The lipid composition of cell wall and plasma membrane of baker's yeast

Chem. Phys. Lipids 4 (1970) 247-256 © North-Holland Publ. Co., Amsterdam.

T H E L I P I D C O M P O S I T I O N OF CELL W A L L AND P L A S M A M E M B R A N E OF BAKER'S YEAST HEIKKI SUOMALAINEN and TIMO NURMINEN Research Laboratories of the State Alcohol Monopoly ( Alko), Helsinki 10, Finland

The fatty acids and phospholipids in the isolated cell envelope, and in a plasma membrane preparation obtained from the cell envelopes by enzymatic digestion, have been compared with those of the whole cells of baker's yeast. Whole cells and cell envelopes were found to have similar total fatty acid compositions. The major fatty acids were CI#:1,Cla:I and C16. C~6:aacid occurred in most abundance in the cell envelopes, but Cls:l predominated in the plasma membrane fraction. The main phospholipid components in whole cells and cell envelopes were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. The whole cells contained phosphatidylcholine in a larger proportion than did the cell envelopes, which were in turn richer in phosphatidylinositol and phosphatidylserine. The principal fatty acids in neutral lipids, and in phospholipids of the whole cells and of the cell envelopes, were C~6:1,Cls:l and Ca6. Phosphatidylinositol and phosphatidylserine contained proportionally less C16:1acid and more C~6 acid than the other phospholipids. During enzymaticdigestion, the original distribution of phospholipids in the cell envelopes remained almost unchanged, which indicates that the phospholipid composition of the plasma membrane corresponds in the main to that of the isolated cell envelopes.

Introduction It has previously been shown l-a) that carefully isolated cell envelopes of yeast contain, besides the ordinary cell wall, fragments of plasma membrane which are enriched in the sedimentable residue obtained by centrifugation after enzymatic digestion of the isolated cell envelopes. In addition to neutral lipids, including sterols, the cell elavelopes contained phospholipids as minor components. Lipid phosphorus was present in the membrane preparation in larger amounts than were the other phosphorus compounds of the cell envelope; the phospholipids are interesting, as their amount correlates with that of the plasma membrane in the preparations. Moreover, it was observed that the percentage of total lipid and protein had increased considerably during enzymatic digestion of the cell envelopes. The results thus indicated that the yeast plasma membrane had a lipoprotein nature. The studies so far reported in connexion with the composition of the yeast plasma membrane have been made with protoplast lysates. Boulton 4) has described the gross composition of two membraneous fractions of Saccharomyces 247

248

H, SUOMALAINEN AND T. NURMINEN

cerevisiae, and Garcia Mendoza and Villanueva 5) have given a brief report on the composition of membranes of a strain of Candida utilis. These preparations also contained protein and lipids as major components. Longley, Rose and Knights 6) have studied the composition of membranes obtained from protoplast lysates of another strain of S. cerevisiae. It is easier to obtain membrane material in sufficient quantity for analysis by starting from a protoplast lysate of a yeast which is easily convertible into protoplasts but by such a method a pure plasma membrane fraction is not obtained. Since the composition of the plasma membrane may differ from that of intracellular membranes of the yeast cell, a different method has now been applied for further clarification of the lipid composition of the plasma membrane and cell wall. A short communication of this work has been presented elsewhere 7).

Experimental Preparation of subcellular fractions The organism used was commercial baker's yeast, obtained from the Rajam/iki Factories of the Finnish State Alcohol Monopoly. For some experiments, the yeast was further cultured on a laboratory scale under aerobic conditions, as described earlier3). The methods employed for the preparation of subcellular fractions were essentially those described previously2,a). Yeast cells were disrupted in a Mickle disintegrator, and the cell envelopes were purified by washing and by differential centrifugation. When larger quantities of cell envelopes were required, a peristaltic pump was used. The mechanical disruption of the cells was effected rapidly, and the number of washings and centrifugations for isolating the cell envelopes was kept at a minimum. The material originating from the interior of the cell was collected at the same time. Enzymatic digestion of the isolated cell envelopes with snail gut enzyme, and the following fractionation of the products, were carried out as described previously a). The resulting digested preparation was collected by centrifugation at 15000 9 for 15 rain and was, after repeated washing and gentle resuspension, further fractionated by differential centrifugation. A heavy sediment (spun down at 1000 g for 5 min) and a light sediment (spun down at 10000 9 for 15 rain) were obtained. It is concluded from the chemical and enzymatic compositions presented earlier 1-3) that the heavy sediment still contains remnants of the cell walls in addition to fragments of plasma membrane, whereas the light sediment consists of fragments of plasma membrane only. After washing, the subceUular fractions, unless used immediately, were lyophilised, and stored in a vacuum desiccator at 4°C until analysed.

LIPIDS OF YEAST CELL ENVELOPE

249

Extraction and estimation of lipid Lipids were usually extracted from lyophilised samples with 10-20 volumes of chloroform-methanol (2: 1, by vol) either for 3 hr while the temperature was raised to 55°C and, after filtration, repeating the extraction for 30 min with a new portion of solvent, or with successive portions of solvent for 12 hr each, and at room temperature. The combined extracts were washed by the method of Folch et al.8). After evaporation the extracted lipid was weighed to constant weight. Reasonable precautions were taken to exclude oxygen and light during these manipulations. A nitrogen atmosphere was used whenever possible,

Separation and determination of lipids The preliminary column fractionation of lipids was carried out on silicic acid, by Letters' methodg). It was found necessary to use step-wise elution with chloroform-methanol mixtures containing increasing amounts of methanol, beginning with pure chloroform and ending with pure methanol. The phospholipids were separated by thin-layer chromatography on silica gel G (E. Merck AG, Darmstadt, Germany) plates, with chloroform-mehanoi-water (65:25:4, by vol) as development solvenO°). The separation, identification and quantitation of phospholipids in small lyophilised samples were accomplished by direct use of the chloroform-methanol extract washed by the Folch procedure 8). The thin-layer chromatograms were developed with two successive solvent systems, chloroform-methanol-water (65:25:4, by vol) for phospholipids and, after drying, hexane-ether (4:1, by vol) for neutral lipids11). For visualisation, the chromatograms were stained with iodine vapour, or sprayed with distilled water for the location of all lipid material, ninhydrin for amino lipids, and acid molybdate for lipids containing phosphorus. The phospholipid components were identified from their RF values in relation to appropriate phospholipid standards. For quantitative evaluation, the individual phospholipid spots or zones were scraped off the plates, and the phospholipid was eluted from the silica gel successively with chloroform-methanol-water (65:25:4, by vol), chloroform-methanol-acetic acid-water (65: 25: 4: 4, by vol) and methanol-acetic acid water (90: 4: 4, by vol). The combined filtrates were concentrated by evaporation, the organic material was digested, and the phosphorus content of each eluted component was determined by the method of Kolb et a/.12). Thin-layer chromatography on Camag (Muttenz, Switzerland) silica gel without calcium sulphate binder was effected with chloroform-methanol-acetic acid-water (25: 15: 4: 2, by vol) as solvent13).

250

H. SUOMALAINEN AND T, NURMINEN

Gas chromatography Lipid samples were prepated for gas chromatography by saponification of the fatty acids followed by methylation. The lipid was refluxed for 3 hr with 0.5 N KOH in absolute methanol. The unsaponified material was removed by extraction with light petroleum (b.p. 40-60°C), the fatty acids were liberated with sulphuric acid and extracted with light petroleum (b.p. 40-60°C). The extract was dried with anhydrous sodium sulphate, and concentrated by evaporation. The fatty acids were esterified with diazomethane x4) and analysed as their methyl esters by gas chromatography employing a 226 Ra detector, and with argon as carrier gas. The column was a glass tube 1.5 m long, packed with acid-washed Celite (10(O120 mesh) coated with 25~ Rheoplex-400 (Fluka AG, Buchs SG, Switzerland). The temperature programme was an isothermal run at 60°C followed by an up-scale rate 4°C/rain to 185°C, then isothermal. Methyl esters of fatty acids were identified by comparison of their retention times on the column with those of standard fatty acid methyl esters in mixtures. The fatty acid composition of the samples was calculated from the peak areas of the chromatograms. Results and discussion

Total lipid and phospholipid content The procedure employed most frequently for the extraction of lipids is that of Folch et a/.S); in this multiple washes are used to free the lipids from non-lipoidal substances. Trevelyan 15) was unable to find lysophosphatides in pressed commercial baker's yeast, but noted their formation in yeast suspended in methanol-water, when yeast phospholipase A was activated. Letters 16) reported that lysophosphatides were present in appreciable amounts in yeast, but one of the solvents he used - also used by Longley eta/. 6) - to extract lipids from the yeast was ethanol which Harrison and Trevelyan 17) found activated the phospholipase A of baker's yeast. Since Trevelyan 1~) could find no evidence that phospholipids were degraded when baker's yeast was extracted with chloroform-methanol (2: 1, by vol), this solvent has been utilised here. The dry weights of total lipid and phospholipid of the isolated cell envelopes were 3.1 and 0.7~o of dry matter respectively. These contents are only about one fifth of those in the cell interior, where the main part of the membranes in aerobic cells is situated. Longley et al. 6) found that about 10~o of the total lipid content of whole cells in a strain of Saccharomyces cererisiae appeared in a bound form and was not extracted by neutral organic solvents. Some of the lipid in yeast is firmly bound to the cell wal118,19) and may have

LIPIDSOFYEASTCELLENVELOPE

251

a structural role. Eddy, 20) however, found that the total lipid content of the cell wails isolated from S. cerevisiae, even after acid hydrolysis, did not exceed 2~. Quite rigorous hydrolytic methods are necessary to extract the firmly bound lipids associated with the cell wall. Consequently, some uncertainty always exists as to how closely the lipid components obtained by such methods resemble the native lipids of the intact cell envelope. As we were more interested in phospholipid components and plasma membrane, no attempt was made to extract the bound lipids. By mechanical treatment, Matile et al. 21) obtained a preparation of membranes of anaerobic S. cerevisiae which contained protein, lipids, and an appreciable amount of polysaccharide composed of mannose. In this preparation, the concentration of total lipids was relatively high, while that of phospholipid-P or sterol was comparatively low. If it is assumed that an average of 4 ~ of a phospholipid is phosphorus, then 15-20~ of the lipid in the protoplast membranes from S. cerevisiae analysed in the study of Longley et al. 6) was phospholipid; the corresponding figure from Boulton's 4) analysis was 25~ and our result for the isolated cell envelopes 23~. Fatty acid composition It was found that, in general, the compositions of fatty acids in whole cells and cell envelopes were similar, although quantitative differences could be observed. The principal fatty acids were found to be palmitoleic acid and oleic acid (table 1). The whole cells contained a somewhat larger proportion ofC16:1 acid than did the cell envelopes. The whole cells of baker's yeast have been found also to contain some short-chain fatty acids with a chain TABLE 1 Fatty acid composition of whole cells, cell envelopes and preparations obtained by enzymatic digestion of the isolated cell envelopes The results are expressed as means of 2-5 experiments Fatty acid

Whole cells

Cell envelopes

Heavy sediment

Light sediment

% of total fatty acids Cio -- Cla C14 C14:1

C15 C16 C16:1 C17 C18 C18:1

C18:2 -~- C18:3

<~

3 4 5

7 6 6

3 6 5

2 6 2

I

--

--

--

7 59 ~ 1 < 1 21 "~ 1

6 51 1 4 18 < 1

6 31 I 50 -

6 24 6 1 52 1

252

H. SUOMALAINEN AND T. NURMINEN

length of 9 carbon atoms or less, 22) but it proved that the short-chain fatty acids were not present in the isolated cell envelopes or in the digested preparations. The C,6:1 acid occurred in most abundance in the isolated cell envelopes, but the C1 s:, acid predominated in the digested preparations. On comparison with the results obtained with the original cell envelopes, a diminished proportion of the C16:, acid and an increased proportion of the C18:, acid were found in the heavy sediment and, even more emphatically, in the light sediment particularly rich in plasma membrane fragments. This phenomenon was always reproducible, although some variation was apparent between parallel experiments, by reason of the shortage of material and difficulties in washing procedures. It should be mentioned that these changes did not occur when the cell envelopes were incubated under the same conditions without the snail gut enzyme.

Phospholipids Silicic acid is favoured as the adsorbent for column chromatography, and analysis of the major phospholipids of yeast by this method has proved successful 23). We have applied the method of Letters 9) for the preliminary column fractionation of yeast lipids on silicic acid, but we found it necessary to apply stepwise elution with chloroform-methanol mixtures containing increasing amounts of methanol, beginning with pure chloroform, and ending in pure methanol. The purification and identification of individual phospholipids on thin layers of silica gel was then effected as is described under Experimental. Because phosphatidylserine and phosphatidylinositol were incompletely resolved combined figures are given for these two phospholipid components. It is also possible that the cardiolipin (diphosphatidylglycerol) contained phosphatidylglycerol. The phospholipid compositions of whole cells, cell envelopes and fractions obtained by enzymatic digestion of the isolated cell envelopes were qualitatively similar (table 2). In all the yeast preparations studied the main phospholipid compounds were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. The whole cells contained phosphatidylcholine in a clearly larger proportion than did the cell envelopes, which in turn were clearly richer in phosphatidylinositol and phosphatidylserine. The results obtained in regard to the phospholipid composition of whole cells are qualitatively in close agreement with those reported previously after studies of whole cells of Saccharomyces cerevisiae.6,15,24) The various quantitative differences may primarily be due not only to varying methods, but to varying yeast strains and growth conditions concerned. Suomalainen and Kedinen22, 25,28) have previously shown that the growth conditions markedly influence the fatty acid composition of whole cells of

253

LIPIDS OF YEAST CELL ENVELOPE

TABLE2 Phospholipid compositions of whole cells, cell envelopes and preparations obtained by enzymatic digestion of the isolated cell envelopes Abbreviations: PI phosphatidylinositol, PS phosphatidylserine, PE phosphatidylethanolamine, PC phosphatidylcholine, PA free acids containing phosphatidic acid, DPG cardiolipin (disphosphatidylglycerol), X unidentified components. The results are expressed as means of 2-5 experiments. Phospholipid

PA DPG X PI÷PS PE PC Recovery, ~

~ of total P in phospholipids Whole cells

Whole envelopes

<5 < 5 5 15 20 55

5 < 5 5 30 15 45

75

80

Digested envelopes

Heavy sediment

Light sediment

5 10 35 15 30

<5 < 5 l0 35 10 45

5 5 10 35 20 20

90

75

95

<5

baker's yeast. Further, Jollow et al. 24) have recently shown that the growth conditions influence the phospholipid composition of whole cells of S. cerevisiae.

On analysis of the phospholipids of the preparations obtained from the isolated cell envelopes by enzymatic digestion, the lipase activities introduce some difficulties. The cell envelope contains an easily extractable phospholipase with p H optimum 3 4 , 27) which at least partly remains in the cell envelopes during the course of preparation28). As it is possible that the phospholipids are unprotected from the action of these phospholipases, the danger arises that prolonged digestion may induce a change in the original phospholipid distribution. The lipase activity appearing in the snail gut enzyme preparations can be removed by gel filtration on a Sephadex column3,29). The influence of yeast lipases is diminished if digestion is carried out at p H 5.8. When the isolated cell envelopes were digested enzymatically to such an extent that their original lipid content had risen to four-fold, it was found that the original composition of phospholipids remained about the same (table 2). It has been stated earlier 1-3) that the fragments of plasma membrane present in carefully isolated cell envelopes are particularly enriched in the light sediment. It may consequently be supposed that the phospholipid composition of the plasma membrane corresponds in the main to that of the isolated cell envelopes. For further characterisation of the lipid components, their content of fatty acids was examined. Neutral lipids and phospholipids of whole cells and

254

H. SUOMALAINEN AND T. NURMINEN

cell envelopes, fractionated by column chromatography on silicic acid, or eluted from thin layer chromatograms, had the approximate fatty acid composition indicated in table 3. The principal fatty acids were again Ca6: a, C,8:, and C16, of which C,6: , acid predominated in every lipid fraction. Macfarlane ~0) has pointed out, that the fatty acid composition is very TABLE 3 Fatty acids in neutral lipids and phospholipids of whole cells and cell envelopes of baker's yeast. Abbreviations: NL neutral lipid, PE phosphatidylethanolamine, PI phosphatidylinositol, PS phosphatidylserine, PC phosphatidylcholine. Lipid fractions were isolated by column chromatography on silicic acid and identified by thin layer chromatography. Fatty acids were analysed by gas chromatography as their methyl esters. The results are expressed as approx, percentages of total fatty acids. Fatty

Whole cells

Envelopes

acid

NL

PE

PI ÷ PS

PC

NL

PE

PI ÷ PS

PC

Clo-C15 C16 C16:1 C~8 Cls:I

10 10 60 < 5 20

10 15 55 ~ 5 20

5 20 35 5 35

10 <10 65 ~ 5 20

5 10 60 5 20

~ 10 15 40 5 35

~ 5 40 40 ~ 5 20

10 10 60 ~ 5 20

similar in the different phospholipids of microorganisms. This situation might facilitate the interchange of acyl glycerols among the various phospholipids. It is, however, demonstrable that the fraction which included phosphatidylinositol and phosphatidylserine contained relatively less of the C,6: 1 acid and more of the C,6 acid than did other lipid fractions. This was found both with whole cells, and even more clearly with cell envelopes. A similar larger proportion of saturated fatty acid residues has been reported by Trevelyan15) in phosphatidylinositol from whole cells of baker's yeast. The results which indicate that isolated cell envelopes contained phosphatidylinositol and phosphatidylserine in a larger proportion than did whole cells (table 2) and that this phospholipid contains less C,6:, acid than do other lipid components (table 3), are in conformity with the smaller content of C~6:, acid found in cell envelopes (table 1). Phosphatidylinositol and phosphatidylserine were the main phospholipids in all the preparations derived from isolated cell envelopes. It is interesting to note that Trevelyan 31) has found complex inositides which contain mannose in baker's yeast, autolysed by means of toluene. The presence of mannose in complex glycosphingolipids, which were resolved into residual phosphatidylinositol and glycolipid components containing mannose, sug-

LIPIDS OF YEAST CELL ENVELOPE

255

gests some c o n n e x i o n with the yeast cell wall, o f which m a n n a n is a m a j o r constituent. It has been p r o p o s e d t h a t the glycosphingolipids m a y influence the h y d r o p h i l i c / h y d r o p h o b i c c h a r a c t e r o f the cell surface if l o c a t e d on the o u t e r side. Nevertheless, if these lipids were a t t a c h e d to the inner side o f the cell wall, they might constitute a link between the p l a s m a m e m b r a n e a n d the cell wall. The present results strongly suggest t h a t in any event an att a c h m e n t exists between the cell wall a n d the p l a s m a m e m b r a n e , as carefully isolated cell envelopes contain fragments o f the p l a s m a m e m b r a n e , a n d , moreover, the digested p r e p a r a t i o n s c o n t a i n e d n o t only p r o t e i n a n d lipids b u t also an a p p r e c i a b l e a m o u n t o f c a r b o h y d r a t e s .

Acknowledgement The a u t h o r s wish to t h a n k Miss P i r k k o Saarinen, M.Sc., a n d Mr. L a u r i J a l k a n e n , M.Sc., for their skilful technical assistance.

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T. Nurminen and H. Suomalainen, unpublished F. B. Anderson and J. W. Millbank, Biochem. J. 99 (1966) 682 M. G. Macfarlane, Advan. Lipid Res. 2 (1964) 91 W. E. Trevelyan, J. Inst. Brewing 74 (1968) 365