The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.

The yeast cell wall contains ␤1,3-glucanase-extractable and ␤1,3-glucanase-resistant mannoproteins. The ␤1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a ␤1,6-glucan moiety, which in its turn is linked to ␤1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The ␤1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of ␤1,6-glucosylated proteins, including Cwp1p, as the ␤1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the ␤1,3-glucanase-resistant fraction through a ␤1,6-glucan moiety. In wild-type cell walls, the ␤1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less ␤1,3-glucan but more chitin, ␤1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased crosslinking of cell wall proteins via ␤1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.The cell wall is crucial for the integrity of Saccharomyces cerevisiae. Its rigid structure maintains the shape of the cell and offers protection against harmful environmental conditions (6, 19). The wall is mainly composed of ␤-glucans and mannoproteins, in addition to smaller amounts of chitin and lipids (6). The glucans, which are interwoven with the chitin fibrils, form the inner skeletal layer of the cell wall, whereas the outer layer consists of mannoproteins. The majority of the cell wall mannoproteins are anchored into the wall through covalent linkages to heteropolymers of ␤1,6-and ␤1,3-glucan (18, 26, 41, 43) (Fig. 1). The ␤1,6-glucosyl moiety of these polymers is phosphodiester-linked to protein as shown by its sensitivity to treatment with ice-cold aqueous hydrofluoric acid (HF) and phosphodiesterases (18). This observation, together with data from other studies, pointed to a glycosylphosphatidylinositol (GPI)-derived structure as the attachment site for ␤1, 6-glucan (3, 23, 40, 42, 45). The ␤1,3/␤1,6-glucan heteropolymer was proposed to be identical to the alkali-soluble ␤1,3/␤1,6-glucan studied by Fleet and Manners (7,8). In S. cerevisiae, this alkali-soluble glucan becomes alkali insoluble through a linkage with chitin (11, 24) via a ␤1,4 bond from the terminal reducing residue of chitin to the nonreducing end of ␤1,3-glucan (20). Furthermore, by digesting cell walls with ␤1,3-glucanase, followed by incubation with exochitinase, a heterogeneous high-molecular-weight complex was isolated (21). Structural studies indicated that in this complex the terminal reducing residue of chitin is linked to ␤1,6-glucan (21). The nonreducing end of the ␤1,6-glucan polymer is bound to the GPI-derived glycan part of a cell wall protein, whereas its reducing terminus is linked to ␤1,3-glucan (...

linkage region between chitin and ␤(133)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with ␤(133)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, ␤(133)-glucan, ␤(136)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to ␤(136)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five ␣-linked mannosyl residues. The ␤(136)-glucan has some ␤(133)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a ␤(134) or ␤(132) linkage. Finally, the reducing end of ␤(136)-glucan is connected to the nonreducing terminal glucose of ␤(133)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and ␤(136)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.Cell walls are essential for the survival of fungal cells. Digestion of cell walls in the absence of an osmotic protector leads to cell lysis due to the high internal turgor pressure. Thus, substances that interfere with cell wall synthesis may be considered as potential antifungal agents (1). Because of its rigidity, the cell wall determines the shape of fungal cells. For that reason, cell wall formation has been used as a model for morphogenesis (1).The major components of fungal cell walls are polysaccharides and glycoproteins (2). In the yeast, Saccharomyces cerevisiae, the cell wall contains ␤(133)-D-glucan, ␤(136)-D-glucan, chitin, and mannoprotein(s) (3). The polysaccharides appear to have a structural function, whereas the mannoprotein(s) may act as "filler" and are important for the permeability of the cell wall (4, 5). How can one explain the strength and resilience of the fungal cell wall? Recent results with S. cerevisiae suggest that the answer may be found in the existence of covalent linkages between the different components of the wall that would give rise to a continuous and consequently stronger fabric. Thus, previous studies in our laboratories showed the presence of linkages between chitin and ␤(133)-glucan (6) as well as among glycoproteins, ␤(136)-glucan, and ␤(133)-glucan (7).The strategy for the investigation of interconnections between chitin and ␤(133)-glucan consisted in the digestion of cell walls with ␤(133)-glucanase, followed by labeling of the exposed reducing ends with borotritide and enzymati...

To isolate the putative linkage region between chitin and beta(1-->3)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(1-->3)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(1-->4) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derived from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(1-->3)-glucan chain by a beta(1-->4) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.

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