria. In addition, yeast also have the mammalian pathways for synthesis of PI, PE and PC, including the methylation of PE to form PC (Chapter 7). Unlike mammalian cells, yeast possess a bacterial-type PS synthase as well as a mitochondrial PS decarboxylase; the latter is also found in mammalian cells. All gene products necessary for the synthesis of DAG, CDP-DAG and PI in yeast are essential for viability. PS synthesis is not essential if growth medium is supplemented with ethanolamine in order to make PE and PC. However, PE is definitely required whereas PC is only required for optimal growth.

No gene products involved in lipid metabolism are encoded by mitochondrial DNA, which in S. cerevisiae encodes eight proteins primarily required for oxidative phosphorylation. Mitochondrial PS is imported from its site of synthesis in the endoplasmic reticulum and converted to PE by the mitochondrial-localised PS decarboxylase (PSD1 gene product). Yeast express nuclear genes that encode a CDP-DAG synthase targeted to the endoplasmic reticulum (CDS1) and one targeted to the mitochondria (TAM41). PG and CL (localised solely to the mitochondria) are synthesised from CDP-DAG via nuclear gene products imported into mitochondria. Null mutants of CRD1 (encodes CL synthase) lack CL but accumulate the immediate precursor PG (normally 10-fold lower than CL) to levels approaching that of CL (ca. 20% of the inner mitochondrial membrane phospholipids). These mutants grow normally on glucose for which mitochondrial ATP formation is not required. However, growth on nonfermentable carbon sources such as glycerol or lactate is considerably slower, indicating a partial defect in oxidative phosphorylation. Therefore, CL appears to be required for optimal mitochondrial function but is not essential for viability. However, lack of PG and CL synthesis due to a null mutation in the PGS1 gene (encodes PG phosphate synthase) results in the inability to utilise nonfermentable carbon sources for growth. Similar effects are seen in mammalian cells with a mutation in the homologous PGS1 gene (Ohtsuka et al., 1993). The surprising consequence of the lack of PG and CL in yeast is the lack of translation of mRNAs of four mitochondria-encoded proteins (cytochrome b and cytochrome c oxidase subunits I–III) as well as cytochrome c oxidase subunit IV (Su and Dowhan, 2006) that is nuclear encoded. These results indicate that some aspects of translation of a subset of mitochondrial proteins (those associated with electron transport complexes in the inner membrane but not ATP metabolism) require PG and/or CL.

5. Role of Lipids in Cell Function

There are at least two ways (White et al., 2001) by which lipids can affect protein structure and thereby cell function. Protein structure is influenced by specific protein–lipid interactions that depend on the chemical and structural anatomy of lipids (head group, backbone, alkyl chain length, degree of unsaturation, chirality, ionisation and chelating properties). However, protein structure is also influenced by the unique self-association properties of lipids that result from the collective properties (bilayer fluidity, thickness, shape, packing properties) of the lipids organised into membranes.

5.1. The Bilayer as a Supermolecular Lipid Matrix

Biophysical studies on membrane lipids coupled with biochemical and genetic manipulation of membrane lipid composition have established that the Lα state of the membrane bilayer is essential for cell viability. However, membranes are made up of a vast array of lipids that have different physical properties (Figure 10), can assume individually different physical arrangements and contribute collectively to the final physical properties of the membrane. Animal cell membranes are exposed to a constant temperature, pressure and solvent environment and therefore do not change their lipid make up dramatically in response to external conditions. The complex membrane lipid composition that includes cholesterol stabilises mammalian cell membranes in the Lα phase over the variation in conditions they encounter. However, there are considerable differences in lipid composition (Table 1) between the various membranes that define the multiple organelles of eukaryotic cells (Drin, 2014). Microorganisms are exposed to a broad range of environmental conditions so have developed systems for changing membrane lipid composition in order to maintain the Lα phase.

5.2. Physical Organisation of the Bilayer

As the growth temperature of E. coli is lowered, the content of unsaturated fatty acids in phospholipids increases to maintain the Lα state and membrane fluidity. Genetic manipulation of phospholipid fatty acid composition in E. coli is possible by introducing mutations in genes required for the synthesis of unsaturated fatty acids (Parsons and Rock, 2013). The mutants require supplementation with unsaturated fatty acids in the growth medium and incorporate these fatty acids to adjust membrane fluidity in response to growth temperature. When mutants with membranes containing a high content of unsaturated fatty acids are grown at low temperature they lyse when raised rapidly to high temperature, probably due to the increased membrane permeability of fluid membranes and a transition from the Lα to the HII phase by PE and/or CL. Conversely, when mutants with membranes containing a high content of saturated fatty acids are grown at high temperatures, growth arrest occurs after a shift to low temperature, due to the reduced fluidity of the membrane. Wild-type cells (not requiring fatty acid supplementation) arrest growth after a temperature shift until fatty acid composition is adjusted to provide favourable membrane fluidity.

Bacterial cells also regulate the ratio of bilayer- to nonbilayer-forming lipids in response to growth conditions (Dowhan, 1997). Bacterial nonbilayer-forming lipids are PE with unsaturated alkyl chains, CL in the presence of divalent cations, and monoglucosyl diacylglycerol (GlcDAG). Extensive studies of lipid polymorphism have been carried out on Acholeplasma laidlawii because this organism alters its ratio of GlcDAG (capable of assuming the HII phase) to GlcGlcDAG (diglucosyl diacylglycerol, which only assumes the Lα phase) in response to growth conditions. High temperature and unsaturation of fatty acids favour the HII phase for GlcDAG. At a given growth temperature the GlcDAG to GlcGlcDAG ratio is inversely proportional to the unsaturated fatty acid content of GlcDAG. As growth temperature is lowered, A. laidlawii either increases the incorporation of unsaturated fatty acids from the medium into GlcDAG or increases the ratio of GlcDAG to GlcGlcDAG to adjust the HII phase potential of its lipids to remain just below the transition from bilayer to nonbilayer. Therefore, the cell maintains the physical properties of the membrane well within that of the Lα phase but with a constant potential to undergo transition to the HII phase.

In contrast to A. laidlawii, E. coli maintains its nonbilayer lipids, CL (in the presence of divalent cations) and PE, within a narrow range and in wild-type cells adjusts the fatty acid content of PE to increase or decrease its nonbilayer potential. In mutants completely lacking PE, the role of nonbilayer lipid appears to be filled by CL, the levels of which rise from <10% to nearly 50% of total phospholipid. The viability of mutants lacking PE is maintained by divalent cations in the same order of effectiveness (Ca²+  >  Mg²+  >  Sr²+, but not Ba²+) for these ions to induce the formation of the nonbilayer phase of CL. The CL content of these mutants varies with the divalent cation used during growth. However, the

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