JOURNAL OF BACrERIOLOGY, Jan. 1972, p. 236-242Copyright 0 1972 American Society for Microbiology

Vol. 109, No. 1Printed in U.S.A.

Transmethylation of Sterols in AerobicallyAdapting Saccharomyces cerevisiae

P. R. STARR' AND L. W. PARKS

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received for publication 9 July 1971

The transmethylation of methyl-'4C-methionine and methyl-'4C-adenosyl-methionine into the nonsaponifiable lipids of anaerobically grown yeast duringadaptation to aerobic conditions was investigated. The rate and extent ofmethyl transfer increased with aeration time and was dependent upon thepresence of a fermentable carbon source and 02. Methionine and adenosylme-thionine uptake rates increased in adaptation buffer but did not seem to be therate-limiting factor for transmethylation under the conditions studied. Thin-layer chromatography of the nonsaponifiable fraction after exposure to labelshowed the labeled product to be ergosterol. Samples taken after short-termexposure to label were composed of two labeled steroidal products, one withkinetics of an ergosterol precursor.

Under aerobic conditions, yeast may synthe-size ergosterol to the extent of 2 to 10% of theirdry weight (8). Although such quantities aremade, little is known of the cellular function ofsterols in yeast. However, much evidence hasbeen presented which would suggest that er-gosterol synthesis is closely related to the de-velopment of aerobic respiration (11, 18).Sterols can be synthesized only under aerobicconditions since conversion of squalene tosterols is dependent upon molecular oxygen(20). Under anaerobic conditions, biosynthesisproceeds only to squalene, which accumulatesuntil aerobic conditions prevail, whereupon itis converted to sterol. Nevertheless, anaerobi-cally growing yeast cells still require vitamin-like quantities of ergosterol (4). If the sterol isnot supplied, anaerobic growth stops after fiveto seven generations, and death begins about24 hr later. Surviving cells are unable to adaptto respiration when exposed to aerobic condi-tions (16). Inability to develop respiratorycompetence in lipid-starved cells may be re-lated to the formation of abnormal promito-chondrial membranes which are deficient inenzyme content (7). Anaerobically grown,lipid-supplemented cultures may synthesizesterols and adapt to respiratory conditionsupon subsequent aeration in resting culture;neither function requires cell growth (10).

The study of the regulation of ergosterol

I Present address: Department of Microbiology, Univer-sity of Illinois, Urbana, Ill. 61801.

synthesis during the shift from anaerobic toaerobic conditions is of importance in the at-tempt to understand the physiological functionof sterols in yeast. Early studies of ergosterolsynthesis employed the Leibermann-Burchardcolorimetric assay (19). The method, however,is not adequate for regulatory studies in that itis not sensitive to small changes in sterol con-centration, the color quickly deteriorates (de-manding close attention to time and tempera-ture during assay), large volumes of cells arerequired, and other nonsaponifiable productsof yeast metabolism interfere with color devel-opment. A specific and very sensitive sterolassay technique measuring the kinetics of theterminal reactions of sterol biosynthesis andtheir regulation would greatly facilitate re-search in this field. Previous results suggestedthat the transfer of radioactively labeledmethyl group from methionine to ergosterolprecursors might satisfy the requirements forsuch a study (1, 2).

All of the carbons in ergosterol arise fromacetate except carbon 28 in the side chain.This carbon is donated from the methyl groupof L-methionine (3), via its activated form, S-adenosylmethionine (AM; 17). Transmethyl-ation occurs after the cyclization of squaleneto lanosterol; however, the identity of the nat-ural methyl acceptor has not been established.Moore and Gaylor (14) have purified a A24_sterol transmethylase which showed stoichiom-etry between the disappearance of zymosteroland AM and the appearance of fecosterol, a

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STEROL TRANSMETHYLATION

methylated zymosterol derivative. Furtherdata suggest that the enzyme has a specificrequirement for an acceptor with a demethyl-ated ring structure (15). However, the isola-tion of 4a-methyl-24-methylene-24,25-dihy-drozymosterol from whole-cell preparations ofyeast suggest that C-28 may be added beforedemethylation of the ring structure (5). Addi-tionally, feeding experiments using 4a-methyl-zymosterol, 4a-methyl-24-methylene-24, 25-dihydrozymosterol, and obtusifoliol showed allto be converted to ergosterol (5). Additionaldata are necessary to clarify the naturalpathway of the terminal reactions of sterolsynthesis. Further progress in determining thissequence beyond lanosterol has been hinderedby the inability to identify ergosterol precur-sors, insolubility of sterols, low uptake rates inwhole cells, and lack of sterol-requiring mu-tants.

Transmethylation reactions in whole cellsmay give indications of rates of synthesis ofnatural precursors and may afford a marker forseparation of those precursors. The steroltransmethylation system in whole cells de-pends upon the uptake of methionine and itsactivation to AM, synthesis of the sterolic pre-cursors, and finally transmethylation to thesterolic substrate. This paper reports the re-sults of our studies on some of the componentsof the transmethylation system and the re-quirements for their expression during aerobicadaptation of anaerobically cultured yeast.

MATERIALS AND METHODSCultures. Haploid strains of Saccharomyces cer-

evisiae used were 22B (met), 3701B (ur), spontaneousrespiratory-deficient mutants (petites) of each, andnys-3, an ergosterol-nonsynthesizing, nystatin-re-sistant mutant obtained from R. Woods (23). MCC,a wild type diploid strain, was used also. Stock cul-tures were maintained on YC slants (19) containingeither 2% (w/v) glucose or galactose and were trans-ferred at monthly intervals.

Conditions for growth and aeration. Cells weregrown anaerobically in 2-liter Erlenmeyer flaskswhich were filled to 1,800 ml with YAF broth (19)made to 2% (w/v) glucose, and into which Bunsenvalves had been fitted to maintain anaerobic condi-tions. After 48 to 60 hr of growth at 30 C, at whichtime stationary phase had been attained, the cul-tures were chilled overnight and then harvested overcrushed ice. Cells were washed twice with 0.1 M po-tassium phosphate buffer (pH 6.6) in a refrigeratedcentrifuge. Washed cells were made to 5 or 10 mg/ml(wet cell weight) in 0.1 M potassium phosphate buffer(pH 6.6) containing 1% (w/v) glucose unless other-wise specified, and were rapidly aerated at 30 C on areciprocating shaker in flasks filled to 0.1 volume.Transmethylation assay. Methyl-( '4C)-L-methi-

onine (60 mCi/mmole) or methyl-( 4C)-S-adenosyl-methionine (58 mCi/mmole) was added to a finalconcentration of 0.025 ztCi/ml unless otherwisenoted, at the times described in the Results section.Samples (5 ml) were chilled for at least 5 min in anice bath and then centrifuged at 650 x g for 3 min;the cell packet was saponified by the methanolic-alkaline pyrogallol method (12). The unsaponifiedlipids were extracted with three 10-ml volumes of n-hexane, dried under a stream of N2, and redissolvedin chloroform or 10 ml of toluene-2,5-diphenyloxa-zole-1, 4-bis-2-(5-phenyloxazolyl)benzene scintilla-tion cocktail (22). Label incorporated into the unsa-ponified lipid fraction was assayed in a Packard Tri-carb liquid scintillation spectrometer.Methionine andAM uptake assay. A 1-ml amount

of the supernatant fluid from the 650 x g centrifuga-tion as described above was added to 10 ml of Bray's(6) solution. The label remaining in the supernatantfluid was assayed in a Packard Tri-carb liquid scin-tillation spectrometer.

Thin-layer chromatography. Glass plates (5 by20 cm) were spread to a thickness of 0.25 mm with45.5% Silica Gel G made up in 4% AgNO3, dried for60 min, and activated at 90 C for 30 min. A 30- to50-Aliter sample of the nonsaponifiable lipids wasapplied as a spot, and the plate was developed for 20to 25 min in benzene-ethyl acetate (5:1, v/v). Thefront moved 13 to 14 cm, and a clear separation oflanosterol, zymosterol, and ergosterol was effectedunder these conditions. The sterols could be ob-served after the plates had been sprayed with Lei-bermann-Burchard reagent (19:1 acetic anhydride-H2SO4) diluted 1:1 (v/v) with chloroform and heatedfor 5 min at 90 C. Labeled spots were detected on aPackard Radiochromatogram Scanner model 7021.

Materials. Zymosterol was obtained fromFleischmann Laboratories, lanosterol from MannResearch Chemicals, New York, N.Y., and ergosterolfrom Calbiochem, Los Angeles, Calif. Labeledmethyl donors were purchased from Amersham/searle, Arlington Heights, Ill. Silica Gel G was pur-chased from Brinkmann Instruments, Inc., West-bury, N.Y.

RESULTSIncorporation of methyl group into the

nonsaponifiable lipids of unadapted cells.When methyl-14C-L-methionine was added toanaerobically grown, washed-cell suspensions,incorporation of label into the nonsaponifiablefraction during aeration was biphasic (Fig. 1).A faster rate of incorporation followed by along period of slow incorporation was ob-served. The maximal amount of label in thenonsaponifiable lipids after 4 hr was only 10%of the total added. These data were not com-patible with results observed by Leibermann-Burchard analysis, which showed sterol syn-thesis to proceed over several hours (19). Todetermine whether the values obtained bytransmethylation were indicative of sterol syn-

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140 were aerated before the label was added, the_* more label was found in the unsaponified

lipids. Thus, the transmethylation step ap-120 peared not to be the rate-limiting reaction,

and the results suggested that the limitingIx0 factor was the availability of sterolic precur-

sors. Identical results were observed for strainsI 3701B, MCC, and nys-3, and for the respira-

80 tory-deficient mutants; therefore, the reactioncE is a general one and not related to the methio-E 60 nine requirement of strain 22B.ti E Rate of methionine uptake during adapta-

40 G tion phase. It is obvious that methyl group40E incorporation into the nonsaponifiable fraction

% ,,.,' is dependent upon entry of the methyl donor20 * -. 20° into the cells. Since the rate of methionine and

t AM uptake increased during the adaptation0 < "_ :. e 0 Cf phase (Table 1), it was necessary to determinel)I2345 6 whether the rate of uptake was limiting the

hn aeration rate of incorporation of label into the sterolFhG.r1.sUptakeandeincooration ofraction. Cultures at 5 mg of wet cells/ml wereFIG. 1. Uptake and incorporation of methyl-'4C_methionine into nonsaponifiable lipids in aerobicallyadapting yeast. Anaerobically grown yeast, made to10 mg/ml in adaptation buffer, was exposed to 0.025,uCi of methyl-'4C-methionine per ml at the onset of 140.aeration. At the arrow, a second addition of 0.025 AiCiof methionine per ml was made. Solid line: counts!minute in nonsaponifiable lipids. Dashed line:counts/minute in medium. 120. X

thetic rates during the slower second phase,the culture was divided after 4 hr of incuba- Ition. More labeled methionine was added to °00one half, and incorporation was measured inboth cultures. Within 15 min, 50% of the label _added to the second culture had been incorpo- c 80rated into the nonsaponifiable lipids. Thus, it 'seemed that the methionine proffered to una- f Adapted cells quickly became unavailable for , /transmethylation of sterols synthesized later in 0 60the adaptation phase.

Effect of aeration time on incorporation of A

methyl group into the nonsaponifiablelipids. Since it was not possible to observe the 40 / A

long-term rate of sterol synthesis during adap-tation by addition of label to an unadaptedculture, short-term rate experiments on cells 20-at various stages of adaptation were per-formed. Accordingly, the kinetics of incorpora-tion of labeled methionine into the nonsaponi- /fiable lipids added after increasing periods of 0 0 1.0 1.5 2.0aeration were determined. Both the rate and Hoursextent of transmethylation of the nonsaponifi- FIG. 2. Effect of aeration on transmethylation ofable fraction increased with increasing aera-

S. cerevisiae 22B. Cells grown anaerobically for 60 hrtion (Fig. 2). A ninefold increase in rate of in- were made to 10 mg/ml in adaptation buffer. At 0 hrcorporation and at least a twofold increase in (@),0.5 hr (A), 1 hr (0), and 2 hr (0), methyl-'4C-me-the amount incorporated were observed during thionine was added to a final concentration of 0.625the 2-hr aeration period. The longer the cells ,Ci/ml.

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STEROL TRANSMETHYLATION

aerated for 1.5 hr, after which time increasingconcentrations of labeled methionine wereadded. Both the rate of uptake and the rate ofincorporation of label into the nonsaponifiablefraction increased with increased concentra-tions of methionine (Table 2). The ratio of therate of incorporation to the rate of uptake di-minished as the concentration of methionineincreased, suggesting that the two were notdirectly linked and that the saturating concen-tration of methionine for the transmethylationstep was lower than that for the uptake step.Uncoupling of uptake mechanism and

transmethylation step. Anaerobically growncells aerated in buffer are being subjected tochanges in two factors. Not only are they sub-

TABLE 1. Effect of aerobic adaptation on rate of S-adenosylmethionine (AM) and methionine uptakea

Uptake rateHr aerated

Methionine AM

Ob (1) 6 x 102 (4) 1.5 x 102(2) 1 x 102 (5) 1.0 x 102(3) 7x102

2c (1) 8 x 103 (4) 5.5 x 103(2) 7.5 x 103 (5) 8.0 x 103(3) 5.5 x 103

a Cell suspensions containing 10 mg of cells per mlwere exposed to 0.025 mCi of methyl-14C-methionineor methyl- '4C-adenosylmethionine per ml. Experi-ment numbers are given in parentheses.

'Rate given as counts per minute disappearingfrom 1 ml of medium during the first 10 min of ex-posure to the label.

cRate given as counts per minute disappearingfrom 1 ml of medium during the first 3 min of expo-sure to the label.

TABLE 2. Effect of concentration of methyl-'4C-methionine on rates of uptake and transmethylationa

methionine Transmethyl- Uptake Transmethyl-(imeti)nn ationbpae

ation/uptake

0.025 3.1 x 103 2.0 x 103 1.550.050 6.3 x 103 4.7 x 103 1.350.075 8.3 x 103 6.7 x 103 1.240.10 9.6 x 103 8.4 x 103 1.14

a Cell suspensions containing 5 mg of cells/mlwere aerated for 1.5 hr before exposure to label.

b Rate given as counts per minute incorporatedinto nonsaponified lipids of 5 ml of cells during thefirst 3 min of exposure to label.

c Rate given as counts per minute disappearingfrom 1 ml of medium during the first 3 min of expo-sure to label.

ject to respiratory induction by imposedaerobic conditions, but they also experienceextreme nutritional step-down. Sparging cellsin adaptation buffer with N2 before exposureto 02 makes it possible to study the effects ofthe two factors separately. Anaerobicallygrown cells were sparged with N2 for 2 hr inadaptation buffer and then exposed to labeledmethionine at the onset of aeration. The ki-netics of uptake and incorporation in thesecells were compared with those of unadaptedcells and those of cells aerated for 2 hr beforeaddition of labeled methionine. The rate ofincorporation of methyl donor into the nonsa-ponifiable fraction in the N2 sparged cells wasonly about 15% of that of the aerated cells(Fig. 3a) and probably only reflected acci-dental exposure to air during manipulation ofthe culture. However, the rate of uptake inthose cells was about 65% of that in cells aer-ated for 2 hr (Fig. 3b). These results suggestthat, whereas the rate of incorporation ofmethyl group into the nonsaponifiable fractionis dependent upon the presence of 02, the in-creased rates of uptake of methyl donor are areflection of nutritional step-down.

Effect of carbon source on rate of trans-methylation of the nonsaponifiable fraction.Development of the transmethylation systemrequired aeration in a fermentable carbonsource. Cells aerated for 2 hr without glucose

al. 0

c

C

0

0 10 20min in label

b24

20.

*16.12

0

*' 12 /E I/

2 8-i<

0 10 20min In label

FIG. 3. Effect of sparging with N2 in adaptationbuffer before aeration. (a) Effect on transmethylation.(b) Effect on methionine uptake. Cells grown anaer-obically for 60 hr were made to 10 mg/ml in adapta-tion buffer, and exposed to 0.025 MCi of methyl-14C_methionine per ml before aeration (-), after 2 hr ofaeration (0), and after 2 hr sparged with N2 (A).

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240 STARR Al

or in 3% ethanol did not exhibit either therapid rate of uptake or of transmethylationwhen exposed to labeled methionine (Fig. 4) orAM. Only those aerated in glucose or galactoseexhibited the increased rates of uptake and ofincorporation into nonsaponifiable lipids.These results agree with data obtained pre-viously by means of the Leibermann-Burchardassay (19).Products of the transmethylation reac-

tdon. To show whether all of the nonsaponifi-able product labeled and isolated by thesemethods was sterolic, unadapted cells of strain22B were aerated for 2 hr in labeled methio-nine, the unsaponified lipids were extracted,and the components were separated by thin-layer chromatography. Although severalstained spots were observed, all of the labelappeared in a spot that turned green whenexposed to Leibermann-Burchard reagent, pre-cipitated with digitonin (13), and had the same

40

30

enc

E 20

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NID PARKS J. BACTERIOL.

mobility as ergosterol (RF 0.13; Fig. 5). Thin-layer scans of other strains tested were iden-tical except in the case of strain nys-3. Thisstrain accumulated a sterolic product that hada higher mobility (RF 0.32), more like that ofzymosterol (RF 0.33). Feeding experimentsshowed that this sterol was converted to ergos-terol (L. W. Parks, unpublished results), andthus may be a natural precursor to ergosterol.To determine whether a similar product couldbe detected in strain 22B, cells were adaptedfor 2 hr before label was added; the culturewas sampled at 3, 20, and 60 min after expo-sure to the label, and the nonsaponifiablelipids were extracted and chromatographed.Since the rate of transmethylation was somuch faster in adapted cells, the possibility ofdetecting the precursor was greater underthese conditions. As shown in Fig. 6, the labelmoved rapidly through a zymosterol-likeproduct and accumulated in ergosterol. After 3min, 50% of the label was in the faster movingcomponent, after 20 min only 15% remained,and after 60 min only ergosterol was labeled.Uptake was complete within 6 min after addi-tion of label.

DISCUSSIONThe synthesis of sterols may be a necessary

2.0

E0L

on I 0

GD2 043 O14 ==

0

FIG. 4. Effect ofCells grown anaerobmg/ml in adaptationgalactose (0, 0) or 30strate addition (-, 0with methyl- 4C-met(closed figures) or

figures).

0 2 4 6 8 10 12 FMIGRATION (cm)

10 2 0 30FIG. 5. Thin-layer chromatography of the non-

saponifiable product of transmethylation in S.m in cerevisiae 22B. Cells grown anaerobically for 60 hr

substrate on transmethylation. were made to 10 mg/ml in adaptation buffer contain-ically for 60 hr were made to 10 ing 0.025 gCi of methyl- 4C-methionine per ml andbuffer containing 1% glucose or aerated for 1 hr. Cells were saponified, and 30 /sliters

Yo ethanol (A, A), and without sub- of the resultant nonsaponified fraction was chromato-1). Each was made to 0.025 ACi/ml graphed, scanned, and sprayed with Leibermann-*hionine at the onset of aeration Burchard reagent. Column 1 = nonsaponified frac-after 2 hr of aeration (open tion of 22B; 2 = digitonin precipitate of nonsaponi-

fied fraction; 3 = ergosterol, 4 = zymosterol.

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STEROL TRANSMETHYLATION

but insufficient prerequisite to the develop-ment of respiratory competence in yeast.Squalene accumulates in anaerobic yeast, butcombines rapidly with molecular 02 at theadvent of aeration to form sterols which be-come a major lipid component of mitochon-dria. It may be speculated that such a reactioncould induce changes that result in the differ-entiation of promitochondria into actively res-piring mitochondria. That it is not also suffi-cient may be shown by the fact that "petites"also synthesize ergosterol which may be foundin their mitochondria-like structures. Sterolsynthesis may be necessary for the maturationof the promitochondrial membrane, but devel-opment of respiratory competence then is de-pendent upon synthesis and placement of res-piratory enzymes into the membrane. It fur-ther may be speculated, since no sterol-re-quiring or deficient mutants have been found,that sterols in aerobic cells exist in integratedstructural units which cannot be supplementedby exogenous sources. For instance, zymosteroldoes not serve as substrate for ergosterol syn-thesis in cell-free preparations (9, 22) unlessendogenous sterols are removed by specialtreatment (14).The nystatin-resistant mutants are the first

ergosterol-deficient isolates of Saccharomycesreported. Even those mutants do not exhibit asterol requirement and still accumulate amethylated sterolic product (21). Thus, thetransmethylation step appears to be an impor-tant cellular reaction, and the product hasnormal function in nys-3 as shown by growthon ethanol and characteristic mitochondrialprofiles in electron micrographs (P. R. Starr,unpublished results).The development of the transmethylation

system in adaptation buffer was dependentupon aeration in the presence of a fermentablecarbon source. An adequate supply of methyldonor was assured under these circumstancesby a concomitant increase in the methionineand AM transport systems. The permeationsystem had increased activity as a result ofnutritional step-down and not as a result ofaerobic adaptation.The transmethylation assay, as described

here, measured activity only over short timeperiods; proffered methionine not used rapidlyfor sterol synthesis became unavailable forlater sterol methylation. In unadapted cells,the rate of transmethylation was low and thepercentage of methionine taken up that waseventually incorporated into the nonsaponifi-able lipids was also low (1 to 6%). In cellsadapted for 2 hr, a higher percentage of the

E

0

0 2 4 6 8 10 12' 14MIGRATION (cm)

FIG. 6. Transmethylation products after 3, 20, and60 min of exposure to methyl- l 4C-methionine. Anaero-bically grown cells, made to 10 mg/ml were aeratedin adaptation medium for 2 hr. The culture was madeto 0.025 ,Ci/ml with methionine. Samples were takenat 3 min (---), 20 mtn (-. -), and 60 min (-), andwere saponified; the nonsaponifiable fraction wasspotted onto thin-layer plates. Each was developedfor 20 min and then scanned. The graph is a com-posite of scans of three different plates.

methionine taken up was incorporated into thesterolic fraction (30 to 40%). Some of the com-petition for methionine could be reduced byaeration in the presence of cycloheximide; inthis case, the rate and extent of methyl groupincorporation into the nonsaponifiable fractionwas increased (P. R. Starr, unpublished re-sults).

Both products of the transmethylation reac-tion of strain 22B isolated by our methods andseparable by thin-layer chromatography weresterolic in nature. The more polar one had ki-netics expected for a precursor of ergosteroland had the thin-layer chromatographic char-acteristics of a methylated zymosterol deriva-tive. The mutant strain, nys-3, did not synthe-size ergosterol, but accumulated a methylatedproduct with the same mobility as the morepolar sterol of strain 22B. This methylatedsterol, identified by nuclear magnetic reso-nance as 8(9),22-ergostadiene 3,B-ol, when fedto strain 22B was transformed into ergosterol(L. W. Parks, unpublished results). Since bothfecosterol, the product of transmethylation inpurified enzyme preparations (14), and 8(9),22-ergostadiene 3,B-ol would most probablyhave the same mobility by the method used, itwas not possible to determine whether the pre-cursor product of strain 22B was one or theother or a mixture of both. The possibility that

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STARR AND PARKS

it was a mixture of both raises interestingspeculation concerning the authenticity ofidentification of any sterol with two doublebonds in the side chain. Ergostatetraene, sug-

gested by Turner and Parks (22) and Katsukiand Block (9) as a precursor of ergosterol, was

not detected.The transmethylation reaction may be used

as a measure of the rate of sterol synthesis inshort-term experiments under the conditionsdescribed. The changes in rates reflect similarchanges in sterol synthesis as shown by theLeibermann-Burchard assay (19). Regulatorystudies in which the labeling technique is usedare now in progress.

ACKNOWLEDGMENTSWe gratefully acknowledge the excellent technical assist-

ance of V. Stromberg. Strain nys-3 was provided by R.Woods.

This investigation was supported by National ScienceFoundation grant GB-8238 and by Public Health Servicegrant AM-05190 from the National Institute of Arthritis andMetabolic Diseases. This report is Oregon Agricultural Ex-periment Station technical paper no. 3131.

LITERATURE CITED

1. Adams, B. G., and L. W. Parks. 1967. Evidence for dualphysiological forms of ergosterol in Saccharomycescerevisiae. J. Cell. Physiol. 70:161-168.

2. Adams, B. G., and L. W. Parks. 1969. Differential effectof respiratory inhibitors on ergosterol synthesis bySaccharomyces cerevisiae during adaptation to ox-

ygen. J. Bacteriol. 100:370-376.3. Alexander, G. J., A. M. Gold, and E. Schwenk. 1957.

The methyl group of methionine as a source of carbon28 in ergosterol. J. Amer. Chem. Soc. 79:2967.

4. Andreason, A. A., and T. J. B. Stier. 1953. Anaerobicnutrition of Saccharomyces cerevisiae. I. Ergosterolrequirement for growth in a defined medium. J. Cell.Comp. Physiol. 41:23-26.

5. Barton, D. H. R., D. M. Harrison, and D. A. Wid-dowson. 1968. 4a-methyl-24-methylene-24,25-dihy-drozymosterol, a new sterol of Saccharomyces cerevi-siae of possible importance in the biosynthesis of er-

gosterol. Chem. Commun. 1968:17-19.6. Bray, G. A. 1960. A simple efficient liquid scintillator

for counting aqueous solutions in a liquid scintillationcounter. Anal. Biochem. 1:279-285.

7. Criddle, S., and G. Schatz. 1969. Promitochondria ofanaerobically grown yeast. I. Isolation and biochem-

ical properties. Biochemistry 8:322-334.8. Dulaney, E. L., E. 0. Stapely, and K. Simpf. 1954. Er-

gosterol production by yeasts. Appl. Microbiol. 2:371-379.

9. Katsuki, H., and K. Bloch. 1969. Studies on the biosyn-thesis of ergosterol in yeast, formation of methylatedintermediates. J. Biol. Chem. 242:222-227.

10. Klein, H. R., N. R. Eaton, and J. C. Murphy. 1954. Netsynthesis of sterols in resting cells of Saccharomycescerevisiae. Biochim. Biophys. Acta 13:591.

11. Kovac, L., J. Subik, G. Russ, and K. Kollar. 1967. Onthe relationship between respiratory activity and lipidcomposition of the yeast cell. Biochim. Biophys. Acta144:94-101.

12. Mahler, H. R., G. Neiss, P. P. Slonimski, and B.Mackler. 1964. Biochemical correlates of respiratorydeficiency. III. The level of some unsaponifiable lipidsin different strains of Baker's yeast. Biochemistry 3:893-895.

13. Monner, D. A., and L. W. Parks. 1968. A method forextraction of sterols from enzymically active cell-freepreparations. Anal. Biochem. 25:61-69.

14. Moore, J. T., Jr., and J. L. Gaylor. 1969. Isolation andpurification of S-adenosylmethionine:A24-sterol meth-yltransferase from yeast. J. Biol. Chem. 244:6334-6340.

15. Moore, J. T., Jr., and J. L. Gaylor. 1970. Investigation ofan S-adenosylmethionine:A24-sterol methyltransfer-ase in ergosterol biosynthesis in yeast. Specificity of

sterol substrates and inhibitors. J. Biol. Chem. 245:4684-4688.

16. Morpurgo, G., G. Serlupi-Crescenzi, G. Tecce, F. Val-ente, and D. Venetacci. 1964. The influence of ergos-terol on the physiology and the ultrastructure of Sac-charomyces cerevisiae. Nature (London) 201:897-899.

17. Parks. L. W. 1958. S-adenosylmethionine and ergosterolsynthesis. J. Amer. Chem. Soc. 80:2023.

18. Parks, L. W., and P. R. Starr. 1963. A relationship be-tween ergosterol and respiratory competency in yeast.J. Cell. Comp. Physiol. 61:61-65.

19. Starr, P. R., and L. W. Parks. 1962. Some factors af-fecting sterol formation in Saccharomyces cerevisiae.J. Bacteriol. 83:1042-1046.

20. Tchen, T. T., and K. Bloch. 1957. On the mechanism ofenzymatic cyclization of squalene. J. Biol. Chem. 226:931-939.

21. Thompson, E. D., P. R. Starr, and L. W. Parks. 1971.Sterol accumulation in a mutant of Saccharomycescerevisiae defective in ergosterol production.Biochem. Biophys. Res. Commun. 43:1304-1309.

22. Turner, J. R., and L. W. Parks. 1965. Transmethylationproducts as intermediates in ergosterol biosynthesis in

yeast. Biochim. Biophys. Acta 98:394-401.23. Woods, R. A., J. Hogg, and L. Miller. 1969. Changes in

the sterol content of nystatin-resistant mutants of

yeast. Heredity 24:516.

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