6533b86dfe1ef96bd12ca93b

RESEARCH PRODUCT

Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents

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The cell wall of Saccharomyces cerevisiae represents some 30% of the total weight of the cell and is made up of β-glucans, mannose-containing glycoproteins (mannoproteins), and small amounts of chitin (9, 15). The mannoproteins can be divided into three groups according to the linkages that bind them to the structure of the cell wall: (i) noncovalently bound, (ii) covalently bound to the structural glucan, and (iii) disulfide bound to other proteins that are themselves covalently bound to the structural glucan of the cell wall (8). Our work has focused on the disulfide-bound mannoproteins, probably the least well known of the three groups mentioned above. Previous work (25) showed that treatment of whole yeast cells with a reducing agent releases four mannoproteins, with molecular masses of 38, 49, 68, and 88 kDa, and a highly polydisperse high-molecular-weight material. Additionally, in the case of cells of sexual mating type a previously treated with α-factor, treatment with a reducing agent releases an O-glycosylated 22-kDa mannoprotein (25). This 22-kDa mannoprotein is one of the subunits of the a-agglutinin and is coded by the AGA2 gene (6). Extraction of whole cells with reducing agents can cause the release of proteins that are not part of the cell wall, and for this reason we chose to use purified cell walls, previously extracted with hot sodium dodecyl sulfate (SDS), as starting material for the β-mercaptoethanol extraction. Also, extraction of the wild-type strain releases mannoproteins that are polydisperse when run in SDS-polyacrylamide gel electrophoresis (PAGE). This polydispersity is due to the fact that some of them are highly glycosylated proteins and thus very difficult to characterize. To minimize this problem, we used both the wild-type strain and an mnn1 mnn9 strain deficient in glycosylation (2, 11). The results presented in this work consist of the characterization of two mannoproteins released from the purified cell walls of the mnn1 mnn9 strain by β-mercaptoethanol.