Yeast Sterol Esters and Theirrelationship to the Growth Of

Yeast Sterol Esters and Their Relationship to the Growth of Yeast'

R. B. BAILEY* AND L. W. PARKS Department 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 acids during cellular growth has been examined. Under all conditions, a constant percentage of sterol esters was maintained during exponential growth. This maintenance level was found to vary with different growth conditions. A sharp increase in the rate of esterification was observed upon entry of the culture into the stationary growth phase. The minor cellular sterol components were found to accumulate after this period of rapid sterol ester synthesis, with a relative decrease in the size of the ergosterol pool. Evidence is presented that sterol esters of ergosterol precursors are unable to be metabolized to ergosterol. Once esterified, the fatty acids do not appear to be scavenged during starvation conditions. It has long been known that much of the tween cellular growth rate and the level of sterol pool of mammalian systems is esterified sterol esters and provides evidence that, once to long-chain fatty acids. In addition, several esterified, a sterol intermediate cannot be fur- investigators have studied the sterol esters of ther converted to ergosterol. higher plants over the past several years (9- 11). Until recently, however, not much atten- MATERIALS AND METHODS tion has been given to the sterol esters offungi. Organisms and cultural conditions. S. cerevisiae The composition and variation of sterol esters 3701B, a haploid uracil auxotroph, was used predom- found in the fungus Phycomyces blakesleeanus inately for this investigation. The organism was have been examined by Mercer and Bartlett (3, grown routinely in a broth medium composed of 1% 15). They studied the fatty acid composition of tryptone and 05% yeast extract. Carbon sources the sterol esters and found predominately C16 were added to 2%, unless otherwise indicated. The and C18 unsaturated species, although at differ- complete chemically defined medium of Wickerham ent compositions than found in the (22) was also used in this study. All cultures were triglyceride incubated at 28 C on a New Brunswick rotary and phospholipid fractions (15). They also indi- shaker. cated that the various sterol moieties of the Growth curves. A 5% inoculum (vol/vol) of cells unesterified sterol fraction maintained a con- grown in glucose from an 18- to 24-h culture was stant level, whereas the esterified forms varied made into fresh medium contained in a Bellco side- considerably in concentration. The sterol esters arm flask. A Klett-Summerson colorimeter with were shown not to be an energy storage product a no. 54 green filter was used to monitor growth. (3). Periodic samples were taken and the percentage of While there are many reports in the litera- sterol esters and/or [14C]methionine incorporation ture involving the study and biosynthesis of into the nonsaponifiable lipids was determined. The sterols in bakers' yeast, procedures for measuring the incorporation of Saccharomyces cerevi- [14C]methionine into the nonsaponifiable lipids have siae (4, 5, 7, 16), there is little known about the been published (19). role the sterol esters play in cellular metabo- Determination of the percentage of sterols es- lism. The sterols have been shown to be esteri- terified. Cell samples were centrifuged and washed fied predominately to unsaturated C16 and C18 once with cold (4 C) water. The cell pack was resus- fatty acids (14). Because of the wealth of infor- pended in 5 to 6 ml of water and frozen. All samples mation on sterol synthesis and function in this were then lyophilized on an Atmo-Vac Labfreeze organism and the ease in culturing and inter- dryer. preting growth data by comparison with Phyco- The dried cells were then treated with 2.0 ml of myces sp., we have elected to use yeast to study dimethyl sulfoxide (Me2SO) for 1 h at 100 C in a the generation and metabolism steamer. After cooling, the total volume was of sterol esters. brought to 15 ml with water, and the lipids were This communication shows a correlation be- extracted twice with 10-ml volumes of petroleum ' Oregon Agricultural Experiment Station technical ether. The combined extracts were evaporated to paper 4041. dryness and loaded onto a miniature, activated alu- 606 VOL. 124, 1975 YEAST STEROL ESTERS 607 mina column with n-hexane. We routinely used lipid-free enzyme preparation (21) for these experi- small funnels as columns, with cotton plugs fitted to ments. support 2 to 3 g of the alumina. The columns were Materials. Both S-adenosyl-L-[methyl-'4C]me- washed with two 5-ml portions of n-hexane. The thionine and L-[methyl-'4C]methionine were prod- sterol esters were eluted with 10 ml of hexane-di- ucts of International Chemical and Nuclear Co. All ethyl ether (1:1, vol/vol), after which the free sterols solvents were purchased from Mallinkrodt as re- were eluted from the column with hexane-ethanol agent grade and were redistilled prior to use. Me2SO (9:1, vol/vol). The eluted fractions were then dried, was purchased from the J. T. Baker Co. All gas and Lieberman-Burchard-positive color (12) was chromatography materials were products of Supelco measured at 625 nm. Inc. The tryptone and yeast extract were bought Preparation and analysis of sterols. Cells were from Difco. Precoated silica gel plates (0.25-mm cultured in 2-liter flasks with 1 liter of a broth thickness), Merck H-254, were products of EM Re- containing 1% tryptone, 0.5% yeast extract, and 2% agents Co. Authentic ergosta-7-en-3,B-ol, ergosta-8- glucose. After the cell pack was harvested and en-3,8-ol, and ergosta-7,22-diene-3/3-ol were gifts washed, it was lyophilized and treated with Me2SO. from A. C. Oehlschlager. All other chemicals were The extracted free sterols were then separated from commercially available and of the highest purity the sterol esters by using thin-layer chromatogra- obtainable. phy or short alumina columns as outlined above. The esterified sterol fraction was saponified under RESULTS reflux for 1 h in 6% KOH in absolute methanol to break the ester bonds. The free sterols were then The treatment of lyophilized cells with extracted into n-hexane and analyzed by thin-layer Me2SO for 1 h at 100 C in a steamer proved to and gas chromatography as described previously be very effective for extracting the sterols from (17). After preliminary separation on silica gel yeast without the danger of destroying the es- plates (cyclohexane-ethyl acetate, 85:15 vol/vol) to ters. The addition of water to the Me2SO prior give 4,4-dimethyl, 4-a-methyl, and 4-demethyl to extraction with petroleum ether prevented bands, the sterols were acetylated overnight in pyri- the extraction of Me2SO with the hexane ex- dine-acetic anhydride (1:2 vol/vol) to give the sterol tracts, which otherwise proved to be a nuis- acetates. Final identification of the sterol acetates was ance. Control experiments were done with made by gas chromatography with glass columns known sterol esters to determine whether they containing 2% SE-30 (6 feet [ca. 1.83 ml by 2 mm) could be hydrolyzed by the Me2SO in a steam and 3% OV-17 (4 feet [ca. 1.22 ml by 2 mm) as the bath, and we found no evidence that they were. liquid phases on an H/P Chrom W (100 to 120 mesh) Our first series of experiments was designed support. The column temperature was maintained to determine whether there was any correlation at 265 C with a helium flow rate of 25 ml/min. For between the degree of esterification of sterols coupled gas chromatography-mass spectrometry, a and the stage of the culture cycle of the orga- Varian MAT, model CH-7, with a Systems Indus- nism. Growth was followed as a function of cell tries 150 data system was used. The separation was mass, and the percentage ofsterols occurring as on 7% OV-17 (4 feet [ca. 1.22 ml) at 285 C. Spectra were taken at 70 ev with a source temperature of esters was determined periodically. We first 175 C. used the rich yeast extract-tryptone medium Thin-layer chromatography was done on silica gel containing 5% glucose as an energy source. As plates (Merck HF-254) of 0.25-mm thickness. The shown in Fig. 1, the degree of esterification separated sterols were visualized with ultraviolet remains at a relatively low level during the light after spraying with berberine in ethanol. The exponential growth phase, but it increases dra- sterols were eluted from the plates through anhy- matically upon entry into late exponential or drous sodium sulfate with methylene chloride. All early stationary growth. With 5% glucose, the solvents used were routinely redistilled. Preparation of substrates and sterol-fatty acid "steady-state" level of sterol esters during expo- esters. Zymosterol was prepared from Fleischman nential growth was very low, ranging from 0 to cake yeast as described previously (20). Zymosteryl- 10%. Two hours after entry into the stationary oleate and other sterol-fatty acid esters were pre- growth phase, however, levels as high as 60% pared by the methods of Knapps and Nicholas (13). esters were obtained, and by 24 h we generally The products were separated from the unreacted found 85 to 90% of the sterols esterified. Figure free sterols by silica gel thin-layer chromatography 1 also shows the total synthesis and accumula- with cyclohexane-ethyl acetate (85:15 vo/vol). Pu- tion ofsterols as measured by the incorporation rity was checked by saponification in alkaline meth- of the "4C-labeled methyl group of methionine anol (6% KOH) and rechromatography of the prod- into the nonsaponifiable lipids. This procedure uct. In all cases, the saponified esters cochromato- graphed with authentic sterol standards. is specific for ergosterol and other sterols with Methyltransferase enzyme preparation. The the C28 methyl group. Sterol synthesis clearly preparation of methyltransferase enzyme is already increases exponentially during the period when a published procedure (20). We used both a crude the percentage of esters is maintained at a very enzyme preparation (20) and a partially purified low level. 608 BAILEY AND PARKS J. BACTERIOL.

50 0 . _0._o--

, 0 100 01 104 0/ A__ // 40

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2 4 6 8 10 12 TIME (hr) FIG. 1. Variation in the level ofsterol esters during fermentative growth. Growth of an aerobically shaken culture in 5% glucose was followed optically with a Klett colorimeter. Samples were taken periodically and assayed for the level of sterol esters and the incorporation of the methyl-'4C group of methionine into the nonsaponifiable lipid fraction. Symbols: 0, Klett units; 0, percent esters; and A, counts per minute (CPM) per 50-ml sample incorporated into the nonsaponifiable lipids. The generation time in this experiment was 90 min.

Running the same experiment, but using 2% growth conditions and always tapered off at ethanol as a carbon source, gave us the data about 90% esters. There also appeared to be shown in Fig. 2. Again there is exponential some correlation between the growth rate and sterol synthesis occurring during a period when the level of esters maintained during exponen- the level of sterol ester is steady, and again tial growth. there is a large increase in esterification after We next looked at the composition of the entry into late exponential or early stationary sterols contained in the esterified fraction. This growth. However, the percentage of esterified was done by harvesting 3 liters of cells each, at sterol is maintained at about 20%, rather than periods of growth calculated to give different at the much lower values found with 5% glu- percent composition of the sterol esters. The cose. In fact, during one such experiment, we sterols were then extracted and analyzed by found a level of 35% esters maintained during thin-layer and gas-liquid chromatography. The exponential growth. Cells cultured on ethanol samples were taken and the ester percentages also approached a value of 90% esters after the studied were 37, 60, and 88% esters, the latter culture had passed into the stationary phase. occurring after 24 h of growth (Table 1). It is Because of the differences in the degree of very apparent that there are significant esterification observed, we repeated these changes in the sterol composition that take growth curves using various carbon sources. place during the growth cycle. Most noticeable Among those tested were 2% glucose, galactose, is the level of ergosterol, which can be seen to and succinate. We also tested these same car- decrease from 92% of the total sterol pool to 40% bon sources in a less rich, chemically defined after 24 h. However, during the same intervals, medium (22). Under all conditions, an abrupt we found the amount of esterified ergosterol to increase in the level of sterol esters occurred as approximate closely the overall degree ofesteri- the cells entered stationary growth. The maxi- fication of all sterols. mal degree of esterification was independent of The other noticeable feature shown by these VOL. 124, 1975 YEAST STEROL ESTERS 609

*S10;0~~~~~~~~~~~~~@<°/> * / 120221032

0~~~~~~~~~

AS ItI 20 I

2 4 6 8 10 12 TIME (hr) FIG. 2. Variation in the level ofsterol esters during respiratory growth. The same procedures as in Fig. 1 were 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 time for 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 95 4a-methyl-zymosterol 0.003 10 <0.001 ND 0.026 ND 4,14-dimethyl-cholesta- 0.014 10 0.026 ND 0.065 100 8,24-diene-3#-ol Ergosta-7,22-diene-3,6-ol 0.008 100 0.032 99 0.049 92 Ergosta-7-ene-3,8-ol 0.017 2 0.117 37 0.018 93 Zymosterol <0.001 ND <0.001 ND 0.162 100 Fecosterol <0.001 ND <0.001 ND 0.059 97 Ergosta-5,7-diene-3,B-ol 0.022 63 0.002 99 0.214 100 Ergosterol 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 extracted after treatment with Me2SO. The free sterols were separated from esterified sterols, and then both fractions were analyzed by thin-layer and gas chromatography. b ND, No sterol detected in ester fraction.

data is the presence of only traces (<0.1%) of that there was much variability in the percent- zymosterol and fecosterol during the earlier age of individual sterols esterified, especially stages of growth, whereas at 24 h (88% esters) during the earlier stages of growth. In some they make up 16 and 6% of the total sterol pool, instances we were unable to detect any of a respectively. Generally, the "minor" sterols be- given sterol in the ester pool, whereas other came more abundant as time progressed, sterols were found only in this fraction. The whereas the ergosterol concentration decreased above cells were all cultured on 5% glucose, but relative to the total sterol pool. We also noticed analyses were also done on ethanol-grown cells, 610 BAILEY AND PARKS J. BACTERIOL. and the composition appears to be independent nential sterol synthesis, it was of interest to see of the added energy source. whether it was in response to one or both of Because of this accumulation of minor ster- these factors. Accordingly, we aerated anaerobi- ols, Wve wondered whether ergosterol precursors cally grown cells in a 0.1 M phosphate buffer, were able to take part in further biosynthetic pH 6.6, containing 1% glucose. Yeast cells so reactions after esterification. One easily testa- incubated rapidly synthesized large amounts of ble reaction is the transmethylation event that sterol (19) but are incapable of dividing. Both takes place in yeast mitochondria (20). We sterol synthesis and the esterification of sterols made a partially purified lipid-free preparation increase very rapidly under such conditions of this enzyme (21) and proceeded to test chemi- (Fig. 3). It is also of interest to compare the cally synthesized (13) zymosteryl-oleate as a rates of sterol synthesis obtained by extracting substrate. Because we have previously reported both Me2SO-treated cells (Lieberman-Bur- three separate enzyme activities for this en- chard-positive color, absorbancy at 625 nm) and zyme (2), we tested all three with similar re- the saponified cells ([methyl-14C]methionine in- sults. Table 2 shows that the esterified form of corporation). The inherent decrease in labeled zymosterol cannot be methylated in this sys- methionine incorporation seen with regular al- tem, whereas free zymosterol is a ready sub- kaline pyrogallol saponification (18) is absent strate. when the cells are lyophilized and treated with We next investigated the effect of incubating Me2SO. This indicates that the latter is a more zymosteryl-oleate with the free sterol in our effective sterol extraction procedure. sterol methyltransferase assay system. Al- To determine the distribution of the esteri- though we expected inhibition of the reaction fied sterols, we utilized a mitochondria isola- by the esterified zymosterol, or perhaps no ef- tion procedure (20) and collected samples of the fect at all, we found that the ester actually cell wall (membranes), mitochondria, and cell stimulated the methyltransferase enzyme. The sap fractions. The total cellular sterol extract at stimulation we observed was maximally 30 to the time of harvest yielded 67% sterol esters. 40% above control reactions containing only We found that the cell wall (membranes) frac- free zymosterol. To determine whether this was tion contained 66% sterol ester, whereas the a general phenomenon of all sterol esters, we mitochondrial fraction had only 17% esters. synthesized C,4, C16, and C,8 esters of lanos- The cell wall and membranes also appeared to terol, ergosterol, and zymosterol. All of the have, by far, the majority of the total sterol pool sterol esters tested stimulated the enzyme sys- (60 to 75%). Although the cell sap gave 44% tem somewhat, but the oleate and stearate es- esters, the total sterol was very small by com- ters (C18) were the most stimulatory. It should parison, and, due to the mechanical cell break- be pointed out that all of these reactions were age and heterogeneity of the fraction, we are done in our partially purified enzyme system hesitant to attach much significance to this that had no detectable ester hydrolase activity. value. Since the increase in esterification during the It was also of interest to determine whether growth cycle coincided with both the entry into the fatty acids could be scavenged by the cell or early stationary phase and the decrease in expo- whether they are left alone once esterified to sterols. Therefore, we harvested an overnight cells and them in 0.1 M TABLE 2. Efficiency ofzymosteryl-oleate as a culture of resuspended substrate for the sterol methyltransferase reactiona phosphate buffer, pH 6.6, and allowed them to incubate 24 h. While the total sterol pool was Counts/min in nonsaponifiable constant on the basis of micrograms of sterol to lipids milligrams of protein, the percentage of esters Substrate tested to after 6 h 0.29 AM 9.3 IAM 93 AM actually increased from 57% 80% S-AM S-AM S-AM and remained constant at that level. Zymosterol 5,630 3,600 3,450 DISCUSSION Zymosteryl-oleate 400 240 290 Control 330 190 170 The rate of esterification of yeast sterols to long-chain fatty acids appears, from these data, a Sterol transmethylation assays were performed, to reflect the physiological state of the cell. A with either zymosterol or zymosteryl-oleate as the culture growing exponentially with a good car- substrate, at each of the three different S-adenosylmethionine (S-AM) concentrations. The control bon source in a rich medium has a very low tubes were run in the absence of any exogenously level of sterols occurring as esters and, in fact, supplied substrate. All sterol substrates were added we were unable to detect any sterol esters un- at a concentration of 200 uM. der some conditions. If a less suitable carbon VOL. 124, 1975 YEAST STEROL ESTERS 611 methyltransferase reaction was stimulated by a 200 80 variety of sterol esters. The stimulation was, at the maximum, only 35 to 40% and was observed 160 60 generally for all tested sterol esters. Since the ~ I enzyme is mitochondrially located (20) and we .E 7° 120 40 ,,, found very low levels of sterol esters associated ~ ~ ~ ~ ~ ~ ~ I with the mitochondrion, this 20- stimulation may 3 be simply fortuitous and of no physiological 80 20 significance. 0 The apparent accumulation of so-called mi- 40 a'i .v0 nor sterols with time is an interesting observa- tion. The cellular ergosterol content decreased from 92 to 40% of the total sterol pool. At the 2 4 6 8 10 same time, zymosterol increased from <0.1 to Hours Aeration 16% of the cellular total sterol pool, and er- FIG. 3. Esterification during aerobic adaptation. gosta-5,7-diene-3,8-ol increased from 2.2 to 21%. Anaerobically grown cells (72 h) were harvested and allowed to incubate in 0.1 M phosphate buffer, pH There were also smaller increases seen in fecos- 6.6, containing 1% glucose. The flask was shaken terol and ergosta-7,22-diene-3(3-ol. These data vigorously. Samples were removed to assay the per- suggest that these other sterols, especially zy- centage ofesters and the radioactive incorporation of mosterol and ergosta-5,7-diene-3/3-ol, might methionine into the nonsaponifiable lipids. Symbols: play an important role in cellular metabolism, A, percent esters; *, total Lieberman-Burchard posi- other than just being intermediates of ergos- tive color, absorbancy at 625 nm, extracted from terol. Otherwise, it is an unreasonable waste of Me1sO-treated cells; and 0, count per minute (CPM) energy in that the esterification process itself incorporated into the nonsaponiflable lipids per 10- requires adenosine 5'-triphosphate (1). ml sample. It has been reported that red blood cells can source is used, such as ethanol or succinate, the freely exchange intracellular sterols, but not level of sterol esters maintained during expo- sterol esters, with those in the surrounding nential growth increases but still remains con- medium (6). If this were also true of yeast, the stant until the culture approaches the station- esterification process would serve to prevent ary phase. Without exception, however, we ob- this exchange and, thereby, keep the sterol pool served a sharp rise in the percentage of sterol of the cell intact during periods when little, if esters upon entry into the stationary phase of any, sterol synthesis is taking place. Also, since growth. Although sterol synthesis also de- we find the majority of the sterol esters present creases at this same time, we feel that the in the cell wall, this might explain the "thicken- esterification process is a response to the over- ing" of the cell wall that has recently been all physiological state ofthe cell, rather than to reported to occur during the stationary phase the decrease in sterol synthesis. This is based (8). on the observations made in the aerobic adapta- The esterification process is obviously not a tion experiment, where sterol synthesis and means by which fatty acids may be stored for esterification both increased rapidly under con- energy until such time as they may be needed. ditions when the cell could not divide. If the We actually observed an increase in esterifica- esterification was due to a decrease in sterol tion to take place under starvation conditions. synthesis alone, one would expect, under these This agrees with the data of Bartlett and Mer- conditions, that esterification would remain at cer (3) for P. blakesleeanus. a constant level until sterol synthesis dimin- The rapid period of esterification seen as the ished. culture approaches the stationary phase may Once sterol intermediates are esterified, they be the consequence of some regulatory interac- are effectively prevented from being further tion. This increase could be due to: (i) a con- metabolized to ergosterol. We have shown that stant rate of esterification during a time when zymosteryl-oleate is not methylated by our in sterol synthesis is decreasing, (ii) release of vitro assay system, although the free sterol is a control of the sterol ester synthetase, (iii) de- ready substrate. Whether this holds true for repression of the sterol ester synthetase, or (iv) other enzymatic reactions in vivo remains un- decreased ester hydrolase activity. Since there tested, but since other sterol intermediates do was a rapid rate of esterification during aerobic accumulate and are generally about 100% ester- adaptation with a concomitant fast rate of ified, it does seem probable. At this point we sterol synthesis, we do not feel that explanation have no proven explanation for why the sterol no. i is satisfactory. All of the other explana- 612 BAILEY AND PARKS J. BACTERIOL. tions do implicate a form of regulatory interac- Changes in the levels and composition of the esteri- of maize seedlings during tion of the esterification process. Work is cur- fied and unesterified sterols germination. Phytochemistry 6:1609-1615. rently being done to clarify this problem. 10. Kemp, R. J., and E. I. Mercer. 1968. The sterol esters of maize seedlings. Biochem. J. 110:111-118. ACKNOWLEDGMENTS 11. Kemp, R. J., and E. J. Mercer. 1968. Studies on the We wish to thank A. C. Oleschlager for generous sup- sterols and sterol esters of the intracellular orga- plies of authentic minor yeast sterols. nelles of maize shoots. Biochem. J. 110:119-125. The work was supported by a grant from the National 12. Klein, H. P., N. R. Eaton, and J. C. Murphy. 1954. Net Science Foundation. synthesis of sterols in resting cells of Saccharomyces cervisiae. Biochim. Biophys. Acta 13:591. LITERATURE CITED 13. Knapps, F. F., and H. J. Nicholas. 1970. Liquid crystal- 1. Anding, C., L. W. Parks, and Guy Ourisson. 1974. line properties of ergosteryl fatty acid esters. Mol. Enzymic modification of cyclopropane sterols in yeast Cryst. Liq. Cryst. 10:170-186. cell-free system. Eur. J. Biochem. 43:459-463. 14. Madyastha, P. B., and L. W. Parks. 1969. The effect of 2. Bailey, R. B., E. D. Thompson, and L. W. Parks. 1974. cultural conditions on the ergosterol ester compo- Kinetic properties of S-adenosylmethionine: A24- nents of yeast. Biochim. Biophys. Acta. 176:858-862. sterol methyltransferase enzyme(s) in mitochondrial 15. Mercer, E. I., and Kim Bartlett. 1974. Sterol esters of structures ofSaccharomyces cerevisiae. Biochim. Bio- Phycomyces blakesleeanus. Phytochemistry 13:1099- phys. Acta 334:127-136. 1105. 3. Bartlett, K., and E. I. Mercer. 1974. Variation in the 16. Parks, L. W., C. Anding, and Guy Ourisson. 1974. levels and composition of the sterols and sterol esters Sterol transmethyltaion during aerobic adaptation of ofPhycomyces blakesleeanus with age of culture. Phy- yeast. Eur. J. Biochem. 43:451-458. tochemistry 13:1115-1121. 17. Parks, L. W., F. T. Bond, E. D. Thompson, and P. R. 4. Barton, D. H. R., J. E. T. Corrie, P. J. Marshall, and D. Starr. 1972. A59'22-Ergostadiene-3j3-ol, an ergosterol A. Widdowson. 1973. Biosynthesis of terpenes and precursor accumulated in wild-type and mutants of steroids. VII. Unified scheme for the biosynthesis of yeast. J. Lipid Res. 13:311-316. ergosterol in Saccharomyces cerevisiae. Bioorg. 18. Starr, P. R., and L. W. Parks. 1962. Some factors affect- Chem. 2:363-373. ing sterol formation in Saccharomyces ceruisiae. J. 5. Barton, D. H. R, U. M. Kempe, and D. A. Widdowson. Bacteriol. 83:1042-1046. 1972. Investigations on the biosynthesis of steroids 19. Starr, P. R., and L. W. Parks. 1972. Transmethylation and terpenoids. VI. The sterols of yeast. J. Chem. of sterols in aerobically adpating Saccharomyces cere- Soc., Perkin Trans. 1, p. 513-522. visiae. J. Bacteriol. 109:236-242. 6. Bruckdorfer, K. R., and C. Green. 1967. The exchange 20. Thompson, E. D., R. B. Bailey, and L. W. Parks. 1974. of unesterified cholesterol between human low-den- Subcellular location of S-adenosylmethionine: A24- sity lipoproteins and rat erythrocyte 'ghosts.' Bio- sterol methyltransferase in Saccharomyces cerevi- chem. J. 104:270-277. siae. Biochim. Biophys. Acta 334:116-126. 7. Fryberg, M., A. C. Oehlschlager, and A. M. Unrau. 21. Thompson, E. D., and L. W. Parks. 1974. Effect of 1972. Biosynthetic routes to ergosterol in yeast. Bio- altered sterol composition on growth characteristics chem. Biophys. Res. Commun. 48:593-597. of Saccharomyces cerevisiae. J. Bacteriol. 120:779- 8. Hammond, S. M., and B. N. Kliger. 1974. Studies on 784. the role of the cell wall of Candida albicans in the 22. Wickerham, L. J. 1946. A critical evaluation of the mode of action of polyene antibiotics. Proc. Soc. Gen. nitrogen assimilation tests commonly used in classifi- Microbiol. 1:45. cation of yeasts. J. Bacteriol. 52:293-301. 9. Kemp, R. J., L. J. Goad, and E. J. Mercer. 1967.