No abstract

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

SummaryLow environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to b1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to b1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with b1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to b1,3-glucan and the alkali-resistant GPI-derived linkage to b1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the b1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to b1,3-glucan was induced by low pH. The low pHinduced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

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 (...