Understanding phospholipid function: Why are there somany lipids?Published, Papers in Press, May 10, 2017, DOI 10.1074/jbc.X117.794891

X William Dowhan1

From the Department of Biochemistry and Molecular Biology, the University of Texas Health Sciences Center, McGovern MedicalSchool, Houston, Texas 77030

Edited by Herbert Tabor

In the 1970s, phospholipids were still considered mere build-ing blocks of the membrane lipid bilayer, but the subsequentrealization that phospholipids could also serve as second mes-sengers brought new interest to the field. My own passion for theunique amphipathic properties of lipids led me to seek other,non-signaling functions for phospholipids, particularly in theirinteractions with membrane proteins. This seemed to be the lastfrontier in protein chemistry and enzymology to be conquered. Iwas fortunate to find my way to Eugene Kennedy’s laboratory,where both membrane proteins and phospholipids were the fociof study, thus providing a jumping-off point for advancing ourfundamental understanding of lipid synthesis, membrane pro-tein biosynthesis, phospholipid and membrane protein traffick-ing, and the cellular roles of phospholipids. After purifying andcharacterizing enzymes of phospholipid biosynthesis in Esche-richia coli and cloning of several of the genes encoding theseenzymes in E. coli and Saccharomyces cerevisiae, I was in a posi-tion to alter phospholipid composition in a systematic mannerduring the cell cycle in these microorganisms. My group wasable to establish, contrary to common assumption (derived fromthe fact that membrane proteins retain activity in detergentextracts) that phospholipid environment is a strong determin-ing factor in the function of membrane proteins. We showedthat molecular genetic alterations in membrane lipid composi-tion result in many phenotypes, and uncovered direct lipid-pro-tein interactions that govern dynamic structural and functionalproperties of membrane proteins. Here I present my personal“reflections” on how our understanding of phospholipid func-tions has evolved.

In the beginning

My contributions to the biological sciences have been depen-dent on my early mentors, my scientific colleagues (students,postdocs, and established investigators), being at the right placeat the right time, opportune evolution of methods and tech-niques, and some scientific insight along with a good amount of

luck. In 1960, I was fortunate to be admitted to Princeton Uni-versity after attending a Detroit suburban high school. Afterinitially majoring in mathematics, I switched to chemistry. I wasfascinated with organic chemistry, which resulted in my life-long interest in the chemistry of living systems. Taking physicalchemistry and later physical organic chemistry from WalterKauzmann formed the foundation and guiding principles formy scientific career. Although Charles Tanford popularized thehydrophobic effect within the biological sciences (1), this con-cept was initially proposed as the hydrophobic bond by Walter(2). The hydrophobic bond/effect is now recognized as the pri-mary driving force for the organization of lipids into a bilayerthat defines the membrane surrounding cells and organellesand results in stabilization of integral membrane proteinswithin the lipid bilayer. Therefore, I recommend a thoroughunderstanding of hydrophobicity, as outlined in Tanford’s book(1), as a basis for all studies involving membrane proteins andlipids. As you will see, there will be several recurring themesduring my career where early exposure to ideas and systemsinfluenced the path of my scientific interests.

I chose to do my senior thesis with Walter Kauzmann, whowas studying the biophysics of protein folding. I still remembertraveling with Walter to a local chicken farm to buy 20 dozenfresh eggs, from which I isolated 50 g of ovalbumin as my modelprotein of study. The smell during this purification preventedme from eating eggs for many years! Like most scientists of thetime, I discarded all the phospholipids that would be the focusof my future studies. I spent many a night in a low-temperatureroom in the basement of the Frick Chemistry Laboratory build-ing using a dated polarimeter taking point-by-point measure-ments to determine the helical content of ovalbumin undervarious denaturing conditions. How I wish I had had accessto a scanning circular dichroism instrument, which were justbecoming available. In fact, Walter acquired one the month Ihanded in my thesis!

In the summer between my junior and senior years, I workedas an analytical chemist at the Ford Motor Company plant out-side of Detroit. We did routine analyses of chemicals providedby suppliers and also of material from the assembly line. Thiswas clearly not what I wanted as a career despite being offered apermanent position upon graduation. I was much more inter-ested in pursuing mechanistic problems. Walter stronglyencouraged me to pursue a Ph.D. in biochemistry and askedwhere I wanted to go. The parents of my wife-to-be, JerilynAtwood, had recently moved to California, so the choice was

This work was supported by NIGMS, National Institutes of Health GrantsGM20478, GM25047, GM35143, GM54273, GM56389, and GM115969 (toW. D.). The author declares that he has no conflicts of interest with thecontents of this article. The content is solely the responsibility of the authorand does not necessarily represent the official views of the National Insti-tutes of Health.

1 Recipient of an endowed chair from the John Dunn Research Foundation. Towhom correspondence should be addressed. E-mail: william.dowhan@uth.tmc.edu.

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obvious. After graduation and our marriage in June of 1964, wemoved to Berkeley so I could begin studies at the University ofCalifornia. The stipend was $2800 a year, a far cry from the$19,000 Ford had offered. Fortunately, Jerilyn worked as a nurseduring most of our years at Berkeley, making our time theremore comfortable. These were of course the wild years ofthe Vietnam War, the free speech movement, and the Reagangovernorship. This was the polar opposite of the social atmo-sphere of the Princeton campus. The military service draft con-stantly made our future uncertain as fellow students, especiallythose with an M.D., disappeared on a regular basis. Most of usconfined our time to the lab but thoroughly enjoyed the colorfulevents that surrounded us.

My first-year courses were in biochemistry (taught byEsmond Snell), a genetics course, and an intensive lab coursewhere we spent all of our free time learning biochemical tech-niques. The genetics course relied heavily on what was knownabout the lac operon in the mid-1960s. This would be a veryimportant 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 ourchoice. I ran across a series of books in the biochemistry librarythat dealt with membranes. At the time, there were still argu-ments concerning the basic structure of membranes. A lipidbilayer as the core of membranes was proposed in 1925 (3),but arguments were still being made for proteins as the orga-nizing framework for membranes. How proteins were orga-nized in the membrane was unknown. A particularly inter-esting section of this book series proposed the organizationof proteins into domains within a lipid bilayer. This was wellbefore Singer-Nicolson proposed the “fluid mosaic model”(4) for membrane structure. Naively, I chose this as my sem-inar topic. The result was a near failure in the course due tomy inability to recognize such “poorly documented scientificstudies.” This was a lesson in the difficulties of challengingcurrent dogma, a practice which I have tried to continue tothis date. Interestingly, my research group later contributedto the body of evidence supporting lipid domains withspecifically associated proteins in bacterial membranes(reviewed in Ref. 5).

I chose to do my Ph.D. thesis under the direction of EsmondSnell because of the opportunity to learn methods of proteinpurification, protein characterization, and enzymology. Esmondwas of course world-renowned for his work on enzymes con-taining pyridoxal phosphate as a cofactor. Esmond’s lab washeavy on postdoctoral fellows each working on their ownenzyme. The presence of experienced postdocs was particularlyimportant during Esmond’s one-year sabbatical, and hisabsence forced me to become independent and resourceful. Igained access to Dan Koshland’s newly arrived Beckman aminoacid analyzer and Howard Schachman’s ultracentrifuge inorder to determine the amino acid composition and molecularweight, respectively, of D-serine dehydratase (6). After devisinga method for producing the apoenzyme followed by reconsti-tution, I used Esmond’s collection of B6 analogues to establishthe structural features of pyridoxal necessity for enzyme bind-ing and catalytic activity (7). While my project progressedsmoothly, the outside world interfered at times. For example,

free speech “terrorists” of the time were blowing up establish-ments of capitalism such as Bank of America branches, and onone occasion, a major electrical tower of Pacific Gas andElectric; the latter event shut down one of my ultracentrifugeruns. I filed my Ph.D. thesis at the Sproul Hall administrationbuilding while it was surrounded with state troopers andstudent demonstrators.

While I was in the lab, Dixon Riley discovered a new pros-thetic group residing in the histidine decarboxylase of Lactoba-cillus 30a (8). An N-terminal pyruvate residue is created by anautocatalyzed serinolysis of the peptide bond N-terminal to aserine residue in the proenzyme. Awareness of this discoverywould be important to my later studies on phosphatidylserine(PS)2 decarboxylase from Escherichia coli (9, 10).

My Ph.D. thesis did not produce any seminal results or earth-shaking principles. However, the training in the scientificmethod and rigorous molecular description of results alongwith a solid background in protein chemistry and enzymologywould serve me well in the years to come. I owe a great debt toEsmond and the faculty at Berkeley for the opportunity to doresearch in their midst.

Making the move to membranes

The earlier encounter with the area of membrane structurein my seminar course, although traumatic, still piqued myinterest, and I was eager to learn more. In the late 1960s, fewintegral membrane proteins had been purified in an activeform. A search of the literature turned up the classic Fred Foxand Eugene (Gene) Kennedy paper (11) describing the identifi-cation of the membrane-associated M protein, which is the lacYgene product LacY. Gene’s group correctly suggested that theM protein carried out both energy-independent equilibrationof substrate across the cell membrane as well as energy-depen-dent accumulation of substrate without modification of thesubstrate (12). This was in stark contrast to the phosphotrans-ferase-dependent systems studied by Saul Roseman (13), whichused metabolic energy to modify the substrate, resulting inaccumulation. These differences in mechanism provided color-ful debates at Gordon Research Conferences for several yearsbetween Saul, Gene, and H. Ronald Kaback as to the mecha-nism by which LacY transports substrate. Although Ron andGene had differences in details, they agreed that a high-energycovalent intermediate was not involved in the LacY mecha-nism. Ron eventually established that an electrochemical pro-ton gradient was the driving force for lactose accumulation,which was the culmination of what Ron termed the “Chemios-motic Wars.” Ron of course went on to establish the detailedmolecular structure and mechanism of LacY (14, 15). It wasduring these Gordon Conferences debates that I got to knowRon (Fig. 1), which resulted in a long and productive scientificfriendship.

After hearing about LacY for so many years, I decided tobring my acquired expertise in protein purification and charac-

2 The abbreviations used are: PS, phosphatidylserine; PG, phosphatidylglyc-erol; PGP, phosphatidylglycerol-P; PE, phosphatidylethanolamine; CL, car-diolipin; PC, phosphatidylcholine; PCTP, phosphatidylcholine transfer pro-tein; PI, phosphatidylinositol; PITP, phosphatidylinositol transfer protein;NAO, 10-N-nonyl acridine orange.

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terization to bear on this protein, and thus make my entry intothe nascent field of membrane proteins. I arrived with my wifeand son David for a postdoctoral position at Harvard MedicalSchool in the late spring of 1969. Gene (Fig. 2) had of coursedelineated most of the metabolic pathways for phospholipidbiosynthesis in mammalian cells and E. coli (summarized inRef. 16). His foray into membrane transport processes was anew 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 lipidmetabolism and membrane biology. Edward Dennis woulddevelop the important surface dilution model to explain thekinetics of integral and peripheral membrane proteins thatused membrane-associated lipid substrates (17). He wouldbecome a leading figure in phospholipase A2 generation of lipidsecond messengers. David Nelson would assume the lead

Figure 1. Gottfried Schatz (left), H. Ronald Kaback (middle), and William Dowhan (right). Taken outside Basel, Switzerland in 1984.

Figure 2. 2003 Gordon Research Conference. Left to right: Dennis Voelker, Anthony Fischl (first doctoral student with George Carman and last postdoctoralfellow with Eugene Kennedy), Eugene Kennedy, William Dowhan, George Carman, Edward Dennis, and Christian Raetz.

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authorship of Lehninger’s classic biochemistry textbook Prin-ciples of Biochemistry. William Wickner would join ArthurKornberg’s lab after medical school, and accomplish the diffi-cult purification of the E. coli DNA polymerase III (18). Hewould return to membrane studies to be a leading figure in thebiosynthesis and assembly of membrane proteins. ChristianRaetz (Fig. 2) would be Harvard’s first M.D./Ph.D. graduate. Hewould combine classical and modern molecular genetics withrigorous mechanistic biochemistry to map mutants in E. coliphospholipid metabolism and delineate the complex pathwaysfor the synthesis of lipid A (19), the core membrane componentof lipopolysaccharide present in Gram-negative bacteria. Car-los Hirschberg would go on to make seminal contributions inglycoprotein biosynthesis. Shortly after my departure, JamesRothman joined the laboratory. He would receive the NobelPrize in physiology in 2013, along with Randy Schekman andThomas C. Südhof, for seminal work on intracellular proteintrafficking. Dennis Voelker would begin to unravel the com-plex process of inter-organelle movement of phospholipids(20) and the role of phosphatidylglycerol (PG) as a lung sur-factant antimicrobial agent (21). Continual interaction withall of these scientists over the years had a profound influenceon my work.

I set out to purify the M protein (i.e. LacY). The assay wasquite crude and based on the original specific radiolabelingdevised by Fred and Gene (11) rather than reconstitution oftransport function in proteoliposomes as later developed byRon (22). I tried all of the available mild non-denaturing deter-gents available at the time with no success. Either the proteinwas not extracted, or if extracted, it was hopelessly aggregated.My scientific disappointments with the purification of LacYwere compounded in the summer of 1969 with my mother’sdiagnosis with terminal pancreatic cancer and the realizationthat my father had advanced Alzheimer’s disease. With thesupport of Gene and the American Cancer Society (whichextended my two-year fellow to three years), I took a nine-month leave from science. My parents both passed away inDecember of 1969, and after settling their affairs, I returned tothe lab in the spring of 1970 to continue with the LacY purifi-cation. However, after several months, it became obvious thatthis project was a failure. Eventual success in purification ofLacY would depend on the availability of octyl glucoside anddodecyl maltoside and the efforts of Hastings Wilson and RonKaback 10 years later (23). However, I would return to LacYsome 20 years later as the model protein in my studies of lipid-protein interactions.

During my absence, Bill Wickner continued his medical stu-dent research rotation in Gene’s laboratory. Bill had made sig-nificant progress in the purification of E. coli PS decarboxylase.Here was an integral membrane protein with a convenientassay that was readily solubilized in an active form by TritonX-100. Bill was a master at large-scale preparation. Membraneswere isolated from a pound of E. coli cell paste followed byextraction and an acetone precipitation step. The first columnwas a 1-liter volume DEAE fractionation with a 10-liter saltgradient in Triton X-100 and 10% glycerol, which was collectedin 250-ml fractions in a modified fraction collection. After a dayin his clinical rotation, Bill would set this up in the evening on

the lab bench behind mine. About 20% of the time, the fractioncollector would malfunction, resulting in the floor becomingcovered in the effluent, which I would have to clean up in themorning. After concentration of the decarboxylase peak and agel filtration column, Bill had a yellow solution with a 500-foldincrease in specific activity. I took over the project at this pointbecause Bill was soon to join Arthur Kornberg at Stanford. AsBill was preparing his oral short presentation on partial isola-tion of a pyridoxal-dependent PS decarboxylase at the SanFrancisco meeting of the Federation of American Societies forExperimental Biology, I decided to subject the sample to asucrose gradient centrifugation step. The result was a yellowpellet at the bottom of the tube and all the activity about half-way down the tube. We either had a newly discoveredpyruvate-dependent decarboxylase or a long way to go beforepurity was attained. It turned out both were true. I completedthe purification over several months starting with 8 kg of E. colicell paste, which after a 3500-fold enrichment yielded about 14mg of active nearly pure enzyme (10). I did not determine theidentity of the prosthetic group; that would wait until Satre andKennedy’s later work, identifying the group as pyruvate boundto a small subunit of the decarboxylase (24), although I did laterreturn to this enzyme to study the mechanism by which theprosthetic group is generated (9). My tenure in Gene’s labafforded the opportunity to study under one of the great scien-tists of the late 20th century, and form lasting personal andscientific friendships for the remainder of my career. Bill and I,with Gene’s careful guidance, had demonstrated that integralmembrane proteins of phospholipid metabolism could be puri-fied to homogeneity in an active form and characterized. Thiswas a seminal accomplishment for the times and provided thefoundation for the next 15 years of my research while stimulat-ing others to carry out similar purifications in bacteria andeukaryotic cells.

Time to find gainful employment

1972 was not a great year to find a job. We were in the middleof the Nixon years of significant cuts in National Science Foun-dation (NSF) and National Institutes of Health (NIH) researchsupport. Most of the new hires were concentrated in the Hous-ton, Texas area, which was undergoing a renaissance in bio-chemistry. George Schroepfer moved from the University ofIllinois to initiate the Department of Biochemistry at Rice Uni-versity. Salih Wakil moved from Duke to Baylor College ofMedicine and was increasing the Department of Biochemistry.Allan Goldstein came from New York to rebuild the Division ofBiochemistry at the University of Texas Medical Branch inGalveston. John (Jack) DeMoss moved from the University ofCalifornian at San Diego to chair the Department of Biochem-istry and Molecular Biology at the newly formed University ofTexas Medical School in Houston. I interviewed at all four insti-tutions and decided to take my chances with Jack in building anew medical school.

When I interviewed in January of 1972, just after the birth ofour son Michael during a December snowstorm, the medicalschool’s first building was a hole in the ground, with laborato-ries in rented space spread over the Texas Medical Center.When I arrived in August, the first two-story building, where

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we taught the first entering class of medical students in Septem-ber, was complete. The five founding members of the depart-ment were housed in cramped rented space on the 13th floor ofthe Center Pavilion Hospital where I shared an office and labo-ratory with Henry Strobel, who came from Minor Coon’sgroup. The building had previously been a hotel, and each roomwas converted to a shared office and laboratory. We were sand-wiched between the hospital below and the drug halfway houseon the floor above. The latter occupants would wander into ourspace and on one occasion collected bottles of radioactive tol-uene scintillation fluid in order to get high. That fall was spentwriting our first grants, setting up our labs, organizing the med-ical school lectures, and designing small group conferences theweek before each meeting with medical students. My first grantwas funded on the initial pass in 1973 despite Nixon seques-tering the NIH budget for a short time. One could still get agrant with a novel idea and a background to support theproject without a lot of preliminary data. My paper on the PSdecarboxylase was not published until 1974 (10). I had dem-onstrated the ability to purify at least one membrane proteinin an active form and wished to purify and characterize a fewmore. Because the grant ran for 43 years, including a 10-yearMERIT Award at the end, its original title of “Structure andFunction of Membrane Proteins” proved to have unexpectedlongevity. Fortunately, after a short grant hiatus, the projectcurrently continues under a new grant number. My recom-mendation has always been to pick a topic in an evolving areathat will set one’s research apart from the crowd. Membraneproteins certainly fitted the bill at that time and remain anactive area of research.

Jack was the model departmental chairman, which I tried toemulate during my chairmanship of the department in the early

1990s. Jack’s approach was to hire bright young faculty, providestrong financial support and guidance, and leave them aloneto develop their unique niche in science. This tradition con-tinues under the strong leadership of Rodney Kellems, thecurrent chair of the department. I like to think that my deci-sion to come to what is now the McGovern Medical Schoolsome 45 years ago and contribute to the research develop-ment, administrative leadership, and teaching mission ofthe school has in some small way contributed to the success-ful development of a strong clinical- and research-basedinstitution.

When I was starting up, Gene had suggested I replicate mysuccess in purification of a bacterial membrane protein inmammalian systems, and so I did spend a few months workingwith beef hearts and livers. However, my passion remained withE. coli lipid biosynthetic enzymes, and so I returned to thistopic. This was a fortuitous decision as the explosion in mod-ern molecular biology and genetics was about to occur. Withmy first graduate student Tim Larson and my first postdocTakashi Hirabayashi, we set out to purify the E. coli PS andphosphatidylglycerol-P (PGP) synthases, the committedsteps to major lipids of E. coli, phosphatidylethanolamine(PE) and PG/cardiolipin (CL), respectively (Fig. 3). Not want-ing to repeat the difficulties of time and material I experiencedwith the PS decarboxylase, we capitalized on new approaches toprotein purification by developing substrate affinity chroma-tography methods. For the PGP synthase, we covalentlyattached the lipid substrate CDP-diacylglycerol to a Sepharosecolumn and eluted the enzyme with its substrate CDP-diacylg-lycerol (25). PS synthase tightly binds to phosphocellulose,which we specifically eluted using CDP-diacylglycerol (26).

Glycerol-3-P

CTP

L-Serine

CO2 Pi

1) cdsA

2) pssA 4) pgsA

3) psd 5) pgpABC

6) clsAB

{clsC}

CDP-DIACYLGLYCEROL

PHOSPHATIDYLSERINE PHOSPHATIDYLGLYCEROL-3-P

PHOSPHATIDYLETHANOLAMINE PHOSPHATIDYLGLYCEROL

CARDIOLIPIN

PHOSPHATIDIC ACIDDIACYLGLYCEROL

ATP

MDO

pre-MDO

7) mdoB

8) dgk

UDP-glucose

UDP-glucose

MONOGLYCOSYL DIACYLGLYCEROL + UDP

DIGLYCOSYL DIACYLGLYCEROL + UDP

9) mgs

10) dgs

Choline

PHOSPHATIDYLCHOLINE + CMP

11) pcs

Inositol

PHOSPHATIDYLINOSITOL+ CMP

12) PIS1Lysyl-tRNA

O-LYSYL PHOSPHATIDYLGLYCEROL+ tRNA

13) mprF

PG{PE}

Glycerol{Ethanolamine}

CMP

PPi

Figure 3. Synthesis of native and foreign phospholipids in E. coli. Pathways native to E. coli are noted with solid arrows, and pathways resulting fromforeign genes introduced into E. coli are noted with dashed arrows. The genes encoding the following enzymes and associated with each biosyntheticstep are listed next to the arrows: 1) CDP-diacylglycerol synthase; 2) PS synthase; 3) PS decarboxylase; 4) PGP synthase; 5) PGP phosphatases; 6) CLsynthases; 7) PG:MDO sn-glycerol-1-P transferase; 8) diacylglycerol kinase; 9) glucosyl diacylglycerol synthase (Acholeplasma laidlawii); 10) diglucosyldiacylglycerol synthase (A. laidlawii); 11) PC synthase (Legionella pneumophila); 12) PI synthase (S. cerevisiae); 13) O-lysyl PG synthase (Staphylococcusaureus). Figure originally published in Ref. 62 (Dowhan, W., et al. (2017) Functional roles of individual membrane phospholipids in Escherichia coli andSaccharomyces cerevisiae. in: Biogenesis of Fatty Acids, Lipids and Membranes (Otto Geiger (ed)), DOI: 10.1007/978-3-319-43676-0_36-1). With kindpermission from Springer International Publishing.

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Chris Raetz and I had a long and productive personal andprofessional relationship until his untimely passing from can-cer in 2011, the same year Gene passed away. Upon completionof his M.D./Ph.D. at Harvard Medical School and a residency atthe Peter Bent Brigham Hospital in Boston, Chris spent twoyears as a postdoctoral fellow with Herb Tabor at the NIH.While we toiled in the cold room purifying enzymes, Chrisdeveloped clever methods for isolating temperature-sensi-tive mutants in PS (27) and PGP (28) synthases followed bymapping of their encoding genes pssA and pgsA gene, respec-tively. The convergence of mutants in phospholipid biosyn-thesis and purification of functional biosynthetic enzymesoccurred fortuitously with the emergence of moleculargenetic engineering of E. coli. While at Stanford, Bill Wick-ner acquired the Clarke and Carbon collection (29) of E. coliDNA fragments carried on a ColE1 plasmid, which Bill sentto Chris at the NIH. Through complementation of the pssAmutant, Chris was able to clone the gene encoding PS syn-thase. We combined forces and used the overproduction ofthe PS synthase coupled with affinity chromatography toisolate about 20 mg of pure enzyme from 350 g of wet weightof E. coli cells (30). This represented one of the first usesof genetic engineering to obtain significant amounts ofenzymes found in very low amounts. We later engineered800-fold overproduction of the enzyme, which yielded 34 mgof enzyme from 20 g of cells (31).

In the mid-1970s to early 1980s, access to DNA ligase and afew restriction enzymes was very limited. I benefited by thearrival to our faculty of Terry Landers from Paul Berg’s lab andCharles McHenry from Arthur Kornberg’s laboratory. Terrybrought the expertise and methodology for isolating severalrestriction enzymes, and Charlie purchased a 350-liter fer-menter that we used to grow cells for isolation of enzymes formanipulation of DNA. With all these resources at hand, wecloned the pgsA gene and amplified its production for effi-cient purification (32). Using the tedious Maxam and Gilbertmethod, we sequenced the pssA (33) and pgsA (34) genes.Chris’s lab identified two genes (pgpA and pgpB) thatencoded enzymes that dephosphorylated several phospholipidsincluding PGP (35). However, double mutants still carried outPG and CL synthesis. We established that a third gene, pgpC,was responsible for anionic phospholipid biosynthesis (36).Chris later cloned pgpC and characterized mutants and thegene product (37). The combined efforts of mapping genesresponsible for lipid biosynthesis, cloning the genes, and puri-fying the resulting gene products validated the lipid biosyn-thetic pathways delineated by our mentor, Gene Kennedy, andbrought lipid metabolism into the modern age of moleculargenetic engineering (summarized in Ref. 16).

In 1976, George Carman (Fig. 2) inquired about a postdoc-toral position in my laboratory. His background was in foodscience, so he had a lot to learn about biochemistry, but what helacked in expertise he made up for in enthusiasm and persever-ance. We set out to apply surface dilution kinetic analysis to PSsynthase to understand how a peripheral membrane enzymecarries out catalysis (38). Later George and Ed Dennis wouldprovide a more detailed description of the method (17). Georgerapidly learned membrane biochemistry and enzymology. I am

pleased that I was able to give George the opportunity to begina very productive research career, which has resulted in himbecoming one of the leaders in the study of lipid metabolism inyeast (39). My most significant contribution to yeast lipid stud-ies was to convince George to stay in academics and turn downthe opportunity to make instant gratin potatoes in the foodprocessing industry.

A faculty leave develops new directions

In 1983, with the luxury of being a tenured full professor withtwo recently renewed NIH grants, I decided to take a facultydevelopment leave for one year in Gottfried (Jeff) Schatz’s (Fig.1) lab at the Biozentrum in Basel, Switzerland to be immersed inyeast genetics and mitochondrial bioenergetics. This would bea dramatic change in the direction of my research program thatwould also benefit my entire family. To get a head start, Iattended the Cold Spring Harbor yeast genetics course inAugust before moving to Basel. Armed with funds from a Gug-genheim Foundation fellowship, my NIH grants, and my insti-tutional support, we arrived in Switzerland in September, 1983.Our son Michael attended 6th grade in the International Schoolof Basel. He developed his French and a great sense of indepen-dence through taking the bus and street car every day to school.Our son David attended the 10th grade at Aiglon College, anEnglish curriculum boarding school in the Swiss Alps city ofChesiéres-Villars on the east side of Lake Geneva; winter phys-ical education included daily skiing. My wife Jerilyn, the kids,and I enjoyed trips throughout Europe and 2 weeks in Egypt.Although there are many personal and professional obsta-cles to sabbatical leaves, I can strongly recommend such anexperience if you have the opportunity, as it enriched ourpersonal lives and my professional career. I also took devel-opment leaves in 1991 and 2010 in Robert Simoni’s labora-tory at Stanford University.

Jeff Schatz was a widely read Renaissance scholar and greatintellect. Besides his seminal studies in mitochondrial bioener-getics, Jeff was an accomplished violinist and a member of theBasel orchestra. During my stay in the lab, I honed my skills inyeast genetics, was introduced to the ease of making geneknockouts in yeast, and acquired a strong background in mito-chondrial bioenergetics, which would change the direction ofmy research once I returned to Houston. The knock-out Iconstructed in the nuclear COX4 gene that encodes a sub-unit of the mitochondrial cytochrome c oxidase respiratoryComplex IV resulted in failure of the multiple subunits of thecomplex to assemble (40). I tried to get Jeff interested inyeast lipids, in particular the role of CL in mitochondrialfunction. However, Jeff, like many at the time, felt that lipidswere not important because the purified respiratory com-plexes were fully active in detergents. My own interest in thisquestion remained, and so I returned to this system in laterstudies on the role of CL in mitochondrial function, discov-ering for example that mutants lacking PG and CL in themitochondria fail to translate the COX4 gene mRNA, thusdemonstrating communication between the mitochondriaand the nucleus (41).

While I was in Gene Kennedy’s lab, one of our weeklyresearch meetings discussed Karel Wirtz’s report of a mamma-

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lian cytoplasmic protein (phosphatidylcholine (PC) transferprotein or PCTP) that catalyzed the exchange of PC betweenmembranes (42). Later a similar activity (PITP) that exchangedphosphatidylinositol (PI) and PC between membranes wasreported (43). These were hypothesized as being involved inmoving phospholipid from the site of synthesis in the endoplas-mic reticulum to other cellular membranes. While in Basel, Iinitiated a partial purification of the latter PITP activity fromSaccharomyces cerevisiae, which formed the basis for an NIHproposal. Shortly after receiving funding upon my return toHouston, Paul van Heusden joined my group from Wirtz’s laband, along with a graduate student, Jackie Aitken, continued thepurification. To my dismay, their first attempt separated thetransfer activity from the protein I had incorrectly proposed asthe PITP. However, persistence resulted in purification of thefraction containing PITP activity to homogeneity, and by usingthe partially determined protein sequence, the encoding genewas identified (44). This paper was recognized as a Journal ofBiological Chemistry Classic (45). Shortly after publication ofthe results, I was contacted by Vytas Bankaitis, who had beenworking on the Sec14 protein, which Randy Schekman hadidentified as a component necessary for movement of proteinsfrom the endoplasmic reticulum to the Golgi of yeast. We col-laborated to demonstrate that Sec14 and PITP are one and thesame (46). This collaboration opened up the whole area of stud-ies pioneered by Bankaitis’ lab on related phospholipid transferproteins, which turned out to be regulatory proteins that inte-grate various cell processes with lipid metabolism.

Why are there so many lipids?

When I returned to Texas, I also returned to studies of phos-pholipid genetics and function in yeast and E. coli. However, myexposure to yeast genetics changed the course of my researchfrom enzymology and protein chemistry to addressing the roleof lipids in cell function. I was fortunate to hire Phil Heacock, avery bright and resourceful technician who would be the pri-mary support of our molecular genetic manipulation of yeastand E. coli over the next 20 years. Outside of the emerging areaof lipids and their derivatives as second messengers, lipids werestill viewed as merely the hydrophobic medium in which mem-brane proteins are embedded. But if a single phospholipid canform a lipid bilayer, why should there be so many lipids (47)?We set out to make null mutants at the branch point of phos-pholipid biosynthesis, i.e. in the pssA and pgsA genes (Fig. 3).This was before the advent of the elegant and efficient meth-ods available today to carry out genetic manipulation andcounterintuitive because Chris Raetz had shown that condi-tional mutations in these genes were lethal. However, Inoticed that the temperature-sensitive mutants pssAts andpsdts were viable, with low PE or high PS and low PE, respec-tively, if grown at the restrictive temperature in the presenceof millimolar concentrations of Mg2�. Extensive analysis ofthe phenotypes of the �pssA mutant, which is viable if grownin the presence of millimolar levels of Ca2�, Sr2�, or Mg2�

(34), has provided fertile research projects to the presentday. Although PE is not absolutely required under controlledgrowth conditions, lack of PE results in rapid cell lysis in theabsence of divalent cations. Under viable conditions, the

mutant displays defects in cell division, energy metabolism,and structural organization of most secondary solute trans-porters (16).

Under the conditions we used, the �pgsA strain we initiallyconstructed was not viable, suggesting that PG is essential (48).The �clsA strain, lacking what turned out to encode the majorCL synthase, was still robustly viable. Later Raetz’s group, witha little help from us, characterized deletions in the remainingtwo clsBC genes, which encode additional CL synthases, anddemonstrated that cells completely lacking CL are viable(49). One could bypass the need for PG specifically by elim-inating the major outer membrane lipoprotein (encoded bylpp) that derives a glycerol moiety from PG. The glycerolforms the diacylglycerol in the thioether linkage to theN-terminal cysteine that tethers the protein to the inner leaf-let of the outer membrane. Failure to make this link results inaccumulation of the nascent protein in the inner membrane,resulting in cell death. However, the �pgsA �lpp strainremains temperature-sensitive for growth (50). Growth athigh temperature was a step we used during the final steps ofconstructing the �pgsA strain, leading to the conclusion thatcells could not survive without PG. These additional prop-erties of the mutant were eventually sorted out, resulting in aviable strain that was used to probe additional roles for ani-onic lipids in cell function.

Ascertaining functional roles for lipids at the molecular levelposes several obstacles. Many early conclusions of lipid func-tion were based on the effects of lipids added to in vitro studiesof cell processes. However, little attention was paid to the com-plex interplay of physical and chemical properties of lipids —defined by both the chemistry of the hydrophilic head groupsand the physical properties of their diverse fatty acid composi-tion — thus leading to artifacts. Because genes encode the lipidmetabolic enzymes and not the lipids directly, mutations affect-ing biosynthetic pathways are indirect in defining function.Loss of function can be highly pleiotropic, especially in eukary-otic cells with multiple membranes. Elimination of a major lipidmay result in loss of cell integrity before a direct requirement ina cell function is revealed. Our approach has been to generateviable but compromised mutants with altered lipid composi-tion followed by detailed characterization of phenotypes incells. In order to establish a direct lipid-cell function relation-ship, the phenotypes have been reconstituted in well-controlledin vitro studies. This approach has resulted in defining at themolecular level previously unrecognized roles for lipids in suchdiverse processes as membrane protein folding, DNA replica-tion, cell division, protein translocation across membranes,energy transduction, and organization of phospholipids intodomains (16).

In 1991, shortly after construction of the above null mutants,two very talented postdoctoral fellows joined my group.Mikhail (Misha) Bogdanov focused on the �pssA strain prop-erties, and Eugenia Mileykovskaya initially characterized the�pgsA strain. Both have become important faculty collabora-tors. Eugenia developed earlier findings showing that, as ineukaryotic cells, membrane phospholipids are not evenlydistributed laterally in the inner membrane bilayer of E. coli.Using 10-N-nonyl acridine orange (NAO), which binds spe-

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cifically to anionic lipids, she was able to visualize anionicphospholipid localization at the poles and septum of dividingcells by fluorescence microscopy (51). A characteristic redshift in NAO’s absorbance indicated that CL was present inthese lipid domains. She and several coauthors further iden-tified a unique role for CL domains in coordinating the loca-tion of the FtsZ-dependent divisome (52) at the center of thecell, adding to our understanding of the importance of phos-pholipids in cell division.

A growing role for yeast

Following our success in E. coli, we extended the approach toS. cerevisiae. Influenced by my experience in Basel with mito-chondrial function, we focused on genes responsible for thesynthesis of PE, PG, and CL in the mitochondria. I chose yeastfor these studies because cells compromised in mitochondrialfunction can still be grown on glucose. Aided by an M.D./Ph.D.student Shao-Chun Chang and a postdoctoral fellow Con-stance Clancey, the yeast nuclear genes PSD1, PGS1, and CRD1that encode the mitochondrial PS decarboxylase (53), PGP syn-thase (54), and CL synthase (55), respectively, were cloned andknocked out. The PSD1 gene was cloned by complementationof the E. coli temperature-sensitive mutant in the PS decarbox-ylase. The PEL1 gene had been cloned and was reported to be asecond PSS (CHO1) gene. We noted that the null mutant lackedPG and CL, and thus surmised correctly that it was the PGS1gene. The null mutant turned out to be respiratory-deficientand displayed structurally aberrant mitochondria. The CRD1gene was uncovered during our initial attempts to find thePGS1 gene by homology searches using the E. coli PGP synthasesequence, which like the yeast CL synthase, contains motifs thatrecognized CDP alcohols such as CDP-diacylglycerol. The�crd1 strain accumulates PG up to 20% in the mitochondria inplace of the normal 20% CL. Surprisingly, PG appears to par-tially substitute for the lack of CL in respiratory function, whichwas unexpected given the exclusive localization of CL to themitochondria.

After extensive studies on the function of anionic phospho-lipids in E. coli and at the beginning of the new millennium,Eugenia and a talented graduate student, Mei Zhang, focusedon the role of CL in mitochondrial function (summarized inRef. 56). Although Chance postulated in 1959 (57) that the indi-vidual mitochondrial electron transport chain complexes wereorganized into a higher-order supercomplex or respirasome,this idea lost favor once individual complexes were isolated infully functional form. The respirasome concept gained favoragain in the early 2000s with the advent of Blue Native-PAGE,which displays large membrane-derived complexes in an activeform. Subjecting a digitonin extract of yeast mitochondria toBlue Native-PAGE showed a supercomplex composed of Com-plexes III (ubiquinol:cytochrome c oxidoreductase) and IV(cytochrome c oxidase) with a molecular weight of about a mil-lion Da. Mei and Eugenia varied expression of the CRD1 geneunder control of a tet promoter, resulting in a proportional levelof CL that correlated with an increase in the ratio of supercom-plex to individual Complexes III and IV. A kinetic analysis ofelectron transfer between Complexes III and IV by cytochromec in whole mitochondria was consistent with the formation of a

supercomplex when CL was present in contrast to the presenceof individual complexes in the absence of CL. Using single-particle electron cryomicroscopy, we further verified that withwild-type CL levels, the supercomplex is composed of ComplexIII (as a dimer) flanked by a Complex IV on each side. When theindividual complexes were isolated in the presence of detergentthat removes all peripheral lipids while maintaining the lipidsintegral to each individual complex, CL was absolutely requiredto reconstitute the supercomplex in vitro. Therefore, we estab-lished that CL “glues” the individual complexes together toform the respirasome with the close contact presumablyincreasing the efficiency of electron transfer and thus allowingmore robust growth of cells containing CL. Current studies arefocused on obtaining a high-resolution structure of the super-complex in order to visualize whether CL is localized in thespaces between Complexes III and IV.

Membrane protein gymnastics in the lipid bilayer

The characterization of the �pssA mutant, lacking PE andcontaining only anionic PG and CL, has been a major focus ofmy research group since the early 1990s (summarized in Ref.58). Phil Heacock developed a set of lipid mutants that repre-sent a tool box of strains useful in defining lipid function in vivo(Fig. 3). The pssA gene was placed under inducer dose-responseregulation so that the level of PE can be controlled or PE syn-thesis turned on and off during the cell growth. Lipids foreign toE. coli were introduced into PE-lacking cells by expression ofgenes from other organisms. PC, mono- and diglucosyl diacyl-glycerol, and lysyl-PG were used to determine what propertiesof PE (net charge, physical properties, or dilution of membranenegative surface charge density) are important for function. Anearly paper in collaboration with Misha and Ron’s lab detailingthe role of PE as a lipochaperone in the folding of LacY (59) wasalso recognized as a Journal of Biological Chemistry Classic (45).Misha and I employed these strains and several secondarytransporters including LacY to establish the Charge BalanceRule (58) for membrane protein organization at the time ofinitial membrane insertion as well as in governing dynamicstructural organization of membrane proteins post-assembly.In wild-type cells, as predicted by the Positive Inside Rule,membrane proteins assemble with positively charged extra-membrane domains facing the cytoplasm. Those domains thatare negatively charged or neutral face the trans side of the mem-brane. The Charge Balance Rule is an extension of the PositiveInside Rule that incorporates the effect of membrane lipid com-position in determination of the orientation of transmembranedomains with respect to the plane of the lipid bilayer. The invivo experiments carried out by Misha and me demonstratethat the membrane lipid composition has a direct effect ontopological organization of membrane proteins at the time ofinitial insertion of transmembrane domains and protein folding(Fig. 4). The presence of PE suppresses the membrane translo-cation potential of negative residues in extramembranedomains containing a mixture of oppositely charged residues.However, in the absence of PE, negative residues become strongtopological determinants, resulting in a change in orientation ofneighboring transmembrane domains. Most dramatic is thattopological organization is not fixed at the time of initial folding

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but can be changed post-folding by a change in lipid environ-ment. The effect of PE and other net neutral or positivelycharged lipids is to dilute the high membrane negative surfacecharge due to PG and CL. Therefore, membrane protein struc-tural organization remains dynamic and responsive to changesin the local lipid environment. Heidi Vitrac, who joined ourresearch group recently, reconstituted the dynamic propertiesof membrane proteins in a totally in vitro system composed ofonly LacY and lipids. She established that the rate of topologicalre-arrangement occurs on a time scale of seconds, making suchchanges of biological significance for any protein in any mem-brane potentially independent of other cellular factors (60). Shealso demonstrated that phosphorylation of a membrane pro-tein extramembrane domain, which alters the charge balancebetween the protein and the membrane surface, results in asimilar rapid change in topological organization (61).

The above studies were carried out using primarily LacY as amodel 12-transmembrane domain-spanning protein, which againillustrates how early studies have influenced my current focus.None of the progress made on the role of phospholipids in deter-mining membrane protein topological organization would havebeen possible without the pioneering work of Ron on LacY and hiswillingness to provide us with LacY derivatives and advice. Ourcurrent studies are focused on defining in molecular terms howmembrane lipid composition and protein phosphorylation governthe dynamic structural organization of membrane proteins.

In 2005, Christian Raetz nominated me as the recipient of theASBMB Avanti Award in Lipids. Walter Shaw, president of AvantiPolar Lipids, presented the ASBMB Avanti Award in Lipids to mein San Diego, California in April of that year (Fig. 5).

In 2014, my former postdoctoral fellows George Carman andWeiming Xia organized a William Dowhan Symposium. This

Figure 4. Topological organization of LacY as a function of membrane lipid composition. Transmembrane domains (Roman numerals) and extramem-brane domains (Arabic numerals) are sequentially numbered from the N terminus to C terminus. LacY orientation with respect to the periplasm and cytoplasmis noted. Net charge of extramembrane domains is shown, and approximate position of positively (red dots) and negatively (green dots) charged residues isshown. Topology of LacY is shown after initial assembly in PE-containing cells (�PE) or after initial assembly in PE-lacking cells (�PE). The interconversion oftopological conformers after assembly in one orientation and the ratio of native to inverted conformer are reversible in both directions in vivo and in vitrodepending on the dynamic level of PE in membranes. At intermediate levels of PE, LacY exists in a mixture of topological forms.

Figure 5. Walter Shaw (left), president of Avanti Polar Lipids, presenting the ASBMB Avanti Award in Lipids to William Dowhan (center) in San Diego,CA, April 2005. Christian Raetz (right) nominated William Dowhan for the award.

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was attended by former lab members, collaborators, and mem-bers of the Texas Medical Center community in Houston,Texas in April of that year (Fig. 6).

Concluding remarks

My scientific career has been continually focused on under-standing how proteins interact with their lipid environment toattain final and dynamic structural organization. There havebeen many recurring themes throughout my career. Earlyexposure to the principles of hydrophobicity, protein chem-istry, LacY, membrane proteins, lipid metabolism, moleculargenetic manipulation, and yeast mitochondria have greatlyshaped and directed my career. I chose not to follow thecrowd but to challenge dogma, convinced that lipids do morethan form bilayers and that detergents do not fully replacelipids. Fortunately, with a good amount of luck, I have beenright more than I have been wrong. Finding conditionswhere null mutants in lipid biosynthetic enzymes compro-mised cell function without destroying membrane barrierproperties was fortunate. Although most secondary trans-porters require PE for full function, only a subset, includingLacY, is dependent on PE for proper native topological ori-entation. The fortuitous selection of LacY as our initialmodel membrane protein lead to the discovery that fullyfolded proteins can undergo topological inversion. The guid-ing principle has always been to understand mechanisms atthe molecular level by following up genetic alteration of cellfunction with detailed in vitro studies. As with all successfulendeavors, progress has relied on contributions from manycollaborators. I was fortunate in my early years to pursuescience at a time when curiosity research was highly valuedand financial support could be gained based on novel ideasthat addressed understanding basic mechanisms in livingsystems. I hope today’s scientists can get the same chance tofollow their curiosity.

Acknowledgments—I am most indebted to my mentors Walter Kauz-mann (Princeton University), Esmond Snell (University of California,Berkeley), Eugene Kennedy (Harvard Medical School), and GottfriedSchatz (Biozentrum, Basel, Switzerland), all of whom unfortunatelyhave passed away. My work has depended on support from collabo-rators William Wickner, Christian Raetz, Ronald Kaback, MikhailBogdanov, Eugenia Mileykovskaya, and Heidi Vitrac. I am grateful toGeorge Carman (Rutgers University) for continuing mutual support,both personal and scientific. Special thanks go to my long-term tech-nician Philip Heacock, 13 graduate students, and 21 postdoctoralfellows who made invaluable contributions to my research program. Igreatly value the support and council of my colleagues John DeMoss,Henry Strobel, Robb Moses, and Charles McHenry, who remain asclose friends. The support of my wife Jerilyn of 53 years has beeninvaluable in balancing my personal and professional life. Myresearch program has been supported by the NIGMS, National Insti-tutes of Health (Grants GM20478, GM25047, GM35143, GM54273,GM56389, and GM115969) since 1973 and an endowed chair fromthe John Dunn Research Foundation since 1998. I am especiallythankful to have had Jean Chin as program officer for many of myNIH-funded years. She has been a strong supporter of lipid and mem-brane research. I wish to acknowledge the American Cancer Societyfor my postdoctoral fellowship awarded in 1969 and the GuggenheimFellowship program for an award in 1983 for my faculty developmentleave. I owe much to the faculty and staff of the Department of Bio-chemistry and Molecular Biology at the McGovern Medical School atthe University of Texas Health Sciences Center in Houston. I owespecial thanks to the Journal of Biological Chemistry for the opportu-nity to serve on its Editorial Board, publishing 57 of my papers, andthe invitation to submit this Reflection.

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Figure 6. William Dowhan Symposium April 2014. A gathering in Houston of former lab members, collaborators and members of the Texas Medical Centercommunity organized by former postdoctoral fellows George Carman and Weiming Xia.

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35. Icho, T., and Raetz, C. R. (1983) Multiple genes for membrane-boundphosphatases in Escherichia coli and their action on phospholipid precur-sors. J. Bacteriol. 153, 722–730

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