894 Q.Y. Yin et al. Fig. 3. MS identiﬁcation of the members of the Pir family. Only the C-terminal regions of the proteins are depicted. The numbers correspond to the positions of amino acid residues in the ORFs of each Pir protein. Identiﬁcation and quantitation cannot be realized on the basis of the tryptic peptides b and c, which occur in all members of the Pir family, or on a and d, which are shared by two members (highlighted). Pir1p–Pir4p contain a unique tryptic peptide (bold and underlined). A unique tryptic peptide from Pir5p or tryptic peptides from the region preceding the conserved four-cysteine (indicated by asterisks) domain have never been observed. Owing to trypsin miscleavage, peptide a appears in two forms: GGILTDGK and GGILTDGKGR. Peptides c and d are generated due to aspeciﬁc enzymatic activity of trypsin (chymotrypsin). of the ASL proteins tend to be lower than the corresponding transcript ratios. As these proteins, at least partially, may end up in the medium (Russo et al., 1992; Kapteyn et al., 2001), this raises the question of whether, in gas1D cells ASL proteins are less efﬁciently incorporated into the walls. Conversely, the protein ratios of Crh2p and Ecm33p are considerably higher than the corresponding transcript ratios. Gas1p, Gas3p and Gas5p are classiﬁed in glycoside hydrolase family 72, and are thought to be directly involved in b-1,3-glucan remodeling (Popolo & Vai, 1999; Mouyna et al., 2000). In gas1D cell walls, a considerable amount of b- 1,3-glucan is present (Ram et al., 1998). This raises the question of whether, in a gas1D mutant, another member of this family may compensate for the loss of Gas1p function in b-1,3-glucan remodeling. Our data suggest that Gas5p but not Gas3p may become more relevant, as in gas1D cells three times more Gas5p is incorporated into the cell wall. Our observation is in agreement with the results of Schuldiner et al. (2005), who found a genetic interaction between GAS1 and GAS5 by epistatic miniarray proﬁling. Interestingly, a search for suppressor genes of the synthetic lethality of a kex2Dgas1D mutant revealed that increased expression of GAS5 was able to rescue the mutant (Tomishige et al., 2005). A recent in vitro study also showed that Gas5p has b-1, 3-transglucosidase activity similar to Gas1p, whereas a similar activity could not be detected for Gas3p under the experimental conditions used (Ragni et al., 2007). Pst1p was here for the ﬁrst time identiﬁed as an authentic covalently linked cell wall protein, probably because its strong upregulation in gas1D cells allowed its identiﬁcation by MS. The increased level of Pst1p in the wall is also consistent with the increased mRNA ratio of 3.87 observed by Lagorce et al. (2003). The level of wall-bound Ecm33p, c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. which is a homolog of Pst1p, is also strongly increased in gas1D cells. This raises the question of whether this protein, which is normally mainly targeted to the plasma membrane, might now function in directly counteracting cell wall damage. Interestingly, similar to deletion of GAS1, deletion of ECM33 is known to result in strong hypersensitivity to the cell wall perturbant Calcoﬂuor White, swollen round cells, and an increased amount of b-1,6-glucosylated pro- teins being secreted into the growth medium (De Groot et al. 2001; Pardo et al., 2004), suggesting that their functions might be related. Crh1p and Crh2p belong to glycoside hydrolase family 16. Their role in cell wall biogenesis has been implicated in linking chitin to the b-1,6-glucan network (Rodrı´quez-Pen˜a et al., 2000; Cabib et al., 2007). In the gas1D cell wall, b-1, 3-glucan levels detected in the alkali-resistant fraction were reduced, although the chitin level was increased as com- pared to the wild type (Ram et al., 1998). The insolubility of b-1,3-glucan in alkali is due to its linkage to chitin. With the limited availability of b-1,3-glucan, there seems to be an increased demand for enzymes that can crosslink chitins to b-1,6-glucan (Kapteyn et al., 1997). This explanation is in agreement with our observation that the levels of both Crh1p and Crh2p have increased about fourfold. Acknowledgements We thank Richard Sprenger (Academic Medical Center, Clinical Proteomics Group) for his technical support. We acknowledge the ﬁnancial support of the Council for Medical Sciences of the Netherlands Organization for Scien- tiﬁc Research (NWO-ZonMW) in purchasing the Q-TOF mass spectrometer, and the Council for Chemical Sciences of The Netherlands Organization for Scientiﬁc Research FEMS Yeast Res 7 (2007) 887–896