Identification of a mannoprotein present in the inner layer of the cell wall of Saccharomyces cerevisiae

Abstract

Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein-which we have named Icwp-in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin. The screening of a gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp. ICWP (EMBL accession number YLR391w, frame ؉3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal. Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan.The cell wall of Saccharomyces cerevisiae is made up of three components, namely, glucans, mannoproteins, and chitin, and represents some 20% of the dry weight of the cell. It consists of a layered structure, with an internal layer made up of ␤-1,3 and ␤-1,6 glucans, small amounts of chitin and mannoproteins, and an outer layer of mannoproteins (13, 23). The inner layer is responsible for the shape and mechanical strength of the wall (19,24,50), while the outer mannoprotein layer determines the surface properties of the cell, such as hydrophobicity, electrical charge, flocculence, and sexual agglutinability, as well as limiting the porosity of the cell wall (8-10, 50).The mannoproteins can be divided into three groups according to the methods used for their extraction from the cell wall: sodium dodecyl sulfate (SDS)-extractable mannoproteins (44), glucanase-extractable mannoproteins, which can be released only after glucanase digestion of the glucan layer (31,44,47), and mannoproteins extractable by reducing agents (35). The glucanase-extractable mannoproteins identified so far have two common characteristics: one is a high serine/threonine content (up to 50% of the C-terminal half of the protein), and the other is the presence of a putative glycosylphosphatidylinositol (GPI) attachment site (23,46). Several of the proteins characterized so far, such as the sexual ␣-agglutinin (27, 29, 30), the anchor subunit of the a-agglutinin (38), and the flocculin encoded by the FLO1 gene, play a role on the surface of the cell (42). In these proteins, the highly O-glycosylated Cterminal half may endow them with a rod-like structure (22) that facilitates the exposure of their ...

Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein

“…The specific growth rates of the yeast form of the parental strain C. albicans CAI4 and the Ura 2 strains 30541-2 and 30542-2 were similar and no differences in morphology were observed, as in S. cerevisiae ssr1 (Moukadiri et al, 1997). No differences in the kinetics of germ tube formation were observed between the mutants and the parental strain.…”

“…1C). All described features are common to other cell-wall proteins found in other fungal species (Lipke et al, 1989;Lu et al, 1994;Moukadiri et al, 1997;Roy et al, 1991;Teunissen et al, 1993;van der Vaart et al, 1995). The difference between the predicted size of CaSsr1p (22?5 kDa) and that deduced from the mobility in SDS-PAGE (70 kDa) could be accounted for by Oglycosylation and GPI modification (Fig.…”

“…More than 100 ORFs were identified as putative cell-wall proteins. A similar analysis has been performed by P. From the ORFs analysed, we selected one (IPF 3054= CaSSR1) that presents a potential GPI region and whose product has high homology (62 %) with S. cerevisiae Ssr1p, an internal cell-wall protein (Moukadiri et al, 1997) (Fig. 1A).…”

“…Proteins of the Pir family can also be extracted from the cell wall by a mild NaOH treatment (Kapteyn et al, 1999;Mrsa et al, 1997). The presence of a GPI anchor has been demonstrated in cell-wall proteins of different fungal species (Frieman et al, 2002;Moukadiri et al, 1997;Staab et al, 1999;Wojciechowicz et al, 1993). The presence of the GPI anchor seems to play an important role in the biology of C. albicans, as mutants are affected in morphogenesis, virulence and cell-wall composition (Richard et al, 2002).…”

“…From all the ORFs identified as potential cell-wall proteins, we selected IPF 3054 for further study. This ORF has a putative GPI-anchor sequence, is rich in Ser/Thr and has a high homology to the Saccharomyces cerevisiae Ssr1p cell-wall protein, which was cloned and studied by our group (Moukadiri et al, 1997). …”