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Understanding phospholipid function: Why are there so many lipids?

2017; Elsevier BV; Volume: 292; Issue: 26 Linguagem: Inglês

10.1074/jbc.x117.794891

1083-351X

William Dowhan,

Bacterial Genetics and Biotechnology

In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved. In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved. My contributions to the biological sciences have been dependent on my early mentors, my scientific colleagues (students, postdocs, and established investigators), being at the right place at the right time, opportune evolution of methods and techniques, and some scientific insight along with a good amount of luck. In 1960, I was fortunate to be admitted to Princeton University after attending a Detroit suburban high school. After initially majoring in mathematics, I switched to chemistry. I was fascinated with organic chemistry, which resulted in my lifelong interest in the chemistry of living systems. Taking physical chemistry and later physical organic chemistry from Walter Kauzmann formed the foundation and guiding principles for my scientific career. Although Charles Tanford popularized the hydrophobic effect within the biological sciences (1Tanford C. The hydrophobic effect: formation of micelles and biological membranes.2nd Ed. Wiley, New York1980Google Scholar), this concept was initially proposed as the hydrophobic bond by Walter (2Kauzmann W. Some factors in the interpretation of protein denaturation.Adv. Protein Chem. 1959; 14: 1-63Crossref PubMed Scopus (4057) Google Scholar). The hydrophobic bond/effect is now recognized as the primary driving force for the organization of lipids into a bilayer that defines the membrane surrounding cells and organelles and results in stabilization of integral membrane proteins within the lipid bilayer. Therefore, I recommend a thorough understanding of hydrophobicity, as outlined in Tanford's book (1Tanford C. The hydrophobic effect: formation of micelles and biological membranes.2nd Ed. Wiley, New York1980Google Scholar), as a basis for all studies involving membrane proteins and lipids. As you will see, there will be several recurring themes during my career where early exposure to ideas and systems influenced the path of my scientific interests. I chose to do my senior thesis with Walter Kauzmann, who was studying the biophysics of protein folding. I still remember traveling with Walter to a local chicken farm to buy 20 dozen fresh eggs, from which I isolated 50 g of ovalbumin as my model protein of study. The smell during this purification prevented me from eating eggs for many years! Like most scientists of the time, I discarded all the phospholipids that would be the focus of my future studies. I spent many a night in a low-temperature room in the basement of the Frick Chemistry Laboratory building using a dated polarimeter taking point-by-point measurements to determine the helical content of ovalbumin under various denaturing conditions. How I wish I had had access to a scanning circular dichroism instrument, which were just becoming available. In fact, Walter acquired one the month I handed in my thesis! In the summer between my junior and senior years, I worked as an analytical chemist at the Ford Motor Company plant outside of Detroit. We did routine analyses of chemicals provided by suppliers and also of material from the assembly line. This was clearly not what I wanted as a career despite being offered a permanent position upon graduation. I was much more interested in pursuing mechanistic problems. Walter strongly encouraged me to pursue a Ph.D. in biochemistry and asked where I wanted to go. The parents of my wife-to-be, Jerilyn Atwood, had recently moved to California, so the choice was obvious. After graduation and our marriage in June of 1964, we moved to Berkeley so I could begin studies at the University of California. The stipend was $2800 a year, a far cry from the $19,000 Ford had offered. Fortunately, Jerilyn worked as a nurse during most of our years at Berkeley, making our time there more comfortable. These were of course the wild years of the Vietnam War, the free speech movement, and the Reagan governorship. This was the polar opposite of the social atmosphere of the Princeton campus. The military service draft constantly made our future uncertain as fellow students, especially those with an M.D., disappeared on a regular basis. Most of us confined our time to the lab but thoroughly enjoyed the colorful events that surrounded us. My first-year courses were in biochemistry (taught by Esmond Snell), a genetics course, and an intensive lab course where we spent all of our free time learning biochemical techniques. The genetics course relied heavily on what was known about the lac operon in the mid-1960s. This would be a very important exposure for my future use of lactose permease (LacY) as a model membrane protein. We also took seminar-based courses where each of us presented on a topic of our choice. I ran across a series of books in the biochemistry library that dealt with membranes. At the time, there were still arguments concerning the basic structure of membranes. A lipid bilayer as the core of membranes was proposed in 1925 (3Gorter E. Grendel F. On bimolecular layers of lipoids on the chromocytes of the blood.J. Exp. Med. 1925; 41: 439-443Crossref PubMed Scopus (440) Google Scholar), but arguments were still being made for proteins as the organizing framework for membranes. How proteins were organized in the membrane was unknown. A particularly interesting section of this book series proposed the organization of proteins into domains within a lipid bilayer. This was well before Singer-Nicolson proposed the "fluid mosaic model" (4Singer S.J. Nicolson G.L. The fluid mosaic model of the structure of cell membranes.Science. 1972; 175: 720-731Crossref PubMed Scopus (6074) Google Scholar) for membrane structure. Naively, I chose this as my seminar topic. The result was a near failure in the course due to my inability to recognize such "poorly documented scientific studies." This was a lesson in the difficulties of challenging current dogma, a practice which I have tried to continue to this date. Interestingly, my research group later contributed to the body of evidence supporting lipid domains with specifically associated proteins in bacterial membranes (reviewed in Ref. 5Matsumoto K. Hara H. Fishov I. Mileykovskaya E. Norris V. The membrane: transertion as an organizing principle in membrane heterogeneity.Front. Microbiol. 2015; 6: 572Crossref PubMed Scopus (40) Google Scholar). I chose to do my Ph.D. thesis under the direction of Esmond Snell because of the opportunity to learn methods of protein purification, protein characterization, and enzymology. Esmond was of course world-renowned for his work on enzymes containing pyridoxal phosphate as a cofactor. Esmond's lab was heavy on postdoctoral fellows each working on their own enzyme. The presence of experienced postdocs was particularly important during Esmond's one-year sabbatical, and his absence forced me to become independent and resourceful. I gained access to Dan Koshland's newly arrived Beckman amino acid analyzer and Howard Schachman's ultracentrifuge in order to determine the amino acid composition and molecular weight, respectively, of d-serine dehydratase (6Dowhan Jr., W. Snell E.E. d-Serine dehydratase from Escherichia coli. II. Analytical studies and subunit structure.J. Biol. Chem. 1970; 245: 4618-4628Abstract Full Text PDF PubMed Google Scholar). After devising a method for producing the apoenzyme followed by reconstitution, I used Esmond's collection of B6 analogues to establish the structural features of pyridoxal necessity for enzyme binding and catalytic activity (7Dowhan Jr., W. Snell E.E. d-Serine dehydratase from Escherichia coli. 3. Resolution of pyridoxal 5′-phosphate and coenzyme specificity.J. Biol. Chem. 1970; 245: 4629-4635Abstract Full Text PDF PubMed Google Scholar). While my project progressed smoothly, the outside world interfered at times. For example, free speech "terrorists" of the time were blowing up establishments of capitalism such as Bank of America branches, and on one occasion, a major electrical tower of Pacific Gas and Electric; the latter event shut down one of my ultracentrifuge runs. I filed my Ph.D. thesis at the Sproul Hall administration building while it was surrounded with state troopers and student demonstrators. While I was in the lab, Dixon Riley discovered a new prosthetic group residing in the histidine decarboxylase of Lactobacillus 30a (8Riley W.D. Snell E.E. Histidine decarboxylase of Lactobacillus 30a. V. Origin of enzyme-bound pyruvate and separation of nonidentical subunits.Biochemistry. 1970; 9: 1485-1491Crossref PubMed Scopus (37) Google Scholar). An N-terminal pyruvate residue is created by an autocatalyzed serinolysis of the peptide bond N-terminal to a serine residue in the proenzyme. Awareness of this discovery would be important to my later studies on phosphatidylserine (PS) 2The abbreviations used are: PSphosphatidylserinePGphosphatidylglycerolPGPphosphatidylglycerol-PPEphosphatidylethanolamineCLcardiolipinPCphosphatidylcholinePCTPphosphatidylcholine transfer proteinPIphosphatidylinositolPITPphosphatidylinositol transfer proteinNAO10-N-nonyl acridine orange. decarboxylase from Escherichia coli (9Li Q.X. Dowhan W. Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli.J. Biol. Chem. 1990; 265: 4111-4115Abstract Full Text PDF PubMed Google Scholar, 10Dowhan W. Wickner W.T. Kennedy E.P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli.J. Biol. Chem. 1974; 249: 3079-3084Abstract Full Text PDF PubMed Google Scholar). phosphatidylserine phosphatidylglycerol phosphatidylglycerol-P phosphatidylethanolamine cardiolipin phosphatidylcholine phosphatidylcholine transfer protein phosphatidylinositol phosphatidylinositol transfer protein 10-N-nonyl acridine orange. My Ph.D. thesis did not produce any seminal results or earthshaking principles. However, the training in the scientific method and rigorous molecular description of results along with a solid background in protein chemistry and enzymology would serve me well in the years to come. I owe a great debt to Esmond and the faculty at Berkeley for the opportunity to do research in their midst. The earlier encounter with the area of membrane structure in my seminar course, although traumatic, still piqued my interest, and I was eager to learn more. In the late 1960s, few integral membrane proteins had been purified in an active form. A search of the literature turned up the classic Fred Fox and Eugene (Gene) Kennedy paper (11Fox C.F. Kennedy E.P. Specific labeling and partial purification of the M protein, a component of the β-galactoside transport system of Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 1965; 54: 891-899Crossref PubMed Scopus (230) Google Scholar) describing the identification of the membrane-associated M protein, which is the lacY gene product LacY. Gene's group correctly suggested that the M protein carried out both energy-independent equilibration of substrate across the cell membrane as well as energy-dependent accumulation of substrate without modification of the substrate (12Kennedy E.P. Fox C.F. Carter J.R. Membrane structure and function.J. Gen. Physiol. 1966; 49: 347-354Crossref PubMed Scopus (2) Google Scholar). This was in stark contrast to the phosphotransferase-dependent systems studied by Saul Roseman (13Roseman S. The transport of carbohydrates by a bacterial phosphotransferase system.J. Gen. Physiol. 1969; 54: 138-184Crossref PubMed Scopus (160) Google Scholar), which used metabolic energy to modify the substrate, resulting in accumulation. These differences in mechanism provided colorful debates at Gordon Research Conferences for several years between Saul, Gene, and H. Ronald Kaback as to the mechanism by which LacY transports substrate. Although Ron and Gene had differences in details, they agreed that a high-energy covalent intermediate was not involved in the LacY mechanism. Ron eventually established that an electrochemical proton gradient was the driving force for lactose accumulation, which was the culmination of what Ron termed the "Chemiosmotic Wars." Ron of course went on to establish the detailed molecular structure and mechanism of LacY (14Kaback H.R. A chemiosmotic mechanism of symport.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 1259-1264Crossref PubMed Scopus (71) Google Scholar, 15Kumar H. Finer-Moore J.S. Kaback H.R. Stroud R.M. Structure of LacY with an α-substituted galactoside: connecting the binding site to the protonation site.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 9004-9009Crossref PubMed Scopus (36) Google Scholar). It was during these Gordon Conferences debates that I got to know Ron (Fig. 1), which resulted in a long and productive scientific friendship. After hearing about LacY for so many years, I decided to bring my acquired expertise in protein purification and characterization to bear on this protein, and thus make my entry into the nascent field of membrane proteins. I arrived with my wife and son David for a postdoctoral position at Harvard Medical School in the late spring of 1969. Gene (Fig. 2) had of course delineated most of the metabolic pathways for phospholipid biosynthesis in mammalian cells and E. coli (summarized in Ref. 16Dowhan W. A retrospective: use of Escherichia coli as a vehicle to study phospholipid synthesis and function.Biochim. Biophys. Acta. 2013; 1831: 471-494Crossref PubMed Scopus (76) Google Scholar). His foray into membrane transport processes was a new direction for the lab, as were attempts to purify membrane-associated proteins. Over the next five years, the Kennedy lab (Fig. 2) filled with what would become the future leaders of lipid metabolism and membrane biology. Edward Dennis would develop the important surface dilution model to explain the kinetics of integral and peripheral membrane proteins that used membrane-associated lipid substrates (17Carman G.M. Deems R.A. Dennis E.A. Lipid signaling enzymes and surface dilution kinetics.J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). He would become a leading figure in phospholipase A2 generation of lipid second messengers. David Nelson would assume the lead authorship of Lehninger's classic biochemistry textbook Principles of Biochemistry. William Wickner would join Arthur Kornberg's lab after medical school, and accomplish the difficult purification of the E. coli DNA polymerase III (18Wickner W. Kornberg A. A holoenzyme form of deoxyribonucleic acid polymerase III: isolation and properties.J. Biol. Chem. 1974; 249: 6244-6249Abstract Full Text PDF PubMed Google Scholar). He would return to membrane studies to be a leading figure in the biosynthesis and assembly of membrane proteins. Christian Raetz (Fig. 2) would be Harvard's first M.D./Ph.D. graduate. He would combine classical and modern molecular genetics with rigorous mechanistic biochemistry to map mutants in E. coli phospholipid metabolism and delineate the complex pathways for the synthesis of lipid A (19Dowhan W. Nikaido H. Stubbe J. Kozarich J.W. Wickner W.T. Russell D.W. Garrett T.A. Brozek K. Modrich P. Christian Raetz: scientist and friend extraordinaire.Annu. Rev. Biochem. 2013; 82: 1-24Crossref PubMed Scopus (3) Google Scholar), the core membrane component of lipopolysaccharide present in Gram-negative bacteria. Carlos Hirschberg would go on to make seminal contributions in glycoprotein biosynthesis. Shortly after my departure, James Rothman joined the laboratory. He would receive the Nobel Prize in physiology in 2013, along with Randy Schekman and Thomas C. Südhof, for seminal work on intracellular protein trafficking. Dennis Voelker would begin to unravel the complex process of inter-organelle movement of phospholipids (20Voelker D.R. Protein and lipid motifs regulate phosphatidylserine traffic in yeast.Biochem. Soc. Trans. 2005; 33: 1141-1145Crossref PubMed Scopus (10) Google Scholar) and the role of phosphatidylglycerol (PG) as a lung surfactant antimicrobial agent (21Numata M. Kandasamy P. Voelker D.R. Anionic pulmonary surfactant lipid regulation of innate immunity.Expert Rev. Respir. Med. 2012; 6: 243-246Crossref PubMed Scopus (24) Google Scholar). Continual interaction with all of these scientists over the years had a profound influence on my work. I set out to purify the M protein (i.e. LacY). The assay was quite crude and based on the original specific radiolabeling devised by Fred and Gene (11Fox C.F. Kennedy E.P. Specific labeling and partial purification of the M protein, a component of the β-galactoside transport system of Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 1965; 54: 891-899Crossref PubMed Scopus (230) Google Scholar) rather than reconstitution of transport function in proteoliposomes as later developed by Ron (22Kaback H.R. Transport studies in bacterial membrane vesicles.Science. 1974; 186: 882-892Crossref PubMed Scopus (170) Google Scholar). I tried all of the available mild non-denaturing detergents available at the time with no success. Either the protein was not extracted, or if extracted, it was hopelessly aggregated. My scientific disappointments with the purification of LacY were compounded in the summer of 1969 with my mother's diagnosis with terminal pancreatic cancer and the realization that my father had advanced Alzheimer's disease. With the support of Gene and the American Cancer Society (which extended my two-year fellow to three years), I took a nine-month leave from science. My parents both passed away in December of 1969, and after settling their affairs, I returned to the lab in the spring of 1970 to continue with the LacY purification. However, after several months, it became obvious that this project was a failure. Eventual success in purification of LacY would depend on the availability of octyl glucoside and dodecyl maltoside and the efforts of Hastings Wilson and Ron Kaback 10 years later (23Newman M.J. Foster D.L. Wilson T.H. Kaback H.R. Purification and reconstitution of functional lactose carrier from Escherichia coli.J. Biol. Chem. 1981; 256: 11804-11808Abstract Full Text PDF PubMed Google Scholar). However, I would return to LacY some 20 years later as the model protein in my studies of lipid-protein interactions. During my absence, Bill Wickner continued his medical student research rotation in Gene's laboratory. Bill had made significant progress in the purification of E. coli PS decarboxylase. Here was an integral membrane protein with a convenient assay that was readily solubilized in an active form by Triton X-100. Bill was a master at large-scale preparation. Membranes were isolated from a pound of E. coli cell paste followed by extraction and an acetone precipitation step. The first column was a 1-liter volume DEAE fractionation with a 10-liter salt gradient in Triton X-100 and 10% glycerol, which was collected in 250-ml fractions in a modified fraction collection. After a day in his clinical rotation, Bill would set this up in the evening on the lab bench behind mine. About 20% of the time, the fraction collector would malfunction, resulting in the floor becoming covered in the effluent, which I would have to clean up in the morning. After concentration of the decarboxylase peak and a gel filtration column, Bill had a yellow solution with a 500-fold increase in specific activity. I took over the project at this point because Bill was soon to join Arthur Kornberg at Stanford. As Bill was preparing his oral short presentation on partial isolation of a pyridoxal-dependent PS decarboxylase at the San Francisco meeting of the Federation of American Societies for Experimental Biology, I decided to subject the sample to a sucrose gradient centrifugation step. The result was a yellow pellet at the bottom of the tube and all the activity about half-way down the tube. We either had a newly discovered pyruvate-dependent decarboxylase or a long way to go before purity was attained. It turned out both were true. I completed the purification over several months starting with 8 kg of E. coli cell paste, which after a 3500-fold enrichment yielded about 14 mg of active nearly pure enzyme (10Dowhan W. Wickner W.T. Kennedy E.P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli.J. Biol. Chem. 1974; 249: 3079-3084Abstract Full Text PDF PubMed Google Scholar). I did not determine the identity of the prosthetic group; that would wait until Satre and Kennedy's later work, identifying the group as pyruvate bound to a small subunit of the decarboxylase (24Satre M. Kennedy E.P. Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.J. Biol. Chem. 1978; 253: 479-483Abstract Full Text PDF PubMed Google Scholar), although I did later return to this enzyme to study the mechanism by which the prosthetic group is generated (9Li Q.X. Dowhan W. Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli.J. Biol. Chem. 1990; 265: 4111-4115Abstract Full Text PDF PubMed Google Scholar). My tenure in Gene's lab afforded the opportunity to study under one of the great scientists of the late 20th century, and form lasting personal and scientific friendships for the remainder of my career. Bill and I, with Gene's careful guidance, had demonstrated that integral membrane proteins of phospholipid metabolism could be purified to homogeneity in an active form and characterized. This was a seminal accomplishment for the times and provided the foundation for the next 15 years of my research while stimulating others to carry out similar purifications in bacteria and eukaryotic cells. 1972 was not a great year to find a job. We were in the middle of the Nixon years of significant cuts in National Science Foundation (NSF) and National Institutes of Health (NIH) research support. Most of the new hires were concentrated in the Houston, Texas area, which was undergoing a renaissance in biochemistry. George Schroepfer moved from the University of Illinois to initiate the Department of Biochemistry at Rice University. Salih Wakil moved from Duke to Baylor College of Medicine and was increasing the Department of Biochemistry. Allan Goldstein came from New York to rebuild the Division of Biochemistry at the University of Texas Medical Branch in Galveston. John (Jack) DeMoss moved from the University of Californian at San Diego to chair the Department of Biochemistry and Molecular Biology at the newly formed University of Texas Medical School in Houston. I interviewed at all four institutions and decided to take my chances with Jack in building a new medical school. When I interviewed in January of 1972, just after the birth of our son Michael during a December snowstorm, the medical school's first building was a hole in the ground, with laboratories in rented space spread over the Texas Medical Center. When I arrived in August, the first two-story building, where we taught the first entering class of medical students in September, was complete. The five founding members of the department were housed in cramped rented space on the 13th floor of the Center Pavilion Hospital where I shared an office and laboratory with Henry Strobel, who came from Minor Coon's group. The building had previously been a hotel, and each room was converted to a shared office and laboratory. We were sandwiched between the hospital below and the drug halfway house on the floor above. The latter occupants would wander into our space and on one occasion collected bottles of radioactive toluene scintillation fluid in order to get high. That fall was spent writing our first grants, setting up our labs, organizing the medical school lectures, and designing small group conferences the week before each meeting with medical students. My first grant was funded on the initial pass in 1973 despite Nixon sequestering the NIH budget for a short time. One could still get a grant with a novel idea and a background to support the project without a lot of preliminary data. My paper on the PS decarboxylase was not published until 1974 (10Dowhan W. Wickner W.T. Kennedy E.P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli.J. Biol. Chem. 1974; 249: 3079-3084Abstract Full Text PDF PubMed Google Scholar). I had demonstrated the ability to purify at least one membrane protein in an active form and wished to purify and characterize a few more. Because the grant ran for 43 years, including a 10-year MERIT Award at the end, its original title of "Structure and Function of Membrane Proteins" proved to have unexpected longevity. Fortunately, after a short grant hiatus, the project currently continues under a new grant number. My recommendation has always been to pick a topic in an evolving area that will set one's research apart from the crowd. Membrane proteins certainly fitted the bill at that time and remain an active area of research. Jack was the model departmental chairman, which I tried to emulate during my chairmanship of the department in the early 1990s. Jack's approach was to hire bright young faculty, provide strong financial support and guidance, and leave them alone to develop their unique niche in science. This tradition continues under the strong leadership of Rodney Kellems, the current chair of the department. I like to think that my decision to come to what is now the McGovern Medical School some 45 years ago and contribute to the research development, administrative leadership, and teaching mission of the school has in some small way contributed to the successful development of a strong clinical- and research-based institution. When I was starting up, Gene had suggested I replicate my success in purification of a bacterial membrane protein in mammalian systems, and so I did spend a few months working with beef hearts and livers. However, my passion remained with E. coli lipid biosynthetic enzymes, and so I returned to this topic. This was a fortuitous decision as the explosion in modern molecular biology and genetics was about to occur. With my first graduate student Tim Larson and my first postdoc Takashi Hirabayashi, we set out to purify the E. coli PS and phosphatidylglycerol-P (PGP) synthases, the committed steps to major lipids of E. coli, phosphatidylethanolamine (PE) and PG/cardiolipin (CL), respectively (Fig. 3). Not wanting to repeat the difficulties of time and mater