Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their Nterminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a ␤1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2⌬. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.The two major components of the cell wall of the yeast Saccharomyces cerevisiae are glucan, which constitutes the inner layer of the cell wall, and mannoproteins, which are embedded in and cover this glucan layer. Chitin is a minor component of the cell wall (13, 18). The mannoproteins can be divided into two groups, the sodium dodecyl sulfate (SDS)-extractable mannoproteins and the glucanase-extractable mannoproteins, which are solubilized by glucanase digestion of the glucan layer. Several glucanase-extractable mannoproteins have been identified. These proteins have two characteristics in common: their C-terminal regions are rich in serine and threonine, and they all contain putative glycosylphosphatidylinositol (GPI) attachment signals. Two of these proteins, ␣-agglutinin (22) and the core subunit of a-agglutinin (33), are involved in mating. The third, which is involved in flocculation, is the product of the FLOI gene (42). Because of the high serine and threonine content of their C-terminal regions, these proteins are probably heavily O glycosylated, which could give the protein a rod-like structure (15). The presence of a GPI anchor has been demonstrated only for the intracellular precursor form of ␣-agglutinin (47). The glucanase-extractable mannoproteins are proposed to be covalently linked to glucan (29,38,45). Several groups have investigated which part of the protein is responsible for anchoring the protein...

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag␣1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing ␣-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag␣1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of ␣-galactosidase-Ag␣1p was sixfold lower than that of ␣-galactosidase-Cwp2p and eightfold lower than that of ␣-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of ␣-galactosidase-Ag␣1p detectable. A lower translation efficiency of the ␣-galactosidase-AG␣1 fusion construct is most likely to be responsible for the low level of protein production. ␣-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.

SummaryWe constructed hybrid proteins containing a plant ␣-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a ␤1,6-glucose-containing sidechain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the ␣-galactosidase-Srp1 fusion proteins, an ␣-galactosidase-Sed1 hybrid protein and an ␣-galactosidase-␣-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an ␣-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of ␤1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.

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The incorporation of mannoproteins in the cell wall of S. cerevisiae and filamentous Ascomycetes Brul, S.; King, A.; van der Vaart, J.M.; Chapman, J.W.; Klis, F.M.; Verrips, C.T. Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Key words: wall, mannoproteins, GPI-anchor, 1,6-glucan, PLC, yeast, fungi AbstractIn yeast, glucanase extractable cell wall proteins are anchored to the plasma membrane at an intermediate stage in their biogenesis via a glycosylphosphatidylinositol (GPI) moiety before they become anchored to the wall glucan via a 1,6-glucan linkage. The mechanism of the membrane processing step of cell wall proteins is not known.Here, we report that Ascomycete filamentous fungi involved in food spoilage such as Aspergillus, Paecilomyces and Penicillium, also contain GPI membrane-anchored proteins some of which are processed by an endogenous phospholipase C activity. Furthermore, similar to the situation in yeast, their cell walls contain mannoproteins which are linked to the glucan backbone through a 1,6-glucan linkage. Interestingly, one mould which contains a significant amount of non covalently linked 1,6-glucosylated cell wall proteins, is much more sensitive towards 1,3-glucanases and membrane perturbing peptides than the others.

Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1, 6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a beta-1, 6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the beta-1, 6-glucan epitope was not linked to the protein part. We conclude that neither N-or O-glycosylation is involved in beta-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable beta-1, 6-glucan attachment site.