Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment are asparagine and glycine.In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions.The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs. 1997 John Wiley & Sons, Ltd.

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...

Deletion of GAS1/GGP1/CWH52 results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

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A heat resistant mutant of E. coli dnaAts46 was isolated, which grows normally only at temperatures above 39 degrees. After a temperature shift from 42 degrees to 32 degrees the mutant overproduces DNA relative to protein. This is due to overinitiation of rounds of chromosome replication at low temperature, as indicated by hybridization and other experiments. The mutation is cotransduced by PI with ilv and could not be separated from dnaAts46 by transduction.