2.1Composition and properties of the cell wall
The cell wall is a sturdy structure providing physical protection and osmotic support . Electron microscopic analysis of the wall using negative staining reveals a layered structure with an electron-transparent internal layer of about 70–100 nm thick depending on growth conditions and genetic background, and an electron-dense outer layer [5,6]. In brewing yeast the electron-transparent inner layer may be as thick as 200 nm . The mechanical strength of the wall is mainly due to the inner layer, which consists of β1,3-glucan and chitin, and represents about 50–60% of the wall dry weight (Table 1). The outer layer, which consists of heavily glycosylated mannoproteins emanating from the cell surface [5,18], is involved among others in cell–cell recognition events [5,19–21]. It also limits the accessibility of the inner part of the wall and the plasma membrane to foreign enzymes such as cell wall-degrading enzymes in plant tissue [22–24]. The carbohydrate side chains of the cell surface proteins contain multiple phosphodiester bridges, resulting in numerous negative charges at the cell surface at physiological pH values . These side chains are responsible for the hydrophilic properties of the wall, and may be involved in water retention and drought protection. The outer protein layer accounts for about one third of the wall dry weight. Cell wall proteins are covalently linked to the β1,3-glucan–chitin network either indirectly through a β1,6-glucan moiety or directly (Sections 2.6 and 2.7). In addition, some proteins are disulfide-bonded to other cell wall proteins [26,27].
Table 1. Cell wall macromolecules in S. cerevisiae
|Macromolecule||Wall dry wt. (%)||Site of synthesis||Mature form||Refs.|
| || || ||DP||Branching|| |
|Mannoproteins||35–40||Secretory pathway||200a||Highly branched|||
The cell wall is highly elastic [28,29]. When yeast cells are transferred to hypertonic solution, they rapidly shrink, and depending on the osmotic stress they may lose more than 60% of their initial volume. This process is reversible. When transferred back to the original medium, the cells immediately expand to their initial volume. This elasticity of the wall is probably due to the elastic properties of the β1,3-glucan chains (see Section 2.2). The elasticity of the wall explains why the wall of living cells is much more permeable than isolated cell walls. Whereas isolated walls are permeable only to molecules of molecular mass up to 760 Da , walls of living cells are permeable to much larger molecules especially under hypotonic conditions and also depending on growth conditions [22,31,32].
2.2The β1,3-glucan network
The mechanical strength of the cell wall is mainly due to β1,3-glucan, which can be specifically stained with aniline blue [33,34]. β1,3-Glucan chains belong to the so-called hollow helix family; in other words, they have a shape comparable to a flexible wire spring that can exist in various states of extension . This property explains the above-mentioned elasticity of the cell wall. Using magic-angle spinning 13C-NMR on living cells, Krainer and co-workers have found that a portion of the β1,3-glucan indeed assumes a helical structure .
β1,3-Glucan is only slightly crystalline in lateral walls . In stationary phase cells, β1,3-glucan molecules were found to consist of about 1500 glucose monomers . This may, however, be an underestimation as a result of partial hydrolysis of glucan chains during the extraction procedure. Considerably higher degrees of polymerization have been reported, depending on the type of acid used for extraction . In addition, the degree of polymerization may depend on environmental conditions, because yeast uses two β1,3-glucan synthase complexes, to an extent that depends on growth phase and carbon source . In their mature form, β1,3-glucan chains of stationary phase cells are moderately branched and contain about 3–4%β1,6-linked glucose residues . Conceivably, the degree of branching of β1,3-glucan may also depend on growth conditions.
The moderate degree of branching in mature β1,3-glucan molecules prevents the extensive crystallization seen on the surface of regenerating spheroplasts [6,37,40]. This is also consistent with the X-ray diffraction data of isolated walls, which point to a low level of crystalline β1,3-glucan . On the other hand, the presence of uninterrupted chain segments of substantial length permits stable interchain association, thus allowing the formation of a three-dimensional network [16,40]. Consistent with this, electron microscopic studies of isolated walls using negative staining reveal a fine network with meshes of about 20–60 nm wide . As isolated walls are in a non-extended state, the pores of the glucan layer in living cells are expected to be wider, explaining why the permeability of the inner wall is only limiting for very large proteins .
The two β1,3-glucan synthase complexes found in yeast contain either Fks1 or Gsc2/Fks2 depending on environmental conditions. Both are multiple-spanning transmembrane proteins that are essential for the synthesis of β1,3-glucan  and the corresponding genes are well conserved among fungi [42,43]. It is generally assumed that Fks1 and Gsc2 represent the catalytic subunits, but this has still to be validated experimentally, because, for example, they lack the known UDP-Glc binding site (K/RXGG) found in glycogen synthase  and in an α1,3-glucan synthase in Schizosaccharomyces pombe. Alternatively, they may represent a pore-forming protein that guides the newly formed chain through the plasma membrane to the outside. An elegant electron microscopic study, using snap-frozen freeze-etched cells, has identified particles in the plasma membrane, from which, on close examination, fibrils with a diameter of 5 nm can be seen emerging . These fibrils disappear into the inner regions of the cell wall, suggesting that they may represent β1,3-glucan fibrils. A follow-up of this older work in combination with the use of molecular genetic tools promises a more complete understanding of in vivo β1,3-glucan synthesis. Interestingly, a plant homologue of FKS1 has been identified in cotton fibers .
It is unknown whether growing β1,3-glucan chains in yeast are extended at the reducing or non-reducing end. However, β1,3-glucan from the fungus Sclerotium rolfsii is extended at the non-reducing end . Also, bacterial cellulose, diatom chitin, and plant homogalacturonan seem to be extended at their non-reducing ends [49–51], suggesting that this is generally the case for processive glycosyltransferases such as β1,3-glucan synthase. This would make the reducing end of the growing chain immediately available for coupling to Pir cell wall proteins (see Section 2.5) and for processing enzymes such as Bgl2 [52,53].
As β1,3-glucan chains are synthesized as linear chains , the presence of branches in the mature molecules implies the existence of β1,3-glucan processing enzymes. Additional enzymes may be needed for integrating newly synthesized β1,3-glucan molecules into the growing wall during isotropic growth. Enzymes potentially involved in these steps are Gas1, an endotransglycosylase that may be involved in extending and rearranging β1,3-glucan chains [55,56], Bgl2, an endotransglycosylase that introduces intrachain β1,6-linkages [52,57], and, possibly, also the Crh1  and the SUN family [57,59]. Their exact function in vivo is, however, still incompletely understood.
2.3Chitin as an intrinsic part of the lateral walls
Chitin is believed to occur as linear chains in the chitin ring, in and around the bud scars and also to a minor extent uniformly dispersed in the lateral walls of the mother cell [60–62]. Chitin isolated from bud scars consists of about 190 N-acetylglucosamine monomers , but it is unknown whether this is also a valid estimate for chitin in the lateral walls. Calcofluor white is a fluorescent, anionic dye that preferentially binds to β1,4-glucans such as chitin, chitosan, and cellulose. It reacts to a lesser extent with β1,3-glucan [63,64]. In S. cerevisiae, Calcofluor white is frequently used to visualize chitin.
Chitin synthesis in S. cerevisiae involves three chitin synthases and is tightly regulated [1,62]. For example, under normal conditions the deposition of chitin in the lateral walls takes place after cytokinesis . This results in a relatively low chitin level in the lateral walls: 0.1–0.2% excluding the bud scar(s) and 1–2% including the bud scars (see also Table 1). In cells with a (genetically) weakened wall, however, chitin synthesis is activated as part of a salvage mechanism and the levels in the lateral walls of those cells may become as high as 20% of the wall dry weight (see also Section 3.4) [10,66]. As to be expected for a salvage mechanism, chitin is then also deposited in the wall of the growing bud . The responsible synthase is encoded by CHS3. Chs3 is a multi-spanning membrane protein with its active site at the cytosolic side of the plasma membrane . Like other fungal chitin synthases, it is a processive enzyme with the diagnostic motif D,D,D,QRRRW . Diatom chitin synthase extends the growing chitin chain at the non-reducing end using UDP-GlcNAc as substrate . Assuming that fungal chitin synthases behave similarly, the reducing end of a chitin chain is directly available for coupling to the acceptor sites of β1,3- and β1,6-glucan molecules [70,71], when it emerges from the pore through the plasma membrane.
β1,6-Glucan is in its mature form a highly branched, water-soluble polymer consisting on average of about 130 glucose monomers . It is unknown whether β1,6-glucan is synthesized as a water-insoluble linear molecule similar to β1,3-glucan , or as a water-soluble branched molecule similar to the mixed β1,3-/β1,6-glucan produced by S. rolfsii. This is particularly relevant regarding the development of an in vitro assay for β1,6-glucan synthase.
β1,6-Glucan is used in the cell wall to connect GPI-dependent cell wall proteins to the β1,3-glucan network (see Fig. 4, Section 2.7). It may also function as acceptor site for chitin, particularly in case of cell wall stress (see Section 2.6 and Fig. 2) [71,73]. The pioneering studies by Bussey and co-workers have identified several genes that affect β1,6-glucan levels in the cell wall . From these studies it has emerged that various ER-resident proteins, Golgi-resident proteins, and cell surface proteins strongly affect β1,6-glucan levels in the cell wall. This might be interpreted in various ways: (i) the biogenesis of β1,6-glucan is a stepwise process that begins in the ER; (ii) the synthesis of β1,6-glucan takes place at the plasma membrane but requires the stepwise synthesis of a primer; or (iii) the synthesis of β1,6-glucan takes place at the plasma membrane but the activity of the synthesizing complex is highly sensitive to various defects in the secretory pathway. Using immunogold labeling, Montijn and co-workers could not detect intracellular β1,6-glucan, not even in a temperature-sensitive mutant that had accumulated secretory vesicles at the restrictive temperature for 2 h . In contrast, plasma membrane-derived vesicles and the cell walls reacted strongly. This seems to exclude the first hypothesis. Because an in vitro assay for β1,6-glucan is not yet available, differentiation between the two other hypotheses is not simple, the more so since they are not mutually exclusive. Summarizing, a clear picture of how β1,6-glucan is synthesized is still lacking.
Figure 4. Putative molecular organization of the cell wall of S. cerevisiae. The mechanical strength of the wall is due to an elastic three-dimensional network of β1,3-glucan molecules kept together by hydrogen bonding between locally aligned chains . As the arrows in the model indicate, the terminal non-reducing ends of the β1,3-glucan side chains are believed to function as acceptor sites for β1,6-glucan and chitin, whereas the reducing end of the β1,3-glucan molecules may be involved in the linkage to Pir-CWPs. The β1,3-glucan layer forms the inner layer of the cell wall, whereas cell wall proteins form the outer layer. The two most abundant CWP–polysaccharide complexes are shown here: GPI-CWP–β1,6-glucan–β1,3-glucan and Pir-CWP–β1,3-glucan. The β1,6-glucan molecules are highly branched and thus water-soluble, tethering GPI-CWPs to the β1,3-glucan network. GPI-CWPs represent the major component of the outer protein layer. The Pir-CWPs are directly linked to the β1,3-glucan network through an alkali-sensitive linkage and are distributed throughout the β1,3-glucan network. This model is based on [2,65,70,71,73,80,115,137]. The model is believed to be valid for C. albicans as well . GPI-CWP, cell wall protein linked through a GPI remnant to β1,6-glucan (β1,6-Glc); Pir-CWP, a cell wall protein from the PIR family linked through an alkali-sensitive linkage to β1,3-glucan.
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Figure 2. Overview of the CWP–polysaccharide complexes in yeast. GPI-CWP, cell wall protein linked through a GPI remnant to β1,6-glucan (β1,6-Glc); Pir-CWP, a cell wall protein from the PIR family linked through an alkali-sensitive linkage to β1,3-glucan (β1,3-Glc). Kollar and co-workers have identified several of the interconnecting linkages between cell wall macromolecules [70,71], but the interpolymer linkages between GPI-CWP and Pir-CWP on the one hand and β1,3-glucan on the other hand are unknown.
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2.5Cell wall proteins (CWPs)
The mannoproteins that form the outer cell wall layer are highly glycosylated with a carbohydrate fraction that often amounts to over 90% (w/w) . Biotinylation of intact cells using a sulfonated derivative of biotin that does not cross the plasma membrane is a convenient tool to distinguish between authentic cell wall proteins and adventitiously bound proteins . The outer layer of mannoproteins is much less permeable to macromolecules than the internal fibrillar layer . This is largely due to the presence of the long and highly branched carbohydrate side chains linked to asparagine residues [1,22] and to the presence of disulfide bridges . Serine and threonine residues, which may carry short oligomannosyl chains, are often clustered, resulting in relatively rigid rod-like regions of the polypeptide backbone [76,77]. Finally, due to phosphodiester bridges in both N- and O-linked mannosyl side chains the cell surface of yeast contains numerous negative charges [1,25]. Interestingly, the sensitivity of yeast cells to the antifungal plant protein osmotin, which has a high isoelectric point and thus is positively charged at physiological pH values, depends on the presence of mannosyl phosphate groups at the cell surface . In addition, the cell wall may bind positively charged, cytosolic proteins originating from lysed cells. Phosphodiester groups can be visualized with Alcian blue .
There are two main classes of proteins covalently coupled to cell wall polysaccharides (Fig. 4, see Section 2.7).
(a) GPI-dependent cell wall proteins (GPI-CWPs). They are generally indirectly linked to β1,3-glucan through a connecting β1,6-glucan moiety. In the genome of S. cerevisiae about 60–70 GPI proteins have been identified (; J.G. De Nobel, unpublished data). About 40 of them are destined for the plasma membrane whereas the others become covalently linked to β1,6-glucan [71,80–84]. They often contain repeats and serine- and threonine-rich regions. The most extensively studied GPI-CWP is Sag1, which is involved in sexual agglutination [5,77,84,85]. Mature proteins only have a remnant of the original GPI anchor, that links them to β1,6-glucan [71,81]. The core structure of this remnant  is formed by –ethanolamine–Pi–(Man)4–, and is probably substituted with additional ethanolamine phosphate groups [86–88]. Interestingly, β1,6-glucan extracted from cell walls by hot acetic acid may contain a minor amount of galactose . Conceivably, in some genetic backgrounds this is part of the GPI anchor remnant of GPI-CWPs . The presence of galactose in the cell wall of S. cerevisiae is consistent with the evidence for a UDP-galactose transporter in bakers’ yeast .
(b) Pir proteins (Pir-CWPs). They are presumably directly linked to β1,3-glucan through an alkali-sensitive linkage. In S. cerevisiae a family of four such proteins has been found [65,91,92]. They are all similarly organized (SP–Kex2–repeat(s)–CX(66)CX(16)CX(12)C), consisting of an N-terminal signal peptide, a Kex2 site, followed by a repeat-containing region with up to 11 repeats, and a highly conserved carboxy-terminal region with four cysteine residues in a conserved spacing pattern. Pir1, Pir2/Hsp150, Pir3, and Pir4/Cis3 all have been localized to the cell wall immunologically [27,93,94]. Several additional proteins, such as Pau1 and its homologues, and Sps100, which is believed to contribute to maturation of the spore wall , and also Ygp1, which is induced by nutrient limitation , are predicted to have an N-terminal signal peptide, but not an addition signal for a GPI anchor. Possibly, their final destination is not the medium, but the cell wall, to which they may become linked in a Pir-CWP-like fashion.
In addition, several cell wall proteins such as Bar1, a protease , Aga2 [5,97], the active subunit of the sexual agglutinin complex in MATa cells, Pir4/Cis3 , and some known or potential cell wall glycanases such as Sun4/Scw3 , can be released from intact cells using a reducing agent. This suggests that they might be disulfide-linked to other cell wall proteins. Reducing agents are also expected to release soluble, intermediate forms of GPI-CWPs . Finally, SDS extraction of isolated walls releases many proteins. With a few exceptions, like the transglucosylase Bgl2  and the chitinase Cts1 [99,100], they are not authentic cell wall proteins and their presence is due to contamination with membrane fragments [11,57, 101].
Members of the Hsp (heat-shock protein) family  and abundant glycolytic enzymes such as Tdh1, Tdh2, and Tdh3  are often found at the cell surface. They can be extracted from intact cells with a reducing agent such as mercaptoethanol under slightly alkaline conditions, suggesting that they are either trapped inside the wall or are ionically bound to cell surface proteins. It is not clear whether these proteins originate from lysed cells or as frequently claimed are exported by a non-conventional secretory mechanism [102–104]. Heat-shock proteins and glycolytic enzymes have also been found in the medium of regenerating spheroplasts, which raises the same issue [104–106].
Cell wall proteins may have various functions, which are summarized in Table 2. In many cases their precise function is unknown. Some proteins, like the very small but presumably abundant GPI-CWPs Ccw12 and Ydr134c with a predicted unprocessed size of 133 and 66 amino acids, respectively, and a codon adaptation index of 0.870 and 0.646, respectively, may be used as a means to present mannan to the cell surface (Fig. 1). Various GPI-CWPs are involved in adhesion events like sexual agglutination and flocculation of yeast cells. Others appear to have an enzymatic function like Crh1, Crh2, and Crr1 . Fig. 2 is required for the normal width of the conjugation tube , suggesting that it may be involved in remodeling of β-glucan in the conjugation tube.
Table 2. Known or putative functions of CWPs
|Presentation of mannan chains||CCW12, YDR134C||Fig. 1|
|Cell wall permeability|| ||[22,24,41]|
|Retention of water|| ||Section 2.1|
|Mating||SAG1, AGA1, AGA2, FIG2||[5,77,107–109]|
|Flocculation||FLO1, FLO5, FLO9, FLO10||[20,109–111]|
|Cell wall processing|
|Glucan remodeling||CRH1, CRH2, CRR1, FIG2 (speculative), BGL2||[52,58,106,108]|
|Cell separation||EGT2, PRY3, CTS1, DSE2, DSE4, SCW11||[57,100,112]|
|Cell wall strengthening||CWP1, PIR1, PIR2/HSP150, PIR3, PIR4/CIS3||[65,113–116]|
|Isotropic growth||PIR1, PIR2/HSP150, PIR3, PIR4/CIS3||Section 3.1; 117|
|Stationary phase||SED1, SPI1||[119,120]|
|Anaerobic growth||DAN1, DAN4, TIP1, TIR1, TIR2, TIR3, TIR4||[121–123]|
|Low temperature||TIP1, TIR1, TIR2, TIR4||[121,122,125]|
|Iron uptake||FIT1, FIT2, FIT3|||
Figure 1. Schematic representation of the GPI-CWP Ccw12. The mature form of Ccw12 is estimated to consist of less than 100 amino acids. Ccw12 has three predicted attachment sites for N-chains. As N-chains may contain up to 200 mannose residues each , they may together have a molecular mass of about 90 kDa. Interestingly, CCW12 has a codon adaptation index (CAI) of 0.87, indicating that it encodes a very abundant protein. Deletion of CCW12 results in a reduced growth rate of about 40% and increased sensitivity to Calcofluor white and Congo red . SP, signal peptide; grey box, GPI anchor addition signal.
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The expression of PIR genes is also up-regulated in case of cell wall stress (see Section 3.3) [113,114], consistent with the idea that their gene products might be involved in cell wall strengthening. Disruption of all four genes results in swollen cells that grow slightly slower and are more sensitive to Calcofluor white and Congo red, indicating that their cell wall is indeed weakened . Interestingly, cells that overexpress PIR2 are more resistant to the plant antifungal protein osmotin whereas cells that lack PIR1, PIR2, and PIR3 are more sensitive to it . Differences in tolerance to osmotin depend on the presence of an intact cell wall, because after spheroplasting the cells are equally sensitive . Possibly, Pir-CWPs make the cell wall less permeable to osmotin and other proteins.
Analysis of CWP–polysaccharide complexes requires a relatively small set of reagents. Recombinant β1,3-glucanase and β1,6-glucanase are now commercially available. For detection, antibodies specific for β1,3-glucan are commercially available, whereas antibodies specific for β1,6-glucan are easy to raise . The lectins concanavalin A for mannoproteins and wheat germ agglutinin for chitin oligomers further complement this list.
Cell wall proteins may be linked to the β1,3-glucan network in various ways (Fig. 2). In cells grown in rich medium the GPI-CWP–β1,6-glucan–β1,3-glucan complex is the most abundant one (Fig. 2A). It consists of cell wall proteins such as Ccw12, Tip1, Ssr1, Cwp1, or Sag1 covalently linked through a GPI remnant to a non-reducing end of β1,6-glucan, which in turn is linked to a non-reducing end of β1,3-glucan [2,71,80,81]. Because the GPI remnant (core structure: –ethanolamine–Pi–(Man)4–) contains a phosphodiester bridge, GPI-CWPs in this complex can be specifically released by using aqueous hydrofluoric acid . Pir-CWP–β1,3-glucan represents another, abundant CWP–polysaccharide complex in which a protein from the PIR family is linked through an alkali-sensitive linkage to β1,3-glucan (Fig. 2D) [65,75,92]. The nature of this linkage is not known, but in view of its sensitivity to mild alkali it is tempting to postulate that it involves an O-linked side chain. Interestingly, immunogold labeling of Pir-CWPs shows that the signal is uniformly dispersed over the chitin–β1,3-glucan layer of the cell wall . In contrast, immunogold labeling of the GPI-CWPs Sag1 and Flo1 results in strong labeling of the outer fibrillar wall layer [5,110]. This indicates that Pir-CWPs are part of the inner layer. A possible interpretation of these observations is presented in Fig. 3, in which it is proposed that a β1,3-glucan molecule is covalently linked through its reducing end to an O-linked side chain of a Pir-CWP, possibly present in the latter's internal repeats.
Figure 3. Hypothetical linkage between a Pir-CWP and a β1,3-glucan polymer. According to this scheme a β1,3-glucan molecule is linked with its reducing end to an oligomannoside, O-linked to a serine or threonine residue of a Pir-CWP. The glycopeptide linkage is alkali-sensitive.
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Some GPI-CWPs such as Cwp1 may also be bound directly to β1,3-glucan through an alkali-sensitive bond presumably in a Pir-CWP-like fashion . As a result, two additional GPI-CWP–polysaccharide complexes can be distinguished, which are either single- or double-linked (Fig. 2B,C) . Importantly, Cwp1 and possibly also other potentially double-linked GPI-CWPs play an important role in the response of cells towards cell wall stress [113,116]. Finally, a fifth complex has been identified in which a GPI-CWP is linked to a β1,6-glucan molecule, which is directly interconnected to chitin (Fig. 2E). Although this type of complex is relatively rare in cells growing on rich medium, under special conditions it can become much more prominent (see Section 3.4 and Table 3) . Interestingly, chitin in the lateral walls, which represents ∼10% of the total chitin, seems to be specifically associated with β1,6-glucan . This may mean that chitin in the lateral walls is predominantly present in the form of the complex GPI-CWP–β1,6-glucan–chitin. Finally, chitin is also directly linked to β1,3-glucan . Chitin is, however, omitted from the first four complexes shown in Fig. 2, because (i) such complexes may exist without a chitin molecule attached to it, for example, in the growing bud, and (ii) a chitin molecule may become linked to another β1,3-glucan molecule than the one shown in the various cell wall protein–polysaccharide complexes, i.e. the linkage between β1,6-glucan and chitin may be quite indirect.
Table 3. Changes in cell wall composition and organization in response to genetically caused cell wall defects
|Less β1,3-glucan||More chitin in the lateral walls||[10,73,116,130,131]|
| ||More GPI-CWP-β1,6-glucan-chitin|| |
|Less β1,6-glucan||More GPI-CWPs in the medium||[65,84]|
| ||More Pir-CWPs in the wall|| |
| ||More β1,3-glucan and chitin in the wall|| |
| ||Possibly, increased usage of the alkali-sensitive linkage between GPI-CWPs and β1,3-glucan|| |
|Defective N- and O-glycosylation||More chitin in the lateral walls||[2,132–135]|
As the synthesis of chitin and β1,3-glucan takes place at the plasma membrane [54,68] and the coupling of GPI-CWPs to β1,6-glucan also occurs outside the plasma membrane [12,84], the interconnections between the cell wall macromolecules also have to be made outside the plasma membrane. This strongly points to the existence of various classes of cell wall assembly enzymes – presumably transglycosylases – responsible for interconnecting (a) GPI-CWPs to β1,6-glucan, (b) β1,6-glucan to β1,3-glucan, (c) chitin to β1,3-glucan, (d) chitin to β1,6-glucan, and (e) cell wall proteins directly to β1,3-glucan through an alkali-sensitive linkage. Interestingly, cells growing in the presence of gentiobiose, which consists of two β1,6-linked glucose monomers, incorporate a GPI-CWP reporter protein less efficiently into the cell wall than wild-type cells, and this is accompanied by increased sensitivity to Zymolyase . This suggests that gentiobiose inhibits a cell wall assembly step involving β1,6-glucan. At present, none of the postulated assembly enzymes has been identified. Also, the sequence of coupling events is largely unknown (see Section 2.7).
2.7Molecular organization of the cell wall
The cell wall forms a bilayered, supramolecular structure that surrounds the entire cell (Fig. 4). Its mechanical strength is largely based on an internal layer of moderately branched β1,3-glucan molecules that form a three-dimensional network that is kept together by hydrogen bonding between laterally associated chains . Methylation analysis has revealed that each β1,3-glucan polymer has multiple side chains . Their terminal non-reducing ends are believed to function as acceptor sites for β1,6-glucan and chitin [70,71], whereas the reducing end of the β1,3-glucan molecules may also be involved in the linkage to Pir-CWPs (see Section 2.6) . Methylation analysis of mature β1,6-glucan has shown that it is a highly branched molecule , which explains why it is water-soluble [15,40]. Chitin is deposited in the lateral walls after cytokinesis has taken place, probably stiffening the cell wall . It represents less than 10% of the total chitin content of the walls and may be coupled to either β1,3-glucan or β1,6-glucan [70,71].
The biosynthetic pathway of the sexual agglutinin Sag1 has been studied in detail and several intermediate glycoforms have been identified including two extracellular glycoforms, namely, a plasma membrane-bound form and a later, soluble form [84,98]. In contrast to the mature wall-bound form, both extracellular intermediates are not yet linked to β1,6-glucan. This suggests that the formation of the GPI-CWP–β1,6-glucan–β1,3-glucan complex proceeds as follows: (i) β1,6-glucan is connected to β1,3-glucan, and (ii) a GPI-CWP is coupled to β1,6-glucan. Nothing is known about how the formation of other CWP–polysaccharide complexes proceeds.
GPI-CWPs, Pir-CWPs, as well as disulfide-linked cell wall proteins all have been successfully used to target fusion proteins to the cell wall for covalent attachment [27,83,85,138–145].
Finally, there is clear evidence for limited release of cell wall proteins into the medium , possibly as a result of remodeling of the cell wall during isotropic growth or when a new bud is formed.
2.8Predictive value of the cell wall model for ascomycotinous yeasts
There is strong evidence that the cell wall of Candida albicans is similarly organized as the wall of S. cerevisiae. The model presented here seems also valid for other Candida species. For example, in Candida glabrata an authentic GPI-CWP involved in adhesion to human epithelial cells has been identified that, after expression in S. cerevisiae cells, permits the latter to efficiently adhere to epithelial cells . Pir-CWPs have been identified in C. albicans[94,148], Kluyveromyces lactis, and in Zygosaccharomyces rouxii. The cell wall model presented here is only partially valid for the fission yeast Schizosaccharomyces pombe. First, unlike S. cerevisiae, the wall of S. pombe contains a considerable amount of α1,3-glucan. Second, Southern analysis did not reveal PIR-like genes in S. pombe. For a more detailed discussion of the cell wall of mycelial fungi from the Ascomycotina, see [42,149].
2.9Identification of cell wall mutants
De Groot and co-workers have shown that cell wall construction in S. cerevisiae is a tightly regulated process and that about 1200 genes directly or indirectly affect formation of the cell wall . Using a hierarchical screening approach, the gene products can be divided in various classes such as proteins involved in the synthesis of cell wall macromolecules, proteins involved in remodeling or interconnecting cell wall polymers and proteins involved in the regulation of cell wall construction. Primary screens for the detection of mutants with an impaired cell wall are often based on increased sensitivity towards compounds that are known to interfere – either directly or indirectly – with normal cell wall construction, such as β1,3-glucanase [151,152], Calcofluor white [150–152] and the related compound Congo red [150,153], SDS, and caffeine . These compounds have in common that their deleterious effect on growth can be alleviated by osmotically stabilizing the medium [23,150,154,155]. Increased sensitivity to sonication [150,156] and hypotonic shock [157,158] has also been used to detect mutants with a cell wall defect. 2-Deoxyglucose, which is incorporated in the cell wall and causes cell lysis in growing areas, may presumably also be effectively used to identify cell wall mutants [159,160]. Alternatively, the culture filtrate may be screened for increased amounts of cell wall proteins or for incomplete CWP–polysaccharide complexes indicative of defective cell wall assembly [84,150]. In general, cell wall perturbants appear to be more effective at 37°C than at 30°C, possibly because of the combined effect of increased turgor pressure on the wall and of defective cell wall construction due to thermal inactivation of cell wall assembly enzymes at 37°C (see Section 3.3).
Hypoglycosylation mutants represent a special category of cell wall-related mutants. They are often more resistant to orthovanadate and hypersensitive to hygromycin, which facilitates their identification . Defects in O-glycosylation can be easily detected using the heavily O-glycosylated endochitinase Cts1 as a marker protein , whereas invertase is a useful marker for defects in N-glycosylation . For a more detailed analysis of O- and N-glycosylation, FACE (fluorophore-assisted carbohydrate electrophoresis) is useful .
Mutants with an endocytotic block show a rare cell wall-related phenotype, namely, the mother cells of such mutants form multiple cell wall layers .