VOL.
YEAST
7: 455- 46 1 ( 199 1 )
Glucan Structure in a Fragile Mutant of Saccharomyces cerevisiae J. HLAGOEVA'. G. STOEVt A N D P. VENKOV lnsiiiute of Moleculrr Biology und tlnstiiute of Orgunic Chemistry. Bulgurian Acudeniy of Sciences, I 1 13 Sofiu.
Bulgciriu
Recened 10 October 1990; revised 19 January I99 I
The phenotype of VY 1160 fragile Sacchuromyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased perrncability of cells growing in osmotically stabilized media. Two mutations. srhl and 1.71. have bccn identified in VY I160 cells and previous studies have shown that the increased permeability is due to the is1 mutation which causes a shortening of mannan side-chains. Here we report that thc srhl mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans providc evidcncc that srbl mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain P ( l 4 ) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, thc srbl cells cannot resist an osmotic shock and, hence. burst immediately. KEY W O R I x --
cell wall mutant; Sacchoromyces c'erevisiue;glucan structure.
INTRODUCTION The cell wall of Succhuromyces cerevisiue consists of almost equal amounts of glucan and mannoproteins and a small amount ofchitin. While glucan supports and maintains the rigidity of the cell wall, the mannoprotein determines its permeability (Zlotnik et ul.. 1984). We have isolated and studied cell wall mutants characterized by cell lysis in hypotonic solutions and increased permeability to substances for which yeasts are known to be impermeable (Venkov et at.. 1974; Waltschewa et at., 1989). The genetic analysis of VY 1 160 fragile mutant identified two mutations, srhf and t s f , responsible for the mutant phenotype (Kozhina et at., 1979). Previous analysis of mannoproteins showed that the VY 1160 mutant cells contain i) two times less mannan, due to a reduction of the polysaccharide part of the mannoprotein complex, and ii) significantly shorter mannan side-chains (Markisch et at., 1983). Experiments with single mutation strains suggested that t s f is the mutation responsible for the structural 'Author
to whom
correspondence should he addressed
0749 503X 9 I 050455 4 7 SO5 00 C 199 I h> John Wiley & Sons Ltd
changes of mannan in VY1160 cells. Recently, it has been demonstrated that permeability in S. cerevisiue cells is limited by the mannan side-chains (De Nobel et al., 1990). Therefore, the increased permeability of VYI 160 mutant cells is due to the t s f mutation which causes a shortening of mannan side-chains. In this communication we report that the other mutation in VY 1160 cells, s r h f , is responsible for quantitative and structural changes of glucan in the mutant cells. In the cell wall of S. cerevisiue there are three types of glucans. The insoluble glucan is a branched p( I -3) polymer with fibrillar structure containing 3% of p( lL6) glycosidic inter-chain linkages and no p( 1 -6) inter-residue linkages. The acid-soluble glucan is a minor component, has a highly branched structure and contains a large proportion of p( 1-6) glycosidic linkages and a smaller proportion of p( lL3) bonds. The alkali-soluble glucan is a major component and has an amorphous structure; i t is mainly p( I - -3) glucan that contains about 10% p( 1-6) linkages and 3% branched residues (LopezRomero and Ruiz-Herrera, 1977).
456
J. BLAGOEVA ET AL.
Table 1.
Yeast strains used
Strain S288C VY1160 2881 ISTL 7SL 191TL AH215
Genotype MATa MATa MATa MATa MATa MATa MATa
gal2 malgal2 ma1 srbl tsl ts2 leu2 tyr- thr- ade- uragal2 ma1 leu2-3 leu2-I12 gal2 mal- srbl tsl leu2-3 leu2-112 gal2 mal- srbl leu2-3 leu2-112 gal2 mal- tsl leu2-3 leu2-112 leu2-3 leu2-112 his3-11 his3-1.5 ~
~
MATERIALS AND METHODS Strains and culture conditions The S . cerevisiae strains used in this study are listed in Table 1. VYI 160 is a fragile mutant obtained from S288C (Venkov et al., 1974). Strains ISTL, 7SL, 191TL were obtained from a cross between VY1160 and AH215. To obtain 288L the stains S288C and AH2 15 were crossed and the zygotes isolated by micromanipulation. All strains, osmotically stable or labile, were cultivated at 30°C in YPD medium (1 YOyeast extract, 2% Bacto peptone, 2% glucose) supplemented with 10% sorbitol, needed as osmotic stabilizer for the fragile cells. The nutrient media components were from Difco. The cells were grown to a culture density of 5 x lo7 cells/ml, washed by centrifugation three times with 10% sorbitol solution and used to isolate glucan fractions. Isolation and guantitation of glucan fractions Glucans were fractionated from cell walls obtained from yeast biomass representing about 5 x 10" cells. Since a complete cell disruption is a prerequisite for the accurate estimation of glucan content per cell, the disruption of cells was followed microscopically for each strain. The cell walls were washed by centrifugation three times with 10 mM-Tris pH = 8.5 and heated for 15min at 60°C to interrupt the action of endogenous glucanases. Alkali-soluble, acid-soluble and insoluble glucans were fractionated by a published procedure (Manners et al., 1973a; Fleet and Manners, 1976). The yield of the different glucan fractions was measured by weight and the amount of glucans was calculated per cell. Determination of molecular weight The molecular weight of the glucan fractions was determined by chromatography on CL Sepharose 6B
Source
G. Fink P. Venkov This study This study This study This study A. Hinnen
(100 cm x 1 cm column). Insoluble glucans were solubilized by treatment with nitrous acid (Mol and Wessels, 1987). The eluant for alkali-soluble and insoluble glucans was 0.1 M-NaOH and the acidsoluble glucans were eluted with 0.5 M-CH~COOH. Sugar content in the column fractions was determined according to Dubois et al. (1956). Analysis of glucan fractions Methylation analysis of glucans was performed according to Hakomori (1964). The degree of methylation was followed by infrared spectrometry. Hydrolysis of methylated glucans was carried out as described (Lindberg, 1972). Methylated sugars were analysed by GLC as methylsylil derivatives using a Perkin-Elmer Sigma 300 gas chromatograph with electronic integrator Shimadzu RC- 1 B. A mixture of methylated and acid-hydrolysed laminarin (linear p(1-3) glucan) and pustulan (linear p( 1-6) glucan) was chromatographed as a control. The identity of the peaks was confirmed by chemical ionization mass spectrometry cpupled with gas chromatography and iso-butan as gas reagent. Smith degradation was performed according to Hay et al. ( 1 965).
RESULTS In preliminary experiments we found differences in the amount and the structure of glucans isolated from parental and mutant cells. Since the amount, purity and structure of glucan fractions were reported to vary among yeast strains and with the isolation procedure used (Duffus et al., 1982), we performed some preliminary steps in order to standardize the experimental procedure. First, we crossed the parental S288C and the mutant VY 1160 cells to a related strain and isolated clones having either only the srhl, or only the tsl mutation, or the wild-type SRBl TS1 alleles (see section on strains and culture conditions). The usage of such isogenic single mutation yeast strains
GLUCAN STRUCTURE IN A FRAGILE MUTANT OF S A C R O M Y C E . 7 CEREVfSfAE
0) C
Q
10
20
30
40
50
Fraction number Figure I . Molecular weight determination of glucans. Gel filtration profiles of insoluble glucans purified from 288L wild-type (solid line) and 7SL fragile mutant (broken 1ine)cells. Thearrows show the positions of molecular weight markers: dextrans of 500 kDa and 264 kDa. The void volume is 27 ml. Fractions of I .6 ml were collected at flow rate of 13.5 mlih.
is a prerequisite to obtain reliable results in comparative studies of glucans. Secondly. the purity of isolated glucan fractions was checked. The elemental analysis of all studied glucans showed a C : H ratio of 1 : 2 and no solid residue was detected after ignition. The analysis of total acid hydrolysates of glucans revealed that they contained only glucose. No peak of mannose was observed in the gas-chromatograms (data not shown). Thirdly, we determined the molecular weights of all three types of glucans and found that alkalisoluble and acid-soluble glucans have equal molecular weight in mutant and wild-type cells. However, significant differences were observed in the study of insoluble glucan. As shown in Figure 1, the fragile mutant cells contained insoluble glucan with markedly reduced molecular weight when compared to the same glucan fraction isolated from wild-type cells. The lower molecular weight is characteristic for the srhl cells while tsl cells have insoluble glucan with a molecular weight equal to that found in wild-type cells. Quantitative analysis of xlucans The rcsults obtained in the study of wild-type and different single mutation isogenic strains are
457
summarized in Table 2. As reported previously (Kozhina et al.. 1979), only srhl cells lyse when suspended in hypotonic solutions. The analysis of glucans showed that srhl cells are characterized by a reduced amount of alkali-soluble glucan. Compared to wild type (288L) or cells having only t s l mutation (191TL), the srhl strains (ISTL and 7SL) contain three to four times less alkali-soluble glucan per cell. While theamount ofacid-soluble glucan is about the same in all studied strains, there is a tendency for an increase in the amount of insoluble glucan in srhl cells. Theamount ofinsoluble glucan was reported to vary significantly with the size ofcells (Katohda et a/., 1976). Since the ISTL and 7SL strains have cells larger than thoseof288Land 191TLstrains, the7SL strain was crossed to an ancestral wild-type strain, the diploid sporulated and several full tetrads with members having equal cell size were isolated. The quantitative determination of insoluble glucan revealed that all members from one tetrad have similar amounts of insoluble glucan per cell irrespective of their genotype (srhl or S R B I ) . However, for all tetrads studied, a strict correlation between srhl genotype and a reduced amount of alkali-soluble glucan was found. The results obtained with one of the tetrads are presented in Table 3. The results obtained in the quantitative determination of glucans showed that the srhl mutation, which is the geneticdeterminant ofcell lysis in hypotonic solutions, caused a decrease in the amount of alkali-soluble glucan to 20- 30% of that found in wild-type cells. The variations in the amount of insoluble glucan do not co-segregate with the fragile phenotype in genetic crosses and most likely reflect the different sizc of cells among strains. Methylation analysis ojglucans We carried out this analysis for alkali-soluble, acid-soluble and insoluble glucans purified from wild-type and different single mutation strains. An example of such an analysis is given in Figure 2. In agreement with literaturedata (Sweeley eta!., 1966). every kind of methylated monomer is presented in the gas chromatogram as a doublet of peaks which correspond to a and p anomers of the glucose. We did not observe differences in the relative molar percentages of the different types of linkages in alkalisoluble and acid-soluble glucans and the values obtained for the wild-type and mutant strains (Table 4) are close to the previously reported data for other wild-type S. cerevisiae strains (Manners et a!., 1973b; Fleet and Manners, 1976). However. significant differences were found in the relative
458
J. BLAGOEVA ET AL.
Table 2. Quantitative analysis of glucans
Strain
Genotype
Percentage lysed cells*
2881, 1STL 7SL 191TL
SRBl TSl srbl tsl srbl TSl SRBl tsl
2 78 64 2
Alkali soluble
Glucan fractions Acid soluble (in 10-'omg/cell)
Insoluble
4.65 f0.45 1.20 f0.2 1 2.32 f0.25 5.51 f0.60
0.35 f0.03 0.42 f0.03 0.30 f0.03 0.37 f0.02
3.70 f0.30 8.49 f0.85 7.06 f0.67 4.84 f0.55
*Determined as described in Venkov et al. (1974).
Table 3. Analysis of glucans in tetrad 16
Tetrad member
Genotype
Percentage lysed cells
SRBl srbl SRBl srbl
1 79 2 68
16a 16b I6c 16d
al
u)
i u)
E L
0
c 0
al
C
0
n
14
16
18
20
22
Ret. time Iminl Figure 2. Methylation analysis of glucans. Gas chromatograms of insoluble glucans purified from 288L wild-type (broken line) and 7SL fragile mutant (solid line) cells.
Glucan fractions Alkali soluble Insoluble (in 10 l o mg/cell) 7.22 0.85 7.50 2.3 1
16.80 12.70 10.80 1505
amounts of p( I-6)-linked glucose residues in the insoluble glucans (Figure 2 and Table 4). Along with some p( 1-6) linkages present as branch points, the insoluble glucan of the fragile lSTL mutant contains about 3% of p( 1-6) inter-residue linkages. The methylation analysis was repeated with the single mutation strains and the data presented in Table 5 clearly show that the appearance of p(1-6) interresidue linkages in the insoluble glucan is an attribute of the srbl mutation. The insoluble glucan purified from t s l cells, like insoluble glucan of wildtype cells, contained p(1-6) linkages only as branch points. An increase of p( 1-6) linkages might appear because of undermethylation. Although the completeness of methylation was checked by infrared spectrometry for every glucan fraction, we performed an additional methylation analysis of insoluble glucans after Smith degradation. The resultsofthesecontrol experiments(Tab1e 6) showed the lack of p( 1-6) linkages after Smith degradation and are additional evidence for the existence of p( 1-6) inter-residue linkages in the insoluble glucan of the fragile mutant cells.
DISCUSSION Although the role of glucan is the main one, the rigidity of S. cerevisiue cells seems to be determined
459
GLUCAN STRUCTURE IN A FRAGILE MUTANT OF SACCHAROMYCES CEREVISIAE
Table 4.
Relative molar percentages of sugar derivatives
Type of linkage
Methylsylil derivatives 2,3,4,6-tetra-O-rnethyl 2,4,6-tri-O-methyl 2,3,4-tri-O-methyl 2.4-di-0-methyl
Table 5.
End groups P(1-3) P(1-6)
Branch points
Alkali soluble 288L lSTL 3.9 84.0 8.8 3.4
2.8 84.3 10.1 2.7
Glucan fractions Acid soluble 288L lSTL 11.8
12.4
7.6 67.8 12.9
5.5
73.6 8.5
Insoluble 288L lSTL 1.4 96.5 0.0 1.8
1.3 94.9 2.6 1.3
Methylation analysis of insoluble glucans
Methylsylil derivatives
Insoluble glucan Strain 7SL (srbl TSl) Strain 191TL (SRBI t s l ) 2.5 93.9 3.2 2.1
2.4 94.2 0.0 3.2
Table 6. Methylation analysis of insoluble glucans after Smith degradation
Methylsylil derivatives
Type of linkage
2,3,4,6-tetra-O-methyl 2,4,6-tri-O-methyl 2,3,4-tri-O-methyl 2,4-di-O-methyl
End groups P(1-3) P( 1-61 Branch points
by the complex structure of the cell wall. For instance, cell lysis occurs in mutants in which the mannoprotein outer chain is greatly shortened (Ballou et al., 1980) or synthesis of chitin is strongly inhibited (Bowers et al., 1974). The antibiotic aculeacin A inhibits the synthesis of insoluble glucan (Mizoguchi et al., 1977) or all types of glucans (Tkacz, 1984) and induces cell lysis in S. cerevisiae. In our studies with the S. cerevisiae fragile mutant VY 1 160 a role for the alkali-soluble glucan in maintaining the integrity of the cell is suggested. Quantitative measurements and genetic studies (Tables 2 and 3) provide evidence that the srbl mutation, which determines the fragile phenotype of the VY 1 160mutant, leads to a decrease of alkali-soluble glucan in the cell wall. In the presence of an osmotic stabilizer in the media the srbl cells multiply exponentially(Venkovetal., 1974),whichsuggests that
Insoluble Smith-degraded glucan Strain 288L (SRBI TSl) Strain lSTL (srbl t s l ) 2.0 96.1 0.0 1.9
1.9 96.0 0.0 2.0
the lower amount of alkali-soluble glucan is not a step limiting the growth rate. However, it seems that the amount of alkali-soluble glucan is critical for maintaining the resistance of S. cerevisiae cells in conditions of osmotic shock because the transfer of the srbl cells to hypotonic solutions is accompanied by the immediate bursting of the cells (Mateeva et al., 1976).Instudiesofotherauthorsamore than 10-fold decrease in the amount ofinsoluble glucan (Katohda et al., 1976) or p(1-6) glucan (Meaden et al., 1990) was found to be without effect on cell rigidity. Contrary to this, a moderate (three- to four-fold) decrease of alkali-soluble glucan found in our studies for srbl cells is accompanied by the appearance of the fragile phenotype. Another observation in this study is the lower molecular weight and the existence of p( 1 4 ) interresidue linkages in the insoluble glucan of the fragile
460
cells. Both wild-type and m u t a n t glucans were isolated in experimental conditions where strong precautions against glucanase action have been taken. Therefore, the lower molecular weight found for the m u t a n t insoluble glucan does not seem to be due to degradation and most likely reflects the existence of shorter glucan fibrils in the fragile cells. In addition, the existence o f p( 1 -6) inter-residue linkages was shown t o prevent the formation o f triple helices a n d their organization into a fibrillar network with appropriate mechanical properties (Kopecka a n d Kreger, 1976). Therefore, the cell wall o f t h e fragile VY I160 m u t a n t contains insoluble glucan characterized by shorter. mechanically unstable fibrils generating a weaker fibrillar network. In addition, there is a shortage o f interstitial substances, alkalisoluble glucan (this study) a n d mannoproteins (Markisch ct al., 1983) in the m u t a n t cells. T h e filling o f the space between the fibrils is highly insufficient a n d the cell wall very fragile. Although growing exponentially in the presence o f osmotic stabilizer. the fragile cells lyse immediately upon transfer t o hypotonic solutions. As shown in previous electron microscopic (Mateeva ct al.. 1976) a n d cell-fusion (Philipovaand Venkov, 1990)studies. t h e l y s i s o f t h e fragile cells represents formation of holes on the cell surface where t h e cell wall is thinner or even missing.
R E FE R E NCES Ballou, L.. Cohen, R. and Ballou, C. (1980). Saccharomycw cerivisiac. mutants tha t make mannoproteins with a truncated carbohydrate outer chain. J . Biol. Chem. 255,5986-5991. Bowers, B., Lcvin, G. and Cabib, E. (1974). Effect of Polyoxin D on chitin synthesis and septum formation in Saccharoniyc*es cerevisiac. J. Bacteriol. 119,564575. De Nobel, J . G . . Klis. F. M.. Priem, J., Munnik, T. and Van den Ende, H. (1990). The glucanase-soluble mannoproteins limit celi wall porosityin Surcharomyces cerevisiae. Yeast 6,49 1-500. Dubois, M.. Gilles. K. A,, Hamilton, J . K., Rebers, P. A. and Smith, F. (1956). Colorimetric method for dctermination of sugars and related substances. Anal. Chem. 28,350-356. DutTus. J.. Levi, C. and Manners. D. (1982).Yeast cell wall glucans. Ad,,. Microhiol. Phjsiol. 23, 15 1-1 8 I . Fleet. G . and Manners, D. (1976). Isolation and composition of an alkali soluble glucan from the cell wall of Sacdiorotnj.ces cerevisiae. J . Gen. Microbiol. 94, 180 - I 92. Hakomori. S. (1964). A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J . Biochem. 55, 205-208.
J. BLAGOEVA E T A L .
Hay, G. W., Lewis, B. A. and Smith, F. (1965). Pcriodate oxidation of polysaccharides: General procedures. Methods Carhohydr. Chem. 5,357- 361. Katohda. S., Abe, N., Matsui, M. and Hayashibc. M. (1976). Polysaccharide composition of the cell wall of bakers yeast with special reference to cell wall obtained from large and small sized cells. Plant a n d C d l Phjsiol. 17,909-919. Kopecka. M. and Kreger. D. (1986). Assembly of microfibrils in vivo and in vitro from (I-3)-P-u-glucan synthesized by protoplasts of Sacchoromyces cerevisiuc. Arch. Microhiol. 143,387.-395. Kozhina, T.. Stateva. L. and Venkov. P. (1979). Genetic analysis of an osmotic sensitive Saccharom~ces ccwvisiue mutant. Molec. Gen. Genet. 170, 351 - 355. Lindberg. B. (1972). Methylation analysis of polysaccharides. In Ginsburg. N . (Ed.). Methods in Enzymology. vol. XXVIII, part B. pp. 178 -195. Lopez-Romero. E. and Ruiz-Herrera, J . (1977). Biosynthesis of 0-glucans by cell free extracts from Saccharom p > sccwvisiae. Biochim. Biophjx Acva 500, 372 384. Manners. D.. Masson, A. and Patterson. J. (1973a). The structure of a P( 1- 3)-wglucan from yeast cell walls. Riochem. J . 135,19-30. Manners. D.. Masson. A,, Patterson, J.. Bjorndal. H . and Lindberg, B. (1973b). The structure of a p( I -6)-uglucan from yeast cell walls. Bbchem. J . 135, 3 1-36. Markisch, U., Reuter, G.. Stateva, L. and Vcnkov, P. (1983). Mannan structure analysis of the fragile Saccharomjws cerevisiae VYI 160 mutant. I n t . J . B i ~ c h e 15, ~ ~ 1373. 1377. Mateeva, Z . , Petrov. P., Venkov, P. and Hadjiolov. A. (1976). Electron microscopic study of the lysis of an osmotic sensitive yeast mutant. J . Microsc. Biol. Cellulairr 26,73-74. Meaden. P., Hill, K., Wagner, J., Slipetz, D., Sommcr. S. and Bussey, H. (1990). The yeast KRES gene encodes a probable cndoplasmic reticulum protein required for ( I 6)P-n-glucan synthesis and normal cell growth. Mol. Cell. Biol. 10, 3013-3019. Mizoguchi. G., Saito, T., Kizuno. Z . and Hayano, K . (1977). On the mode of action of a new antifungal antibiotic aculeacin A: inhibition of cell wall synthesis in Sacdiaromyces cerevisiae. J . Anribiot. 30,308-3 13. Mol. P. and Wessels, J . (1987). Linkage bctwecn glucosaminoglycan and glucan determine alkali insolubility of the glucan in walls of Sacchoromyces cerevisiae. F E M S Microbiol. Letters 41,95 -99. Philipova, D. and Vendov. P. (1990). Cell fusion of Succhoromyces wrevisiae fragile mutants. Yeast 6, 205 212. Sweeley, C. C.. Wells. W. W. and Bentley. R. (1966). Gas chromatography of carbohydrates. In Ginsburg. N. (Ed.). Methods in Enzymology, vol. VIII, pp. 95-108. Tkacz. J. Z. (1984). I n viro synthesis of p(1-6) glucan in Sucdiaroniyes cereuisiac.. In Nombela, C . (Ed.), Microbial Cell W d l Synthesis and Autoljsis. Elsevier Sci. Publishers, Amsterdam, pp. 287-296.
CLUCAN STRUCTURE IN A FRAGILE MUTANT OF SACCHAROMYCES CEREVISIAE
Venkov, P., Hadjiolov, A,, Battaner, E. and Schlessinger, D. ( 1974).Saccharomyces cerevisiae sorbitol dependent fragile mutants. Biochem. Biophys. Res. Commun. 56, 559-604. Waltschewa, L., Philipova, D. and Venkov, P. (1989). Increased extracellular secretion in fragile mutants of Sarcharomyces cerevisiae. Yeast 5,3 13-320.
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Zlotnik, H., Fernandez, M., Bowers, B. and Cabib, E. (1984). Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J. Bacteriol. 159, 1018-1026.
YEAST
7: 455- 46 1 ( 199 1 )
Glucan Structure in a Fragile Mutant of Saccharomyces cerevisiae J. HLAGOEVA'. G. STOEVt A N D P. VENKOV lnsiiiute of Moleculrr Biology und tlnstiiute of Orgunic Chemistry. Bulgurian Acudeniy of Sciences, I 1 13 Sofiu.
Bulgciriu
Recened 10 October 1990; revised 19 January I99 I
The phenotype of VY 1160 fragile Sacchuromyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased perrncability of cells growing in osmotically stabilized media. Two mutations. srhl and 1.71. have bccn identified in VY I160 cells and previous studies have shown that the increased permeability is due to the is1 mutation which causes a shortening of mannan side-chains. Here we report that thc srhl mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans providc evidcncc that srbl mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain P ( l 4 ) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, thc srbl cells cannot resist an osmotic shock and, hence. burst immediately. KEY W O R I x --
cell wall mutant; Sacchoromyces c'erevisiue;glucan structure.
INTRODUCTION The cell wall of Succhuromyces cerevisiue consists of almost equal amounts of glucan and mannoproteins and a small amount ofchitin. While glucan supports and maintains the rigidity of the cell wall, the mannoprotein determines its permeability (Zlotnik et ul.. 1984). We have isolated and studied cell wall mutants characterized by cell lysis in hypotonic solutions and increased permeability to substances for which yeasts are known to be impermeable (Venkov et at.. 1974; Waltschewa et at., 1989). The genetic analysis of VY 1 160 fragile mutant identified two mutations, srhf and t s f , responsible for the mutant phenotype (Kozhina et at., 1979). Previous analysis of mannoproteins showed that the VY 1160 mutant cells contain i) two times less mannan, due to a reduction of the polysaccharide part of the mannoprotein complex, and ii) significantly shorter mannan side-chains (Markisch et at., 1983). Experiments with single mutation strains suggested that t s f is the mutation responsible for the structural 'Author
to whom
correspondence should he addressed
0749 503X 9 I 050455 4 7 SO5 00 C 199 I h> John Wiley & Sons Ltd
changes of mannan in VY1160 cells. Recently, it has been demonstrated that permeability in S. cerevisiue cells is limited by the mannan side-chains (De Nobel et al., 1990). Therefore, the increased permeability of VYI 160 mutant cells is due to the t s f mutation which causes a shortening of mannan side-chains. In this communication we report that the other mutation in VY 1160 cells, s r h f , is responsible for quantitative and structural changes of glucan in the mutant cells. In the cell wall of S. cerevisiue there are three types of glucans. The insoluble glucan is a branched p( I -3) polymer with fibrillar structure containing 3% of p( lL6) glycosidic inter-chain linkages and no p( 1 -6) inter-residue linkages. The acid-soluble glucan is a minor component, has a highly branched structure and contains a large proportion of p( 1-6) glycosidic linkages and a smaller proportion of p( lL3) bonds. The alkali-soluble glucan is a major component and has an amorphous structure; i t is mainly p( I - -3) glucan that contains about 10% p( 1-6) linkages and 3% branched residues (LopezRomero and Ruiz-Herrera, 1977).
456
J. BLAGOEVA ET AL.
Table 1.
Yeast strains used
Strain S288C VY1160 2881 ISTL 7SL 191TL AH215
Genotype MATa MATa MATa MATa MATa MATa MATa
gal2 malgal2 ma1 srbl tsl ts2 leu2 tyr- thr- ade- uragal2 ma1 leu2-3 leu2-I12 gal2 mal- srbl tsl leu2-3 leu2-112 gal2 mal- srbl leu2-3 leu2-112 gal2 mal- tsl leu2-3 leu2-112 leu2-3 leu2-112 his3-11 his3-1.5 ~
~
MATERIALS AND METHODS Strains and culture conditions The S . cerevisiae strains used in this study are listed in Table 1. VYI 160 is a fragile mutant obtained from S288C (Venkov et al., 1974). Strains ISTL, 7SL, 191TL were obtained from a cross between VY1160 and AH215. To obtain 288L the stains S288C and AH2 15 were crossed and the zygotes isolated by micromanipulation. All strains, osmotically stable or labile, were cultivated at 30°C in YPD medium (1 YOyeast extract, 2% Bacto peptone, 2% glucose) supplemented with 10% sorbitol, needed as osmotic stabilizer for the fragile cells. The nutrient media components were from Difco. The cells were grown to a culture density of 5 x lo7 cells/ml, washed by centrifugation three times with 10% sorbitol solution and used to isolate glucan fractions. Isolation and guantitation of glucan fractions Glucans were fractionated from cell walls obtained from yeast biomass representing about 5 x 10" cells. Since a complete cell disruption is a prerequisite for the accurate estimation of glucan content per cell, the disruption of cells was followed microscopically for each strain. The cell walls were washed by centrifugation three times with 10 mM-Tris pH = 8.5 and heated for 15min at 60°C to interrupt the action of endogenous glucanases. Alkali-soluble, acid-soluble and insoluble glucans were fractionated by a published procedure (Manners et al., 1973a; Fleet and Manners, 1976). The yield of the different glucan fractions was measured by weight and the amount of glucans was calculated per cell. Determination of molecular weight The molecular weight of the glucan fractions was determined by chromatography on CL Sepharose 6B
Source
G. Fink P. Venkov This study This study This study This study A. Hinnen
(100 cm x 1 cm column). Insoluble glucans were solubilized by treatment with nitrous acid (Mol and Wessels, 1987). The eluant for alkali-soluble and insoluble glucans was 0.1 M-NaOH and the acidsoluble glucans were eluted with 0.5 M-CH~COOH. Sugar content in the column fractions was determined according to Dubois et al. (1956). Analysis of glucan fractions Methylation analysis of glucans was performed according to Hakomori (1964). The degree of methylation was followed by infrared spectrometry. Hydrolysis of methylated glucans was carried out as described (Lindberg, 1972). Methylated sugars were analysed by GLC as methylsylil derivatives using a Perkin-Elmer Sigma 300 gas chromatograph with electronic integrator Shimadzu RC- 1 B. A mixture of methylated and acid-hydrolysed laminarin (linear p(1-3) glucan) and pustulan (linear p( 1-6) glucan) was chromatographed as a control. The identity of the peaks was confirmed by chemical ionization mass spectrometry cpupled with gas chromatography and iso-butan as gas reagent. Smith degradation was performed according to Hay et al. ( 1 965).
RESULTS In preliminary experiments we found differences in the amount and the structure of glucans isolated from parental and mutant cells. Since the amount, purity and structure of glucan fractions were reported to vary among yeast strains and with the isolation procedure used (Duffus et al., 1982), we performed some preliminary steps in order to standardize the experimental procedure. First, we crossed the parental S288C and the mutant VY 1160 cells to a related strain and isolated clones having either only the srhl, or only the tsl mutation, or the wild-type SRBl TS1 alleles (see section on strains and culture conditions). The usage of such isogenic single mutation yeast strains
GLUCAN STRUCTURE IN A FRAGILE MUTANT OF S A C R O M Y C E . 7 CEREVfSfAE
0) C
Q
10
20
30
40
50
Fraction number Figure I . Molecular weight determination of glucans. Gel filtration profiles of insoluble glucans purified from 288L wild-type (solid line) and 7SL fragile mutant (broken 1ine)cells. Thearrows show the positions of molecular weight markers: dextrans of 500 kDa and 264 kDa. The void volume is 27 ml. Fractions of I .6 ml were collected at flow rate of 13.5 mlih.
is a prerequisite to obtain reliable results in comparative studies of glucans. Secondly. the purity of isolated glucan fractions was checked. The elemental analysis of all studied glucans showed a C : H ratio of 1 : 2 and no solid residue was detected after ignition. The analysis of total acid hydrolysates of glucans revealed that they contained only glucose. No peak of mannose was observed in the gas-chromatograms (data not shown). Thirdly, we determined the molecular weights of all three types of glucans and found that alkalisoluble and acid-soluble glucans have equal molecular weight in mutant and wild-type cells. However, significant differences were observed in the study of insoluble glucan. As shown in Figure 1, the fragile mutant cells contained insoluble glucan with markedly reduced molecular weight when compared to the same glucan fraction isolated from wild-type cells. The lower molecular weight is characteristic for the srhl cells while tsl cells have insoluble glucan with a molecular weight equal to that found in wild-type cells. Quantitative analysis of xlucans The rcsults obtained in the study of wild-type and different single mutation isogenic strains are
457
summarized in Table 2. As reported previously (Kozhina et al.. 1979), only srhl cells lyse when suspended in hypotonic solutions. The analysis of glucans showed that srhl cells are characterized by a reduced amount of alkali-soluble glucan. Compared to wild type (288L) or cells having only t s l mutation (191TL), the srhl strains (ISTL and 7SL) contain three to four times less alkali-soluble glucan per cell. While theamount ofacid-soluble glucan is about the same in all studied strains, there is a tendency for an increase in the amount of insoluble glucan in srhl cells. Theamount ofinsoluble glucan was reported to vary significantly with the size ofcells (Katohda et a/., 1976). Since the ISTL and 7SL strains have cells larger than thoseof288Land 191TLstrains, the7SL strain was crossed to an ancestral wild-type strain, the diploid sporulated and several full tetrads with members having equal cell size were isolated. The quantitative determination of insoluble glucan revealed that all members from one tetrad have similar amounts of insoluble glucan per cell irrespective of their genotype (srhl or S R B I ) . However, for all tetrads studied, a strict correlation between srhl genotype and a reduced amount of alkali-soluble glucan was found. The results obtained with one of the tetrads are presented in Table 3. The results obtained in the quantitative determination of glucans showed that the srhl mutation, which is the geneticdeterminant ofcell lysis in hypotonic solutions, caused a decrease in the amount of alkali-soluble glucan to 20- 30% of that found in wild-type cells. The variations in the amount of insoluble glucan do not co-segregate with the fragile phenotype in genetic crosses and most likely reflect the different sizc of cells among strains. Methylation analysis ojglucans We carried out this analysis for alkali-soluble, acid-soluble and insoluble glucans purified from wild-type and different single mutation strains. An example of such an analysis is given in Figure 2. In agreement with literaturedata (Sweeley eta!., 1966). every kind of methylated monomer is presented in the gas chromatogram as a doublet of peaks which correspond to a and p anomers of the glucose. We did not observe differences in the relative molar percentages of the different types of linkages in alkalisoluble and acid-soluble glucans and the values obtained for the wild-type and mutant strains (Table 4) are close to the previously reported data for other wild-type S. cerevisiae strains (Manners et a!., 1973b; Fleet and Manners, 1976). However. significant differences were found in the relative
458
J. BLAGOEVA ET AL.
Table 2. Quantitative analysis of glucans
Strain
Genotype
Percentage lysed cells*
2881, 1STL 7SL 191TL
SRBl TSl srbl tsl srbl TSl SRBl tsl
2 78 64 2
Alkali soluble
Glucan fractions Acid soluble (in 10-'omg/cell)
Insoluble
4.65 f0.45 1.20 f0.2 1 2.32 f0.25 5.51 f0.60
0.35 f0.03 0.42 f0.03 0.30 f0.03 0.37 f0.02
3.70 f0.30 8.49 f0.85 7.06 f0.67 4.84 f0.55
*Determined as described in Venkov et al. (1974).
Table 3. Analysis of glucans in tetrad 16
Tetrad member
Genotype
Percentage lysed cells
SRBl srbl SRBl srbl
1 79 2 68
16a 16b I6c 16d
al
u)
i u)
E L
0
c 0
al
C
0
n
14
16
18
20
22
Ret. time Iminl Figure 2. Methylation analysis of glucans. Gas chromatograms of insoluble glucans purified from 288L wild-type (broken line) and 7SL fragile mutant (solid line) cells.
Glucan fractions Alkali soluble Insoluble (in 10 l o mg/cell) 7.22 0.85 7.50 2.3 1
16.80 12.70 10.80 1505
amounts of p( I-6)-linked glucose residues in the insoluble glucans (Figure 2 and Table 4). Along with some p( 1-6) linkages present as branch points, the insoluble glucan of the fragile lSTL mutant contains about 3% of p( 1-6) inter-residue linkages. The methylation analysis was repeated with the single mutation strains and the data presented in Table 5 clearly show that the appearance of p(1-6) interresidue linkages in the insoluble glucan is an attribute of the srbl mutation. The insoluble glucan purified from t s l cells, like insoluble glucan of wildtype cells, contained p(1-6) linkages only as branch points. An increase of p( 1-6) linkages might appear because of undermethylation. Although the completeness of methylation was checked by infrared spectrometry for every glucan fraction, we performed an additional methylation analysis of insoluble glucans after Smith degradation. The resultsofthesecontrol experiments(Tab1e 6) showed the lack of p( 1-6) linkages after Smith degradation and are additional evidence for the existence of p( 1-6) inter-residue linkages in the insoluble glucan of the fragile mutant cells.
DISCUSSION Although the role of glucan is the main one, the rigidity of S. cerevisiue cells seems to be determined
459
GLUCAN STRUCTURE IN A FRAGILE MUTANT OF SACCHAROMYCES CEREVISIAE
Table 4.
Relative molar percentages of sugar derivatives
Type of linkage
Methylsylil derivatives 2,3,4,6-tetra-O-rnethyl 2,4,6-tri-O-methyl 2,3,4-tri-O-methyl 2.4-di-0-methyl
Table 5.
End groups P(1-3) P(1-6)
Branch points
Alkali soluble 288L lSTL 3.9 84.0 8.8 3.4
2.8 84.3 10.1 2.7
Glucan fractions Acid soluble 288L lSTL 11.8
12.4
7.6 67.8 12.9
5.5
73.6 8.5
Insoluble 288L lSTL 1.4 96.5 0.0 1.8
1.3 94.9 2.6 1.3
Methylation analysis of insoluble glucans
Methylsylil derivatives
Insoluble glucan Strain 7SL (srbl TSl) Strain 191TL (SRBI t s l ) 2.5 93.9 3.2 2.1
2.4 94.2 0.0 3.2
Table 6. Methylation analysis of insoluble glucans after Smith degradation
Methylsylil derivatives
Type of linkage
2,3,4,6-tetra-O-methyl 2,4,6-tri-O-methyl 2,3,4-tri-O-methyl 2,4-di-O-methyl
End groups P(1-3) P( 1-61 Branch points
by the complex structure of the cell wall. For instance, cell lysis occurs in mutants in which the mannoprotein outer chain is greatly shortened (Ballou et al., 1980) or synthesis of chitin is strongly inhibited (Bowers et al., 1974). The antibiotic aculeacin A inhibits the synthesis of insoluble glucan (Mizoguchi et al., 1977) or all types of glucans (Tkacz, 1984) and induces cell lysis in S. cerevisiae. In our studies with the S. cerevisiae fragile mutant VY 1 160 a role for the alkali-soluble glucan in maintaining the integrity of the cell is suggested. Quantitative measurements and genetic studies (Tables 2 and 3) provide evidence that the srbl mutation, which determines the fragile phenotype of the VY 1 160mutant, leads to a decrease of alkali-soluble glucan in the cell wall. In the presence of an osmotic stabilizer in the media the srbl cells multiply exponentially(Venkovetal., 1974),whichsuggests that
Insoluble Smith-degraded glucan Strain 288L (SRBI TSl) Strain lSTL (srbl t s l ) 2.0 96.1 0.0 1.9
1.9 96.0 0.0 2.0
the lower amount of alkali-soluble glucan is not a step limiting the growth rate. However, it seems that the amount of alkali-soluble glucan is critical for maintaining the resistance of S. cerevisiae cells in conditions of osmotic shock because the transfer of the srbl cells to hypotonic solutions is accompanied by the immediate bursting of the cells (Mateeva et al., 1976).Instudiesofotherauthorsamore than 10-fold decrease in the amount ofinsoluble glucan (Katohda et al., 1976) or p(1-6) glucan (Meaden et al., 1990) was found to be without effect on cell rigidity. Contrary to this, a moderate (three- to four-fold) decrease of alkali-soluble glucan found in our studies for srbl cells is accompanied by the appearance of the fragile phenotype. Another observation in this study is the lower molecular weight and the existence of p( 1 4 ) interresidue linkages in the insoluble glucan of the fragile
460
cells. Both wild-type and m u t a n t glucans were isolated in experimental conditions where strong precautions against glucanase action have been taken. Therefore, the lower molecular weight found for the m u t a n t insoluble glucan does not seem to be due to degradation and most likely reflects the existence of shorter glucan fibrils in the fragile cells. In addition, the existence o f p( 1 -6) inter-residue linkages was shown t o prevent the formation o f triple helices a n d their organization into a fibrillar network with appropriate mechanical properties (Kopecka a n d Kreger, 1976). Therefore, the cell wall o f t h e fragile VY I160 m u t a n t contains insoluble glucan characterized by shorter. mechanically unstable fibrils generating a weaker fibrillar network. In addition, there is a shortage o f interstitial substances, alkalisoluble glucan (this study) a n d mannoproteins (Markisch ct al., 1983) in the m u t a n t cells. T h e filling o f the space between the fibrils is highly insufficient a n d the cell wall very fragile. Although growing exponentially in the presence o f osmotic stabilizer. the fragile cells lyse immediately upon transfer t o hypotonic solutions. As shown in previous electron microscopic (Mateeva ct al.. 1976) a n d cell-fusion (Philipovaand Venkov, 1990)studies. t h e l y s i s o f t h e fragile cells represents formation of holes on the cell surface where t h e cell wall is thinner or even missing.
R E FE R E NCES Ballou, L.. Cohen, R. and Ballou, C. (1980). Saccharomycw cerivisiac. mutants tha t make mannoproteins with a truncated carbohydrate outer chain. J . Biol. Chem. 255,5986-5991. Bowers, B., Lcvin, G. and Cabib, E. (1974). Effect of Polyoxin D on chitin synthesis and septum formation in Saccharoniyc*es cerevisiac. J. Bacteriol. 119,564575. De Nobel, J . G . . Klis. F. M.. Priem, J., Munnik, T. and Van den Ende, H. (1990). The glucanase-soluble mannoproteins limit celi wall porosityin Surcharomyces cerevisiae. Yeast 6,49 1-500. Dubois, M.. Gilles. K. A,, Hamilton, J . K., Rebers, P. A. and Smith, F. (1956). Colorimetric method for dctermination of sugars and related substances. Anal. Chem. 28,350-356. DutTus. J.. Levi, C. and Manners. D. (1982).Yeast cell wall glucans. Ad,,. Microhiol. Phjsiol. 23, 15 1-1 8 I . Fleet. G . and Manners, D. (1976). Isolation and composition of an alkali soluble glucan from the cell wall of Sacdiorotnj.ces cerevisiae. J . Gen. Microbiol. 94, 180 - I 92. Hakomori. S. (1964). A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J . Biochem. 55, 205-208.
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Hay, G. W., Lewis, B. A. and Smith, F. (1965). Pcriodate oxidation of polysaccharides: General procedures. Methods Carhohydr. Chem. 5,357- 361. Katohda. S., Abe, N., Matsui, M. and Hayashibc. M. (1976). Polysaccharide composition of the cell wall of bakers yeast with special reference to cell wall obtained from large and small sized cells. Plant a n d C d l Phjsiol. 17,909-919. Kopecka. M. and Kreger. D. (1986). Assembly of microfibrils in vivo and in vitro from (I-3)-P-u-glucan synthesized by protoplasts of Sacchoromyces cerevisiuc. Arch. Microhiol. 143,387.-395. Kozhina, T.. Stateva. L. and Venkov. P. (1979). Genetic analysis of an osmotic sensitive Saccharom~ces ccwvisiue mutant. Molec. Gen. Genet. 170, 351 - 355. Lindberg. B. (1972). Methylation analysis of polysaccharides. In Ginsburg. N . (Ed.). Methods in Enzymology. vol. XXVIII, part B. pp. 178 -195. Lopez-Romero. E. and Ruiz-Herrera, J . (1977). Biosynthesis of 0-glucans by cell free extracts from Saccharom p > sccwvisiae. Biochim. Biophjx Acva 500, 372 384. Manners. D.. Masson, A. and Patterson. J. (1973a). The structure of a P( 1- 3)-wglucan from yeast cell walls. Riochem. J . 135,19-30. Manners. D.. Masson. A,, Patterson, J.. Bjorndal. H . and Lindberg, B. (1973b). The structure of a p( I -6)-uglucan from yeast cell walls. Bbchem. J . 135, 3 1-36. Markisch, U., Reuter, G.. Stateva, L. and Vcnkov, P. (1983). Mannan structure analysis of the fragile Saccharomjws cerevisiae VYI 160 mutant. I n t . J . B i ~ c h e 15, ~ ~ 1373. 1377. Mateeva, Z . , Petrov. P., Venkov, P. and Hadjiolov. A. (1976). Electron microscopic study of the lysis of an osmotic sensitive yeast mutant. J . Microsc. Biol. Cellulairr 26,73-74. Meaden. P., Hill, K., Wagner, J., Slipetz, D., Sommcr. S. and Bussey, H. (1990). The yeast KRES gene encodes a probable cndoplasmic reticulum protein required for ( I 6)P-n-glucan synthesis and normal cell growth. Mol. Cell. Biol. 10, 3013-3019. Mizoguchi. G., Saito, T., Kizuno. Z . and Hayano, K . (1977). On the mode of action of a new antifungal antibiotic aculeacin A: inhibition of cell wall synthesis in Sacdiaromyces cerevisiae. J . Anribiot. 30,308-3 13. Mol. P. and Wessels, J . (1987). Linkage bctwecn glucosaminoglycan and glucan determine alkali insolubility of the glucan in walls of Sacchoromyces cerevisiae. F E M S Microbiol. Letters 41,95 -99. Philipova, D. and Vendov. P. (1990). Cell fusion of Succhoromyces wrevisiae fragile mutants. Yeast 6, 205 212. Sweeley, C. C.. Wells. W. W. and Bentley. R. (1966). Gas chromatography of carbohydrates. In Ginsburg. N. (Ed.). Methods in Enzymology, vol. VIII, pp. 95-108. Tkacz. J. Z. (1984). I n viro synthesis of p(1-6) glucan in Sucdiaroniyes cereuisiac.. In Nombela, C . (Ed.), Microbial Cell W d l Synthesis and Autoljsis. Elsevier Sci. Publishers, Amsterdam, pp. 287-296.
CLUCAN STRUCTURE IN A FRAGILE MUTANT OF SACCHAROMYCES CEREVISIAE
Venkov, P., Hadjiolov, A,, Battaner, E. and Schlessinger, D. ( 1974).Saccharomyces cerevisiae sorbitol dependent fragile mutants. Biochem. Biophys. Res. Commun. 56, 559-604. Waltschewa, L., Philipova, D. and Venkov, P. (1989). Increased extracellular secretion in fragile mutants of Sarcharomyces cerevisiae. Yeast 5,3 13-320.
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Zlotnik, H., Fernandez, M., Bowers, B. and Cabib, E. (1984). Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J. Bacteriol. 159, 1018-1026.
