Yeast Cell-Wall Glucans

Yeast Cell-Wall Glucans J. H. DUFFUS, CAROLYN LEV1 and D. J. MANNERS Department of Brewing and Biological Sciences, Heriot- Watt Universiiy, Edinburgh EH I I HX, Scotland I. Introduction . . , . . . . . . 11. Structural analysis of yeast glucans . . . . A. General methods . . . . . . . B. Glucans in walls of Sucrhoromycrs rerevisiue . . C . Glucans from other Sacchuromvces species . . D. Glucans in walls of Sc~ii=o.succliurom~ce.~ pnmhe . E. Glucans from Candidu species. . . . . F. Other yeast glucans . . . . . . . 111. Yeast wall glucan synthesis A. Introduction . . . . . . . . B. Studies with inhibitors of glucan synthesis . . C. Glucan synthetases of whole and fractionated cells . D. Glucan synthesis and glucan synthetases in protoplasts IV. Physiological control of glucan content . . . . . . . A. Introduction . B. The cell cycle . . . . . . . . C. Yeast-mycelium interconversion . . . . D. Effects of nutrient limitation . . . . . E. Effects of metabolic inhibitors. . . . . F. Miscellaneous . . . . . . . . V. Acknowledgements . . . . . . . References . . . . . . . . .

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I 52 152 155 160 162 162 165 166 166 166 167 169 171 171 171 174 174 176 177 177 178

I. Introduction Although the chemistry of the yeast cell wall has been the subject of continued investigation in several laboratories, overall progress has been relatively slow. This arises partly from the complex nature of the wall which may contain three, four or even more different polysaccharide components, in addition to protein, phosphorus and lipids, and partly from the relative insolubility

152

J. H. DUFFUS, CAROLYN LEV1 AND D. J . MANNERS

of some of these components which renders purification and structural analysis by conventional methods extremely difficult. Thanks to the sustained work of C. E. Ballou and his coworkers, a substantial amount of information on the structure and metabolism of yeast mannans is now available (for reviews, see Ballou, 1974, 1976). The purpose of this review is to describe the present state of knowledge on the structure and metabolism of the related glucan components of yeast cell walls. Many of the earlier structural studies involved commercial samples of pressed baker’s yeast (i.e. various strains of Succhuromyces cerevisiue) and, although these enabled the main structural features to be identified, they were of limited value from a metabolic point of view. With recent improvements in polysaccharide fractionation and in the methods of structural analysis, it is now possible to use smaller samples of yeast from laboratory-scale cultures as the experimental material, and hence, to relate the changes in cell-wall structure with cell growth. The structural analysis of glucan preparations isolated from various species of yeast will be described in the next section, where all of the constituent monosaccharide residues will be assumed to have the D-configuration. The following section will describe synthesis of glucan from nucleoside diphosphate sugar compounds by a variety of yeast enzyme preparations, and the final section deals with the physiological control of the glucan content of yeast cell walls during various conditions of growth. The literature survey for this review has been selective rather than comprehensive. Much of the early work has been summarized elsewhere, e.g. Phaff (1963, 1971, 1977). MacWilliam (1970), and other current assessments of the literature have been provided by Bacon (1981) and by Fleet and Phaff (1981).

11. Structural Analysis of Yeast Glucans A. G E N E R A L M E T H O D S

1. Prepurution

of Yeust Glucuns

The early work on yeast glucan was carried out using whole pressed cells of baker’s yeast, which were extracted with hot alkali to remove mannan. This was then precipitated as the copper complex after addition of Fehling’s solution. The alkali-extracted cells still contained some glycogen, which was removed by extraction with warm acetic acid, o r by autoclaving with water, and the final residue was regarded as “yeast glucan”. It was characteristically insoluble, a property which prevented examination of its homogeneity by

YEAST CELL-WALL GLUCANS

153

standard methods, and hindered the subsequent structural analysis. The overall composition of the yeast cell wall was usually described (e.g. Northcote, 1962) as glucan (about 30%), mannan (about 30%), protein (about 10-15%), lipid (8-9%) and chitin (1-273, the wall representing about 15% of the dry weight of the cell. The above results do not take account of the presence of about 20% of an alkali-soluble glucan (see Section II.B.2, p. 158), which has only been recognized in more recent years. The true glucan content of many cell walls is therefore about 50%. However, it should be emphasized that all of these analytical results will vary with the species and strains of yeast, its conditions of growth, and with the methods used for the preparation of the cell walls and their fractionation. As an alternative to using pressed cells, the yeast can be disrupted mechanically to give cell-wall preparations which, after being washed with various solvents, are a suitable starting material (e.g. Northcote and Horne, 1952). However, it has recently been realized that the cell walls contain endogenous 8-glucanases, which can partially hydrolyse cell-wall components, causing autolysis. It is therefore essential that these enzymes should be inactivated during the preparation of cell walls. Treatment with Tris buffer or sodium phosphate buffer at pH 8.5 has been recommended (Fleet and Phaff, 1973; Fleet and Manners, 1976). Bacon and his coworkers (1969) reported that glucan is more easily extracted by alkali from isolated cell walls than from intact cells. This difference is discussed in detail by these workers, but the reasons are not yet clearly understood. It is therefore obvious that caution is required in comparing and interpreting the results of alkaline extractions of cell-wall preparations and of intact cells.

2. Analytical Methods We have already noted that the structural analysis of the glucans is hampered by their relative insolubility. As a simple example, total or partial acid hydrolysis by mineral acid is incomplete, without a prior treatment with formic acid. Methylation analysis remains an important technique in polysaccharide studies. With glucans, the proportion of 2, 3, 4, 6-tetra-0-methyl glucose represents the non-reducing terminal residues, whereas the amount of 2, 4, 6- and 2, 3, 4-tri-0-methyl glucose indicates the proportion of (1 + 3)- and (1 -+ 6)-inter-residue linkages. The presence of 2, 4-di-0-methyl glucose, assuming complete methylation, indicates residues linked at C-1 , C-3 and C-6, i.e. branch points, and the amount should be equivalent to that of tetra-0-methyl glucose. However, yeast glucan is notoriously difficult to methylate with the conventional Haworth reagents (dimethyl sulphate

1 54

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

and sodium hydroxide). Bell and Northcote (1950) found 3 1 methylations gave a methoxyl content of 41.7% (theoretical 45.6%), whereas Manners and Patterson (1966) obtained a methoxyl content of 41.5% after 16 treatments. The introduction of improved reagents (e.g. Hakomori, 1964) has, to some extent, facilitated methylation, but doubts about the completeness of the etherification of insoluble glucans still remain. Periodate oxidation provides a useful alternative method of aralysis. The amount of periodate reduced per glucose residue (expressed as molecular proportions, or mol. prop.) is determined by the presence of diol and trio1 groups. The latter arise from (a) terminal glucose residues and (b) ( 1 -6)linked glucose residues. A linear (1 + 3)-/?-glucan should reduce less than 0.1 mol. prop. of periodate, whereas the presence of several ( 1 -6)-linked residues could easily increase this value to 0 4 0 . 6 mol. prop. The amount of formic acid released is also a measure of the relative proportion of nonreducing terminal residues and of (1 + 6)-linked residues. Subsequent analysis of periodate-oxidized glucans involving borohydride reduction and partial acid hydrolysis (i.e. a Smith degradation), or borohydride reduction and total acid hydrolysis, also provides useful information on the location of ( 1 -+ 6)-linked residues within a (1 -+ 3)-P-glucan macromolecule. Readers should consult the series “Methods in Carbohydrate Chemistry” edited by R. L. Whistler and J. N. BeMiller, and published by Academic Press for further details of these methods. A further method of analysis involves selective enzymolysis. Ideally, purified enzymes of known action pattern should be used, so that structural analysis of the end-products gives information on the structure of the enzymically resistant material and also on the nature of the linkages hydrolysed. It should be realized that the specificity of some P-glucanases is controlled not by the linkage which is hydrolysed (b), but by an adjacent linkage (a): (a) (b) - G - G - G Hence, some endo-( 1 -+3)-P-glucanases may hydrolyse (1 -4) or (1 -6)linkages in substrates containing both (1 -3)- and a second type of glucosidic linkage (Perlin and Suzuki, 1962; Cunningham and Manners, 1964). For example, Rombouts and his coworkers (1978) have examined two extracellular ( I -6)-P-glucanases produced by Bacillus circuluns WL- 12, which differ in their ability to lyse yeast cell walls. The non-lytic enzyme hydrolysed ( 1 -6)-P-glucans in an endo fashion, but did not hydrolyse branched (1 -3)-P-glucans containing ( 1 -6)-interchain linkages. By contrast, the lytic enzyme showed both endo-( 1 -6)-p-glucanase activity and activity towards branched (1 3)-P-glucans having (1 -6)-interchain linkages. This indicates an ability of the lytic enzyme to hydrolyse certain ( 1 -3)-linkages in the

-

YEAST CELL-WALL GLUCANS

155

vicinity of a (1 -6)-interchain linkage. In some instances, degradation of a glucan by an unpurified enzyme preparation can be used, provided that deductions are confined to the structure of the products of enzyme action, and do not include the nature and site of the susceptible linkages. Such an experiment provides a means of partial hydrolysis of the P-glucan which is less random than the use of dilute acid. The presence of endogenous j?-glucanases in yeast cell walls has already been mentioned; see for example Abd-el-al and Phaff (1969), Notario et al. (1975) and Villa et al. (1979). Although a detailed discussion of their action falls outside the scope of this review, we should note that their action on substrates in vivo enables some conclusions to be drawn on the molecular structure of the latter. A retrospective and current view of these enzymes has been provided by Phaff (1979). B . G L U C A N S IN W A L L S O F S A C C H A R O M Y C E S C E R E V I S I A E

1. Alkali-Insoluble Glucans

During the period 1950-1960, differences of opinion developed on the structure of the alkali-insoluble glucan from baker’s yeast. On the basis of a methylation analysis, Bell and Northcote (1950) suggested it had a highly branched structure with relatively short chains of (1 -3)-linked glucose residues interlinked by about 11% of ( 1 -2)-glucosidic linkages. The average chain length, confirmed by periodate oxidation, was nine glucose residues. By contrast, Peat and his coworkers (1958a), using partial acid hydrolysis, concluded that the glucan was linear and contained certain sequences of ( 1 -3)- and (1 -6)-linked j?-glucose residues. The presence of about 10-20% of (1 -6)-linkages was confirmed by tosylation followed by iodination of primary hydroxyl groups (Peat et al., 1958b). In attempts to resolve these differences, Manners and Patterson (1966) carried out further methylation, periodate oxidation and also enzymic degradation studies, and concluded that yeast glucan had a branched structure, containing main chains of (1 -6)-linked P-glucose residues, to which were attached linear side chains of ( 1 -+3)-linked /?-glucose residues. An alternative structure was proposed by Misaki et al. (1968) on the basis of their chemical studies (see Fig. la) which was similar, in some respects, to that proposed by Manners and Patterson (1966). In all of these experiments (19501968), the yeast had been successively extracted with hot alkali, and with acetic acid, and/or autoclaving in water to remove mannan and glycogen respectively, and the residual material was regarded as the “yeast glucan”. A key observation by Bacon and Farmer ( I 968) eventually led to a solution of the problem. These workers showed that yeast glucan prepared as already

156

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

(a)

+6)-[GIy-

-~1-~6)-[Gl,-(1-~6)-G-(1-~3)-G-(l-6)-G-(1

I

I

h

3

j.

6

7 ....G-(l-t3)-G--(l~3)-G.... FIG. 1 . Structure of yeast glucan. (a) Shows the partial structure of yeast glucan as proposed by Misaki er al. (1968). G denotes a /?-D-glucopyranose residue, and x y = 40-50. (b) Shows the partial structure of a segment of yeast /?-(1 -+3)-glucan. a + b + c comprise about 60 glucose residues, although the exact lengths of a, b and c are unknown.

+

described was heterogeneous, and contained an acetic acid-soluble polysaccharide which was shown by partial hydrolysis and infrared spectroscopy to be a (1 +6)-P-glucan. Their suggestion that “yeast glucan” was, in fact, a mixture of a major (1 -+3)-j?-glucan component and a minor (1 +6)-/3-glucan component provided an explanation for many of the earlier experimental results. This significant discovery by Bacon and Farmer (1968) led to a detailed fractionation of bakers yeast, and a chemical characterization of the resulting (1 -+ 3)- and ( 1 + 6)-P-glucan components (Manners et a/, 1973 a, b). The fractionation scheme is outlined in Fig. 2 . The major component (about 85%) was a lightly branched (1 -+ 3)-,!3-glucanof high molecular weight (minimum DP value 1450 f 150, equivalent to a molecular weight of about 240,000) and containing about 3% of (1 -+ 6)-P-glucosidic interchain linkages (Manners et al., 1973a). The minor component (about 15%) had a minimum DP value of 130-140 (equivalent to a molecular weight of about 22,000), and contained a high proportion of (1 -+6)-linkages (65%), together with a smaller number of (1 -+3)-linkages which were present as both interresidue (5%) and interchain (14%) linkages (Manners et af., 1973b). The molecule was highly branched, possessing 16% of non-reducing terminal groups. These analytical results apply only to the glucans from one particular sample of baker’s yeast, and the authors emphasized that glucan preparations from different batches of

157

YEAST CELL-WALL GLUCANS

with 3",, sodium hydroxide at 75 C for 6 hours

Alkali-soluble carbohydrate

Alkali-insoluble carbohydrate

Treated with Fehling's solution

(mannan) (mannan)

denatured denatured Iprotein)

Extracted 27 times with

0.5~ acetic acid at 90 C

glucan glucan

1

Soluble Soluble glucans glucans (glycogen and (1 + 6)$D-glucan) I

iodine solution

Iucanase

Precipitated iodinecomplex (glycogen)

Purified

FIG. 2 . Fractionation of baker's yeast to give purified ( 1 and alkali-soluble glucan.

Soluble glucan

Treated with z-amy lase

(1 -,6 ) - 8 - ~ -

3)- and ( 1 -+ 6)-8-~-ghcan.

yeast may differ in the relative proportion of the two components, and that the individual components might vary considerably in degree of branching and other structural parameters. This structural analysis of the major component did not indicate whether single or multiple branching was present, or whether the molecule had a laminated, comb or tree-type structure (or some variant of these) of the kinds

158

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

suggested for the amylopectin component of starch (see Fig. Ib, p. 156). The major ( 1 +3)-/?-glucan is. now generally regarded as providing an insoluble “envelope” which is responsible for the rigidity of the cell wall. The molecular weight of about 240,000 represents a minimum value, and is based on the assumption that each molecule contains one free reducing glucose residue. The validity of this assumption is not yet known, but no other method is available for determining the molecular size of such an insoluble macromolecule. In spite of this uncertainty in the size of the (1 +3)-P-glucan component, it seems clear that numerous individual molecules would be required per cell. The mode of association of the molecules is not known, but the overall degree of branching (about 3%) is so low that some of the linear segments of chains could be held together in triple helix conformations, to give a macromolecular structure with a substantial degree of rigidity and insolubility in water (Rees, 1973). The presence of small but significant amounts of (1 -+6)-/?-glucan in the baker’s yeast cell wall poses questions regarding its biological function, and the control of the relative rates of synthesis of the two types of /?-glucan. Since the proportion of the two types of linkage in the (1 -+6)-/?-glucan are so different from those in the major ( 1 -+3)-/?-glucan, the former is unlikely to be an intermediate in the biosynthesis of the latter. As will be described in more detail later in this section (p. 163), ( 1 -+ 6)-/?-glucan components have been detected in several different yeasts (Manners et al., 1974), so that the questions posed above are of general interest in considering yeast cell-wall glucans as a whole. Since the ( 1 -+6)-/?-glucan is soluble under non-alkaline conditions and has a relatively low molecular weight, it might be considered as a reserve rather than a structural material, although of course, this would duplicate the function normally assigned to glycogen, with which it is closely associated (Evans and Manners, 1971). Alternatively, the glucan could function as a plasticizer or filling material in the relatively rigid envelope constituted by the major ( 1 +3)-/?-glucan, where its function could be to prevent excessive aggregation of linear segments of chains, so that some measure of flexibility is retained in the wall, as would be required for wall expansion during growth (Manners et al., 1974). 2. Alkali-Soluble Glucans

The earlier literature contains several reports on the presence of alkali-soluble glucan in cell-wall preparations of baker’s yeast and Sacch. cerevisiue (Roelofsen, 1953; Kessler and Nickerson, 1959) but its molecular structure was unknown. Fleet and Manners (1976, 1977) accordingly carried out a detailed study of this glucan.

159

YEAST CELL-WALL GLUCANS

The polysaccharide was extracted from cell walls of Sacch. cerevisiar NCYC I 109 and baker’s yeast with cold (4°C) dilute sodium hydroxide under nitrogen for six days. The glucan (GI),which amounted to about 20:4 of the dry weight of the cell wall, precipitated as a gel on neutralization of the alkaline extract (Fleet and Manners, 1976) and the monosaccharide composition is given in Table 1. The GI preparations were insoluble in water, but soluble in 1M sodium hydroxide and dimethyl sulphoxide. Physicochemical studies showed the preparations to be homogeneous. Partial acid hydrolysis showed the presence of both (1 3)- and (1 -+6)-B-glucosidic linkages, and methylation analysis indicated that the latter were present as both interresidue and interchain linkages (Table 1). Electron microscopic examination of the cell walls showed that the alkaline extraction had removed an amorphous surface layer, revealing many bud-scar structures. -+

TABLE 1. Properties of alkali-soluble glucans (GI) from Succhuromyces cerevisiue 1109 and from baker’s yeast Property Composition Carbohydrate Nitrogen Hexosamine Glucose Mannose Molecular size Degree of polymerization Methylation analysis“ 2,3,4,6-tetra2,4,6-tri2,3,4-tri2,4-di “0-methyl

D-glUCitOl

Glucan G , from Succharomyces cerevisiue 1 109

Glucan G I from baker’s yeast

99.0 0.13 0.0 98.5 1.5

98.0 0.26 0.0 97.0 3.0

1330

1810

3.7 84.7 8.3 3.3

4.7 79.3 12.0 4. I

derivatives expressed as mol per 100 mol

In attempts to examine the fine structure of G I , it was subjected to enzymic degradation (Fleet and Manners, 1977). The overall structure that emerged was of a macromolecule with a (1 + 3)-/?-glucan “core” having a low degree of branching (about 2.073, and also containing occasional ( I -+ 6)-linked residues. To this “core” were attached various side chains containing mainly (1 +3)-linked residues, or mainly ( 1 +6)-linked residues, or a mixture of both. Glucan G I always contained a mannan fragment which could be released by the action of an endo-( 1+6)-P-glucanase. This would imply that part of the cell-wall mannan is held in place by some ( 1 -+6)-linkedglucose residues,

160

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

although the nature of this association (i.e. covalent or otherwise) has not yet been established. The biological relationship between the alkali-insoluble glucans and the alkali-soluble glucan has also not been established, but Katohda et al. (1976) have shown that cell walls prepared from large cells of baker’s yeast contained a higher amount of alkali-insoluble glucan, whereas cell walls from small cells contained more alkali-soluble glucan. C. G L U C A N S F R O M O T H E R S A C C H A R O M Y C E S S P E C I E S

The majority of investigators have examined glucan preparations from Sacch. cerevisiae, and the literature contains only a limited number of references to structural studies on related polysaccharides from other species of yeast. These give the overall impression that they are not greatly different from the glucans of Sacch. cerevisiae, bearing in mind the previous comments on the possible variation in the relative amounts of the three different glucans, and possible variations in molecular size, degree of branching and the relative proportion of (1 -3)- and (1 +6)-fi-glucosidic linkages. Table 2 contains some comparative data on the yields of acetic acidsoluble glucan (B), hot water-soluble material (C) and insoluble glucan (D) from various yeasts, and Table 3 shows the properties of some of these glucan D preparations. The latter consist mainly of ( 1 +3)-P-glucan, with some (1 +6)-glucan in a rather greater amount than was present in baker’s yeast. Related data from Schizosaccharomycespombe (see Section 1I.D)are included for comparative purposes. Partial acid hydrolysates of all the acetic acid extracts B contained gentiosaccharides and maltosaccharides. The latter were removed by treatment of the extracts with a-amylase, prior to partial acid hydrolysis. Fraction B is therefore a mixture of glycogen and a (1 -6)-P-glucan. All of the glucan samples D, on periodate oxidation, reduced more periodate and produced more formic acid than a purified (1 -3)-P-glucan, thus confirming that fraction D was a mixture of a (1 -+ 3)-P-glucan, with small amounts of (1 + 6)-P-glucan. The glucans from Sacch. carlsbergensis are of special interest, since they provided the starting material for one of the first studies on alkali-soluble glucan. Eddy and Woodhead (1968) suspended cell walls in 3% sodium hydroxide solution at 4°C under nitrogen for up to nine days. About 20% of the cell wall dissolved. The soluble polysaccharide was a glucan, with an [a], value of +9 in 3% sodium hydroxide, which was resistant to a- and P-amylases, and it had a molecular weight of about 5. lo5 by sedimentation measurements. This work confirmed earlier observations in the literature (Phaff, 1963) that alkali dissolves some of the glucose residues in the yeast

YEAST CELL-WALL GLUCANS

161

TABLE 2. Fractionation of yeast wall preparations from various yeast species From Manners et a/. (1974) Weight of fractions obtained from 150 g washed organisms

Yeast

Acetic acidsoluble glucan (mg)

Water-soluble material 0%)

Insoluble glucan (g)

460

23

2.80

360 120

61 69

3.60 2.7 I

Saccharomyces jrugilis" (NCYC 100) Saccharomyces ,fermentati (NCYC 161) Schizosuccharomyces pomhe

"This yeast is also known as Klu.vveromyces ,frugilis.

TABLE 3. Properties of insoluble glucan preparations from various yeast species From Manners et al. (1974)

Periodate oxidation Enzymic degradation

Reduction of periodate (molar proportions)

Trio1 groups

Number of glucose residues divided by the number of trio1 groups

25

0.48

19.5

5.1

26

0.48

18.4

5.4

32

0.27

12.7

7.9

38

0.12

3.2

24

032

22.8

(%I

(%)

Yeast Saccharomyces jragilis" Saccharomyces ,fermentati Saccharomy ces cerevisiae Purified ( 1 + 3)$glucan from Saccharomyces cerevisiae Schizosaccharomyces pomhe

"This yeast is also known as Kluvveromyce.s.fra~ilis

31 4.4

162

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

cell wall, and was the starting point of the detailed analysis of Sacch. cerevisiae described in Section II.B.2 (p. 158). D. G L U C A N S I N W A L L S O F S C H I Z O S A C C H A R O M Y C E S P O M B E

The fission yeast Schiz. pombe differs chemically from budding yeasts in that a-mannan and chitin are absent from their cell walls, and that about onethird of the glucan contains (I -*3)-a-linked glucose residues (Bacon et al., 1968). This finding provides an explanation for the apparent resistance of walls of Schiz. pombe to extensive hydrolysis by (1 -3)- and (1 +6)-flglucanases. Bush et al. (1974) examined the wall structure of Schiz. pombe by a variety of techniques. The major components were galactomannan (9-14%), a-glucan (28%) and fl-glucan. The presence of the last polymer was revealed by digestion of the walls with an exo-( 1 -+3)-fl-glucanase, which gave 42% conversion into reducing sugars, 96% of which was glucose. Gentiobiose (3.5%) and laminaribiose (0.5%) were also produced. The a-glucan had a mainly linear structure with (1 -* 3)-linkages, but there was also some evidence for the presence of about 77{ of ( 1 -+4)-linkages. The b-glucan was heterogeneous and contained three different components (Manners and Meyer, 1977), namely a lightly branched (1 +3)-j?-glucan (R-1) which was insoluble in a!kali and acetic acid, a highly branched (l+6)-bglucan, and an alkali-soluble (1 -,3)-fl-glucan (G-I) which has a different structure from glucan R-I. The properties of two of these glucans are summarized in Tables 4 and 5, together with similar data from Sacch. cerevisiae. In general, the alkali-soluble glucan from Schiz. pombe, has a smaller molecular size, and a higher degree ofbranching than the samples from Sacch. cerevisiae. The ( I -+6)-~-glucans also show significant differences. The alkali-insoluble but acetic acid-soluble and alkali-soluble (G-I) glucans comprise about 2 and 24% of the cell wall, respectively. This study also confirmed the presence of a (1 --.3)-linked a-glucan, for which a D P value of 207 k 20 was reported. Schizosaccharomycespornbe thus contains four distinct glucans, in addition to a galactomannan. The investigation of pathways for biosynthesis of these closely related glucans poses a formidable problem for the biochemist. E. GLUCANS FROM CANDIDA SPECIES

The cell wall of the pathogenic yeast Candida albicans has been o f special interest, although it is now known that mannans rather than glucans are the major antigenic materials (Yu et al., 1967). A detailed chemical study of alkalisoluble glucan preparations from C. albicans serotypes A and B, and from

163

YEAST CELL-WALL GLUCANS

TABLE 4. A comparison of the properties of some alkali-soluble ( 1 from yeasts From Manners and Meyer (1977)

Schizosucc~iuromyces Succhuromyces pombe cere visiue Fraction G-I NCYC 1109 Baker's yeast

Property Approximate yield

+ 3)-p-glucans

(yo)

[a], in I M sodium

hydroxide

Degree of polymerization Methylation analysis" 2,3.4,6-tetra2,4,6-tri2,3,4-tri2,4-di-

24

22

13

+ 20

n.d.

n.d.

809

1330

1810

15.0

3.7 84.7 8.3 3.3

4.7 79.3 12.0 4.1

66.3 4.2 14.6

"Expressed as 0-methyl D-glucitol derivatives, mol per 100 mol. n.d. indicates the value was not determined.

TABLE 5. A comparison of the properties of some alkali-insoluble acetic acid-soluble (1 + 6)-p-glucans from yeasts From Manners and Meyer (1977) Property Approximate yield, from alkaliinsoluble residue (%) [aID in water

Degree of polymerization Methylation analysis" 2,3.4,6-tetra2,4,6-tri2,3.4-tri2.4-di-

Yeast Schizosucchuromyces pombe

Succhuromyces cere visiue

23

13

- 27"

-

32"

186

140

44 5 9 43

16

5 65 14

"Expressed as 0-methyl D-glUCitOl derivatives, mol per 100 mol.

C. purupsilosis was carried out by Bishop et al. (1960) and Yu et al. (1967), and their results are summarized in Table 6. All of the glucan preparations were of relatively low molecular weight, were highly branched, and contained a high proportion of (1 +6)-linked residues. The mono-0-methyl glucose noted in Table 6 is most probably the result of undermethylation, and is not structurally significant. The glucan from C. albicans serotype A differs from the others in having a higher degree of branching and few, if any, ( 1 +3)-

164

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

TABLE 6. Properties of /I-glucans from Candida ulbicans and Cundida parapsifasis From Bishop et al. (1960) and Yu et al. (1967) Property

Candidu albicans Serotype A Serotype B

D

30

-25'

2"

30 -t 3b

1.60

1.52

1.58

0.72

0.79

0.78

26 48 trace 22

10 61 17 6

11 63 15 7 5 2

-

Degree of polymerization Periodate oxidation Periodate reduced (mol. prop.) Production of formic acid (mol. prop.) Methylation analysis' 2,3,4,6-tetra2,3,4-tri2,4,6-tri2.4-di2,3-di2-mono-

Candida parapsilosis

30

-

-

4

- 35

33

* 3b

Determined by hypoiodite oxidation. bDetermined by osmometry of the methylated glucans in chloroform. 'Expressed as 0-methyl o-glucose derivatives, mol per 100 mol.

linked glucose residues. In many respects, these glucans are similar to the alkali-insoluble acetic acid-soluble glucans isolated from Sacch. cerevisiae and certain other yeasts. (see Sections I1.B and ILC, pp. 156 and 163) The above work does ,not, however, represent a complete account of glucans from C. albicans. Firstly, it was carried out before the heterogeneity of yeast glucans had been fully appreciated, so that any alkali-insoluble glucan was not included in the studies. Secondly, the organism shows dimorphism, and exists in blastospore and mycelial forms, the latter being mainly responsible for its pathogenicity (Chattaway et al., 1968). These workers examined cell walls from the mycelial and blastospore forms and separated them into alkali-soluble and alkali-insoluble fractions. The alkali-insoluble fraction of the mycelial form contained three times as much chitin as the blastospore form, but only one-third of the protein. The glucan contents of the two forms were similar (4547%) but differed in fine structure since pglucanase treatment of the mycelium and blastospore released 19 and 39% of glucose, respectively. It is evident that the dimorphism involves many changes in the biosynthetic pathways for glucan, protein and chitin. Candida albicans can be treated with polyene antibiotics, such as amphotericin, but stationary-phase organisms show greater resistance than exponentially grown organisms. This resistance is associated with the cell wall

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as protoplasts prepared from resistant cultures are sensitive (Gale et al., 1975). However, there are no significant differences in the penetrability of the cell wall to poly(ethy1ene glycols) of different molecular sizes at the end of the growth phase and after prolonged incubation in the stationary phase (Cope, 1980). Cytochemical studies have shown the presence of (1 + 6)-glucan and mannan at the outer surface of the cell walls and, in the case of exponentially growing organisms, there was less staining in the inner regions where ( 1 + 3)glucan and chitin are believed to be more abundant (Cassone et ul., 1979). By contrast, walls from stationary-phase cultures gave a more uniform staining reaction, indicating a greater intermixing of the four polysaccharide components. Treatment of stationary-phase organisms with various hydrolytic enzymes, particularly an endo-( I +3)-/?-glucanase, caused a decrease in the resistance to amphotericin, suggesting that the ( 1 -3)-P-glucan could play a major role in the phenotypic resistance (Gale et al., 1980). Chitinase, trypsin and cr-mannosidase showed similar but less pronounced effects. It is clear that detailed structural studies on all the cell-wall components of C. alhicans are required. Other species of Candida also appear to contain a mixture of glucans in their cell walls but, in general, detailed structural studies have not been reported. One exception is a Canclida sp. which was grown as a possible source of singlecell protein. The cell walls contained both alkali-soluble and alkali-insoluble glucans, composed of varying proportions of (1 +3)- and (1 +6)-/?-glucosidic linkages (Martin, 1982). However, this yeast did not appear to produce a predominantly ( I +6)-linked /?-glucan soluble in acetic acid, but insoluble in alkali, of the type described in Table 3 (p. 161). F . OTHER YEAST G L U C A N S

The literature contains many reports on cell-wall components of yeasts, but many of these are concerned with the taxonomy, serology or physiology of the organisms, rather than with their detailed molecular structures. I t seems probable that many yeasts will contain one or more glucan components, which will differ in solubility in alkali, and that these will contain a high proportion of (1 +3)-/3-glucosidic linkages. In some instances, (1 -+6)-8glucan components may also be present. For example, with Kfoeckera apiculata, the alkali-insoluble fraction reduced 0.47 mol. prop. of periodate, contained 18.4% of trio1 groups and, on partial acid hydrolysis, yielded gentiosaccharides (Manners et al., 1974). A detailed and systematic study of many of these glucan preparations would be rewarding. There also exist several dimorphic organisms which exist in either mycelial or yeast-like forms, and represent an interesting biological system for the study of biochemical events governing this type of interconversion. For

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example, the pathogenic fungus Paracoccidioides brasiliensis produces a (I-,3)-~-glucan in the yeast form and a mixture of a (I+3)-P-glucan and a galactomannan in the mycelial form (San-Blas and San-Blas, 1977). Biochemically, the interconversion of an cr-glucan and a /3-glucan is unusual, and indeed, remarkable. In addition, both forms contain chitin. The cellwall structure is not stable, but changes according to the environment, especially the temperature at which the organism is grown. Since the cellwall composition appears to correlate with the degree of virulence, it has been suggested (San-Blas and San-Blas, 1977) that the cell-wall polysaccharides, and particularly the ( 1 -+3)-a-glucan, play a role in the active protection of the organism against the defence mechanism of the host. Further detailed chemical and biochemical studies on those organisms that are clinically important is warranted.

111. Yeast Wall Glucan Synthesis A. INTRODUCTION

This section is intended to review the information available on the identification and regulation of the enzymes of yeast cell-wall glucan synthesis. As Farkis (1979) has pointed out, yeast cell-wall glucan synthetases have been very difficult to study. The enzymes responsible for synthesis of (1 -+3)-aand (I+6)-P-linkages are not yet understood in any detail, nor are the mechanisms that determine the proportion of different linkages in wall glucans. However, some of the properties of a (1-+3)-P-glucan synthetase have recently been elucidated (Shematek et af., 1980; Shematek and Cabib, 1980). B. S T U D I E S W I T H I N H I B I T O R S O F G L U C A N S Y N T H E S I S

Glucose 1-phosphate was implicated as a precursor in the pathway of yeast glucan synthesis by Chung and Nickerson (1954). They found that fluoride inhibition of yeast growth, ascribed to inhibition of phosphoglucomutase and hexose phosphate isomerase, was associated with a decrease in the synthesis of glycogen, mannan and glucan. 2-Deoxyglucose has been used in many studies of synthesis in the yeast wall, although it has multiple effects on cell metabolism (Kratky et a/., 1975). 2-Deoxyglucose appears to cause lysis in growing Schiz. pombe by wall breakage at the sites of wall growth (Megnet, 1965). A hexokinase-deficient mutant was found to be resistant to 2deoxyglucose, implying the necessity of 2-deoxyglucose 6-phosphate synthe-

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sis for toxicity and for inhibition of wall synthesis (Megnet, 1965). Johnson ( 1 968) confirmed that the sites of wall damage by 2-deoxyglucose in Schiz. pombe, Pichia farinosa and Sacch. cerevisiae were in the regions of wall glucan synthesis. He proposed that wall growth occurred by enzymic breakage of glucan chains, followed by extension of the chains by a glucan synthetase which utilized U D P-glucose. U DP-2-Deoxyglucose, synthesized from 2-deoxyglucose, would prevent chain elongation, but not the initial breakage (Johnson, 1968). However, if (1 3)-/?-glucan chains are extruded through the plasma membrane (see Section III.D, p. 169), as is chitin in Sacch. cerevisiae (Duran et al., 1975), elongation of already externalized chains would require an as yet unidentified glucan synthetase, i.e. not the (1-3)p-glucan synthetase located on the cytoplasmic side of the plasma membrane by Shematek et al. (1980). Biely et a1 (1971) have shown that some 2-deoxyglucose is incorporated into walls of Succh. cerevisiae, which supports the idea that a site of inhibition of wall synthesis by 2-deoxyglucose is at glucan chain elongation (see also Section IV.E, p. 176, in this review). The antibiotic aculeacin A inhibits 14C-glucoseincorporation into the acidinsoluble wall fraction of Sacch. cerevisiae (Mizoguchi et al., 1977). Cell lysis occurs at the growing bud tips (Mizoguchi et al., 1977). The amphophilic antibiotics papulacandin B and echinocandin B (Baguley et al., 1979) specifically inhibit glucose incorporation into glucans synthesized by whole cells and by sphaeroplasts of Sacch. cerevisiae and C . albicans. Synthesis of the alkaliinsoluble glucans by the sphaeroplasts was specifically blocked, whereas synthesis of the alkali-soluble glucans and of mannan was relatively unaffected. It can be inferred from this that alkali-soluble and insoluble glucans are made by different synthetases, or that a secondary factor involved in regulating synthesis of alkali-soluble glucan, possibly the environments of the synthetases in the plasma membrane, is the site of action of these antibiotics.

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C. G L U C A N S Y N T H E T A S E S O F W H O L E A N D F R A C T I O N A T E D C E L L S

More direct evidence that UDP-glucose was a precursor of wall glucans was obtained by Sentandreu et al. (1 9 7 9 , who fed toluene-treated Sacch. cerevisiae with UDP-[ 14C]glucose.( 1 + 3)-/?-Glucan, as well as some glycogen and chitin, were labelled. Most of the label was recovered in membrane particles of “intracytoplasmic origin”. No lipids were labelled in whole cells, and bacitracin had no effect, indicating a lack of involvement of lipid intermediates. At ImM, A T P inhibited incorporation of label by 50%. Incorporation was stimulated by Mg2+.Sentandreu et al. ( 1 975) proposed that glucan synthesis occurred in cytoplasmic vesicles which fused with the plasmamembrane to externalize the nascent wall glucan.

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Synthesis of B-glucan by a particulate preparation from Sacch. cerevisiae was obtained by B a h t et al. (1976). A membrane fraction incorporated [ ‘‘C]glucose from both UDP-[ ‘‘C]glucose and GDP-[ ‘‘C]glucose into glucans with (1 -+ 3)-8- and (1 -+ 6)-P-linkages. Incorporation from UDPglucose and GDP-glucose was additive. The products from UDP-glucose were more susceptible to digestion by an endo-( 1 -+ 6)-p-glucanase than the products from GDP-glucose. They concluded that the product from UDPglucose was predominantly (1 -+ 3)-P-linked, with some (1 -+ 6)-B-linkages, whereas the product from GDP-glucose was predominantly (1 6)-B-linked and included (1 -+ 3)-B-linkages as side chains. Balint et al. (1976) also suggested that much of the product from GDP-glucose was linked to protein, since it could be solubilized under conditions of B-elimination. Elorza et al. (1976) used a temperature-sensitive mutant of Sacch. cerevisiue blocked in RNA transport into the cytoplasm to study cell-wall synthesis. Mannan synthesis was inhibited more rapidly than glucan synthesis at the restrictive temperature. Cycloheximide also inhibited mannan synthesis, but glucan synthesis continued for at least five hours after application of the inhibitor. Either the mRNA species for synthesis of cell-wall glucan are long lived, as Elorza et al. (1976) concluded, or else the glucan synthetases are long lived and are not constituents of vesicles released into the extracytoplasmic space. Cell-free extracts of mid-exponential phase cells of Sacch. cerevisiae were also used by Lopez-Romero and Ruiz-Herrera (1 977) to demonstrate glucan synthesis from nucleoside diphosphate sugars. UDP-Glucose was the glucosyl donor for glucan synthesis in a particulate “mixed membrane” fraction (0.127 nmol min-’ mg protein-’), and in a fraction containing cell walls (0.49 nmol min- mg protein- l ) . There was no soluble activity. GDP-Glucose was less effective as a glucosyl donor than UDP-glucose (1050 pmol min-’ mg protein-’). The product of the “mixed membrane” fraction was predominantly (1 + 3)-B-linked, but contained 0.6% of (1 -+ 6)j?-linkages and was more soluble in alkali. Lopez-Romero and Ruiz-Herrera (1977) pointed out that the enzymes responsible for the incorporation of GDP-glucose may not be those involved in (1 3)-P-glucan synthesis, but may be the same as those studied by B a h t et al. (1976), since the activity of the glucan synthetase studied by Balint et u1. (1976), with GDP-glucose, was only 0.5-1 pmol min- mg protein- and produced a (1 -+ 6)-B-linked glucan, probably also bound to protein. Further characterization of glucan synthetase activity from the “mixed membrane” fraction (Lopez-Romero and Ruiz-Herrera, 1978), showed that the synthetase had a pH optimum of 6.7, a K , for UDP-glucose of 0.12 mM, and was stimulated by divalent cations. Uridine diphosphate and glucono-b-lactone were inhibitory. There was no apparent requirement for a primer. -+

’

-+

’

’

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D . G L U C A N S Y N T H E S I S A N D G L U C A N S Y N T H E T A S E S IN P R O T O P L A S T S

One approach to studying synthesis of cell-wall glucan has been to prepare yeast protoplasts or sphaeroplasts by digestion of the cell wall with snail-gut enzyme in the presence of osmoticum (Peberdy, 1979) and follow the course of wall regeneration. Protoplasts of Sacch. cerevisiae form a network of fibrils, without forming a complete wall, when incubated in liquid media; but if embedded in gel, a “fibrillar network” and an “amorphous matrix” are synthesized together, followed by regeneration of a complete wall (NeEas, 1971). Protoplasts from Schiz. pombe form a complete wall in liquid media. A fibrillar network forms and increases in density for 15 hours. This is followed by the appearance of amorphous material and then wall regeneration (NeEas, 1971). Nadsonia elongata regeneration proceeds like that in Schiz. pombe. The nets of Schiz. pombe differ from those of Sacch. cerevisiae, in that microfibrils of ( 1 -+ 3)-fi-glucan are formed, followed by the formation of fibrils of (1 -+ 3)a-glucan (Kreger and Kopecka, 1978). NeEas (1971) suggested that a diffusible factor is required for full wall reversion and that a gel medium, or the very dense fibrillar network formed by Schiz. pombe, serves to retain this unknown substance. Fibrils from Sacch. cqrevisiae are labelled by [3H]glucose (NeEas et al., 1970). P u l s e 4 h a s e experiments have shown that networks are formed by “interposition of new fibrils” all around the protoplast surface (NeEas et al., 1970). The nets are composed of long chain (1+3)-fi-linked glucans arranged in crystalline microfibrils, which are about 40% alkali-soluble (Kreger and Kopecka, 1973, 1975). The lack.of (1 -+6)-fi-linkages makes it appear as though the (1 -+6)-fi-synthetase is inactivated or lost from the protoplast by the procedure used to prepare these structures (Kreger and Kopecka, 1973, 1975). Yeast protoplasts prepared using snail-gut enzyme are leaky to some soluble compounds (Oura et al., 1970). The leakiness and loss of the ( 1 +6)-fi-synthetase may be only part of the damage to the cell surface caused by protoplast formation. The lag period observed before regeneration of cell walls by protoplasts of leaf cells from Antirrhinum mums is a function of the conditions used to prepare protoplasts and is not lengthened by subsequent treatment with wall-dissolving enzymes (Burgess et al., 1978), thus implying that damage to the synthetic machinery during protoplast formation is more critical in delaying regeneration than accumulation of surface polysaccharides. The alkali-insoluble portions of the nets of Sacch. cerevisiae and C . albicans are susceptible to papulacandin B (Baguley et a[., 1979), suggesting that the soluble and insoluble glucans are synthesized by different enzymes or in different environments in the plasma membrane. Synthesis of glucan microfibrils is the most sensitive site of action of 2-

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deoxy-2-fluoro-~-glucosefound by Biely et al. (1973b). Shematek et al. (1980) and Shematek and Cabib (1980) carried out a detailed study of glucan synthesis by protoplast membranes of Sacch. cerevisiae. The rates of UDP[ 14C]glucoseincorporation into acid-precipitable polysaccharide were 15 to 35nmolglucose - ’ mgprotein-I (Shemateketal., 1980).Neither ADP-glucose nor GDP-glucose could substitute for UDP-glucose (Shematek et al., 1980) and either ATP or GTP, bovine serum albumin and glycerol are required for activity (Shematek et al., 1980). The product was an unbranched (1 +3)P-glucan and was formed de novo. Lipids were not labelled, dolichol phosphate did not stimulate glucan synthesis and [ 14C]glucosylphosphoryldolichol did not label the glucan. Thus, lipid intermediates are not involved in synthesis of this glucan (Shematek et al., 1980). Glucan synthetase was found to be associated with the concanavalin Astabilized plasmalemma fraction which can be isolated from protoplasts of Sacch. cerevisiae. Pretreatment of lysed, but not of intact, protoplasts with glutaraldehyde inactivated the synthetases. Shematek et al. (1 980) concluded that the (1 +3)-P-glucan synthetase is located on or in the inner surface of the plasma membrane. This does not remove the possibility that synthetase originally on the outside of the plasmalemma is lost in the process of protoplast formation, as is apparently the case with (1 +6)-P-glucan synthetase. The (1 +3)-P-glucan synthetase was subject to regulation; ATP and GTP stimulated activity, but the characteristics of activation by ATP and GTP differed (Shematek and Cabib, 1980). Estimation of the K , values for ATP and GTP were complicated by the presence of phosphatases in the membrane preparation, but appeared to be of the order of 24 ,UM for ATP and 0.16 ,UM for GTP. Activation by ATP was enhanced by time-dependent preincubation with ATP at 30°C but not at O T , and required the presence of a heat-stable, dialysable, “supernatant factor”. Activation by GTP is neither time-, nor temperature-dependent. Magnesium ions are inhibitory, especially to the ATP-activated enzyme, as is EDTA. However, GTP-dependent activation requires EDTA in the initial protoplast lysis, apparently to protect against degradation by phosphatases. On the basis of these results, Shematek and Cabib (1980) have proposed a model whereby, in addition to activation by GTP, the glucan synthetase may be activated by the phosphorylated form of the “supernatant factor” and regulated by its reversible phosphorylation and dephosphorylation. Phosphorylation would require ATP, and dephosphorylation would require endogenous phosphatases and Mg2 . Protection against Mg2 +-dependant phosphate hydrolysis would then be provided by EDTA, but would also inhibit ATP-dependent phosphorylation. Farkis (1 979) has proposed that wall synthesis may be inhibited by the presence of one or more proteinaceous inhibitors. These would be kept in contact with the plasma membrane by an intact cell wall. Loosening of the +

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wall by endogenous glucanases during normal growth, or by snail-gut enzymes during protoplast formation, would cause loss of the inhibitor(s) and result in activation of polysaccharide synthetases. Acid phosphatase, a low pH-optimum ATPase and a MgZ+-dependent ATPase are found in the cell envelope of Sacch. cerevisiae (Suomalainen and Nurminen, 1973). On the basis of the model of regulation of the ( 1 -+3)-8glucan synthetase described by Shematek and Cabib (1980), at least one of these phosphatases may be a good candidate for a “proteinaceous inhibitor”. However, this could account for only part of the regulation of wall synthesis since, as pointed out by NeEas (1971), complete reversion of yeast protoplasts appears to require a protein- or polysaccharide-retaining network (a gel medium for Sacch. cerevisiue and a dense microfibrillar network for Schiz. pombe).

IV. Physiological Control of Glucan Content A.

INTRODUCTION

In recent years, an increasing proportion of yeast research has been devoted to elucidating the changes that occur in yeast cells during normal growth and division, yeast-mycelial interconversion, and exposure to growth media of different compositions. This research has not only added to our understanding of fundamental biology, but has opened the way to greater control of yeast growth which may enable us to optimize yeast properties of commercial importance. It is possible that studies of yeast glucans in the physiological context have lagged behind other studies and it is hoped that the following review of the current literature will stimulate further progress. B. T H E C E L L C Y C L E

The first study of bulk glucan changes in relation to the cell cycle appears to be that of Dawson (1967) using Candida utilis in what he called a “continuous phased culture” (Dawson, 1969). It is clear that division was synchronous in this culture but there was no control to establish that the cells were otherwise normal and not subject to the treatment-induced changes that have complicated cell-cycle analysis with synchronous cultures (Mitchison, 1977). However, Dawson (1967, 1969) found that the g1ucan:mannan ratio was constant with a value of 0.5 throughout the cycle in cells using 3% or 5% glucose as the carbon source. In cells using 1% glucose as the carbon

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source, the ratio rose from about 0.3 to about 0.5 during the cycle. Unfortunately only one cycle was studied. Kuenzi and Fiechter (1972) studied changes in the carbohydrate composition of Sacch. cerevisiae during the cell cycle. To achieve synchronization, the yeast was grown in a chemostat and, after six generation times, the medium supply was interrupted. After seven hours, fresh medium was supplied at a dilution rate that ensured complete oxidative growth on glucose. This process induced a cyclic partially synchronized budding. As with Dawson's method, there is no way of knowing what cell abnormalities have been induced by the drastic treatment. In this case, the g1ucan:mannan ratio remains fairly constant, even when budding is taking place, with a ratio of about 0.7 at a dilution rate of 0.05 and about 0.9 at a dilution rate of 0.1. Later, Sierra et al. (1973) investigated the biosynthesis of alkali- and acidinsoluble glucan using synchronized cultures of two strains of Sacch. cerevisiae. The cells of one strain, LK2GI 2, were synchronized by harvesting an asynchronous culture, immersing the cells in ice water to chill them, selectingcells of about the same size from a Ficoll gradient, and re-inoculating them into growth medium. Thus, this strain was subjected to temperature shock, osmotic shock and nutrient depletion, all processes that can modify metabolism dramatically. The other strain, a temperature-sensitive mutant, was transferred from its permissive temperature (25°C) to a restrictive temperature (37°C) for 120 minutes before returning the temperature to 25°C. Again, the inherent assumption that the synchronization of cell division attained must be associated with a normal cell cycle is unlikely to be true. Despite this, Sierra et al. (1973) concluded that both glucan and mannan are synthesized continuously and in an exponential fashion through the whole cell cycle. Recently, Biely (1978) recalculated the results of Sierra et al. (1973) to obtain average rates of glucan and mannan synthesis at 15-minute intervals, and showed that the rates of glucan and mannan synthesis decline during budding. At about the same time, Biely, et al. (1973a) described an electronmicroscope autoradiographic study of cell-wall formation in yeast. These authors found that the pattern and extent of labelling of cell walls of Sacch. cerevisiae with tritium from tritiated glucose varied through the cell cycle. They used exponentially growing asynchronous cultures of the yeast subjected to 15-minute pulses of radioactive glucose. The results, therefore, relate to a cell cycle which has suffered a minimum of distortion. Again it appears that the rate of incorporation of new glucan into a growing bud increases with the size of the bud to a maximum before division begins, when the rate decreases substantially until new bud initiation occurs. Budd (1975) studied incorporation of [U-'4C]glucose, maltose and maltotriose into the polysaccharides of Sacch. cerevisiae in synchronous culture during fermentation of brewer's wort. Synchrony was induced by repeated

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subculture of the yeast cells skimmed from the surface of previous fermentations. This would appear to be a technique for selecting out the smallest cells, i.e. those that have just budded from a parent. If the wort into which they are inoculated is not substantially different from that from which they were isolated, such cells should not be greatly perturbed. On the other hand, any such surface yeast population tends to contain a proportion of abnormal cells, and it is possible that the decrease in oxygen availability accompanying transfer of cells from a surface culture to a totally immersed culture may induce biochemical changes and oscillations not associated with the normal cell cycle. Budd’s (1975) conclusion was that incorporation of 14C into wall glucan was continuous and stable. Hayashibe et a / . (1977) looked at the mode of increase in cell-wall polysaccharides in synchronous cultures of Sacch. cerevisiae prepared using yet another method. Commercial baker’s yeast cells were fractionated into small- and large-sized cells by repeated centrifugation in de-ionized water and the large-sized cells were inoculated into fresh growth medium to give a culture which is highly synchronous with respect to division (Hayashibe and Sando, 1970). Although this is apparently a selection method, establishing synchrony by selecting cells on the point of budding, it is likely that there is a large component of induction brought about by nutrient depletion during the centrifugation, and perhaps also by temperature as the centrifuge temperature is not stated. Thus, abnormal induced fluctations in cell components may occur. The results obtained indicate that the increase in total glucan is almost linear throughout the cell cycle with a decrease in accumulation about the time of cell division. Alkali-soluble glucan accumulates mainly as the buds grow whereas the alkali-insoluble glucan tends to accumulate around the time of cell division and bud initiation. Biely (1978) re-evaluated the results of Biely et a / . (1973a), Sierra et a / . (1973) and Hayashibe et al. (1977) described above, and pointed out that they all agree in showing that the rate of glucan and mannan synthesis is markedly decreased during cell division and budding initiation. He also pointed out that the possibility that it ceases completely cannot be ruled out because of the relatively long sampling intervals used in these studies. In this connection, it may be important to note the work of Maddox and Hough (1971) who observed that bud initiation in Sacch. cerevisiae is accompanied by intensive glucanase and mannanase activity. In conclusion, it may be said that most of the cell-cycle studies have used methods for establishing synchronous cultures that are idiosyncratic in the sense that their use has been restricted to specific laboratories, and hence they lack the background of independent assessment that more widely used techniques would have. This makes it important to repeat and extend these studies with synchronous cultures established by other means. All methods of preparing synchronous cultures have drawbacks, but these can be overcome,

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to some extent, by use of appropriate control cultures. A notable feature of the work described above is the absence of such controls and these must be included in any future investigations.

C. YE A S T-M Y C E L I U M I N TER C 0 N V E R S I O N

Yeast-mycelium interconversion has been reviewed on a number of occasions (Scherr and Weaver, 1953; Romano, 1966; Phaff, 1971) and work covered in these reviews will be described only in so far as it is directly relevant to yeastwall glucans. In 1969, Kanetsuna et al. investigated the yeast and mycelial cell walls of Paracoccidioides brasiliensis. They found that the yeast-wall glucan was almost entirely soluble in alkali ( I M NaOH) but that the mycelial glucan was only 60 to 65% alkali soluble. The alkali-soluble glucan gave a weakly positive response to the periodic acid-Schiff reaction, was not hydrolysed by snail digestive juice and had a-glucosidic linkages. The alkali-insoluble glucan gave a clearly positive response to the periodic acid-Schiff reaction and was hydrolysed by snail digestive juice. Subsequently, it was established that the alkali-soluble glucan from the yeast form of the organism had the properties of a (1 +3)-a-glucan (Kanetsuna and Carbonell, 1970), unlike the equivalent fraction from the mycelial form which proved to be a variable mixture of a- and j-glucans. Electron microscopy showed that the a-glucan occurred on the outer surface of the yeast phase in the form of short fibres (Carbonell et a/., 1970). Synthesis of this glucan can be induced by adding foetal calf serum to the growth medium (San-Blas and Vernet, 1977). Similar studies on Blastornyces derrnatidis showed that the yeast form of this organism had a cell wall composed of 95% a-glucan and 5% P-glucan, whereas the mycelial cell wall was 60% a-glucan and 40% P-glucan (Kanetsuna and Carbonell, 197I). Histoplasma capsulaturn has also been investigated and in this organism the yeast cell walls contain about 47% a-glucans, mainly (1 -,3)-linked, and 31% P-glucan, mainly (1 -3)-linked, whereas the mycelial cell wall has essentially no a-glucan and only 19% P-glucans, again with mainly (1-3)P-linkages (Kanetsuna et al., 1974). Again, electron microscopic studies show the a-glucan to be located on the outer part of the yeast cell wall.

D. E F F E C T S O F N U T R I E N T L I M I T A T I O N

Nutrient limitation has long been known to lead to altered composition of yeast cell walls. This is obvious in batch cultures where exponential-phase cells are much more susceptible to protoplast formation than are stationary-phase cells (Eddy and Williamson, 1957, 1959). Unfortunately, only a few nutrient

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deficiencies have been investigated to any extent, namely deficiencies in inositol, biotin, nitrogen, carbohydrate, phosphate and oxygen. In 1960, Ghosh et ul. studied the effects of inositol deficiency on Sacch. curlsbergensis. They found that daughter buds failed to separate from parent cells and aggregates of up to 50 cells were formed. The cell walls under these conditions contained up to three times as much glucan as walls from cells growing with an adequate inositol supply. Similar results were obtained in experiments with Sacch. cerevisiue (Challinor et al., 1964; Power and Challinor, 1969). When grown in the complete absence of inositol, Sacch. cerevisiae had a much weaker cell wall than when grown in a complete medium, and the wall had a greater percentage of glucan and hexosamine. These results were confirmed by Dominguez et al. (1978) who further demonstrated that the change in wall composition was due to the relative insensitivity of glucan synthesis to inositol deficiency when compared with mannan synthesis. The effects of biotin deficiency on yeast glucan are similar to those of inositol. When Sacch. cerevisiae was grown in biotin-deficient media supplemented with aspartate, the ratio of glucan to mannan increased three-fold (Dunwell et al., 196I). This change is associated with a marked thickening and pronounced multiple layering of the cell walls (Dixon and Rose, 1964). Failure to divide in such media seems to be due to inability to produce a cross septum. The increased ratio of glucan to mannan is not seen in all cells grown in biotin-deficient media (Mizunaga, et al., 1971). At least one strain of Saccharomyces shows a slight decrease in the glucan :mannan ratio when grown in biotin-free medium supplemented with aspartate. Similarly, wall thickening is not always seen. The same strain, which does show wall thickening in a medium containing ammonium ions as the source of nitrogen, shows none in a medium where the nitrogen is supplied as amino acids, even though the glucose: mannan ratio is increased. It was further observed by Mizunaga et al. (1 97 1) that enrichment of glucan occurs in their yeast strain with age of the batch culture, irrespective of the quantity of biotin supplied. Clearly there is a need for such-studies of the effects of nutrient deficiency to be carried out under chemostat conditions. McMurrough and Rose (1967) used continuous culture to study the effects of growth rate and nitrogen and carbohydrate nutrient limitation on the composition and structure of the cell wall of Sacch. cerevisiue. They found that the contents of total glucan and mannan were little affected by growth rate, although the distribution among wall fractions varied. Yeast cells grown at any rate under nitrogen limitation were longer and thinner than those grown at the same rate under carbohydrate limitation, and the electron microscope showed that the walls of these long, thin cells were more porous than normal. In general, there appeared to be a direct correlation between the amount of glucose in the medium and the amount of glucan in the walls. The effects of carbohydrate and nitrogen deficiency on yeast glycogen

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synthesis have also been studied (Becker et al., 1979). Cells of Sacch. carlsbergensis growing in batch culture under either carbohydrate or nitrogen limitation initially deplete their glycogen and this is resynthesized only in later exponential phase. Cells harvested in early logarithmic phase cannot synthesize glycogen, even in glucose-phosphate buffer which supports glycogen

synthesis in stationary-phase cells. Lack of oxygen or addition of ammonia

slows down glycogen synthesis in cells grown under carbohydrate or nitrogen limitation. The sensitive control of glycogen metabolism in yeast cells is shown by the rapid commencement of glycogen synthesis within one minute of addition of glucose to a starved cell suspension, and in the equally rapid decrease in glycogen content when glucose is removed from cells that have synthesized glycogen in glucose-phosphate buffer. Another study on yeast glycogen content, this time in Sacch. cerevisiae, showed that the glycogen content of this yeast was higher in anaerobic 1% glucose media than in the same media under aerobic conditions, and that it increased further when the glucose content of the media was increased to 8%. The rest of the yeast glucan behaved similarly but increased only slightly with increasing glucose concentration (Chester and Byrne, 1968). Phosphate limitation affects the glucan content of walls of Sacch. cerevisiae lowering it from 45% to 27% in cells grown in continuous chemostat culture when the medium phosphate concentration is decreased from 3 g-' to 81.6 mg-' at 30°C (Ramsay and Douglas, 1979). E. E F F E C T S O F M E T A B O L I C I N H I B I T O R S

There are a number of reports of the action of inhibitors on yeast glucan accumulation. Cycloheximide, which inhibits protein synthesis at the ribosome, selectively inhibits synthesis of mannan protein without affecting the formation of glucan microfibrils (Netas et al., 1968; Elorza and Sentandreu, 1969; Elorza et al., 1976). On the other hand, lomofungin, thiolutin and 8hydroxyquinoline at concentrations that inhibit RNA synthesis inhibit both glucan and mannan synthetases (Elorza et al., 1976). This is probably due to their making magnesium and manganese unavailable 'by chelation. Lomofungin was used at a concentration of 50pg ml-. In a subsequent study by Kopecka and Farkgs (1979), lomofungin was found not to inhibit the synthesis of the (1 +3)-/?-glucan network in regenerating protoplasts of Sacch. cerevisiae at a concentration of 20 pg ml-l. 2-Deoxyglucose is probably the inhibitor that affects glucan synthesis most directly. Johnson (1968) reported that it caused lysis of cell walls of Schiz. pombe, Pichia farinosa and Sacch. cerevisiae at sites that co-incided with the regions of growth of their glucan layers. Biely et al. (1971) made similar observations on Sacch. cerevisiae and produced evidence for incorporation of

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2-deoxyglucose into the cell wall. Interestingly, the glucose content of the cell walls from treated cells was unaltered, though the mannose content had fallen by more than a third. Subsequently, Biely et ul. (1974) studied the effects of 2-deoxyglucose on Schiz. pornbe. They observed that formation of the cell plate was more sensitive to the inhibitor than was synthesis of lateral walls. Further, cycloheximide and nalidixic acid could suppress 2-deoxyglucoseinduced lysis suggesting that inhibition of glucan synthesis is not the whole explanation of this phenomenon. Biely et al. (1973b) used the related compound, 2-deoxy-2-fluoro-~-g~ucose, to inhibit synthesis in Sacch. crrevisiae and cause extensive cell lysis. Cells resistant to this compound proved to be defective in the final steps of cell division. Schizosaccharomyces pombe was similarly affected, but was more resistant to the inhibitor than was Sacch. cerevisiae. Three other inhibitors affecting yeast glucan synthesis have recently been reported. 2-Deoxy-~-arabinohexosehas been found to increase the relative amounts of chitin and glucan in the cell wall of Rhodosporidium tordoides (Sipicki and FarkBs, 1979). This is associated with defective cell division and separation of daughter cells, which remain attached to the mother cells giving multicellular aggregates or, growing on yeast extract agar, pseudomycelia. F. M I S C E L L A N E O U S

Little has been said in this section about yeast glycogen because it is not clear where it is located in the cell. However, Gunja-Smith et al. (1977) have shown that a proportion of the water-insoluble glycogen fraction is associated with a fi-glucan-like polysaccharide which may be a cell-wall component. They also showed that the amounts of yeast glycogen fractions vary during growth in batch culture, and so one may conclude that there are physiological controls which should be further investigated. It might be supposed that changes in cell-wall glucan would accompany yeast conjugation but the evidence from studies of the basidiomycete Tremellu mesenterica seems to indicate that gross changes in wall composition do not occur (Reid and Bartnicki-Garcia, 1976). Finally, the point must be made that many of the physiologically induced changes in yeast-wall glucan probably result from the action of endogenous glucanases. For recent reviews of the role of these enzymes, the reader is referred to the articles by Phaff (1 977, 1979). V. Acknowledgements

The authors are indebted to the Science Research Council for financial

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support (Grant GR/A/7056-4); one of us (D.J.M.) wishes to acknowledge many helpful discussions with J. S. D. Bacon and G. H. Fleet which have contributed significantly to the progress made in this laboratory.

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