Vol. 141, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1980, p. 558-5640021-9193/80/02-0558/07$02.00/0

Yeast Mutants Auxotrophic for Choline or EthanolamineKATHARINE D. ATKINSON,' BRUCE JENSEN,' ANITA I. KOLAT,2 ELIZABETH M. STORM,2

SUSAN A. HENRY,23* AND SEYMOUR FOGEL'Department of Genetics, University of California, Berkeley, California 94720,' and Departments of Genetics2

and Molecular Biology,3 Albert Einstein College ofMedicine, Bronx, New York 10461

Three mutants of the yeast Saccharomyces cerevisiae which require exogenousethanolamine or choline were isolated. The mutants map to a single locus (chol)on chromosome V. The lipid composition suggests that chol mutants do notsynthesize phosphatidylserine under any growth conditions. If phosphatidyleth-anolamine or phosphatidylcholine, which are usually derived from phosphatidyl-serine, were synthesized from exogenous ethanolamine or choline, the mutantsgrew and divided relatively normally. However, mitochondrial abnormalities wereevident even when ethanolamine and choline were supplied. Diploids homozygousfor the chol mutation were defective in sporulation. Growth on nonfermentablecarbon sources was slow, and a high proportion of respiratory-deficient (petite)cells were generated in chol cultures.

Mutants unable to synthesize specific mole-cules may be utilized to examine the roles ofindividual membrane components in cell growthand membrane functions. A genetic approach tothe molecular requirements for membrane as-sembly, growth, and function is readily devel-oped in microorganisms in which conditionalmutants may be obtained. Bacterial mutantsauxotrophic for various phospholipid precursorshave been isolated, as have temperature-sensi-tive mutants with defects not readily correctedby exogenous supplementation (2, 9, 10, 14, 23,32). These mutants provide information regard-ing the effects of altered lipid composition onprocaryotic membrane assembly and function.Fungal mutants provide the potential for similarinvestigations of the roles played by specificphospholipids in the biogenesis of the complexarray of eucaryotic membranous organelles.Fungal mutants autotrophic for the phospho-lipid precursors fatty acids (12, 13, 24, 26) andinositol (15, 27) have been described. However,in studies pertaining to membrane properties,the fungal mutants have not been exploited tothe same degree as have bacterial mutants (16,28).This report concerns Saccharomyces cerevi-

siae mutants which require either ethanolamineor choline for growth. The mutants' propertiesprovide some startling insights into eucaryoticcellular requirements for certain membranephospholipids.

MATERIALS AND METHODSYeast strains. S. cerevisiae 2180-IA (a gal2

SUC2), from the Berkeley strain collection, was theparental strain from which choline-requiring mutantswere derived. Strains 2180-1B (a gal2 SUC2) from the

Berkeley collection, G599-1OB (a ura3 hom3 hisl)provided by John Game, and S5 (a adel) from H. 0.Halvorson were used in crosses. Strain MC13 (a ino1-13 Iys2 canl), described by Culbertson and Henry (3),was used in crosses and as a related choline-independ-ent (CHO') strain in comparative studies.Media and growth conditions. Strains were

maintained on YEPD plates (1% yeast extract, 2%peptone, 2% glucose, and 2% agar). Respiratory suffi-ciency was tested on YPGE plates, with 2% glyceroland 2% ethanol in place of glucose. Synthetic completemedium contained 2% glucose; 0.67% Difco Yeast Ni-trogen Base without amino acids; the amino acids (inmg/liter) lysine (20), arginine (10), leucine (10), me-thionine (10), threonine (60), tryptophan, (10), histi-dine (10), adenine (10), uracil (10), and myo-inositol(3 mg/liter, to total 5 mg/liter with the inositol con-tained by Yeast Nitrogen Base); and one of the follow-ing supplements: 1 mM choline, 1 mM ethanolamine,1 mM serine, 1 mM N,N-dimethylethanolamine (allfrom Sigma Chemical Co.), or 1 mM N-methyletha-nolamine (from Eastman Chemical Co.). Auxotrophicmarkers were scored on media lacking a single com-ponent of the complete medium. Inositol-less mediumwas prepared using Difco Vitamin-Free Yeast Base asdescribed by Culbertson and Henry (3).

Colonies were grown on agar plates at 25 or 30°C asindicated. Liquid cultures were pregrown overnight inYEPD at 30°C. Log-phase cells were harvested bycentrifugation, washed twice with synthetic medium,suspended in synthetic medium containing the varioussupplements at a density of 1 x 106 to 2 x 10' cells perml, and incubated at 25 or 30°C as indicated. Opticaldensity of cultures was determined in a Klett spectro-photometer with a red filter. Viable cells were deter-mined by plating appropriate dilutions onto YEPDplates. To minimize aggregation of cells, the first cul-ture dilution before plating was briefly sonicated. Cellnumbers obtained by counting the colonies comparedwell with cell counts obtained by hemacytometer,indicating that cellular aggregation was not a signifi-cant problem in plating experiments.

558

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

VOL. 141, 1980

Mutagenesis and genetic analysis. Strain 2180-1A was treated with the mutagen ethyl methane sul-fonate by the method of Lindegren et al. (18). Coloniesfrom treated cells grown on YEPD plates were replica-plated to synthetic complete medium supplementedwith 1 mM choline and to medium with no supple-ment. Colonies that failed to grow without addedcholine were isolated.Mutant isolates were crossed to strain 2180-IB, and

the diploids were induced to sporulate on acetatemedium (6). Asci were digested with 10% glusulase(Endo Laboratories) and dissected by micromanipu-lation on a YEPD plate (5). Spore colonies were rep-lica-plated to the various media to determine thesegregation of auxotrophic requirements. Mating typewas determined by spraying plates with a water sus-pension of cells of each mating type (5). After severalhours, the formation of diploids was determined bymicroscopic observation of zygotes, or, in other crosses,by replica-plating to minimal medium where only dip-loids with complementing genetic markers could grow.Choline-requiring segregants of each mating type wereselected. Isolates derived from each original mutantwere crossed to each other to determine the comple-mentation pattern.

Lipid composition analysis. Cells were labeledwith [32P]orthophosphate (New England NuclearCorp.) in synthetic complete medium supplementedwith ethanolamine or choline or with no supplement.The final specific activity of the phosphate in thegrowth medium was 0.544 mCi/mmol. The cells weregrown at 30°C for five to six generations in the pres-ence of the label. The cells were harvested in logarith-mic growth at a cell density of 1 x 107 to 2 x 107 cellsper ml. Labeling for additional generations producedno change in the percentage distribution of the labelinto lipid classes or in the specific activity of the lipidextracted, so it is assumed that a steady-state labelingcondition was achieved. To determine the phospho-lipid composition of cells starved for ethanolamine,cultures of strain KA101 were grown for five to sixgenerations in the presence of 32P as described above.The cells were then shifted, after washing with 2volumes of ethanolamine-deficient medium, to me-dium lacking ethanolamine but containing 32P at thesame specific activity as in the initial growth period.The cells were harvested after 6 h of additional label-ing in the ethanolamine-deficient medium, and thelipid composition was determined as described below.

Lipids were extracted from spheroplasts by a single-step modification of the Folch extraction method (7).Spheroplasts were prepared by harvesting cells bycentrifugation, washing twice with water, and suspend-ing at a density of 5 x 107 to 1 x 108 cells per ml in adigestion mixture consisting of: 1.2 M glycerol, 100mM sodium thioglycolate, 50 mM Tris-S04 (pH 7.5),and 0.5 mg of Zymolyase 5000 (Kirin Breweries, Tak-asaki, Gumma, Japan) per ml. This mixture was de-vised to address difficulties in cell wall removal follow-ing periods of starvation for lipid precursors. Unu-sually high osmotic support and sulfhydryl-reducingagent are required to remove cell walls in some circum-stances and do not appear to produce damage tonormal cells. After 15 min of incubation at 25°C, lessthan 1% osmotically insensitive whole cells remain,and all spheroplasts are maintained intact. Sphero-

YEAST CHOLINE AUXOTROPHS 559

plasts were separated from the digestion mixture bycentrifugation at 1,000 x g for 5 min. The supernatantwas drawn off as completely as possible because thepresence of glycerol in the subsequent lipid extractsdistorts the chromatographic separation. Spheroplastpellets were suspended in chloroform-methanol (2:1)for 1 h and washed with 0.2 volume of water. Theorganic phase was drawn out, and a sample wascounted in a scintillation counter to determine totallabeled lipid. The remainder was dried under nitrogenand suspended in a small volume of chloroform-meth-anol for chromatography. The method of lipid extrac-tion used results in rapid and efficient extraction ofthe major yeast phospholipids, including those of pri-mary interest in this study: phosphatidylserine, -eth-anolamine, and -choline. The method does not effi-ciently extract several polar lipids, including the majoryeast sphingolipids, which are extractable by othermethods (31). The residual polar phospholipids in ourextracts remain close to the origin of the chromatog-raphy system employed and are not resolved. Sincethey are not of primary interest in this analysis, theywere simply tabulated together and are listed as "polarlipids" in the composition analysis. They include CDP-diglyceride and residual sphingolipids (30, 31).

Lipids were separated by the two-dimensional paperchromatography system of Steiner and Lester (30, 31),using EDTA-treated Whatman SG81 paper, dimen-sion 1 (chloroform-methanol-30% ammonium hydrox-ide-water; 66:27:3:0.8), and dimension 2 (chloroform-methanol-glacial acetic acid-water; 32:4:5:1). Radio-activity was located by autoradiography with KodakNo-Screen X-ray film. Spots were cut from the chro-matogram and counted in a scintillation counter. Insome cases, chromatograms were sprayed with nin-hydrin (4) or acid molybdate, followed by UV irradia-tion (8). Lipids were identified by comparison with themigration of standards purchased from GIBCO: phos-phatidylinositol, phosphatidylserine, phosphatidyleth-anolamine, phosphatidylcholine, cardiolipin, andphosphatidic acid.

In some cases lipids were subjected to mild alkalinehydrolysis by the method of Ames (1), and the prod-ucts were separated on Whatman no. I paper in thesolvent methanol-98% formic acid-water (80:13:7) orthe solvent phenol saturated with water-glacial aceticacid-ethanol (10:1:1.2) (29). Labeled hydrolysis prod-ucts were identified by autoradiography, and unla-beled products or standards were identified by spray-ing with ninhydrin or molybdate.

RESULTSGenetics. Three independent choline-requir-

ing mutants were obtained. In each case thecholine requirement was recessive in diploidsand segregated as a single nuclear gene mutation(2:2 in tetrads). The three mutants failed tocomplement one another.

Diploids homozygous for the chol mutationfailed to sporulate on potassium acetate sporu-lation medium, as described in Materials andMethods, whether supplemented with choline orwith ethanolamine. The defect in sporulationmay be the result of the mitochondrial deficien-

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

560 ATKINSON ET AL.

cies described below. Because these crossescould not be sporulated, recombination analysiswas not possible. However, on the basis of thecomplementation data, the mutants would allappear to be alleles of the same locus. In addi-tion, a series of ethanolamine auxotrophs inde-pendently isolated by V. Letts and I. Dawesappear to be alleles of this same locus (V. Letts,personal communication).One of the original isolates was crossed to

strain 2180-1B, and a derivative was crossed tostrain MC13. A derivative of this cross was back-crossed once again to MC13 to derive strainKA101 (a chol-1 inol-13 Iys2 canl). This straingrows more vigorously than the original mutantisolate and, unlike the 2180 strains, tends not toform multicellular aggregates in liquid growthmedia.

Strain KA101, crossed to strain G599-1OB,yielded meiotic linkage information which lo-cates chol on the right arm of chromosome V.Table 1 indicates the tetrad classes obtained andthe recombination frequencies calculated by theformula of Perkins (22). Very likely, this is thesame locus on chromosome V identified origi-nally by C. Lindegren (personal communication)as the map location of a series of choline auxo-trophs. (These mutants have been lost.)Growth requirements of chol mutants.

The three original mutants grow on syntheticcomplete plates supplemented with either 1 mMethanolamine, 1 mM N-methylethanolamine, 1mM N,N-dimethylethanolamine, or 1 mM cho-line. In the absence of at least one of thesesupplements, the mutants do not grow. Furtherstudies reported here were conducted with strainKA101, often together with strain MC13 as aclosely related choline-independent (CHO')strain.

Figures 1 and 2 indicate growth of strainKA101 in liquid synthetic complete mediumwith and without the various supplements. In

TABLE 1. Meiotic segregation ofgenetic markers instrain KA159"'

Non-Gene pair Parental paren- Tetra- Map dis-

ditypes tal di- types tancetypes

cho-can 19 8 76 60.2cho-ura 58 4 44 32.1cho-hom 48 1 57 29.7cho-his 41 0 64 30.5hom-his 102 0 6 2.8

"KA101 a canl + chol + + inol Iys2G599-10B a + ura3 + hom3 hisl + +

Conversion tetrads: cho4 (3.6%), canl (3.6%), ural(0.9%), homl (0.9%), his2 (1.8%).

U,,z oD 100 _

-- - - --_ _ -= =

40

30

202 4 6 8 24

HOURSFIG. 1. Growth requirement of the chol mutant.

Growth in terms of culture optical density was deter-mined in KA1OI cells grown in synthetic completemedium at 30°C with no supplement (A), with ) mMserine (-), or with ) mM ethanolamine (0).

400r300

200U,z

J.

.1." -&

-0

//., ".11"

/// 11.1 la

ol.1

.1-I

11ol

."" .1

10,.0,

.1.,

.e

.11

1001

40

36--

242 4 6 8HOURS

FIG. 2. Growth rates with different supplements.Growth in terms of culture optical density was deter-mined in KAIOI cells grown in synthetic completemedium at 30°C with I mM ethanolamine (0), I mMN-methylethanolamine (A), I mM N,N-dimethyl-ethanolamine (5), or I mM choline (0).

the absence of any supplement, strain KA101stopped growing after approximately one dou-bling in optical density. Supplementation withseine did not enhance growth, whereas supple-mentation with ethanolamine permitted contin-uous growth of the mutant strain. Ethanolamineprovided the most favorable supplementationfor chol mutants, compared to supplementationwith its methylated derivatives (Fig. 2). KA101cultures grown at 300C in synthetic completemedium supplemented with 1 mM ethanolaminegrew with a doubling time of 3 h and showeddoubling times of nearly 3 h with 1 mM N-methylethanolamine, 3 h and 40 min with cho-line, and 5 h with N,N-dimethylethanolamine.

J. BACTERIOL.

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

VOL. 141,1980

By comparison, parallel cultures of the CHO'strain MC13 (not shown) grew equally well, witha doubling time of 2 h, in media with or withouteach of the four supplements. Optimal supple-mentation was achieved with 1 mM concentra-tions of each supplement. With choline, higherconcentrations did not affect growth rate or sta-tionary culture density. Supplementation withmore than 1 mM ethanolamine was less effectivethan 1 mM in terms of these two growth param-eters. Ethanolamine is quite alkaline, and theinhibitory effects of higher concentrations wereovercome by adjusting the medium pH to 5 orless.Viability during starvation for ethanol-

amine, choline, or inositol. Starvation for thephospholipid precursors ethanolamine and cho-line resulted in the cessation of cell growth, butdid not impair cell viability (Fig. 3). In KA101cultures grown at 300C in synthetic completemedium lacking supplements, virtually all cellsretained viability and resumed growth when eth-anolamine was restored after up to 10 h ofgrowth. By contrast, KA101 cells starved of ino-sitol undergo logarithmic viability loss compa-rable to that observed in strain MC13 (11).

Effect of chol on respiratory sufficiency.A high proportion of respiratory-deficient (pe-tite) cells are found in KA101 cultures. Theproportion of petites is markedly affected bygrowth temperature. Since petites produce col-onies with a different morphology from respira-tory-sufficient colonies, this effect of chol wasfirst observed on agar plates. KA101 cells spreadon YEPD agar plates and incubated at 250Cyield colonies that are white in color, small, andperfectly round in shape. These colonies arepetite: they do not grow when replica-plated toYPGE plates containing nonfermentable carbonsources, at either 25 or 300C. Most colonies from

107

106

YEAST CHOLINE AUXOTROPHS

KA101 cells grown at 300C are different: theyare creamy in color, large, and ragged in shape.These colonies grow on YPGE plates at 300C,but not as fast as similarly treated colonies ofthe CHO' strain MC13. Furthermore, mostKA101 colonies grown at 300C have petite sec-tors that fail to grow on YPGE. These sectorsprobably explain the ragged shape of the colo-nies.The accumulation of respiratory-deficient

cells during logarithmic growth is shown in Fig.4. Cultures were established from a respiratory-sufficient KA101 colony that had been grown at300C. Parallel cultures in YEPD medium weregrown at 25 or 300C for several days, with suc-cessive diluting to maintain log-phase cultures.At intervals, cell samples were spread on YEPDplates and incubated at 300C, and the resultantcolonies were tested for respiratory sufficiency.The proportion ofrespiratory-deficient cells rosein cultures grown at both temperatures. At 250C,virtually 100% of the cultures became respira-tory deficient after 4 days of growth, whereasthe proportion of petites leveled off at approxi-mately 50% in the culture growing at 300C.The tendency to generate petites, particularly

at a low growth temperature, cosegregates withthe chol mutation, as illustrated in Table 2.Meiotic spore clones were grown at 300C, and

'°°r-90g-

801

701

60[VP- 501

40

301

20

10'

2 4 6 8 12 24HOURS

FIG. 3. Viability of strain KAIOl. Cell viabilitywas determined in KAIOI cultures grown in syntheticcomplete medium at 300C with inositol and ethanol-amine (0), with no inositol (0), or with no ethanol-amine (A).

/0 ,c __o

0 10 20 30 40 50HOURS

FIG. 4. Accumulation of petites in chol cultures.KA1OI cultures in YEPD medium were grown ateither 25°C (0) or 30°C (0) for several days. Cultureswere maintained in log phase, from 106 to 5 x 107cells per ml, by repeated dilution with fresh YEPDmedium. At intervals, cells were spread on YEPDplates and incubated at 30°C. Thepercentage of cellsthat yielded respiratory-deficient (petite or p-) colo-nies is shown.

561

'WI

VIJi

ULU

-6

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

562 ATKINSON ET AL.

auxotrophic markers were tested by replica-plat-ing. A suspension of cells from each spore clonewas prepared and spread on YEPD agar plates,which were incubated at 25 and 30°C. The res-piratory sufficiency of resultant colonies wastested on YPGE plates. The CHO' spore cloneseach yielded 90% or more respiratory-sufficient(p+) colonies at both growth temperatures. Allcholine-requiring clones yielded 100% petite(p-) colonies when grown at 250C and up to 50%petite colonies when grown at 300C, along with

TABLE 2. Cosegregation of chol and generation ofpetites (p-)"

Phenotype of

Tet- Spore cho lys i,~, ade Mat- coloniesTrad ,Spore cho lyts ino ade ing

type at at250C 300C

1 A - + - + a p- p-/p+B - + + - a P- P-/P+C + - + - a p+ p+D + - - + a p+ p+

2 A - - - - a p- p-/p+B + + - - a P+ P+C + + + + a p+ p+D - - + + a p- p-/p+

3 A + + + - a p+ p+B + - - - a p+ p+C - + + + a p- p-/p+D - - - + a p- p-/P+

Genotype of the parental diploid:KA101 a chol inol lys2 canl +

KA102: S5 a + + + + adel

respiratory-sufficient colonies which grew on

YPGE but distinctly more slowly than CHO'colonies.Phospholipid composition. The steady-

state phospholipid composition of strain KA101was examined under different growth conditions.Table 3 indicates lipid compositions determinedby [:32P]orthophosphate labeling over five to sixgenerations of growth in synthetic media withand without ethanolamine or choline. The com-

position of strain MC13 grown with ethanol-amine is shown beside that of strain KA101grown with ethanolamine or with choline. Alsoshown is the composition after several hours ofcontinued labeling in the absence of any supple-ment. In all KA101 extracts, no phosphatidyl-serine label was detected above background lev-els. In these experiments, background countswere 50 cpm and the total lipid label in eachchromatogram was 5 x 104 cpm. Phosphatidyl-serine labeling was therefore less than 0.1% oftotal lipid label in KA101 cells. Compared to thenormal level of approximately 8%, observed inMC13 cells, the chol mutant contains less than1/80 the normal amount of phosphatidylserine.Hydrolysis of lipids from KA101 failed to pro-duce detectable glycerophosphorylserine, and noninhydrin-reacting material could be detectedon chromatograms in the position where phos-phatidylserine migrates. The only ninhydrin-positive phospholipid from strain KA101 wasphosphatidylethanolamine.The absence of phosphatidylserine is but one

difference between the lipid compositions ofchol and CHO' strains. The proportion of phos-phatidylinositol is nearly 50% greater in KA101

TABLE 3. Phospholipid composition"Totalnmol of

Strain Medium supplement PL PI PS PC PE PMME PDME CL PA lipid Pper 107cells

KAIOI Ethanolamine (1 4.6 32.8 h 41.7 15.0 1.0 1.1 1.2 2.6 13.7mM)

KAIOI Choline (1 mM) 3.2 29.4 - 57.8 4.8 - - 1.8 3.0 13.3MC13a Ethanolamine (1 4.9 22.7 8.1 31.8 23.3 1.9 2.5 1.6 3.2 13.8

mM)KAIOI1 Pregrowth: 6.3 42.3 - 43.3 2.0 - - 2.7 2.5 12.9

ethanolamine (1mM)

6-h final growth: nosupplement

"Lipid abbreviations: PL, polar lipids; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidyl-choline; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine; PDME, phosphati-dyldimethylethanolamine; CL, cardiolipin; PA, phosphatidic acid. Numbers given in the body of the table arepercentages of total lipid phosphorus determined for each lipid.

No label detected above background.During the ethanolamine starvation experiment, 0`P was maintained at a uniform specific activity before

and after transfer to ethanolamine-deficient medium as described in the text.

J. BACTrERIOL.

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

VOL. 141, 190

cebls than in MC13 cells. Additionally, there is agreater proportion of phosphatidylcholine andless phosphatidylethanolamine. The proportionsof these two lipids is abnormal even in cellsgrown with ethanolamine, and even more so incells grown with choline. The phosphatidyleth-anolamine content drops to about 5% of totalphospholipid over five to six generations ofgrowth with choline, and does not drop furtherwith loager growth under these conditions. Thelipid composition detected after several hours ofdeprivation of both supplements (Table 3) isnearly the same as the highly unusual composi-tion of cells growing with choline. The net effectupon composition in unsupplemented cells maybe seen in Table 3.

DISCUSSIONThe growth and phospholipid composition

data tend to support the hypothesis that thechol defect lies in phosphatidylserine synthesis.The chol mutants grow if supplemented withethanolamine or one of its methylated deriva-tives. The original mutant selection was done inthe presence of choline in the expectation thatthe choline auxotrophs isolated would be similarto the Neurospora choline auxotrophs, whichare defective in the methyl transferases (15, 25)that catalyze the conversion of phosphatidyleth-anolamine to phosphatidylcholine. However, thefact that ethanolamine is as effective in support-ing growth as choline is makes this expectationunlikely. The growth characteristics of the yeastchol mutants are consistent with a defect in theconversion of phosphatidylserine to phosphati-dylethanolamine. However, the phospholipidcomposition data do not support this hypothesis.An Escherichia coli mutant defective in thisstep accumulates phosphatidylserine (9). Theyeast mutants described here are completelydeficient in this lipid. The finding that phospha-tidylserine is absent in the chol mutant raisesthe possibility that the defect lies in the synthe-sis of phosphatidylserine. Obviously, a clear un-derstanding of the nature of the defect will re-quire rigorous biochemical analysis. These stud-ies are in progress. Whatever the true nature ofthe defect, it is clear that growth of the mutantdepends upon the utilization of exogenous cho-line, ethanolamine, or one of its methylated de-rivates. In yeast, these precursors have beenshown to be directly incorporated into phospho-lipids (33-35) via the pathways originally de-scribed by Kennedy and Weiss (17).A striking conclusion that may be drawn from

properties of the yeast chol mutants is that thepresence of detectable phosphatidylserine is notrequired for continuing vegetative growth and

YEAST CHOLINE AUXOTROPHS 563

division of this eucaryotic organism. Bacterialmutants with a temperature-sensitive defect inthe phosphatidylserine synthetase are unable togrow at the nonpermissive temperature (reviewby Raetz [23]). However, these bacteria lack analternative route, like the pathway described byKennedy and Weiss (17), to synthesize phospha-tidylethanolamine. Furthermore, they do notmake phosphatidylcholine. Thus, comparablemutant defects in yeast and bacteria result indifferent phospholipid deficiencies and indicatedifferent lipid requirements for growth. Bacteriarequire phosphatidylserine synthesis, either it-self or as a precursor to phosphatidylethanola-mine. The results obtained with the chol mu-tants show that in yeast phosphatidylserine isnot strictly required, nor are normal levels ofphosphatidylethanolamine.

Phosphatidylcholine synthesis and accumu-lation are required for growth of the chol mu-tant. Since none of the supplementation condi-tions employed permits synthesis of phosphati-dylethanolamine but not phosphatidylcholine, itcannot be concluded that phosphatidylcholine isspecifically required for all yeast growth. Phos-phatidylethanolamine is largely dispensable, asshown by the mutants' lipid composition whilegrowing with choline supplementation (Table 3).Although phosphatidylcholine does not appearto be dispensable, interruption of its synthesisand accumulation does not lead to cell death(Fig. 3). In contrast, starvation for the phospho-lipid precursor, inositol, results in drastic viabil-ity loss in yeast inositol mutants (Fig. 3; 11).The chol mutants exhibit several properties

that are different from CHO' strains and arepresumed to be due to phospholipid abnormali-ties. In comparison with the CHO' strain MC13,the chol strain KA101 grows more slowly evenwith optimal supplementation, grows poorly onnonfermentable carbon sources, and displays acold-sensitive tendency to lose mitochondrialfunction completely, and its homozygous dip-loids fail to sporulate. All of these characteristicscould be due to defects of the mitochondrialmembranes. Mitochondrial membranes may bemore sensitive to compositional changes of thetype found in chol mutants than are the func-tions of other membranes that are required forgrowth and division. There are many phospho-lipid alterations evident in chol cells. Thoseresponsible for mitochondrial abnormalities arenot yet resolved. Possibilities include a specificrequirement for phosphatidylserine. Alterna-tively, abnormalities caused by differences in thelipids synthesized by the Kennedy pathwaycould lead to physiological difficulties. The fattyacid compositions of phosphatidylcholines made

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

564 ATKINSON ET AL.

by the de novo and Kennedy pathways differ(34). These alterations, or more subtle changesin the balance of the different cellular phospho-lipids, could be the cause of observed mitochon-drial deficiencies in chol mutants.The chol defect in phosphatidylserine synthe-

sis and the resultant abnormal pattern of phos-pholipid metabolism make these mutants idealsubjects for manipulating the relative propor-tions of membrane phospholipids and for ex-amining the roles of these lipids in the functionsof eucaryotic membranes.

ACKNOWLEDGMENTSWe are indebted in many ways to Alec Keith and Robert

Lester for techniques and ideas used in these investigations.K.D.A. is a fellow of the Damon Runyon-Walter Winchell

Cancer Fund, grant DRG-167-F. S.A.H. is the recipient ofPublic Health Service research career development awardGM-00024 and research grants GM-19629 and GM-19100 fromthe National Institutes of Health. S.F. is supported by PublicHealth Service grant GM-17317 from the National Institutesof Health.

LITERATURE CITED

1. Ames, G. F. 1968. Lipids of Salnonella typhimurium andEscherichia coli: structure and metabolism. J. Bacte-riol. 95:833-843.

2. Cronan, J. E. 1978. Molecular biology of bacterial mem-brane lipids. Annu. Rev. Biochem. 47:163-189.

3. Culbertson, M. R., and S. A. Henry. 1975. Inositolrequiring mutants of Saccharomyces cerevisiae. Ge-netics 80:23-40.

4. Dittmer, J. C., and R. L Lester. 1964. A single specificspray for the detection of phospholipids on thin-layerchromatograms. J. Lipid Res. 5:126-128.

5. Fogel, S., and D. D. Hurst. 1967. Meiotic gene conver-sion in yeast tetrads and the theory of recombination.Genetics 57:45-481.

6. Fogel, S., R. K. Mortimer, K. Lusnak, and F. Tavaras.1979. Meiotic gene conversion-a signal of the basicrecombination event in yeast. Cold Spring HarborSymp. Quant. Biol. 43:1325-1341.

7. Folch, J., M. Lees, and G. H. S. Stanley. 1957. A simplemethod for the isolation and purification of total lipidsfrom animal tissues. J. Biol. Chem. 226:497-509.

8. Hanes, C. A., and F. A. Isherwood. 1949. Separation ofthe phosphoric esters on filter paper chromatogram.Nature (London) 164:1107-1112.

9. Hawrot, E., and E. P. Kennedy. 1978. Phospholipidcomposition and membrane function in phosphatidyl-serine decarboxylase mutants of Escherichia coli. J.Biol. Chem. 253:8213-8220.

10. Hennen, P. E., H. B. Carter, and W. D. Nunn. 1978.Changes in macromolecular synthesis and nucleosidetriphosphate levels during glycerol-induced growthstasis of Escherichia coli. J. Bacteriol. 136:929-935.

11. Henry, S. A., K. D. Atkinson, A. I. Kolat, and M. R.Culbertson. 1977. Growth and metabolism of inositol-starved Saccharomyces cerevisiae. J. Bacteriol. 130:472-484.

12. Henry, S. A., and S. Fogel. 1971. Saturated fatty acidmutants in yeast. Mol. Gen. Genet. 113:1-19.

13. Henry, S. A., and A. D. Keith. 1971. Membrane prop-erties of saturated fatty acid mutants of yeast revealedby spin labels. Chem. Phys. Lipids 7:284-304.

14. Holmgren, E. 1978. A mutant of Bacillus subtilis with

temperature-sensitive synthesis of fatty acids. FEMSMicrobiol. Lett. 3:327-329.

15. Hubbard, S. C., and S. Brody. 1975. Glycerophospholi-pid variation in choline and inositol auxotrophs of Neu-rospora crassa. J. Biol. Chem. 250:7173-7181.

16. Keith, A. D., B. J. Wisnieski, J. C. Williams, and S.A. Henry. 1973. Membranes of yeast and Neurospora:lipid mutants and physical studies, p. 259-321. In J. A.Erwin (ed.), Lipids and biomembranes of eukaryoticmicroorganisms. Academic Press Inc., New York.

17. Kennedy, E. P., and S. B. Weiss. 1956. The function ofcytidine coenzymes in the biosynthesis of phospholip-ids. J. Biol. Chem. 222:193-214.

18. Lindegren, G., Y. L Hwang, Y. Oshima, and C. C.Lindegren. 1965. Genetical mutants induced by ethyl-methanesulfonate in Saccharomyces. Can. J. Genet.Cytol. 7:491499.

19. Linneman, W. A. M., P. Boers, and P. F. Elbers. 1977.Localization of acid phosphatase in Saccharomyces cer-evisiae: a clue to cell wall formation. J. Bacteriol. 131:638-644.

20. Moor, H. 1967. Endoplasmic reticulum as the initiator ofbud formation in yeast (S. cerevisiae). Arch. Mikrobiol.57:135-146.

21. Papahadpopoulos, D., and A. Portia. 1978. Calcium-induced lipid phase transitions and membrane fusion.Ann. N.Y. Acad. Sci. 308:50-66.

22. Perkins, D. D. 1949. Biochemical mutants in smut fungusUstilago maydis. Genetics 34:607-626.

23. Raetz, C. R. H. 1978. Enzymology, genetics, and regula-tion of membrane phospholipid synthesis in Esche-richia coli. Microbiol. Rev. 42:614-659.

24. Resnick, M. R., and R. K. Mortimer. 1966. Unsaturatedfatty acid mutants ofSaccharomyces cerevisiae. J. Bac-teriol. 92:597-600.

25. Scarborough, G. A., and J. F. Nye. 1967. Methylationof ethanolamine phosphatides by microsomes from nor-mal and mutant strains of Neurospora crassa. J. Biol.Chem. 242:238-242.

26. Schweizer, E., and H. Bolling. 1970. A Saccharomycescerevisiae mutant defective in saturated fatty acid bio-synthesis. Proc. Natl. Acad. Sci. U.S.A. 67:660-666.

27. Shatkin, A. J., and E. L. Tatum. 1961. The relationshipof m-inositol to morphology in Neurospora crassa. Am.J. Bot. 48:760-771.

28. Silbert, D. F. 1975. Genetic modification of membranelipid. Annu. Rev. Biochem. 44:315-339.

29. Silbert, D. F., J. E. Cronan, I. R. Beacham, and M. E.Harder. 1974. Genetics engineering of membrane lipid.Fed. Proc. 33:1725-1732.

30. Steiner, S., and R. L. Lester. 1972. Metabolism of di-phosphoinositide and triphosphoinositide in Saccharo-myces cerevisiae. Biochim. Biophys. Acta 260:82-87.

31. Steiner, S., and R. L. Lester. 1972. Studies on thediversity of inositol-containing yeast phospholipids: in-corporation of 2-deoxyglucose into lipid. J. Bacteriol.109:81-88.

32. Tyhach, R. J., E. Hawrot, M. Satre, and E. P, Ken-nedy. 1979. Increased synthesis of phosphatidylserinedecarboxylase in a strain of Escherichia coli bearing ahybrid plasmid. J. Biol. Chem. 254:627-633.

33. Waechter, C. J., and R. L. Lester. 1971. Regulation ofphosphatidylcholine biosynthesis in Saccharomycescerevisiae. J. Bacteriol. 105:837-843.

34. Waechter, C. J., and R. L. Lester. 1973. Differentialregulation of the N-methyltransferases responsible forphosphatidylcholine synthesis in Saccharomyces cere-visiae. Arch. Biochem. Biophys. 158:401410.

35. Waechter, C. J., M. R. Steiner, and R. L. Lester. 1969.Regulation of phosphatidylcholine biosynthesis by themethylation pathway in Saccharomyces cerevisiae. J.Biol. Chem. 244:3419-3422.

J. BACTERIOL.

on Decem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from