JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 606-612Copyright ©) 1975 American Society for Microbiology

Vol. 124, No. 2Printed in U.S.A.

Yeast Sterol Esters and Their Relationship to the Growth ofYeast'

R. B. BAILEY* AND L. W. PARKSDepartment of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received for publication 16 June 1975

Variation in the percentage of sterols esterified to long-chain fatty acidsduring cellular growth has been examined. Under all conditions, a constantpercentage of sterol esters was maintained during exponential growth. Thismaintenance level was found to vary with different growth conditions. A sharpincrease in the rate of esterification was observed upon entry of the culture intothe stationary growth phase. The minor cellular sterol components were foundto accumulate after this period of rapid sterol ester synthesis, with a relativedecrease in the size of the ergosterol pool. Evidence is presented that sterolesters of ergosterol precursors are unable to be metabolized to ergosterol. Onceesterified, the fatty acids do not appear to be scavenged during starvationconditions.

It has long been known that much of thesterol pool of mammalian systems is esterifiedto long-chain fatty acids. In addition, severalinvestigators have studied the sterol esters ofhigher plants over the past several years (9-11). Until recently, however, not much atten-tion has been given to the sterol esters of fungi.The composition and variation of sterol estersfound in the fungus Phycomyces blakesleeanushave been examined by Mercer and Bartlett (3,15). They studied the fatty acid composition ofthe sterol esters and found predominately C16and C18 unsaturated species, although at differ-ent compositions than found in the triglycerideand phospholipid fractions (15). They also indi-cated that the various sterol moieties of theunesterified sterol fraction maintained a con-stant level, whereas the esterified forms variedconsiderably in concentration. The sterol esterswere shown not to be an energy storage product(3).While there are many reports in the litera-

ture involving the study and biosynthesis ofsterols in bakers' yeast, Saccharomyces cerevi-siae (4, 5, 7, 16), there is little known about therole the sterol esters play in cellular metabo-lism. The sterols have been shown to be esteri-fied predominately to unsaturated C16 and C18fatty acids (14). Because of the wealth of infor-mation on sterol synthesis and function in thisorganism and the ease in culturing and inter-preting growth data by comparison with Phyco-myces sp., we have elected to use yeast to studythe generation and metabolism of sterol esters.This communication shows a correlation be-

' Oregon Agricultural Experiment Station technicalpaper 4041.

606

tween cellular growth rate and the level ofsterol esters and provides evidence that, onceesterified, a sterol intermediate cannot be fur-ther converted to ergosterol.

MATERIALS AND METHODSOrganisms and cultural conditions. S. cerevisiae

3701B, a haploid uracil auxotroph, was used predom-inately for this investigation. The organism wasgrown routinely in a broth medium composed of 1%tryptone and 05% yeast extract. Carbon sourceswere added to 2%, unless otherwise indicated. Thecomplete chemically defined medium of Wickerham(22) was also used in this study. All cultures wereincubated at 28 C on a New Brunswick rotaryshaker.

Growth curves. A 5% inoculum (vol/vol) of cellsgrown in glucose from an 18- to 24-h culture wasmade into fresh medium contained in a Bellco side-arm flask. A Klett-Summerson colorimeter witha no. 54 green filter was used to monitor growth.Periodic samples were taken and the percentage ofsterol esters and/or [14C]methionine incorporationinto the nonsaponifiable lipids was determined. Theprocedures for measuring the incorporation of[14C]methionine into the nonsaponifiable lipids havebeen published (19).

Determination of the percentage of sterols es-terified. Cell samples were centrifuged and washedonce with cold (4 C) water. The cell pack was resus-pended in 5 to 6 ml of water and frozen. All sampleswere then lyophilized on an Atmo-Vac Labfreezedryer.

The dried cells were then treated with 2.0 ml ofdimethyl sulfoxide (Me2SO) for 1 h at 100 C in asteamer. After cooling, the total volume wasbrought to 15 ml with water, and the lipids wereextracted twice with 10-ml volumes of petroleumether. The combined extracts were evaporated todryness and loaded onto a miniature, activated alu-

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YEAST STEROL ESTERS 607

mina column with n-hexane. We routinely usedsmall funnels as columns, with cotton plugs fitted tosupport 2 to 3 g of the alumina. The columns werewashed with two 5-ml portions of n-hexane. Thesterol esters were eluted with 10 ml of hexane-di-ethyl ether (1:1, vol/vol), after which the free sterolswere eluted from the column with hexane-ethanol(9:1, vol/vol). The eluted fractions were then dried,and Lieberman-Burchard-positive color (12) wasmeasured at 625 nm.

Preparation and analysis of sterols. Cells werecultured in 2-liter flasks with 1 liter of a brothcontaining 1% tryptone, 0.5% yeast extract, and 2%glucose. After the cell pack was harvested andwashed, it was lyophilized and treated with Me2SO.The extracted free sterols were then separated fromthe sterol esters by using thin-layer chromatogra-phy or short alumina columns as outlined above.The esterified sterol fraction was saponified underreflux for 1 h in 6% KOH in absolute methanol tobreak the ester bonds. The free sterols were thenextracted into n-hexane and analyzed by thin-layerand gas chromatography as described previously(17). After preliminary separation on silica gelplates (cyclohexane-ethyl acetate, 85:15 vol/vol) togive 4,4-dimethyl, 4-a-methyl, and 4-demethylbands, the sterols were acetylated overnight in pyri-dine-acetic anhydride (1:2 vol/vol) to give the sterolacetates.

Final identification of the sterol acetates wasmade by gas chromatography with glass columnscontaining 2% SE-30 (6 feet [ca. 1.83 ml by 2 mm)and 3% OV-17 (4 feet [ca. 1.22 ml by 2 mm) as theliquid phases on an H/P Chrom W (100 to 120 mesh)support. The column temperature was maintainedat 265 C with a helium flow rate of 25 ml/min. Forcoupled gas chromatography-mass spectrometry, aVarian MAT, model CH-7, with a Systems Indus-tries 150 data system was used. The separation wason 7% OV-17 (4 feet [ca. 1.22 ml) at 285 C. Spectrawere taken at 70 ev with a source temperature of175 C.

Thin-layer chromatography was done on silica gelplates (Merck HF-254) of 0.25-mm thickness. Theseparated sterols were visualized with ultravioletlight after spraying with berberine in ethanol. Thesterols were eluted from the plates through anhy-drous sodium sulfate with methylene chloride. Allsolvents used were routinely redistilled.

Preparation of substrates and sterol-fatty acidesters. Zymosterol was prepared from Fleischmancake yeast as described previously (20). Zymosteryl-oleate and other sterol-fatty acid esters were pre-pared by the methods of Knapps and Nicholas (13).The products were separated from the unreactedfree sterols by silica gel thin-layer chromatographywith cyclohexane-ethyl acetate (85:15 vo/vol). Pu-rity was checked by saponification in alkaline meth-anol (6% KOH) and rechromatography of the prod-uct. In all cases, the saponified esters cochromato-graphed with authentic sterol standards.

Methyltransferase enzyme preparation. Thepreparation of methyltransferase enzyme is alreadya published procedure (20). We used both a crudeenzyme preparation (20) and a partially purified

lipid-free enzyme preparation (21) for these experi-ments.

Materials. Both S-adenosyl-L-[methyl-'4C]me-thionine and L-[methyl-'4C]methionine were prod-ucts of International Chemical and Nuclear Co. Allsolvents were purchased from Mallinkrodt as re-agent grade and were redistilled prior to use. Me2SOwas purchased from the J. T. Baker Co. All gaschromatography materials were products of SupelcoInc. The tryptone and yeast extract were boughtfrom Difco. Precoated silica gel plates (0.25-mmthickness), Merck H-254, were products of EM Re-agents Co. Authentic ergosta-7-en-3,B-ol, ergosta-8-en-3,8-ol, and ergosta-7,22-diene-3/3-ol were giftsfrom A. C. Oehlschlager. All other chemicals werecommercially available and of the highest purityobtainable.

RESULTSThe treatment of lyophilized cells with

Me2SO for 1 h at 100 C in a steamer proved tobe very effective for extracting the sterols fromyeast without the danger of destroying the es-ters. The addition of water to the Me2SO priorto extraction with petroleum ether preventedthe extraction of Me2SO with the hexane ex-tracts, which otherwise proved to be a nuis-ance. Control experiments were done withknown sterol esters to determine whether theycould be hydrolyzed by the Me2SO in a steambath, and we found no evidence that they were.Our first series of experiments was designed

to determine whether there was any correlationbetween the degree of esterification of sterolsand the stage of the culture cycle of the orga-nism. Growth was followed as a function of cellmass, and the percentage of sterols occurring asesters was determined periodically. We firstused the rich yeast extract-tryptone mediumcontaining 5% glucose as an energy source. Asshown in Fig. 1, the degree of esterificationremains at a relatively low level during theexponential growth phase, but it increases dra-matically upon entry into late exponential orearly stationary growth. With 5% glucose, the"steady-state" level of sterol esters during expo-nential growth was very low, ranging from 0 to10%. Two hours after entry into the stationarygrowth phase, however, levels as high as 60%esters were obtained, and by 24 h we generallyfound 85 to 90% of the sterols esterified. Figure1 also shows the total synthesis and accumula-tion of sterols as measured by the incorporationof the "4C-labeled methyl group of methionineinto the nonsaponifiable lipids. This procedureis specific for ergosterol and other sterols withthe C28 methyl group. Sterol synthesis clearlyincreases exponentially during the period whenthe percentage of esters is maintained at a verylow level.

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608 BAILEY AND PARKS

5% Glucose

/o0 /~~~~~/00/ /

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, 001

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2 4 6 8 10 12TIME (hr)

FIG. 1. Variation in the level of sterol esters during fermentative growth. Growth ofan aerobically shakenculture in 5% glucose was followed optically with a Klett colorimeter. Samples were taken periodically andassayed for the level of sterol esters and the incorporation of the methyl-'4C group of methionine into thenonsaponifiable lipid fraction. Symbols: 0, Klett units; 0, percent esters; and A, counts per minute (CPM) per50-ml sample incorporated into the nonsaponifiable lipids. The generation time in this experiment was 90 min.

Running the same experiment, but using 2%ethanol as a carbon source, gave us the datashown in Fig. 2. Again there is exponentialsterol synthesis occurring during a period whenthe level of sterol ester is steady, and againthere is a large increase in esterification afterentry into late exponential or early stationarygrowth. However, the percentage of esterifiedsterol is maintained at about 20%, rather thanat the much lower values found with 5% glu-cose. In fact, during one such experiment, wefound a level of 35% esters maintained duringexponential growth. Cells cultured on ethanolalso approached a value of 90% esters after theculture had passed into the stationary phase.

Because of the differences in the degree ofesterification observed, we repeated thesegrowth curves using various carbon sources.Among those tested were 2% glucose, galactose,and succinate. We also tested these same car-bon sources in a less rich, chemically definedmedium (22). Under all conditions, an abruptincrease in the level of sterol esters occurred asthe cells entered stationary growth. The maxi-mal degree of esterification was independent of

growth conditions and always tapered off atabout 90% esters. There also appeared to besome correlation between the growth rate andthe level of esters maintained during exponen-tial growth.We next looked at the composition of the

sterols contained in the esterified fraction. Thiswas done by harvesting 3 liters of cells each, atperiods of growth calculated to give differentpercent composition of the sterol esters. Thesterols were then extracted and analyzed bythin-layer and gas-liquid chromatography. Thesamples were taken and the ester percentagesstudied were 37, 60, and 88% esters, the latteroccurring after 24 h of growth (Table 1). It isvery apparent that there are significantchanges in the sterol composition that takeplace during the growth cycle. Most noticeableis the level of ergosterol, which can be seen todecrease from 92% of the total sterol pool to 40%after 24 h. However, during the same intervals,we found the amount of esterified ergosterol toapproximate closely the overall degree of esteri-fication of all sterols.The other noticeable feature shown by these

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YEAST STEROL ESTERS 609

*S10;@<°/> * / 120221032

AS 20It II

0~~~~~~~~~~~~~

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2 4 6 8 10 12TIME (hr)

FIG. 2. Variation in the level of sterol esters during respiratory growth. The same procedures as in Fig. 1were used except the cells were cultured in 2% ethanol. Symbols: 0, Klett units; *, percent esters; and A,counts per minute (CPM) per 50-ml sample incorporated into the nonsaponifiable lipids. The generation timefor this experiment was 145 min.

TABLE 1. Fluctuation of the cellular sterol ester pool during growtha

37% ester 60% ester 88% ester

Sterol component Fraction of total % Sterol Fraction of total % Sterol Fraction of total % Sterol

sterol pool esterified sterol pool esterified sterol pool esterified

Lanosterol <0.001 NDb 0.001 ND <0.001 954a-methyl-zymosterol 0.003 10 <0.001 ND 0.026 ND4,14-dimethyl-cholesta- 0.014 10 0.026 ND 0.065 100

8,24-diene-3#-olErgosta-7,22-diene-3,6-ol 0.008 100 0.032 99 0.049 92Ergosta-7-ene-3,8-ol 0.017 2 0.117 37 0.018 93Zymosterol <0.001 ND <0.001 ND 0.162 100Fecosterol <0.001 ND <0.001 ND 0.059 97Ergosta-5,7-diene-3,B-ol 0.022 63 0.002 99 0.214 100Ergosterol 0.921 37 0.820 62 0.402 73

a At levels of approximately 30, 60, and 90% ester, the cells were harvested and sterols were extractedafter treatment with Me2SO. The free sterols were separated from esterified sterols, and then both fractionswere analyzed by thin-layer and gas chromatography.

b ND, No sterol detected in ester fraction.

data is the presence of only traces (<0.1%) ofzymosterol and fecosterol during the earlierstages of growth, whereas at 24 h (88% esters)they make up 16 and 6% of the total sterol pool,respectively. Generally, the "minor" sterols be-came more abundant as time progressed,whereas the ergosterol concentration decreasedrelative to the total sterol pool. We also noticed

that there was much variability in the percent-age of individual sterols esterified, especiallyduring the earlier stages of growth. In someinstances we were unable to detect any of agiven sterol in the ester pool, whereas othersterols were found only in this fraction. Theabove cells were all cultured on 5% glucose, butanalyses were also done on ethanol-grown cells,

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610 BAILEY AND PARKS

and the composition appears to be independentof the added energy source.

Because of this accumulation of minor ster-ols, Wve wondered whether ergosterol precursorswere able to take part in further biosyntheticreactions after esterification. One easily testa-ble reaction is the transmethylation event thattakes place in yeast mitochondria (20). Wemade a partially purified lipid-free preparationof this enzyme (21) and proceeded to test chemi-cally synthesized (13) zymosteryl-oleate as asubstrate. Because we have previously reportedthree separate enzyme activities for this en-zyme (2), we tested all three with similar re-sults. Table 2 shows that the esterified form ofzymosterol cannot be methylated in this sys-tem, whereas free zymosterol is a ready sub-strate.We next investigated the effect of incubating

zymosteryl-oleate with the free sterol in oursterol methyltransferase assay system. Al-though we expected inhibition of the reactionby the esterified zymosterol, or perhaps no ef-fect at all, we found that the ester actuallystimulated the methyltransferase enzyme. Thestimulation we observed was maximally 30 to40% above control reactions containing onlyfree zymosterol. To determine whether this wasa general phenomenon of all sterol esters, wesynthesized C,4, C16, and C,8 esters of lanos-terol, ergosterol, and zymosterol. All of thesterol esters tested stimulated the enzyme sys-tem somewhat, but the oleate and stearate es-ters (C18) were the most stimulatory. It shouldbe pointed out that all of these reactions weredone in our partially purified enzyme systemthat had no detectable ester hydrolase activity.

Since the increase in esterification during thegrowth cycle coincided with both the entry intoearly stationary phase and the decrease in expo-

TABLE 2. Efficiency of zymosteryl-oleate as asubstrate for the sterol methyltransferase reactiona

Counts/min in nonsaponifiablelipids

Substrate tested0.29 AM 9.3 IAM 93 AMS-AM S-AM S-AM

Zymosterol 5,630 3,600 3,450Zymosteryl-oleate 400 240 290Control 330 190 170

a Sterol transmethylation assays were performed,with either zymosterol or zymosteryl-oleate as thesubstrate, at each of the three different S-adenosyl-methionine (S-AM) concentrations. The controltubes were run in the absence of any exogenouslysupplied substrate. All sterol substrates were addedat a concentration of 200 uM.

nential sterol synthesis, it was of interest to seewhether it was in response to one or both ofthese factors. Accordingly, we aerated anaerobi-cally grown cells in a 0.1 M phosphate buffer,pH 6.6, containing 1% glucose. Yeast cells soincubated rapidly synthesized large amounts ofsterol (19) but are incapable of dividing. Bothsterol synthesis and the esterification of sterolsincrease very rapidly under such conditions(Fig. 3). It is also of interest to compare therates of sterol synthesis obtained by extractingboth Me2SO-treated cells (Lieberman-Bur-chard-positive color, absorbancy at 625 nm) andthe saponified cells ([methyl-14C]methionine in-corporation). The inherent decrease in labeledmethionine incorporation seen with regular al-kaline pyrogallol saponification (18) is absentwhen the cells are lyophilized and treated withMe2SO. This indicates that the latter is a moreeffective sterol extraction procedure.To determine the distribution of the esteri-

fied sterols, we utilized a mitochondria isola-tion procedure (20) and collected samples of thecell wall (membranes), mitochondria, and cellsap fractions. The total cellular sterol extract atthe time of harvest yielded 67% sterol esters.We found that the cell wall (membranes) frac-tion contained 66% sterol ester, whereas themitochondrial fraction had only 17% esters.The cell wall and membranes also appeared tohave, by far, the majority of the total sterol pool(60 to 75%). Although the cell sap gave 44%esters, the total sterol was very small by com-parison, and, due to the mechanical cell break-age and heterogeneity of the fraction, we arehesitant to attach much significance to thisvalue.

It was also of interest to determine whetherthe fatty acids could be scavenged by the cell orwhether they are left alone once esterified tosterols. Therefore, we harvested an overnightculture of cells and resuspended them in 0.1 Mphosphate buffer, pH 6.6, and allowed them toincubate 24 h. While the total sterol pool wasconstant on the basis of micrograms of sterol tomilligrams of protein, the percentage of estersactually increased from 57% to 80% after 6 hand remained constant at that level.

DISCUSSIONThe rate of esterification of yeast sterols to

long-chain fatty acids appears, from these data,to reflect the physiological state of the cell. Aculture growing exponentially with a good car-bon source in a rich medium has a very lowlevel of sterols occurring as esters and, in fact,we were unable to detect any sterol esters un-der some conditions. If a less suitable carbon

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YEAST STEROL ESTERS 611

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FIG. 3. Esterification during aerobic adaptation.Anaerobically grown cells (72 h) were harvested andallowed to incubate in 0.1 M phosphate buffer, pH6.6, containing 1% glucose. The flask was shakenvigorously. Samples were removed to assay the per-

centage of esters and the radioactive incorporation ofmethionine into the nonsaponifiable lipids. Symbols:A, percent esters; *, total Lieberman-Burchard posi-tive color, absorbancy at 625 nm, extracted fromMe1sO-treated cells; and 0, count per minute (CPM)incorporated into the nonsaponiflable lipids per 10-ml sample.

source is used, such as ethanol or succinate, thelevel of sterol esters maintained during expo-

nential growth increases but still remains con-

stant until the culture approaches the station-ary phase. Without exception, however, we ob-served a sharp rise in the percentage of sterolesters upon entry into the stationary phase ofgrowth. Although sterol synthesis also de-creases at this same time, we feel that theesterification process is a response to the over-all physiological state of the cell, rather than tothe decrease in sterol synthesis. This is basedon the observations made in the aerobic adapta-tion experiment, where sterol synthesis andesterification both increased rapidly under con-ditions when the cell could not divide. If theesterification was due to a decrease in sterolsynthesis alone, one would expect, under theseconditions, that esterification would remain ata constant level until sterol synthesis dimin-ished.Once sterol intermediates are esterified, they

are effectively prevented from being furthermetabolized to ergosterol. We have shown thatzymosteryl-oleate is not methylated by our invitro assay system, although the free sterol is aready substrate. Whether this holds true forother enzymatic reactions in vivo remains un-

tested, but since other sterol intermediates doaccumulate and are generally about 100% ester-ified, it does seem probable. At this point we

have no proven explanation for why the sterol

methyltransferase reaction was stimulated by avariety of sterol esters. The stimulation was, atthe maximum, only 35 to 40% and was observedgenerally for all tested sterol esters. Since theenzyme is mitochondrially located (20) and wefound very low levels of sterol esters associatedwith the mitochondrion, this stimulation maybe simply fortuitous and of no physiologicalsignificance.The apparent accumulation of so-called mi-

nor sterols with time is an interesting observa-tion. The cellular ergosterol content decreasedfrom 92 to 40% of the total sterol pool. At thesame time, zymosterol increased from <0.1 to16% of the cellular total sterol pool, and er-gosta-5,7-diene-3,8-ol increased from 2.2 to 21%.There were also smaller increases seen in fecos-terol and ergosta-7,22-diene-3(3-ol. These datasuggest that these other sterols, especially zy-mosterol and ergosta-5,7-diene-3/3-ol, mightplay an important role in cellular metabolism,other than just being intermediates of ergos-terol. Otherwise, it is an unreasonable waste ofenergy in that the esterification process itselfrequires adenosine 5'-triphosphate (1).

It has been reported that red blood cells canfreely exchange intracellular sterols, but notsterol esters, with those in the surroundingmedium (6). If this were also true of yeast, theesterification process would serve to preventthis exchange and, thereby, keep the sterol poolof the cell intact during periods when little, ifany, sterol synthesis is taking place. Also, sincewe find the majority of the sterol esters presentin the cell wall, this might explain the "thicken-ing" of the cell wall that has recently beenreported to occur during the stationary phase(8).The esterification process is obviously not a

means by which fatty acids may be stored forenergy until such time as they may be needed.We actually observed an increase in esterifica-tion to take place under starvation conditions.This agrees with the data of Bartlett and Mer-cer (3) for P. blakesleeanus.The rapid period of esterification seen as the

culture approaches the stationary phase maybe the consequence of some regulatory interac-tion. This increase could be due to: (i) a con-stant rate of esterification during a time whensterol synthesis is decreasing, (ii) release ofcontrol of the sterol ester synthetase, (iii) de-repression of the sterol ester synthetase, or (iv)decreased ester hydrolase activity. Since therewas a rapid rate of esterification during aerobicadaptation with a concomitant fast rate ofsterol synthesis, we do not feel that explanationno. i is satisfactory. All of the other explana-

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612 BAILEY AND PARKS

tions do implicate a form of regulatory interac-tion of the esterification process. Work is cur-

rently being done to clarify this problem.

ACKNOWLEDGMENTS

We wish to thank A. C. Oleschlager for generous sup-

plies of authentic minor yeast sterols.The work was supported by a grant from the National

Science Foundation.

LITERATURE CITED

1. Anding, C., L. W. Parks, and Guy Ourisson. 1974.Enzymic modification of cyclopropane sterols in yeastcell-free system. Eur. J. Biochem. 43:459-463.

2. Bailey, R. B., E. D. Thompson, and L. W. Parks. 1974.Kinetic properties of S-adenosylmethionine: A24-

sterol methyltransferase enzyme(s) in mitochondrialstructures ofSaccharomyces cerevisiae. Biochim. Bio-phys. Acta 334:127-136.

3. Bartlett, K., and E. I. Mercer. 1974. Variation in thelevels and composition of the sterols and sterol estersofPhycomyces blakesleeanus with age of culture. Phy-tochemistry 13:1115-1121.

4. Barton, D. H. R., J. E. T. Corrie, P. J. Marshall, and D.A. Widdowson. 1973. Biosynthesis of terpenes andsteroids. VII. Unified scheme for the biosynthesis ofergosterol in Saccharomyces cerevisiae. Bioorg.Chem. 2:363-373.

5. Barton, D. H. R, U. M. Kempe, and D. A. Widdowson.1972. Investigations on the biosynthesis of steroidsand terpenoids. VI. The sterols of yeast. J. Chem.Soc., Perkin Trans. 1, p. 513-522.

6. Bruckdorfer, K. R., and C. Green. 1967. The exchangeof unesterified cholesterol between human low-den-sity lipoproteins and rat erythrocyte 'ghosts.' Bio-chem. J. 104:270-277.

7. Fryberg, M., A. C. Oehlschlager, and A. M. Unrau.1972. Biosynthetic routes to ergosterol in yeast. Bio-chem. Biophys. Res. Commun. 48:593-597.

8. Hammond, S. M., and B. N. Kliger. 1974. Studies on

the role of the cell wall of Candida albicans in themode of action of polyene antibiotics. Proc. Soc. Gen.Microbiol. 1:45.

9. Kemp, R. J., L. J. Goad, and E. J. Mercer. 1967.

Changes in the levels and composition of the esteri-fied and unesterified sterols of maize seedlings duringgermination. Phytochemistry 6:1609-1615.

10. Kemp, R. J., and E. I. Mercer. 1968. The sterol esters ofmaize seedlings. Biochem. J. 110:111-118.

11. Kemp, R. J., and E. J. Mercer. 1968. Studies on thesterols and sterol esters of the intracellular orga-

nelles of maize shoots. Biochem. J. 110:119-125.12. Klein, H. P., N. R. Eaton, and J. C. Murphy. 1954. Net

synthesis of sterols in resting cells of Saccharomycescervisiae. Biochim. Biophys. Acta 13:591.

13. Knapps, F. F., and H. J. Nicholas. 1970. Liquid crystal-line properties of ergosteryl fatty acid esters. Mol.Cryst. Liq. Cryst. 10:170-186.

14. Madyastha, P. B., and L. W. Parks. 1969. The effect ofcultural conditions on the ergosterol ester compo-

nents of yeast. Biochim. Biophys. Acta. 176:858-862.15. Mercer, E. I., and Kim Bartlett. 1974. Sterol esters of

Phycomyces blakesleeanus. Phytochemistry 13:1099-1105.

16. Parks, L. W., C. Anding, and Guy Ourisson. 1974.Sterol transmethyltaion during aerobic adaptation ofyeast. Eur. J. Biochem. 43:451-458.

17. Parks, L. W., F. T. Bond, E. D. Thompson, and P. R.Starr. 1972. A59'22-Ergostadiene-3j3-ol, an ergosterolprecursor accumulated in wild-type and mutants ofyeast. J. Lipid Res. 13:311-316.

18. Starr, P. R., and L. W. Parks. 1962. Some factors affect-ing sterol formation in Saccharomyces ceruisiae. J.Bacteriol. 83:1042-1046.

19. Starr, P. R., and L. W. Parks. 1972. Transmethylationof sterols in aerobically adpating Saccharomyces cere-

visiae. J. Bacteriol. 109:236-242.20. Thompson, E. D., R. B. Bailey, and L. W. Parks. 1974.

Subcellular location of S-adenosylmethionine: A24-

sterol methyltransferase in Saccharomyces cerevi-siae. Biochim. Biophys. Acta 334:116-126.

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