The Functional Importance of Structural Features of Ergosterol in Yeast*

(Keceived for publication, February 21, 1978)

William R. Nes, Bernard C. Sekula, W. David Nes, and John H. Adler

From the Department of Biological Sciences, Drexel Uniuersity, Philadelphia, Pennsylvania 19104

As an approach to the study of the relationship be- tween the structure of sterols and their capacity to function in the lipid leaflet of membranes, various ste- rols were examined for their ability to support the growth of anaerobic Saccharomyces cereuisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24P-methyl-A”- grouping > 24fi-methyl group > A’s7-diene system = k’- bond = no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic proc- esses of removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C- 22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol proba- bly acts functionally in the form of its preferred con- former in which C-22 is to the right (“right-handed”) in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.

There are substantial reasons for believing that dominant sterolsl play their principal role as architectural components of membranes (3-5). In an attempt to shed further light on the relationship between the sterol’s structure and its ability to function in this manner we have used the yeast, Saccha- romyces cereuisiae, grown anaerobically as an experimental system. Other cells which have been used in uiuo for structure- activity correlations include the Pythiaceous fungi (6, 7), choleplasmas (8-ll), yeast mutants (4, 12), and pleuropneu- monia-like organisms (13). Studies of model systems, valuable as they have proven to be (5, 14-16), have not yet progressed

* This work was supported by Grant AM-12172 from the National Institutes of Health. Certain aspects have been the subjects of prelim- inary communications (1, 2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The “dominant” sterols are those comprising approximately 90% of the mixture in question (3, 4). Thus, the dominant sterols of living systems as a whole appear to be unsaturated derivatives of cholestanol and 24-alkylcholestanol, while in man there is a single dominant sterol, cholesterol.

to the point where all the components of a given membrane can be assembled artificially.

S. cerevisiae has a number of particularly advantageous characteristics for research of this kind. In the first place, although it is eukaryotic, elimination of oxygen prevents the conversion of squalene to sterol without inducing death due to deprivation of energy. Sterols with various structures can therefore be administered and the effects on growth and other parameters measured without contamination from endoge- nous sterol. Most of the other eukaryotic cells, notably those of vertebrates, having an obligatory requirement for the op- eration of the cytochrome system, will not tolerate anaerobi- osis. The ability of anaerobic yeast to respond favorably to a sterol supplement was discovered in the early 1950’s by An- dreasen and Stier (17) who also presented suggestive evidence for the fact that cholesterol will not satisfactorily replace ergosterol. The yeast system has been exploited in the labo- ratories of Linnane and co-workers (18) and Hossack and Rose (19). However, unlike Andreasen and Stier (17), the more recent authors (18, 19) have concluded that yeast has little specificity in its sterol requirement. Our own preliminary work (2) corroborated the original findings (17), and the purpose of the present paper is to document this in detail and to expand the results to a number of other sterols. The sterols used in our investigation have been chosen for their ability to reveal the importance of precise molecular features. The structural difference, for instance, between cholesterol and ergosterol is multifaceted involving the presence and absence of a methyl group as well as of two different double bonds. We made these changes in a step-wise manner so that the contribution of each could be assessed. In addition, we examined the impor- tance of the configurations at C-20 and C-24, the significance of the conformation about the 17(20)-bond, and the effect of nuclear methyl groups.

A second reason for choosing yeast was that the structure of its sterol (ergosterol) has been extremely well defined both by classical methods (20) and more recently with the help of proton magnetic resonance spectroscopy which has clarified a number of configurational questions (21). These various investigations lead to structure la (Scheme 1) for ergosterol. It is a A~,7,U~-lranS -triene bearing a 24P-methyl group, BOcu-H- atom, and 3/3-hydroxyl group. No epimers accompany ergos- terol in S. cerevisiae (21). The only real uncertainties left about the structure are precise positioning of C-atoms about the 20(22)-, 23(24p, and 24(25)-bonds in the preferred confor- mations and the effect, if any, of complexing with phospholip- ids when interdigitation occurs to form a lipid leaflet in membranes. In the absence of complexing, the saturated car- bon atoms probably exist primarily in the staggered confor- mation as shown by structure la which ignores the stereo- chemistry at C-25. With respect to the latter, two staggered conformations are possible and are depicted by structures lb and lc. Of these, we prefer lb, because, as more readily seen

6218

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Importance of Structural Features of Ergosterol in Yeast 6219

H

C-26 C-27 HmC-28 HmC-28

C-23 C-23

Id le SCHEME 1. Structures of ergosterol (la; preferred conformer

about 17(20)-bond) and 20-isoergosterol (2, not preferred conformer at 17(20)-bond), and conformational possibilities for the end of the side chain (lb and Ic). The Newman projections (Id and le) refer, respectively, to structures lb and Zc. The arrow (lb to Ic) implies 120’ rotation of C-25 relative to C-24 in the direction taking C-26 behind the plane of the paper. Conformer lb would have less methyl- methyl interaction and is probably the preferred one.

from the Newman projections (Id and le) below the structures in Scheme 1, the H-atoms disperse the C-atoms more effec- tively in lb. Structure lb (equivalent to Id) possesses a 25p- H-atom, and lc (equivalent to le) has a 25a-H-atom. The nomenclature’ is discussed in detail elsewhere (4, 22).

A third, less crucial, but nevertheless important reason for studying S. cerevisiae is that it can be considered represent- ative of a substantial number of eukaryotic organisms based on the presence in many of them of ergosterol or a closely related sterol, e.g. one of its dihydro derivatives. Ergosterol itself, named for its discovery (23) in ergots produced by the fungus Claviceps, is the dominant sterol’ of all yeasts, many other fungi, especially Ascomycetes and Basidiomycetes, some algae, and certain other microorganisms as reviewed in detail elsewhere (4, 22, 24, 25). It also forms a part of the sterol mixture of the primitive tracheophyte, Lycopodium complan- atum (26, 27), and the fruit of a citrus tree (28). Thus, structure-activity correlations with yeast probably do not constitute an isolated relationship. Moreover, certain of the chemical features we have examined are common to all of the usual sterols including cholesterol. In particular, the stereo- chemistry at C-20 is the same not only in animal, fungal, and algal sterols but also in those of vascular plants (21, 26, 29). Similarly, in all living systems biosynthesizing sterol, the sterol is derived from a 4,4,14-trimethylsterol (4).

’ Conventional use of the Greek letters of the alphabet (a and ,0) for configurations is reversed in the nucleus and the side chain. Substituents on the front side of the nucleus are designated p, those on the back 01. In the side chain, when it is oriented in the staggered right-handed conformation as shown in la (Scheme I), substituents in front are designated (Y and those in back p.

EXPERIMENTAL PROCEDURES

Materials-Cholesterol, 5oc-cholestan-SD-01, and 5a-cholestane were recrystallized commercial samples. Campesterol and ergosterol were also obtained commercially. That they are configurationally pure samples was demonstrated by nuclear magnetic resonance spec- troscopy (26, 30). “Campesterol” from most sources, however, is an epimeric mixture (30). Brassicasterol, derived from rape seed oil, was the gift of Dr. Henry Kircher, University of Arizona. 7-Dehydrocho- lesterol was prepared chemically from cholesterol, and 24p-methyl- cholesterol was synthesized from ergosterol (31). I’regnenolone served as the starting material for the synthesis of 20oc-hydroxycholesterol (32) as well as for (E)-17(20)-, and (Z)-17(20)-dehydrocholesterol (33). 20-Methylcholesterol was prepared as described in the literature (34). Cycloartenol was isolated from Strychnos nux vomica (35, 36). Lan- osterol was purified from commercial “lanosterol” derived from sheep’s wool. Commercial “lanosterol” contains roughly equal amounts of lanosterol and 24.dihydrolanosterol together with a few per cent each of agnosterol (lanost-7,9(11),24-trien-3/%01) and 24. dihydroagnosterol (37). We found that on lipophilic Sephadex (Lipi- dex-5000 developed with 5% methanol in hexane) agnosterol moves the fastest followed by a mixture of the lanosterol and 24-dihydroag- nosterol with the 24-dihydrolanosterol moving the slowest. Samples of lanosterol and 24-dihydrolanosterol obtained in this way were used as substrates for the yeast experiments. Lanosterol purified by argen- tation chromatography had about the same purity as did the one from Sephadex separation. The gas-liquid and other chromatographic properties, melting points, and mass and nuclear magnetic resonance spectra of the various compounds were consistent with the expected structures and with a high degree of purity. Lanosterol was approxi- mately 95% pure; the purity of the other sterols approached 100%

Recovery of Sterok from Yeast-Yeast cells, collected by centrif- ugation, were extracted in a Soxhlet apparatus for 24 h. The resulting material was saponified (10% KOH in methanol at the boiling tem- perature for 10 min) and the neutral lipid obtained by dilution with water and extraction into ether was chromatographed on a thin layer of Silica Gel G in ether:benzene (1:9). Sterols lacking methyl groups at C-4 move somewhat slower than those bearing methyl groups at this position under these conditions. Cholesterol and lanosterol, placed on the edges of the plate, were used to mark the approximate positions where the 4-desmethyl- and 4,4-dimethylsterols should ap- pear in the chromatogram. The exact regions were made visible by the use of a spray of 2’,7’-dichlorofluoroscein. The sterols were eluted with ether and analyzed by gas-liquid chromatography on XE-60 at 235°C in a 6-foot U-tube equipped with a flame ionization detector. Cholesterol was the standard for determination of the relative reten- tion times (RRT). Examples of RRT are: 7-dehydrocholesterol, 1.20; 24P-methylcholesterol, 1.29; ergosterol, 1.35; lanosterol, 1.46; 24.di- hydrolanosterol, 1.25; and cycloartenol, 1.80. The presence or absence of a A”,‘-diene system was determined by UV spectroscopy in ethanol on a Cary spectrophotometer, model 15.

Anaerobic Yeast Culture-A wild type diploid Saccharomyces cereuisiae (ATCC 18790) was grown in continuous culture at 27°C in a synthetically compounded yeast nitrogen base as previously de- scribed (17) except that only the following vitamins were included: biotin, calcium pantothenate, nicotinamide, and pyridoxine and thia- min hydrochlorides. Transfers into fresh medium were made every few days. The apparatus has been described by others (38). The atmosphere above the culture was commercial nitrogen which was passed through two solutions of alkaline pyrogallol (39). The medium contained 1.0 mg of ergosterol per liter and the sterol was added in Tween-80. The Tween-80, which amounted to 15 ml/liter of medium, not only emulsified the st.erol but provided a source of unsaturated fatty acid which is necessary for anaerobic growth (40). These con- ditions were always used for continuous culture. However, we have found that 1.0 mg/liter of ergosterol is actually too small a concentra- tion to support growth under more thoroughly anaerobic conditions (N, scrubbed with CrCL (39)), and in continuous culture the cells must partially or wholly use sterol derived endogenously. The effect of sterol concentration is one of several parameters to be presented later in a more detailed consideration of the means for and effects of anaerobiosis. Continuous culture in the manner just described is not regarded by us as growth under anaerobic conditions but rather as a situation in which the availability of molecular oxygen has been severely limited. In this situation yeast was found to undergo a marked change with time. When cells were transferred to an atmos- phere of Nz scrubbed with CrClz, the cell count with cholesterol depended on t.he time the cells had been kept in continuous culture.

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6220 Importance of Structural Features of Ergosterol in Yeast

The ceII count increased with increasing time of prior continuous culture as will be described in greater detail elsewhere. Cells grown in continuous culture for 2 to 6 weeks, which we describe as “new,” were the ones used, except where noted, in the present report. Our ability to repeat results with a new culture is illustrated in Table I. With six different cultures examined over 2 years, cholesterol-induced growth averaged 23% of the ergosterol-induced growth. The highest value was 34% at 5.0 weeks and the lowest value was 11% at 2.5 weeks which reflects some adaptation during the 2.5-week interval. A plot of the data in Table I gives average values of 13, 20, 28, and 36 at 2,3,4, and 5 weeks, respectively, with an error of 10%. Cells grown in continuous culture for several months or more are designated as “old.” Choles- terol-induced growth with old cells reached approximately 80% of the ergosterol-induced growth.

For the analysis of sterol-induced growth, cells from the continuous culture were taken at log phase. An inoculum of 2.0 ml was injected into a l-liter bottle in an atmosphere of commercial nitrogen of the highest purity passed through two towers of 0.4 M CrCL. This yielded an initial population of about 1.0 x lo” cells/ml. The same medium as used for the continuous culture was used except that the sterol concentration was 5 to 20 mg/liter. Growth showed little, if any, dependence on the sterol concentration in this range, and in the case of ergosterol it gave a maximal cell count (1 X 10’ cells/ml) at stationary phase. Stationary phase was reached by 72 h at 27°C with ergosterol or cholesterol; the quantitative data reported were obtained at this time and temperature unless otherwise noted. Six experimental vessels were used simultaneously. One always contained ergosterol and one no sterol as controls. The nitrogen for all six vessels came from the same source, i.e. from the same tank and scrubbing towers. Crude N, was passed through the vessel filled with sterilized medium for 17 h at 50 ml/min. CrC&crubbed Na of the highest purity was then passed through the vessel at 35 ml/min for 7 h just prior to inoculation after which no gas was allowed to enter. The experimental vessels were closed with a rubber stopper which contained an injection port, an inlet-tube for Na, and an outlet-tube for CO2 passing under ethanol to prevent back-diffusion of air. All places where the stopper was in contact with the experimental vessel, injection port, or tubing were sealed with molten paraffin. The injection port was pierced for the inoculum and then paraffin-sealed. The paraffin sealing was found to be essential in order for no growth to occur in the vessel with no sterol. A condition of no growth was achieved with this system in the absence of sterol, while growth proceeded in the presence of ergos- terol. We are unaware of this degree of anaerobiosis having been obtained after so long a period of time by other investigators, and difficulty in achieving consistent results has already been noted in the literature (17, 41). Some cell counts reported earlier are for much shorter times (18) than in our case which does not obviate the problem.

Cell Counting and Sizing-Cells were counted in two ways. The number of individual cells per ml at or approaching maturity (as large as or more than one-half the size of the parent cell) whether attached to, aggregated with, or independent of other cells was determined microscopically. Cells were also counted and sized with a Coulter Counter model TArr equipped with a population accessory having sixteen channels (size ranges). The instrument automatically calcu- lated and plotted both the size distribution in terms of the per cent of the cells having a given range of diameters (assuming the shape of a sphere) and the volume distribution in terms of the per cent of a given range of volumes relative to the total volume. Since the volume increases exponentially with respect to the diameter, the two distri- butions are not identical. A few very large particles will affect the latter curve more than the former. The Coulter Counter senses aggregates as single individuals, while the visual count discriminates single individuals in an aggregate. Operationally we refer to Coulter data as “particle counts” and visual data as “cell counts.”

RESULTS AND DISCUSSION

The plan of research in its simplest form was to examine

pairs of substrates differing structurally from one another by

a single feature (moiety) and then in effect to construct a list

of relative contributions which each moiety makes to the

activity. In order to do this, we wanted to use sterols which

did not necessarily possess the methyl group at C-24. The

absence of the methyl group would allow,us to assemble a

group of substrates which had the precise variations desired

without always having to cope synthetically or otherwise

(isolations, etc.) with an asymmetric carbon atom. If, of course, the 24/3-methyl group were essential for any biological activity

at all, then a different course of action would have become

necessary. Happily this was not the case. The easiest molecule

to obtain which resembles ergosterol most closely without its

actually having the methyl group was 7-dehydrocholesterol.

This sterol also lacks a Az2-bond but retains the A537-diene

system found in ergosterol. 7-Dehydrocholesterol is in effect

ergosterol’s 24-desmethyl-22-dihydro derivative. When it was

used as substrate, the yeast grew to 23% of the level induced

by ergosterol (Table II). This immediately demonstrated that

the biological behavior of sterols is, with respect to structural

moieties, not necessarily a simple “all-or-nothing” phenome-

TABLE I

Anaerobic yeast cell stationary populations

Culture Age of in- Date of cell Visual cell count” Percent-

NO. OCUlUlIl” count age Cholesterol Ergo&ml growth’

I 2.0 l/27/76 I 3.0 2/2/76 I 4.5 2/13/76

II 4.0 8/9/76 II 5.0 8/16/76

III 3.0 6/17/77 III 3.5 6/20/77 IV 2.5 8/12/77 IV 3.0 8/15/77

v 3.5 10/26/77 VI 3.0 12/12/77

cells/ml

14 x IO” 82 x 10” 17 24 x 10” 75 x 10” 32 25 x 10” 101 x 10” 25 26 x 10” 93 x 10” 28 38 x 10” 113 x 10” 34 15 x 10” 113 x 10” 13 29 x 10” 89 x 10” 33 13 x 10” 115 x 10” 11 24 x 10” 101 x 10” 24 19 x 10” 93 x 10” 20 14 x 10” 105 x 10” 13

U The inoculum was derived from a continuous culture in which transfers of fresh medium were made once or twice a week. The age is the time from initial inoculation with a standing test tube culture maintained in air.

‘Cell counts were obtained after 72 h of growth in the sealed experimental vessels.

‘ Cell count in cholesterol-containing medium divided by that in ergosterol-containing medium times 100.

TABLE II

Effect of sterol structure on the growth of new cells of anaerobic yeast

stem1 Per cent”

5n-Cholestane t1

5a-Cholestan-3,&ol 23

Cholesterol 23

Lathosterol 38

7-Dehydrocholesterol 23

24a-Methylcholesterol 32

24,&Methylcholesterol 75

Brassicasterol 100

Ergosterol 100

Lanosterol tl

24.Dihydrolanosterol t1

Cvcloartenol <I

” The per cent is the cell count after 72 h (in the sealed experimen- tal vessels) divided by the count obtained with ergosterol controls. The values are averages of at least two experiments. Deviation from the average rarely exceeded 5%. The maximum deviation (10%) was obtained with cholesterol for which more extensive data were ob- tained (~6 Table I). The age of the cells used varied from 2 to 5 weeks.

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Importance of Structural Features of Ergosterol in Yeast 6221

non as suggested by others (18) but rather that the effect of the sterol is a quantitative phenomenon. In order to examine the problem further it was next necessary to determine whether removal of the diene system (A”,‘) in ring B would abolish activity. In particular, would the change from 7-de- hydrocholesterol to cholesterol prevent growth? Examination of this point was necessary to establish whether we could do our further studies in the a”-series or whether all substrates would necessarily have to be converted to their a”,7-deriva- tives. Fortunately, from an experimental point of view, cho- lesterol also induced some growth. The number of cells (Table II) was similar to what was found with 7-dehydrocholesterol. This indicated that little difference is associated with the degree of unsaturation in ring B and allowed us to proceed to an examination of other features.

The contribution which addition of a 24P-methyl group makes was assessed through the use of 24P-methylcholesterol as the substrate. The yeast not only grew very much better than with cholesterol, but the response (Table II) was nearly as good as with ergosterol. When a double bond was added at C-22 to give 22-trans-dehydro-24P-methylcholesterol (brassi- casterol; 7-dihydroergosterol), the yeast grew in a manner nearly indistinguishable from that obtained with ergosterol (Table II). The precise character of ergosterol’s side chain consequently appears to be quantitatively more important than the degree of unsaturation in ring B, but an exacting analysis of the significance of the configuration and size of a group at C-24 is complicated by a rotational problem. How- ever, if we empirically restrict ourselves for the time being to the question as to whether or not the configuration at C-24 is important in the case of a methyl group, the answer can be given; yes, it is important. We have incubated 24a-methylcho- lesterol (campesterol) with the yeast and found the growth- response to be poor. Campesterol gave a cell count (Table II) less than half the value found with its epimer (24P-methyl- cholesterol). A more incisive examination of the effect of changing the environment at C-24 will be the subject of a subsequent paper.

An interplay between configuration and conformation also exists at C-20. Since rotation about the 17(20)-bond will place the three groups on C-20 one by one in opposition to C-18 (the so-called “angular” or axial methyl group between rings C and D) in each of the three skew conformations, there will be increasing stability to these rotameric conditions as the size of the group opposing the angular methyl group decreases. The smallest is the 20a-H-atom, and, when this is in front and opposing (in a 1,3-diaxial relationship with) the angular methyl group, C-22 is to the right (trans-oriented to C-13) in the usual view of the molecule. We refer to a sterol with C-22 to the right as being “right-handed.” If the configuration at C-20 is inverted, C-22 will have to be on the left (“left- handed”) in order for the H-atom, now with a P-configuration, to be in front. As a first approximation one would guess that the conformer associated with membranes would be the one which is intrinsically the most stable. This assumption would lead to the following predictions. Since ergosterol is a sterol with a 20a-H-atom, the “right-handed” conformer (la in

Scheme 1) would be the one supporting the growth of yeast. Its 20P-epimer (20-isoergosterol) would be inactive, but only if bimolecular complexing (with phospholipid or protein) has other stereochemical components. 20-Isoergosterol can in fact assume a “right-handed” skew conformation (2 in Scheme 1) even though it is not preferred. In this condition the H-atom and C-21 have been exchanged compared to the natural sterol. The methyl group (C-21) will now be in front. I f space is not available at this point in the molecule with which the sterol complexes, the 208-epimer would be inactive, while the pres-

ence of space for the methyl group ought to permit fit and therefore activity. We have been able to sort out these various factors in the AC-series by fixing C-22 rigidly to the right or left and by replacing the H-atom at C-20 with other groups.

The sterol side chain is attached to ring D through a single u-bond which permits rotation of the two carbon atoms (C-17 and C-20) relative to one another, unless bulk prohibits it which does not appear to be the case (29, 42, 43). When a double bond is present at the juncture, i.e. a A’7(20’-bond, rotation is prevented, and two isomers (cis and tram, 3 and 4, Scheme 2) are possible which are not interconvertible. We have incubated the yeast with the two 17(20)-dehydrocholes- terols (3 and 4, Scheme 2) using an old culture. We anticipated that one isomer might and the other probably would not induce growth, if the double bond itself did not destroy activity. In one case (4, Scheme 2, the (E)- or trans-isomer, with C-22 to the right), the side chain is approximately in the same position (“right-handed”) as in sterols with a 20a-H- atom, while in the other (3, Scheme 2, the (Z)- or c&isomer) C-22 is on the same side as C-13 (“left-handed”). We have recently demonstrated a profound difference in the fate of these isomers in the protozoan Tetrahymena pyriformis (1, 44). Only the “right-handed” isomer is metabolized, the “left- handed” one being recovered unchanged. The same influence of stereochemistry was found in growth support given to anaerobic yeast (Table III). The “right-handed” isomer in- duced almost as much growth as did the analogous sterol, cholesterol, with the 17(20)-bond saturated, but the “left- handed” isomer promoted no growth at all. The failure of the “left-handed” isomer to induce growth in cells from an old culture is especially significant, since cells from an old culture grow more profusely than do those from a new one. We interpret the results to imply that C-22 is on the right side when sterols complex with other constituents of the mem- brane. Placing C-22 in front or in back ought to make an even bigger steric difference than placing it on the left where it is found to be inconsistent with activity. It follows from the

5 R

4

OH \ . , -1

-R -\

ti

5

7

6

SCHEME 2. Structures of sterols in the As-series with varying ster- eochemistry at the A”““‘- bond or different substituents or stereo- chemistry at C-20. R = (CH&CH-(CH.,)2; 3 = (Z)-17(20)-dehydro- cholesterol; 4 = (E)-17(20)-dehydrocholesterok 5 = 20whydroxycho- lesterol; 6 = 20.methylcholesterol; 7 = 20-isocholesterol.

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6222 Importance of Structural Features of Ergosterol in Yeast

TABLE III

Effect of stereochemistry at C-20 of sterols on the growth of old cells of anaerobic yeast

Stero! Per cent"

Cholesterol 100

20-Isocholesterol t1

(E)-17(20)-Dehydrocholesterol 69

(Z)-17(20)-Dehydrocholesterol <l

Boa-Hydroxycholesterol <I

20.Methylcholesterol <I

” The per cent is the cell count after 72 h (in the sealed experimen- tal vessels) divided by the count obtamed with cholesterol controls. The values are averages of duplicate experiments with deviations from the average of 1%.

results that the A17”“‘-bond per se does not prevent activity and consequently that C-20 does not have to bear a substituent (even the Boa-H-atom) in the plane perpendicular to the molecule.

When, instead of being eliminated, the 20Lu-H-atom was replaced with an OH- or CH:l-group, giving 20a-hydroxycho- lesterol (5, Scheme 2) and 20-methylcholesterol (6, Scheme 2), no growth occurred (Table III). In the “right-handed” conformer of these sterols (Scheme 2), which from the work with the A’7’““‘-sterols should be the one required for complex- ing, an OH- or CHZi-group projects in front. Similarly, in 20- isocholesterol ( 7, Scheme 2), also inactive (Table III), a CHa- group (C-21) is in front in the “right-handed” conformer. The abolition of activity by placing a group in front regardless of its polarity (OH or CH:,), regardless of whether the “right- handed” conformer is probably preferred (20a-hydroxy- and 20-methylcholesterol) or not preferred (20-isocholesterol), and regardless of whether there remained (in 20a-hydroxy- and 20-methylcholesterol) or did not remain (in 20-isocholesterol) a group on the left suggests a very close approach of some molecule (phospholipid, etc.) to the front face of the sterol when complexing occurs (presumably in the membrane).

A very close fit of the sterol when it complexes was also indicated by the lack of activity (Table II) of lanosterol and 24-dihydrolanosterol. They have overall nuclear shapes very closely related to those of cholesterol, ergosterol, etc., except that methyl groups project axially to the rear (the 14a-methyl group), axially in front (4/3-methyl group), and equatorially toward the bottom (4cr-methyl group). One or more of these methyl groups must be thoroughly inconsistent with fit,” since the side chain, being the same as in cholesterol cannot have prohibited growth. Nor can the Ax-bond have prohibited growth, since several viable yeast mutants have been produced (45-47) which have AH-sterols, as in the case of Nys-300 with 90% of the sterol mixture being 24-methylenecholest-8-en-3P- 01 (fecosterol). Similarly, the inactivity we observed when the nuclear methyl groups were introduced is corroborated by the fact that yeast mutants lacking the ability to demethylate and accumulating sterols with one or more of the methyl groups require an exogenous source of sterol (48-50). In the case of one of these mutants (48), incidentally, cholesterol was found to be less efficient in supporting growth than was ergosterol which parallels our own findings about the importance of

,’ An alternative but related view would be that improper rather than no fit occurs leading to a complex with sufficiently altered geometry to disrupt the membrane so severely that growth is not possible.

alkylation at C-24. The detrimental effect which one or more of the nuclear methyl groups has explains why yeast has a complicated enzymatic system for their removal and why yeast does not normally have a large steady-state concentra- tion of such methylated sterols as lanosterol. Since 4,4,14- trimethylsterols quite generally are in low concentration and are converted to their 4,4,14+isdesmethyl derivatives in living systems, it would appear that some of the steric requirements for membranous architecture are the same from one organism to the other. Sterols retaining just the equatorial 4a-methyl group have been found more often than those with just a 14a- methyl group (4, 22), and sterols with just a 4/?-methyl group probably do not exist (4,22). The data on occurrence probably reflect relative concentrations and therefore also suggest that fit of the sterol into membranes depends on the steric condi- tion of the face(s) of the molecule.

We have also examined the ability of cycloartenol to support the growth of anaerobic yeast. Cycloartenol is the intermedi- ate between squalene oxide and 4,4,14-trisdesmethylsterols in photosynthetic plants (4, 22) and is not metabolized by yeast (51). Based on the work with lanosterol, it should not have and experimentally did not (Table II) give any support to growth.

A variety of studies have shown that the hydroxyl group at C-3 cannot be removed with retention of membranous activity in a sterol. For instance, while 5cu-cholestan-S/Lo1 will support growth of choleplasmas (8, 52), where the sterol is known to be in the membrane, the corresponding hydrocarbon (5a- cholestane) will not act in a positive manner (52). Similarly, yeast deprived of oxygen has been reported to respond to the stanol but not to the hydrocarbon (18). We have reexamined the yeast case with the same results (Table II). Growth was induced by 5Lu-cholestan-3P-01, and the growth-response to the sterol was quantitatively, but not qualitatively, similar to that with cholesterol. The cell counts were similar, but dead

cells seen microscopically with cholesterol were not observed with the stanol. The corresponding hydrocarbon (5cy-choles- tane) did not induce any growth at all.

Growth-support which some of the sterols give to yeast could conceivably be the result of a metabolite of the sterol. To assess this point, we selected five representative cases to examine for metabolism. Cells were grown in batches of 6 liters each in the presence of ergosterol to obtain enough sterol (6 mg) from the lyophilized cells to examine well. Isolation of the sterol from the neutral lipids was accomplished on a column of AleO:l deactivated with 3% water. An eluent of ether graded into hexane was used. Both the total neutral lipids and the sterol fraction from the chromatogram showed a single sterol peak (1% or less of other sterols) when submit- ted to gas-liquid chromatography. The position of the peak (RRT 1.33) was the same as that of authentic ergosterol. No lanosterol was detected in the 4,4-dimethyl region of the chromatogram. The ultraviolet spectrum of the reisolated ergosterol showed A,,,, 272, 282, and 294 nm with a shoulder near 265 nm and a minimum at 230 nm. These characteristics are typical of b’,7-sterols. The mass spectrum showed, as expected for ergoeterol (27), m/e 396 (M’, lOO%), 381 (M’ - CH:s, 9%), 363 (M’ - CH:, - HZO, 93%), 337 (M+ - H,O - CnH5, 43%), 271 (M” - side chain, 25%), 253 (M+ - HZ0 - side chain, 55%), and 211 (M” - Hz0 - side chain - CZIHs, 34%). The data prove that the administered ergosterol was present in the cells in the absence of hydrogenated derivatives or other simple metabolites, e.g. more highly dehydrogenated ones, from which it is reasonable to believe that it was ergos- terol itself which was acting functionally. Although as exten- sive an analysis was not made with other sterols, we did isolate sterol from l-liter cultures of yeast grown in the presence of

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Importance of Structural Features of Ergosterol in Yeast 6223

FIG. I. Distributions of sizes and vol- umes of anaerobic yeast cells grown on 24-desmethylsterols as measured on a Coulter Counter after growth for 72 h at 27°C. The ‘%B of Population is the number of particles with a given diameter divided by the total number of particles. The %’ of Total Volume is the volume of parti- cles of a given diameter divided by the total volume of all particles. A = B = cholesterol; C = D = lathosterol; and E = F = 7-dehydrocholesterol.

l0i

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I 2A2.5 524A5AdA ‘,‘,‘I’,’ 10 l3 16 2o

I 25

cholesterol, lathosterol, 7-dehydrocholesterol, and 24P-meth- ylcholesterol. Purification of the neutral lipid fractions was achieved by thin layer chromatography. In the cases of cho- lesterol, 7-dehydrocholesterol and 24P-methylcholesterol, the rates of movement (RRT 1.00, 1.20, and 1.27, respectively) in gas-liquid chromatography coincided with expectations for the three substrates. The peaks obtained indicated less than 2% of other sterols in the cases of cholesterol and 24/$meth- ylcholesterol and less than 7% in the case of 7-dehydrocholes- terol. When lathosterol (the AT-analog of cholesterol; cholesta- 7-en-3P-01) was used as the sterol-supplement, it also was recovered (RRT 1.12) but was accompanied by a substance with a clearly defined peak (RRT 1.04) representing as much as 11% of the total reisolated desmethylsterol. It is not certain whether this was a sterol, but it probably was. In view of this uncertainty, the question of metabolism as well as of growth response in the case of lathosterol cannot be considered defin- itive, but tentatively it appears as if lathosterol does support growth to some degree (Table II). This is consistent with the fact that A7-st,erols comprise 80% of the mixture from some viable nystatin-resistant yeasts (Nys-30 and Nys-200) which do not require exogenous sterol (46).

Ergosterol-induced growth yielded single cells at stationary phase appearing round under the microscope. Such cells are among those to be seen in our previously published photo- micrograph (2) of yeast grown in the presence of cholesterol. Thus, some of the cholesterol-induced cells seem to be normal, but this condition is illusory, because many of the cells are quite abnormal. Abnormal character is evident from dark and apparently dead cells, from the failure of buds to separate from the parents (pseudomycelia), and from dark fragments. 7-Dehydrocholesterol induced a population of cells exhibiting the same characteristics.

The morphologic effect was quantitated through the use of a Coulter Counter (Figs. I and 2). Ergosterol-induced cells had a narrow distribution of sizes. In other words, the popu- lation tended to be homogenous with the most often encoun- tered diameter being near 6 pm. As the structure of ergosterol was changed successively through removal of the A7- and A”-

DIAMETER (urn)

bonds, as shown in Fig. 2, the number of larger particles increased. When the 24/3-methyl group was removed (Fig. l), both smaller and larger particles became important contrib- utors to the distribution. In addition, particles of very small diameter (t3 pm) appeared, and the distribution became bimodal with a minimum between 3.2 and 4.0 pm. Actually, there could have been particles even smaller than this, but for instrumental reasons they were not measurable. We believe the small particles represent the fragments seen microscopi- cally. The very large particles with diameters in excess of about 10 pm were probably the pseudomycelia evident from the visual examination. The particle count for cholesterol- grown cells was also always lower (less than half) than the cell count. This can be seen from the growth curves in the earlier paper (2). We believe this difference in counts parallels the size distribution and reflects the presence of pseudomy- Celia in which we visually counted several cells of an aggregate as individuals, while it is the aggregate (particles) which should have been sensed by the Coulter Counter as an indi- vidual. Similar divergence in the particle and cell counts was found with cells grown on 7-dehydrocholesterol, which we interpret as was done for the cholesterol case.

In summary, each feature of ergosterol seems to have some functional significance in yeast, although the contribution which each makes to viability does not appear to be quanti- tatively the same. The hydroxyl group at C-3, configuration at C-20, and the absence of one or more nuclear methyl groups appear to be the most crucial of the features examined, and ergosterol seems to act biologically in the form of its right- handed conformer (C-22 trans-oriented to C-13). The degree of unsaturation in ring I3 plays a minor role. The presence of a methyl group at C-24 proved to be the next most important feature, with the natural ,&configuration being better than the epimeric condition. Without the methyl group, ignoring a small contribution of the A”‘-bond, sterols had severely low- ered activity; cells could not maintain their normal ability to separate from parents, and death and fragmentation pro- ceeded. A lowered cell count which also occurred with sterols lacking the methyl group could in principle be derived from

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6224

5

Importance of Structural Features of Ergosterol in Yeast

50 A

T B

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’ 5.0 ’ 8D;013 ,;202;

DIAMETER (urn)

premature death which would have the effect of negating a logarithmic increase in the cell numbers. However, slower reproduction itself may also be involved. Ergosterol and cer- tain other sterols were recovered from the anaerobic cells unchanged after having been administered exogenously in the medium. This indicates ergosterol is used functionally without being metabolically altered which is consistent with its normal presence in yeast as the dominant sterol. When yeast was grown on other sterols, e.g. cholesterol, no ergosterol or other sterol was found in the anaerobic cells. This is taken to mean inconsequential endogenous biosynthesis of sterol occurred under our conditions.

The failure of earlier workers (18, 19) to find a significant difference in the response of yeast to different sterols, e.g. cholesterol, ergosterol, and lanosterol, all of which are re- ported to be active, leading to the suggestion that yeast lacks molecular specificity in regard to its sterol requirement (18),

probably has its origin in the experimental difficulties which one has in obtaining operationally anaerobic conditions. That oxygen was far from absent in one study (19) is evident from the reported presence of substantial quantities (approximately 30%) of ergosterol in the sterol mixture derived from cells which had been cultured “anaerobically” in the presence of sterols other than ergosterol. The lack of differential response in that study (19) to the sterols which had been presented in the medium could be explained by an overriding effect of the endogenously biosynthesized sterol.

Achnowledgments-We thank Mr. M. Young and Mr. K. Krevitz for technical assistance.

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FIG. 2. Distributions of sizes and vol- umes of anaerobic yeast cells grown on 24b-methylsterols as measured on a Coulter Counter after growth for 72 h at 27°C. The Qx of Population is the number of particles with a given diameter divided by the total number of particles. The 9’ of Total Volume is the volume of parti- cles of a given diameter divided by the total volume of all particles. A = B = 24P-methylcholesterol; C = D = 22. trans.dehydro-24/j-methylcholesterol (brassicasterol); and E = F = 7,%2-truns- bisdehydro-24P-meth,ylcholesterol (er- gosterol).

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W R Nes, B C Sekula, W D Nes and J H AdlerThe functional importance of structural features of ergosterol in yeast.

1978, 253:6218-6225.J. Biol. Chem. 

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