Archives of
Hicrobiology
Arch. Microbiol. 117, 239-245 (1978)
9 by Springer-Verlag 1978
Plasma-Membrane Lipid Composition and Ethanol Tolerance in Saccharomyces cerevisiae D. Susan Thomas, J. A. Hossack, and A. H. Rose Zymology Laboratory, School of Biological Sciences, Bath University, Claverton Down, Bath BA2 7AY, Avon, England
Abstract. Populations of cells suspended anaerobically in buffered (pH4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration. Key words: Plasma membrane Lipids Saccharomyces cerevisiae - Ethanol tolerance Sterols - Fatty-acyl residues.
m
Although many strains of yeast remain viable in the presence of concentrations of ethanol (3 - 4 ~ w/v) that are lethal to other micro-organismS, little is known of the molecular basis of this tolerance. Gray (1941) found that ethanol tolerance is not confined to any one genus or species of yeast, and went on to report (Gray, 1945) that induced tolerance of glucose in Saccharomyces cerevisiae, brought about by sequential transfer into media containing higher concentrations of glucose, was accompanied by a decrease in ethanol tolerance. Examination of a range of yeast species subsequently revealed that those which tolerate high concentrations of ethanol store less lipid and carbohydrate than less tolerant strains (Gray, 1948). Troyer (1953, 1955) largely confirmed the results of Gray (1948), and further reported that methanol is generally less toxic than ethanol. Thereafter, ethanol tolerance in yeasts
failed to attract research interest until Hayashida et al. (1974) and Hayashida and Hongo (1976) reported that sak6 yeasts (strains of S. cerevisiae) acquired enhanced tolerance to ethanol when grown in the presence of a fraction from the envelope of Aspergillus oryzae containing unsaturated fatty-acyl residues. The present paper reports on the manner in which both the sterol and phospholipid fatty-acyl composition of the plasma membrane of S. cerevisiae NCYC 366 influence the ability of this yeast to remain viable in buffered suspensions containing ethanol. The rationale behind the study was that the plasma membrane is the first sensitive organelle to make contact with ethanol when cells are suspended in ethanol solutions, and that since ethanol is an amphipathic compound the lipid composition of the plasma membrane may have an important effect on ethanol tolerance. The lipid composition of the plasma membrane in S. cerevisiae NCYC 366 was altered by exploiting the anaerobically-induced requirement of this microbe for a sterol and an unsaturated fatty acid (Andreasen and Stier, 1953, 1954), both of which requirements are fairly non-specific (Light et al., 1962; Proudlock et al., 1968), as described by Hossack and Rose (1976).
Materials and Methods Experimental Cultures. Saccharomyces cerevisiae NCYC366 was grown anaerobically in medium supplemented with an unsaturated fatty acid (30 rag/l) and campesterol, cholesterol, ergosterol or stigmasterol (5 rag/l), and harvested from mid exponential-phase cultures (0.24-0.26 mg dry wt/ml) as described by Hossack and Rose (1976) and Hossack et al. (1977). When cells were to be analysed for lipids, or converted into sphaeroplasts, 2 ml of a solution containing 10 mg each of chloramphenicol and cycloheximide were injected into the cultures through a Suba seal on the side of the flask (Alterthum and Rose, 1973), and the culture incubated for a further 15 min before harvesting. Cells to be analysed for lipid were washed twice with water at 4~ freeze dried, and stored at - 20~C over silica gel. Cells to be converted into sphaeroplasts were washed twice with 1.2 M sorbitol containing 10 mM
0302-8933/78/0117/0239/$01.40
240 MgC12 and 10 mM imidazole hydrochloride (pH 6.0). Those which were to be incubated in buffer for viability studies were washed twice in 67 mM KH2PO4 at 4~ all vessels used being flushed with oxygen-free nitrogen gas.
Viability Measurements. Viability of yeast populations was measured by plate counts and by staining with methylene blue. Yeast suspension (1.25rag dry wt/ml) was diluted in 67raM KHzPO 4 (pH4.5) containing where indicated 1.0 M ethanol, to a concentration of 50 ~tg dry wt/ml. Cell suspensions (500 ml) were incubated anaerobically as described by Alterthum and Rose (1973) at 30~ with stirring. Portions (2 ml) were removed at intervals with a hypodermic syringe 9 through the Suba seal, and diluted with 67 mM KHzPO 4 such that 0.1 ml of the diluted suspension contained 100-300 viable cells. Portions (0.1 ml) of the diluted suspension were spread onto each of three plates (8.5 cm diam.) of malt extract (0.3 % w/v) - yeast extract (0.3%, w/v)-glucose (1.0%, w/v)-mycological peptone (0.5%, w/v) - agar (2.0 %, w/v) medium (Wickerham, 1951) and incubated aerobically at 30 ~ C for 48 h, when the number of colonies on each plate was counted. Staining with methylene blue (Fink and Kfihles, 1933) gave a rapid assessment of the viability of yeast populations. A portion (1.0ml) of the undiluted suspension was stained with methylene blue solution (0.01%, w/v, methylene blue containing 2 %, w/v, sodium citrate) for 5 rain, and concentrated by filtering through a Millipore filter (0.45 ~m pore size). Cells were resuspended in 0.1 ml water, wet preparations examined under the microscope, and 1000 cells scored for viability, live cells remaining colourless.
Lipid Analyses. Lipids were extracted from freeze-dried cells by a modification of the method of Letters (1968) described by Hossack and Rose (1976) except that the residues were suspended in methanol for 15rain instead of 10rain. Classes of lipid were separated by quantitative thin-layer chromatography on plates coated with a layer (0.4ram thick) of Kieselgel HF254+366 (Merck). Plates were developed with petroleum spirit ( 4 0 - 6 0 ~ C)-diethylether-acetic acid (70: 30:2, v/v/v) containing butylated hydroxytoluene (0.005 %, w/v). Total phospholipid and individual phospholipids were assayed as described by Hossack and Rose (1976). Phospholipids were eluted from the silica gel with two portions (3 ml) of chloroform-methanolwater (5:5:1, v/v/v), followed by 3ml of methanol and 3 ml of methanol-acetic acid-water (95:1:5, v/v/v). Phospholipid was determined by assaying the phosphorus content of the extract, using the method of Chen et al. (1956). Values for phosphorus contents were multiplied by 25 to give the total phospholipid content. Sterol acetates (Kuksis, 1967) were prepared as described by Hunter and Rose (1972) and separated by gas-liquid chromatography on columns of OV-17. Fatty-acyl residues in phospholipids and plasmamembrane preparations were converted into methyl esters and separated by gas-liquid chromatography on a column of EGSS-X, as described by Hunter and Rose (1972) except that the detector temperature was 250 ~C. Peak areas were measured, and relative amounts of compounds calculated, by the method of Pecsok (1959). Isolation of Plasma Membranes. Purified preparations of plasma membrane were isolated by a modification of the Hossack and Rose (1976) technique using sphaeroplasts, which were prepared as described by Cartledge and Rose (1973). Glass vessels and centrifuge tubes used in the preparation of sphaeroplasts were flushed with oxygen-free nitrogen before use. Throughout the preparation, chloramphenicol and cycloheximide were included in all reaction mixtures each at 0.2 mg/ml. The outer surface of sphaeroplasts was labelled by lactoperoxidase-catalysed iodination (Marchalonis, 1969; Phillips and Morrison, 1970), the sphaeroplasts lysed, and plasma membranes isolated by isopycnic density-gradient centrifugation. Iodination was carried out by a modification of the method of Schibeci et al. (1973) as reported by Hossack and Rose (1976). The label indicated the position on the gradient at which plasma membrane accumulated. Purified plasma membranes were removed
Arch. Microbiol., Vol. 117 (1978) from the corresponding position on a gradient on which components oflysed unlabelled sphaeroplasts were separated because of the effect ofiodination on phospholipid fatty-acyl residues (Mersel et al., 1976).
Measurement of Ethanol Concentration and Ethanol Dehydrogenase Activity. Ethanol concentrations in culture filtrates that had been filtered through a membrane filter (Millipore; 0.45gin pore size; 2:5 cm diam.) were determined using a Biochemica Test Combination (Boehringer, Mannheim, W. Germany). The method is based on the ethanol dehydrogenase assay of Bficher and Redetzki (1951), and it was established that the method recommended for the kit did not need modification for use with culture filtrates. Culture filtrates were diluted 10 fold with water before assay. Ethanol dehydrogenase activity of cell extracts was measured by the method of Vallee and Hoch (1955) based on formation ofNADH. After centrifugation of culture, cells (about 320 mg dry wt) were washed with water at 4 ~ C, and suspended in about 15ml 0.05 M potassium phosphate buffer (pH 7.7) and 40g glass beads (0.170.18 mm diam.; Glasperlen, B. Braun, Melsungen, W. Germany) added to the suspension which was then treated in a homogenisor (B. Braun, Melsungen, W. Germany) for 4 rain at maximum speed. Beads were removed by passing the suspension through a sintered glass filter (No. 1) and the protein content of the disrupted cell suspension measured (Lowry et al., 1951). Suspensions of disrupted cells were diluted with bovine serum albumin (0.1%, w/v in 0.01%, w/v, potassium dihydrogen phosphate, pH 7.5) to decrease the cell protein content to 1 mg/ml. A quartz cuvette (1 cm path length) was charged with 1.5 ml 0.032 M sodium pyrophosphate (pH 8,8), 0.5 ml 2 M ethanol and 1.0ml 25mM NAD +, and pre-incubated at 25~ suspensions of disrupted cells were also pre-incubated at 25 ~C. The reaction was started by adding 0.1 ml disrupted cell suspension to the cuvette, and the extinction at 340nm measured after 15 and 45 s. In control reaction mixtures substrate solution or enzyme extract was replaced by water. Ethanol dehydrogenase activity is expressed as gmoles NADH formed/rain, mg protein.
Chemicals. All chemicals used were reagent grade or of the highest purity available commercially. Chloroform, ethanol and methanol were redistilled before use. Palmitoleic acid (C16:1, A9 eis-hexadecenoic acid), oleic acid (C18:1, A9 eis-octadecenoic acid), linoleic acid (C18:2, A9'12 cis,cis-octadecadienoic acid), e-linolenic acid (C18:3, A 9&z'15 cis,eis,cis-octadecatrienoic acid), and cetoleic acid (C20:h A11 cis-eicosaenoic acid) were supplied by Sigma Chemical Co. (London) Ltd., as were cholesterol and ergosterol. Campesterol and stigmasterol were purchased from Applied Science Laboratories through Field Instruments Ltd. (U.K.), and OV-17 on Gas Chrom Q and EGSS-X from Phase Separations, Rock Ferry, Cheshire, England. Sterols and fatty acids were used without further purification.
Results Ethanol Tolerance as A f f e c t e d by Sterol and F a t t y - A c y l Composition o f Cells W h e n s u s p e n d e d i n b u f f e r ( p H 4.5) c o n t a i n i n g 1.0 M e t h a n o l , p o p u l a t i o n s o f cells e n r i c h e d i n l i n o l e y l residues remained viable to a greater extent than those e n r i c h e d i n oleyl r e s i d u e s , i r r e s p e c t i v e o f t h e n a t u r e o f t h e s t e r o l e n r i c h m e n t (Fig. 1). A c o n c e n t r a t i o n o f 1.0 M ethanol was chosen since aerobic growth of Saccharomyces cerevisiae N C Y C 366 is c o m p l e t e l y inhibited when the ethanol concentration in defined m e d i u m e x c e e d s 1.2 M . H o w e v e r , p o p u l a t i o n s e n r i c h e d
241
D. S. Thomas et al. : Lipids and Ethanol Tolerancein Saccharomyces cerevisiae 100
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100
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la--d. Decreasein viability of populations ofSaccharomyces cerevisiae NCYC 366 enriched in different sterols and either oleyl (closed symbols) or linoleyl residues (open symbols), when incubated at 30~ in 67 mM KHzPO4 (pH 4.5) containing (continuous lines) or lacking (dotted lines) 1.0 M ethanol. Viability of populations enriched in campesterol is indicated by inverted triangles (a), cholesterol by upright triangles (b), ergosterol by squares (c), and stigmasterol by circles (d). Vertical bars indicate 95 ~ confidencelimits
Fig.
in ergosterol or stigmasterol, and linoleyl residues, were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues (Fig. 1). Populations of cells enriched in ergosterol and ~-linolenyl residues retained viability very similarly to those enriched in the same sterol and linoleyl residues. Populations suspended in ethanol-free buffer, irrespective of the nature of the sterol and fatty-acyl enrichments, remained almost completely viable over the duration of the incubation (Fig.l). Filtrates from cultures supplemented with ergosterol or cholesterol, and oleic or linoleic acid, and containing 0.32 mg dry wt/ml were assayed for ethanol after filtration through Millipore filters (0.45 ~tm pore size). At the 5 ~ level [points of variance-ratio (F) distribution], there was no
significant difference in the ethanol concentration in the filtrates (average 3.19 mg/ml). Assuming that one mole of ATP was produced for each mole of ethanol excreted, the Yare value for the cultures is 4.57. Lipid and Plasma-Membrane Composition of Cells Grown in the Presence o f Different Sterols and Fatty Acids Phospholipids from S. cerevisiae NCYC366, grown anaerobically in the presence of cholesterol or ergosterol, and oleic or linoleic acid, were enriched to the extent of 5 5 - 6 2 ~ with the exogenously provided fatty-acyl residue (Table 1). The degree of enrichment with linoleyl residues was slightly lower than with oleyl
242 Table 1
Fatty-acyl composition of phospholipids in
Saccharomyces cerevisiae NCYC 366 grown
Arch. Microbiol., Vol. 117 (1978)
Fatty-acyl residue
anaerobically in medium supplemented with cholesterol or ergosterol and oleic or linoleic acid
Percentage composition of fatty-acyl residues in phospholipids from cells grown in medium supplemented with Cholesterol Oleic acid
Values indicated are +_ standard errors of the mean values from two independent analyses. Values for unsaturation (A/mole) were calculated as described by Kates and Hagen (1964)
C12:0 C13:0 C14:0 C16:o Ca8:0 C18:1 C18:2 A/mole
residues. Plasma membranes from cells grown anaerobically in medium supplemented with ergosterol and oleic or linoleic acid also were enriched comparably with the exogenously provided fatty-acyl residue (Table 2). Enrichment with linoleyl rather than oleyl residues did not cause significant changes in the composition of the free sterol fraction in cells (Table 3). The phospholipid content of cells grown in the presence of oleic or linoleic acid did not differ depending on whether the sterol supplement was cholesterol or ergosterol (Table 1).
Ethanol Dehydrogenase Activities of Cells Enriched in Ergosterol and Oleyl or Linoleyl Residues Extracts of cells grown anaerobically in the presence of ergosterol and oleic acid had an ethanol dehydrogenase activity of 1.25 _+ 0.16 (S.E.M.) ~tmoles NADH formed/rain- mg protein, while extracts of cells grown in medium supplemented with the same sterol and linoleic acid had an activity of 1.69 _+ 0.22 (S.E.M.) gmoles/min, mg protein. These values are not significantly different at the 10 ~ level when analysed by the Student t-test. When cells grown in the presence of ergosterol and oleic or linoleic acid were suspended in buffer containing M ethanol for 8 or 24 h, there was no change in the ethanol concentration in the buffer.
Ethanol Tolerance of Cells Enriched in Ergosterol and Unsaturated Fatty-Acyl Residues of Different Chain Length To assess the importance of fatty-acyl chain length in conferring ethanol tolerance on S. cerevisiae NCYC 366, ceils were also grown in media supplemented with ergosterol and palmitoleic or cetoleic acid and
0.4_+0.1 3.1 • 0.4 3.7 • 0.5 27.6• 4.0 • 0.1 61.2 • 0.3 0.0 0.61
Table 2.
Ergosterol Linoleic acid
Oleic acid
1.7_+0.1 2.3 • 0.1 5.1 • 0.2 31.7• 4.3 • 0.2 0.0 54.9 • 0.6
1.9_+0.3 2.3 • 0.2 2.6 • 0.3 28.2_+0.2 2.7 • 0.2 62.3 • 0.2 0.0
1.09
Fatty-acyl
0.62
composition
of
plasma
Linoleic acid 2.0_+0.2 1.0 • 0.2 4.0 • 0.1 30.1• 5.3 _+ 0.0 0.0 57.5 • 1.9 1.15
membrane
of
Saccharomyces cerevisiae NCYC 366 grown anaerobically in media supplemented with ergosterol and oleic or linoleic acid Fatty-acyl residue
Percentage composition of membranes from cells grown in the presence of Oleic acid
C12:o C13:o C14:0 C15:o C16:o C16:1 C18:o C18:1 C18:2 A/mole value
5.5 3.8 13.2 0.0 23.5 0.0 0.0 54.0 0.0 0.54
Linoleic acid 2.4 1.8 4.9 0.3 31.9 0.4 1.7 2.8 53.8 1.1
The A/mole value was calculated as described by Kates and Hagen (1964)
their ability to remain viable in buffered 1 M ethanol compared with cells grown in medium supplemented with ergosterol and oleic acid. Cells grew at similar rates in media supplemented with any one of these three unsaturated fatty acids. Populations of cells grown in medium supplemented with ergosterol and myristoleic acid (C14:1) were not included in the comparison since these cultures grew at a very much slower rate, and cells from the cultures might therefore contain components that were different in concentration because of growthrate effects on cell composition (Brown and Rose, 1969). Populations of cells grown in the presence of cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of palmitoleic acid were more resistant to ethanol (Fig. 2).
D. S. Thomas et al. : Lipids and Ethanol Tolerance in Saceharomyces cerevisiae Table 3
Free sterol composition of Saccharomyces cerev&iae NCYC 366 grown anaerobically in medium supplemented with ergosterol or cholesterol, and oleic or linoleic acid
Medium supplements
The values quoted are means _+ standard errors of the mean from two independent analyses. nd indicates that the sterol could not be detected
Ergosterol Cholesterol
lO(1
Cholesterol Zymosterol 24(28) Dehydroergosterol
oleic acid linoleic acid
78.1 _+2.0 72.3 _+2.3
rtd nd
6.1 +_ 1.4 8.3 +_ 1.5
16.8 + 1.5 19.4 + 3.8
oleicacid linoleic acid
nd nd
79.1 + 0.6 78.0 + 1.9
nd nd
20.9 + 0,6 22.0 + 1,9
20
2
3
4
Time
5
6
7
8
9
(h)
Fig. 2. Decrease in viability of populations of Saccharomyces cerevisiae NCYC 366 grown in the presenceof ergosterol and palmitoleic
(9 oleic (e) or cetoleic acid (,) when incubated at 30~C in 67 mM KH2PO4 containing (continuous lines) or lacking (dotted lines) 1.0 M ethanol. Vertical bars indicate 95 % confidence limits
Discussion
Alterthum
and
Rose
(1973)
reported
Content (percent of total) of Ergosterol
...................... @'""'
1
243
that
Saccharomyces cerevisiae grown anaerobically in the
presence of ergosterol and oleic, linoleic or c~-linolenic acid, contained lipids enriched (65, 57 and 54%, respectively, of the total fatty-acyl residues) with the exogenously supplied acid. The present paper describes a comparable enrichment in phospholipids and plasma membranes isolated from cells grown in the presence of ergosterol and oleic or linoleic acids. Furthermore, a 70 % enrichment in the free-sterol fraction of plasma membranes isolated from cells grown in the presence of oleic acid and cholesterol or stigmasterol was demonstrated by Hossack and Rose (1976), while data reported in the present paper show that the extent of enrichment in free sterols is not changed when linoleic rather than oleic acid is included in the medium. Finally, incorporating cholesterol or ergosterol in the medium, along with oleic or linoleic acid, is not
accompanied by changes in the content or composition of cellular phospholipids. Assuming that the toxic effect of ethanol results from inhibition of intracellular enzymes, these analytical data suggest that differences in the capacity of S. cerevisiae to remain viable in buffered ethanol are associated with the presence of specific sterols and fatty-acyl residues in the yeast plasma membrane. The possibility that populations remain viable in the presence of ethanol longer because they are able to detoxify the alcohol can be dismissed in view of the comparable ethanol dehydrogenase activities of cells grown in the presence of ergosterol and either oleic or linoleic acid, and their inability to lower the ethanol concentration when suspended in buffered ethanol for up to 24h. Tolerance to ethanol is probably explained in part at least by the creation of a more effective barrier to entry of the alcohol into cells when the membrane contains sterols with a A 22 unsaturated alkyl chain (ergosterol and stigmasterol) rather than a saturated chain at C-17 (cholesterol and campesterol). The ability of the plasma membrane of S. cerevisiae N C Y C 366 to resist stretching, a property associated with a diminished tendency for phospholipid polar headgroups to move apart in the plane of the membrane, was reported by Hossack and Rose (1976) to be greater in membranes enriched with sterols that have A 22 unsaturated alkyl chain at C-17, irrespective of the presence of alkyl groups (methyl or ethyl) at C-24 or the configuration (R or S) of these groups. Phillips and his colleagues (Phillips, 1972; Phillips and Finer, 1974), from nuclear magnetic resonance and electron spin resonance studies on cholesterol-lecithin dispersions, concluded that, when a cholesterol molecule lies alongside a lecithin molecule with the polar headgroup of the lecithin juxtaposed with the C-3 hydroxyl group of the sterol, movement of the first eight to nine methylene groups of the fatty-acyl chains on the lecithin molecule is constrained by the sterol nucleus. This part of the fatty-awl chain is in the gel phase while the remainder of the chain, not similarly constrained, is in the liquid phase. Hossack and Rose (1976) suggested that the
244 presence of a double bond at C-22 in the sterol side chain increased stabilization in the distal part of the fatty-acyl chain that makes contact with the sterol side chain. We now propose that this extra stabilization could lead to formation of a more effective barrier to entry of ethanol molecules into yeast cells. The barrier effect could be even greater if there exists an asymmetrical distribution of sterol molecules in the yeast membrane. Allotopy, or asymmetrical distribution of phospholipids between the two monolayers in membranes, has been demonstrated in several animal-cell membranes (Bretscher, 1972; Patton and Keenan, 1975; Tsai and Lenard, 1975). However, the phenomenon has yet to be demonstrated in membranes from eukaryotic micro-organisms. Asymmetrical distribution of sterols is known to occur in the erythrocyte membrane where there is a preferential distribution (2: 1) in the outer monotayer (Fisher, 1975). A similar preferential distribution of sterols with an unsaturated side chain in the outer monolayer of the yeast plasma membrane would considerably enhance its capacity to exclude ethanol, since this would lower the phospholipid: sterol ratio to a value that would increase the condensing effect on phospholipids in the monolayer (Demel et al., 1972). The phospholipid: sterol ratio in plasma membranes of S. cerevisiae N C Y C 366 grown anaerobically in the presence of stigmasterol and oleic acid is 8: I (Hossack and Rose, 1976). One possible explanation of the ability o f p h o s p h o lipids with linoleyl rather than oleyl residues to confer resistance to ethanol on S. cerevisiae is that a membrane containing multiply unsaturated fatty-acyl residues, because of its greater fluidity, is better able to accommodate ethanol molecules that penetrate the outer monolayer. At the same time, those ethanol molecules inside the membrane would less easily enter the cytosol when the inner monolayer contains A 22 sterols. This concept of increased dissolution may also explain why populations of cells grown anaerobically in medium supplemented with ergosterol and palmitoleic acid retained viability to a greater extent than those grown in the presence of the same sterol and oleic or cetoleic acids, for the presence of shorter (C16:~) rather than longer (Cls:~, C20:1) residues could also lead to an increased solution of ethanol in the membrane. One alternative explanation of the increased ethanol tolerance of cells with plasma membranes enriched in multiply unsaturated fatty-acyl residues is that the presence of increased fluidity in the membrane compensates for a decreased repulsion between polar head groups on phospholipids that would arise when water molecules surrounding these headgroups were replaced by ethanol molecules. Another is that the presence of multiply unsaturated residues increases the stability of membrane-bound enzymes. Finally, if ethanol too-
Arch. Microbiol., Vol. 117 (1978) lecules were located in the hydrophobic interior of the membrane, they could restrict movement of fatty-acyl chains, a restriction which would be less extensive if the membrane contained multiply unsaturated fatty-acyl residues. The enhanced ethanol tolerance of yeast that have plasma membranes with multiply unsaturated fatty-acyl residues is in agreement with the observations of Hayashida et al. (1974) and Hayashida and H o n g o (1976) on increased ethanol tolerance in sakb yeasts grown in the presence of a fraction from Aspergillus oryzae containing unsaturated fatty-acyl residues. Interestingly, Ingram (1976), in a study of the effect of growth in the presence of ethanol (4 ~ , v/v) on the lipid composition of Escherichia coIi, reported that this caused an increased synthesis of phospholipids with CIs:I residues. It would be interesting to discover whether S. cerevisiae can similarly adapt to high concentrations of ethanol in growth media. The manner in which ethanol kills S. cerevisiae is unclear, although the most likely explanation is that death results from denaturation of intracellular enzymes following passage of ethanol through the plasma membrane. Such a mechanism would explain why differences in the ability of cells to remain viable in buffered ethanol may be attributed to inequalities in the barrier properties of the plasma membrane. Acknowledgements. The work reported in this paper was supported by the Science Research Council (U.K.) under grant B/RG/73016. D.S.T. also thanks the Council for the award of a research studentship.
References Alterthum, F., Rose, A. H.: Osmotic lysis of sphaeroplasts from Saccharomyees cerevisiaegrown anaerobicallyin media containing different unsaturated fatty acids. J. Gen. Microbiol. 77, 371 - 382 (1973) Andreasen, A. A., Stier, T. J. B.: Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J. Cell. Comp. Physiol. 41, 23-36 (1953) Andreasen, A. A., Stier, T. J. B.: Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. J. Cell. Comp. Physiol. 43, 271-281 (1954) Bretscher, M. S. : Phosphatidyl ethanolamine: differentiallabellingin intact cells and cell ghosts of human erythrocytes by a membrane-impermeable reagent. J. Mol. Biol. 71, 523--528 (1972) Brown, C. M., Rose, A. H. : Fatty-acid composition ofCandida utilis as affectedbygrowth temperature and dissolved-oxygentension. J. Bacteriol. 99, 371-378 (1969) Bficher, Th., Redetzki, H. : A specificphotometric method for ethyl alcohol through fermentation. Klin. Wochr. 29, 615- 616 (1951) Cartledge, T. G., Rose, A. H. : Properties of low densityvesiclesfrom Saeeharomyees eerevisiae. In: Proc. 3rd Int. Spee.Symp.Yeasts, (H. Suomalainen, C. Waller, eds.), pp. 251-259. Helsinki: Oy 1973
D. S. Thomas et al. : Lipids and Ethanol Tolerance in Saceharomyces cerevisiae Chen, P. S., Toribara, Y. Y., Warner, H.: Micro-determination of phosphorus. Anal. Chem. 28, 1756-1758 (1956) Demel, R. A., Bruckdorfer, K. R., van Deenen, L. L. M. : Structural requirements of sterols for the interaction with lecithin at the airwater interface. Biochim. Biophys. Acta 255, 3 1 1 - 320 (1972) Fink, H., Kfihles, R. : Beitrfige zur Methylenblauf'firbung der Hefezel len und Studien fiber die Permeabilitfit der Hefezelhnembran. II. Mitteilung. Eine verbesserte Fftrbeflfissigkeit zur Erkennung yon toten Hefezellen. Hoppe Seyler's Z. Physiol. Chem. 218, 6 5 - 6 6 (1933) Fisher, K. A. : Analysis of membrane halves: cholesterol. Proc. Nat. Acad. Sci. Wash. 73, 173-176 (1975) Gray, W. D. : Studies on the alcohol tolerance of yeasts. J. Bacteriol. 42, 561-574 (1941) Gray, W. D. : The sugar tolerance of four strains of distiller's yeast. J. Bacteriol. 49, 4 4 5 - 452 (1945) Gray, W. D.: Further studies on the alcohol tolerance of yeast; its relationship to cell storage products. J. Bacteriol. 55, 5 3 - 5 9 (1948) Hayashida, S., Feng, D. D., Hongo, M.: Function of the high concentration alcohol-producing factor. Agr. Biol. Chem. 38, 2001-2006 (1974) Hayashida, S., Hongo, M.: The mechanism of formation of high concentration alcohol in Sakfi brewing. Abstr. 5th Int. Ferm. Syrup. (H. Dellweg, ed.), p. 384. Berlin: Versuchs- und Lehranstalt fiir Spiritusfabrikation und Fermentationstechnologie 1976 Hossack, J. A., Belk, D. M., Rose, A. H. : Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis. Arch. Microbiol. 114, 137-142 (1977) Hossack, J. A., Rose, A. H.: Fragility of plasma membranes in Saccharomyces eerevisiae enriched with different sterols. J. Bacteriol. 127, 6 7 - 7 5 (1976) Hunter, K., Rose, A. H.: Lipid composition of Saccharomyces eerevisiae as influenced by growth temperature. Biochim. Biophys. Acta 260, 639-653 (1972) Ingrain, L. O.: Adaptation of membrane lipids to alcohols. J. Bacteriol. 125, 670-678 (1976) Kates, M., Hagen, P. O.: Influence of temperature on fatty acid composition of psychrophilic and mesophilic Serratia s!~pCan. J. Biochem. 42, 481-488 (1964) Kuksis, A.: Gas chromatography of neutral glycerides. In: Lipid chromatographic analysis, Vol. 1 (G. V. Marinetti, ed.), pp. 239-337. London: Arnold 1967 Letters, R. : Phospholipids of yeasts. In: Aspects of yeast metabolism (A. K. Mills, ed.), pp. 303-319. Oxford: Blackwells 1968
245
Light, R. J., Lennarz, W. J., Bloch, K.: The metabolism of hydroxystearic acids in yeast. J. Biol. Chem. 237, 1793-1800 (1962) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Marchalonis, J. J. : An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113,299 - 305 (1969) Mersel, M., Benenson, A., Doljanski, F. : Lactoperoxidase-catalysed iodination of surface membrane lipids. Biochem. Biophys. Res. Commun. 70, 1166--1171 (1976) Patton, S., Keenan, T. W. : The milk fat globule membrane. Biochim Biophys. Acta 415, 273-309 (1975) Pecsok, R. L. : Analytical methods. In: Principles and practice of gas chromatography (R. L. Pecsok, ed.), pp. 135 - 150. New York: Wiley 1959 Phillips, D. R., Morrison, M.: The arrangement of proteins in the human erythrocyte membrane. Biochem. Biophys. Res. Commun. 41), 284-289 (1970) Phillips, M. C. : The physical state of phospholipids and cholesterol in monolayers, bilayers and membranes. Prog. Surf. Membr. Sci. 5, 139-221 (1972) Phillips, M. C., Finer, E. G. : The stoichiometry and dynamics of lecithin-cholesterol clusters in bilayer membranes. Biochim. Biophys. Acta 356, 199-206 (1974) Proudlock, J. W., Wheeldon, L. W., Jollow, D. J., Linnane, A. W. : Role of sterols in Saccharomycex cerevisiae. Biochim. Biophys. Acta 152, 434-437 (1968) Schibeci, A., Rattray, J. B. M., Kidby, D. K.: Isolation and identification of yeast plasma membrane. Biochim. Biophys. Acta 311, 1 5 - 2 5 (1973) Troyer, J. R. : A relationship between cell multiplication and alcohol tolerance in yeasts. Mycologia 45, 2 0 - 3 9 (1953) Troyer, J. R. : Methanol tolerance of three strains of Saceharomyces eerevisiae. Ohio J. Sci. 55, 185-187 (1955) Tsai, K. H., Lenard, J. : Asymmetry of influenza virus membrane bilayer demonstrated with phospholipase C. Nature (Lond.) 253, 554-555 (1975) Vallee, B. L., Hoch, F. L.: Zinc, a component of yeast alcohol dehydrogenase. Proc. Nat. Acad. Sci. Wash. 4 1 , 3 2 7 - 338 (1955) Wickerham, L. J. : Taxonomy of yeasts. I. Techniques of classification. US Dept. Agric. Tech. Bull. no. 1029, US Dept. Agric., Washington D.C. (1951)
Received November 24, 1977
Hicrobiology
Arch. Microbiol. 117, 239-245 (1978)
9 by Springer-Verlag 1978
Plasma-Membrane Lipid Composition and Ethanol Tolerance in Saccharomyces cerevisiae D. Susan Thomas, J. A. Hossack, and A. H. Rose Zymology Laboratory, School of Biological Sciences, Bath University, Claverton Down, Bath BA2 7AY, Avon, England
Abstract. Populations of cells suspended anaerobically in buffered (pH4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration. Key words: Plasma membrane Lipids Saccharomyces cerevisiae - Ethanol tolerance Sterols - Fatty-acyl residues.
m
Although many strains of yeast remain viable in the presence of concentrations of ethanol (3 - 4 ~ w/v) that are lethal to other micro-organismS, little is known of the molecular basis of this tolerance. Gray (1941) found that ethanol tolerance is not confined to any one genus or species of yeast, and went on to report (Gray, 1945) that induced tolerance of glucose in Saccharomyces cerevisiae, brought about by sequential transfer into media containing higher concentrations of glucose, was accompanied by a decrease in ethanol tolerance. Examination of a range of yeast species subsequently revealed that those which tolerate high concentrations of ethanol store less lipid and carbohydrate than less tolerant strains (Gray, 1948). Troyer (1953, 1955) largely confirmed the results of Gray (1948), and further reported that methanol is generally less toxic than ethanol. Thereafter, ethanol tolerance in yeasts
failed to attract research interest until Hayashida et al. (1974) and Hayashida and Hongo (1976) reported that sak6 yeasts (strains of S. cerevisiae) acquired enhanced tolerance to ethanol when grown in the presence of a fraction from the envelope of Aspergillus oryzae containing unsaturated fatty-acyl residues. The present paper reports on the manner in which both the sterol and phospholipid fatty-acyl composition of the plasma membrane of S. cerevisiae NCYC 366 influence the ability of this yeast to remain viable in buffered suspensions containing ethanol. The rationale behind the study was that the plasma membrane is the first sensitive organelle to make contact with ethanol when cells are suspended in ethanol solutions, and that since ethanol is an amphipathic compound the lipid composition of the plasma membrane may have an important effect on ethanol tolerance. The lipid composition of the plasma membrane in S. cerevisiae NCYC 366 was altered by exploiting the anaerobically-induced requirement of this microbe for a sterol and an unsaturated fatty acid (Andreasen and Stier, 1953, 1954), both of which requirements are fairly non-specific (Light et al., 1962; Proudlock et al., 1968), as described by Hossack and Rose (1976).
Materials and Methods Experimental Cultures. Saccharomyces cerevisiae NCYC366 was grown anaerobically in medium supplemented with an unsaturated fatty acid (30 rag/l) and campesterol, cholesterol, ergosterol or stigmasterol (5 rag/l), and harvested from mid exponential-phase cultures (0.24-0.26 mg dry wt/ml) as described by Hossack and Rose (1976) and Hossack et al. (1977). When cells were to be analysed for lipids, or converted into sphaeroplasts, 2 ml of a solution containing 10 mg each of chloramphenicol and cycloheximide were injected into the cultures through a Suba seal on the side of the flask (Alterthum and Rose, 1973), and the culture incubated for a further 15 min before harvesting. Cells to be analysed for lipid were washed twice with water at 4~ freeze dried, and stored at - 20~C over silica gel. Cells to be converted into sphaeroplasts were washed twice with 1.2 M sorbitol containing 10 mM
0302-8933/78/0117/0239/$01.40
240 MgC12 and 10 mM imidazole hydrochloride (pH 6.0). Those which were to be incubated in buffer for viability studies were washed twice in 67 mM KH2PO4 at 4~ all vessels used being flushed with oxygen-free nitrogen gas.
Viability Measurements. Viability of yeast populations was measured by plate counts and by staining with methylene blue. Yeast suspension (1.25rag dry wt/ml) was diluted in 67raM KHzPO 4 (pH4.5) containing where indicated 1.0 M ethanol, to a concentration of 50 ~tg dry wt/ml. Cell suspensions (500 ml) were incubated anaerobically as described by Alterthum and Rose (1973) at 30~ with stirring. Portions (2 ml) were removed at intervals with a hypodermic syringe 9 through the Suba seal, and diluted with 67 mM KHzPO 4 such that 0.1 ml of the diluted suspension contained 100-300 viable cells. Portions (0.1 ml) of the diluted suspension were spread onto each of three plates (8.5 cm diam.) of malt extract (0.3 % w/v) - yeast extract (0.3%, w/v)-glucose (1.0%, w/v)-mycological peptone (0.5%, w/v) - agar (2.0 %, w/v) medium (Wickerham, 1951) and incubated aerobically at 30 ~ C for 48 h, when the number of colonies on each plate was counted. Staining with methylene blue (Fink and Kfihles, 1933) gave a rapid assessment of the viability of yeast populations. A portion (1.0ml) of the undiluted suspension was stained with methylene blue solution (0.01%, w/v, methylene blue containing 2 %, w/v, sodium citrate) for 5 rain, and concentrated by filtering through a Millipore filter (0.45 ~m pore size). Cells were resuspended in 0.1 ml water, wet preparations examined under the microscope, and 1000 cells scored for viability, live cells remaining colourless.
Lipid Analyses. Lipids were extracted from freeze-dried cells by a modification of the method of Letters (1968) described by Hossack and Rose (1976) except that the residues were suspended in methanol for 15rain instead of 10rain. Classes of lipid were separated by quantitative thin-layer chromatography on plates coated with a layer (0.4ram thick) of Kieselgel HF254+366 (Merck). Plates were developed with petroleum spirit ( 4 0 - 6 0 ~ C)-diethylether-acetic acid (70: 30:2, v/v/v) containing butylated hydroxytoluene (0.005 %, w/v). Total phospholipid and individual phospholipids were assayed as described by Hossack and Rose (1976). Phospholipids were eluted from the silica gel with two portions (3 ml) of chloroform-methanolwater (5:5:1, v/v/v), followed by 3ml of methanol and 3 ml of methanol-acetic acid-water (95:1:5, v/v/v). Phospholipid was determined by assaying the phosphorus content of the extract, using the method of Chen et al. (1956). Values for phosphorus contents were multiplied by 25 to give the total phospholipid content. Sterol acetates (Kuksis, 1967) were prepared as described by Hunter and Rose (1972) and separated by gas-liquid chromatography on columns of OV-17. Fatty-acyl residues in phospholipids and plasmamembrane preparations were converted into methyl esters and separated by gas-liquid chromatography on a column of EGSS-X, as described by Hunter and Rose (1972) except that the detector temperature was 250 ~C. Peak areas were measured, and relative amounts of compounds calculated, by the method of Pecsok (1959). Isolation of Plasma Membranes. Purified preparations of plasma membrane were isolated by a modification of the Hossack and Rose (1976) technique using sphaeroplasts, which were prepared as described by Cartledge and Rose (1973). Glass vessels and centrifuge tubes used in the preparation of sphaeroplasts were flushed with oxygen-free nitrogen before use. Throughout the preparation, chloramphenicol and cycloheximide were included in all reaction mixtures each at 0.2 mg/ml. The outer surface of sphaeroplasts was labelled by lactoperoxidase-catalysed iodination (Marchalonis, 1969; Phillips and Morrison, 1970), the sphaeroplasts lysed, and plasma membranes isolated by isopycnic density-gradient centrifugation. Iodination was carried out by a modification of the method of Schibeci et al. (1973) as reported by Hossack and Rose (1976). The label indicated the position on the gradient at which plasma membrane accumulated. Purified plasma membranes were removed
Arch. Microbiol., Vol. 117 (1978) from the corresponding position on a gradient on which components oflysed unlabelled sphaeroplasts were separated because of the effect ofiodination on phospholipid fatty-acyl residues (Mersel et al., 1976).
Measurement of Ethanol Concentration and Ethanol Dehydrogenase Activity. Ethanol concentrations in culture filtrates that had been filtered through a membrane filter (Millipore; 0.45gin pore size; 2:5 cm diam.) were determined using a Biochemica Test Combination (Boehringer, Mannheim, W. Germany). The method is based on the ethanol dehydrogenase assay of Bficher and Redetzki (1951), and it was established that the method recommended for the kit did not need modification for use with culture filtrates. Culture filtrates were diluted 10 fold with water before assay. Ethanol dehydrogenase activity of cell extracts was measured by the method of Vallee and Hoch (1955) based on formation ofNADH. After centrifugation of culture, cells (about 320 mg dry wt) were washed with water at 4 ~ C, and suspended in about 15ml 0.05 M potassium phosphate buffer (pH 7.7) and 40g glass beads (0.170.18 mm diam.; Glasperlen, B. Braun, Melsungen, W. Germany) added to the suspension which was then treated in a homogenisor (B. Braun, Melsungen, W. Germany) for 4 rain at maximum speed. Beads were removed by passing the suspension through a sintered glass filter (No. 1) and the protein content of the disrupted cell suspension measured (Lowry et al., 1951). Suspensions of disrupted cells were diluted with bovine serum albumin (0.1%, w/v in 0.01%, w/v, potassium dihydrogen phosphate, pH 7.5) to decrease the cell protein content to 1 mg/ml. A quartz cuvette (1 cm path length) was charged with 1.5 ml 0.032 M sodium pyrophosphate (pH 8,8), 0.5 ml 2 M ethanol and 1.0ml 25mM NAD +, and pre-incubated at 25~ suspensions of disrupted cells were also pre-incubated at 25 ~C. The reaction was started by adding 0.1 ml disrupted cell suspension to the cuvette, and the extinction at 340nm measured after 15 and 45 s. In control reaction mixtures substrate solution or enzyme extract was replaced by water. Ethanol dehydrogenase activity is expressed as gmoles NADH formed/rain, mg protein.
Chemicals. All chemicals used were reagent grade or of the highest purity available commercially. Chloroform, ethanol and methanol were redistilled before use. Palmitoleic acid (C16:1, A9 eis-hexadecenoic acid), oleic acid (C18:1, A9 eis-octadecenoic acid), linoleic acid (C18:2, A9'12 cis,cis-octadecadienoic acid), e-linolenic acid (C18:3, A 9&z'15 cis,eis,cis-octadecatrienoic acid), and cetoleic acid (C20:h A11 cis-eicosaenoic acid) were supplied by Sigma Chemical Co. (London) Ltd., as were cholesterol and ergosterol. Campesterol and stigmasterol were purchased from Applied Science Laboratories through Field Instruments Ltd. (U.K.), and OV-17 on Gas Chrom Q and EGSS-X from Phase Separations, Rock Ferry, Cheshire, England. Sterols and fatty acids were used without further purification.
Results Ethanol Tolerance as A f f e c t e d by Sterol and F a t t y - A c y l Composition o f Cells W h e n s u s p e n d e d i n b u f f e r ( p H 4.5) c o n t a i n i n g 1.0 M e t h a n o l , p o p u l a t i o n s o f cells e n r i c h e d i n l i n o l e y l residues remained viable to a greater extent than those e n r i c h e d i n oleyl r e s i d u e s , i r r e s p e c t i v e o f t h e n a t u r e o f t h e s t e r o l e n r i c h m e n t (Fig. 1). A c o n c e n t r a t i o n o f 1.0 M ethanol was chosen since aerobic growth of Saccharomyces cerevisiae N C Y C 366 is c o m p l e t e l y inhibited when the ethanol concentration in defined m e d i u m e x c e e d s 1.2 M . H o w e v e r , p o p u l a t i o n s e n r i c h e d
241
D. S. Thomas et al. : Lipids and Ethanol Tolerancein Saccharomyces cerevisiae 100
....................
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
100
.......................................... ~ . ~
80
80
6o
60 g
4o
40
20
2o
ta
0
5
10 Time (h)
20
ol
0
5
10 Time (h)
15
20
100
100
zZ
15
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
80
80
60
60
ca 40
20 9
2O
0
0
5
10 Time
15
20
(h)
0
~ C18=~1~ 1,
5
10 Time (h)
,
15
20
b
la--d. Decreasein viability of populations ofSaccharomyces cerevisiae NCYC 366 enriched in different sterols and either oleyl (closed symbols) or linoleyl residues (open symbols), when incubated at 30~ in 67 mM KHzPO4 (pH 4.5) containing (continuous lines) or lacking (dotted lines) 1.0 M ethanol. Viability of populations enriched in campesterol is indicated by inverted triangles (a), cholesterol by upright triangles (b), ergosterol by squares (c), and stigmasterol by circles (d). Vertical bars indicate 95 ~ confidencelimits
Fig.
in ergosterol or stigmasterol, and linoleyl residues, were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues (Fig. 1). Populations of cells enriched in ergosterol and ~-linolenyl residues retained viability very similarly to those enriched in the same sterol and linoleyl residues. Populations suspended in ethanol-free buffer, irrespective of the nature of the sterol and fatty-acyl enrichments, remained almost completely viable over the duration of the incubation (Fig.l). Filtrates from cultures supplemented with ergosterol or cholesterol, and oleic or linoleic acid, and containing 0.32 mg dry wt/ml were assayed for ethanol after filtration through Millipore filters (0.45 ~tm pore size). At the 5 ~ level [points of variance-ratio (F) distribution], there was no
significant difference in the ethanol concentration in the filtrates (average 3.19 mg/ml). Assuming that one mole of ATP was produced for each mole of ethanol excreted, the Yare value for the cultures is 4.57. Lipid and Plasma-Membrane Composition of Cells Grown in the Presence o f Different Sterols and Fatty Acids Phospholipids from S. cerevisiae NCYC366, grown anaerobically in the presence of cholesterol or ergosterol, and oleic or linoleic acid, were enriched to the extent of 5 5 - 6 2 ~ with the exogenously provided fatty-acyl residue (Table 1). The degree of enrichment with linoleyl residues was slightly lower than with oleyl
242 Table 1
Fatty-acyl composition of phospholipids in
Saccharomyces cerevisiae NCYC 366 grown
Arch. Microbiol., Vol. 117 (1978)
Fatty-acyl residue
anaerobically in medium supplemented with cholesterol or ergosterol and oleic or linoleic acid
Percentage composition of fatty-acyl residues in phospholipids from cells grown in medium supplemented with Cholesterol Oleic acid
Values indicated are +_ standard errors of the mean values from two independent analyses. Values for unsaturation (A/mole) were calculated as described by Kates and Hagen (1964)
C12:0 C13:0 C14:0 C16:o Ca8:0 C18:1 C18:2 A/mole
residues. Plasma membranes from cells grown anaerobically in medium supplemented with ergosterol and oleic or linoleic acid also were enriched comparably with the exogenously provided fatty-acyl residue (Table 2). Enrichment with linoleyl rather than oleyl residues did not cause significant changes in the composition of the free sterol fraction in cells (Table 3). The phospholipid content of cells grown in the presence of oleic or linoleic acid did not differ depending on whether the sterol supplement was cholesterol or ergosterol (Table 1).
Ethanol Dehydrogenase Activities of Cells Enriched in Ergosterol and Oleyl or Linoleyl Residues Extracts of cells grown anaerobically in the presence of ergosterol and oleic acid had an ethanol dehydrogenase activity of 1.25 _+ 0.16 (S.E.M.) ~tmoles NADH formed/rain- mg protein, while extracts of cells grown in medium supplemented with the same sterol and linoleic acid had an activity of 1.69 _+ 0.22 (S.E.M.) gmoles/min, mg protein. These values are not significantly different at the 10 ~ level when analysed by the Student t-test. When cells grown in the presence of ergosterol and oleic or linoleic acid were suspended in buffer containing M ethanol for 8 or 24 h, there was no change in the ethanol concentration in the buffer.
Ethanol Tolerance of Cells Enriched in Ergosterol and Unsaturated Fatty-Acyl Residues of Different Chain Length To assess the importance of fatty-acyl chain length in conferring ethanol tolerance on S. cerevisiae NCYC 366, ceils were also grown in media supplemented with ergosterol and palmitoleic or cetoleic acid and
0.4_+0.1 3.1 • 0.4 3.7 • 0.5 27.6• 4.0 • 0.1 61.2 • 0.3 0.0 0.61
Table 2.
Ergosterol Linoleic acid
Oleic acid
1.7_+0.1 2.3 • 0.1 5.1 • 0.2 31.7• 4.3 • 0.2 0.0 54.9 • 0.6
1.9_+0.3 2.3 • 0.2 2.6 • 0.3 28.2_+0.2 2.7 • 0.2 62.3 • 0.2 0.0
1.09
Fatty-acyl
0.62
composition
of
plasma
Linoleic acid 2.0_+0.2 1.0 • 0.2 4.0 • 0.1 30.1• 5.3 _+ 0.0 0.0 57.5 • 1.9 1.15
membrane
of
Saccharomyces cerevisiae NCYC 366 grown anaerobically in media supplemented with ergosterol and oleic or linoleic acid Fatty-acyl residue
Percentage composition of membranes from cells grown in the presence of Oleic acid
C12:o C13:o C14:0 C15:o C16:o C16:1 C18:o C18:1 C18:2 A/mole value
5.5 3.8 13.2 0.0 23.5 0.0 0.0 54.0 0.0 0.54
Linoleic acid 2.4 1.8 4.9 0.3 31.9 0.4 1.7 2.8 53.8 1.1
The A/mole value was calculated as described by Kates and Hagen (1964)
their ability to remain viable in buffered 1 M ethanol compared with cells grown in medium supplemented with ergosterol and oleic acid. Cells grew at similar rates in media supplemented with any one of these three unsaturated fatty acids. Populations of cells grown in medium supplemented with ergosterol and myristoleic acid (C14:1) were not included in the comparison since these cultures grew at a very much slower rate, and cells from the cultures might therefore contain components that were different in concentration because of growthrate effects on cell composition (Brown and Rose, 1969). Populations of cells grown in the presence of cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of palmitoleic acid were more resistant to ethanol (Fig. 2).
D. S. Thomas et al. : Lipids and Ethanol Tolerance in Saceharomyces cerevisiae Table 3
Free sterol composition of Saccharomyces cerev&iae NCYC 366 grown anaerobically in medium supplemented with ergosterol or cholesterol, and oleic or linoleic acid
Medium supplements
The values quoted are means _+ standard errors of the mean from two independent analyses. nd indicates that the sterol could not be detected
Ergosterol Cholesterol
lO(1
Cholesterol Zymosterol 24(28) Dehydroergosterol
oleic acid linoleic acid
78.1 _+2.0 72.3 _+2.3
rtd nd
6.1 +_ 1.4 8.3 +_ 1.5
16.8 + 1.5 19.4 + 3.8
oleicacid linoleic acid
nd nd
79.1 + 0.6 78.0 + 1.9
nd nd
20.9 + 0,6 22.0 + 1,9
20
2
3
4
Time
5
6
7
8
9
(h)
Fig. 2. Decrease in viability of populations of Saccharomyces cerevisiae NCYC 366 grown in the presenceof ergosterol and palmitoleic
(9 oleic (e) or cetoleic acid (,) when incubated at 30~C in 67 mM KH2PO4 containing (continuous lines) or lacking (dotted lines) 1.0 M ethanol. Vertical bars indicate 95 % confidence limits
Discussion
Alterthum
and
Rose
(1973)
reported
Content (percent of total) of Ergosterol
...................... @'""'
1
243
that
Saccharomyces cerevisiae grown anaerobically in the
presence of ergosterol and oleic, linoleic or c~-linolenic acid, contained lipids enriched (65, 57 and 54%, respectively, of the total fatty-acyl residues) with the exogenously supplied acid. The present paper describes a comparable enrichment in phospholipids and plasma membranes isolated from cells grown in the presence of ergosterol and oleic or linoleic acids. Furthermore, a 70 % enrichment in the free-sterol fraction of plasma membranes isolated from cells grown in the presence of oleic acid and cholesterol or stigmasterol was demonstrated by Hossack and Rose (1976), while data reported in the present paper show that the extent of enrichment in free sterols is not changed when linoleic rather than oleic acid is included in the medium. Finally, incorporating cholesterol or ergosterol in the medium, along with oleic or linoleic acid, is not
accompanied by changes in the content or composition of cellular phospholipids. Assuming that the toxic effect of ethanol results from inhibition of intracellular enzymes, these analytical data suggest that differences in the capacity of S. cerevisiae to remain viable in buffered ethanol are associated with the presence of specific sterols and fatty-acyl residues in the yeast plasma membrane. The possibility that populations remain viable in the presence of ethanol longer because they are able to detoxify the alcohol can be dismissed in view of the comparable ethanol dehydrogenase activities of cells grown in the presence of ergosterol and either oleic or linoleic acid, and their inability to lower the ethanol concentration when suspended in buffered ethanol for up to 24h. Tolerance to ethanol is probably explained in part at least by the creation of a more effective barrier to entry of the alcohol into cells when the membrane contains sterols with a A 22 unsaturated alkyl chain (ergosterol and stigmasterol) rather than a saturated chain at C-17 (cholesterol and campesterol). The ability of the plasma membrane of S. cerevisiae N C Y C 366 to resist stretching, a property associated with a diminished tendency for phospholipid polar headgroups to move apart in the plane of the membrane, was reported by Hossack and Rose (1976) to be greater in membranes enriched with sterols that have A 22 unsaturated alkyl chain at C-17, irrespective of the presence of alkyl groups (methyl or ethyl) at C-24 or the configuration (R or S) of these groups. Phillips and his colleagues (Phillips, 1972; Phillips and Finer, 1974), from nuclear magnetic resonance and electron spin resonance studies on cholesterol-lecithin dispersions, concluded that, when a cholesterol molecule lies alongside a lecithin molecule with the polar headgroup of the lecithin juxtaposed with the C-3 hydroxyl group of the sterol, movement of the first eight to nine methylene groups of the fatty-acyl chains on the lecithin molecule is constrained by the sterol nucleus. This part of the fatty-awl chain is in the gel phase while the remainder of the chain, not similarly constrained, is in the liquid phase. Hossack and Rose (1976) suggested that the
244 presence of a double bond at C-22 in the sterol side chain increased stabilization in the distal part of the fatty-acyl chain that makes contact with the sterol side chain. We now propose that this extra stabilization could lead to formation of a more effective barrier to entry of ethanol molecules into yeast cells. The barrier effect could be even greater if there exists an asymmetrical distribution of sterol molecules in the yeast membrane. Allotopy, or asymmetrical distribution of phospholipids between the two monolayers in membranes, has been demonstrated in several animal-cell membranes (Bretscher, 1972; Patton and Keenan, 1975; Tsai and Lenard, 1975). However, the phenomenon has yet to be demonstrated in membranes from eukaryotic micro-organisms. Asymmetrical distribution of sterols is known to occur in the erythrocyte membrane where there is a preferential distribution (2: 1) in the outer monotayer (Fisher, 1975). A similar preferential distribution of sterols with an unsaturated side chain in the outer monolayer of the yeast plasma membrane would considerably enhance its capacity to exclude ethanol, since this would lower the phospholipid: sterol ratio to a value that would increase the condensing effect on phospholipids in the monolayer (Demel et al., 1972). The phospholipid: sterol ratio in plasma membranes of S. cerevisiae N C Y C 366 grown anaerobically in the presence of stigmasterol and oleic acid is 8: I (Hossack and Rose, 1976). One possible explanation of the ability o f p h o s p h o lipids with linoleyl rather than oleyl residues to confer resistance to ethanol on S. cerevisiae is that a membrane containing multiply unsaturated fatty-acyl residues, because of its greater fluidity, is better able to accommodate ethanol molecules that penetrate the outer monolayer. At the same time, those ethanol molecules inside the membrane would less easily enter the cytosol when the inner monolayer contains A 22 sterols. This concept of increased dissolution may also explain why populations of cells grown anaerobically in medium supplemented with ergosterol and palmitoleic acid retained viability to a greater extent than those grown in the presence of the same sterol and oleic or cetoleic acids, for the presence of shorter (C16:~) rather than longer (Cls:~, C20:1) residues could also lead to an increased solution of ethanol in the membrane. One alternative explanation of the increased ethanol tolerance of cells with plasma membranes enriched in multiply unsaturated fatty-acyl residues is that the presence of increased fluidity in the membrane compensates for a decreased repulsion between polar head groups on phospholipids that would arise when water molecules surrounding these headgroups were replaced by ethanol molecules. Another is that the presence of multiply unsaturated residues increases the stability of membrane-bound enzymes. Finally, if ethanol too-
Arch. Microbiol., Vol. 117 (1978) lecules were located in the hydrophobic interior of the membrane, they could restrict movement of fatty-acyl chains, a restriction which would be less extensive if the membrane contained multiply unsaturated fatty-acyl residues. The enhanced ethanol tolerance of yeast that have plasma membranes with multiply unsaturated fatty-acyl residues is in agreement with the observations of Hayashida et al. (1974) and Hayashida and H o n g o (1976) on increased ethanol tolerance in sakb yeasts grown in the presence of a fraction from Aspergillus oryzae containing unsaturated fatty-acyl residues. Interestingly, Ingram (1976), in a study of the effect of growth in the presence of ethanol (4 ~ , v/v) on the lipid composition of Escherichia coIi, reported that this caused an increased synthesis of phospholipids with CIs:I residues. It would be interesting to discover whether S. cerevisiae can similarly adapt to high concentrations of ethanol in growth media. The manner in which ethanol kills S. cerevisiae is unclear, although the most likely explanation is that death results from denaturation of intracellular enzymes following passage of ethanol through the plasma membrane. Such a mechanism would explain why differences in the ability of cells to remain viable in buffered ethanol may be attributed to inequalities in the barrier properties of the plasma membrane. Acknowledgements. The work reported in this paper was supported by the Science Research Council (U.K.) under grant B/RG/73016. D.S.T. also thanks the Council for the award of a research studentship.
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Received November 24, 1977
