Cell-cell contact during mating in the yeast Saccharomyces cerevisiae is mediated by cell surface adhesion glycoproteins, called a- and alpha- agglutinin, which are expressed by cells of a and alpha mating type, respectively. Alpha-agglutinin is predicted to have a structure similar to the immunoglobulin superfamily of mammalian cell adhesion proteins, suggesting that elucidation of adhesion protein interactions in yeast will have implications for higher eukaryotes. An important goal of the proposed research is to characterize the adhesion protein interactions. Mutagenesis of the alpha-agglutinin adhesion domain, based on information provided by a structural model, is being used to identify residues involved in adhesion and in the structure of the protein. This approach will be extended to the alpha-agglutinin adhesion subunit. The specificity of adhesion protein interactions in higher eukaryotes is critical for specific viral-cell, cell-cell, and cell-extracellular matrix interactions. Other species of budding yeast express agglutinins that share biochemical features with the S. cerevisiae agglutinins; however agglutinin interactions show species specificity. The cloning of agglutinin structural genes from the yeast S. kluyveri will facilitate structural modeling of the adhesion proteins and studies of species specificity. Hybrid S. cerevisiae/S. kluyveri genes will be constructed and tested for function and specificity, and the genes will be mutagenized and screened for change-of-specificity mutants to identify residues involved in specificity. The final goal of the proposed research involves an analysis of the mechanism of cell wall localization of the agglutinins. Alpha-agglutinin is transported to the plasma membrane through linkage to a glycosyl phosphatidylinositol anchor, after which a novel mechanism acts to transfer it to the cell wall. The nature of the alpha-agglutinin-cell wall linkage will be characterized, and the alpha-agglutinin sequences involved in this targeting mechanism will be identified. In addition, mutant screens will be undertaken to identify enzymes involved in this novel mechanism. This mechanism of cell wall attachment of alpha- agglutinin is implicated in general cell wall mannoprotein localization. Because cell wall structure in other fungi is similar to yeast, investigation of the mechanism of cell wall biogenesis in S. cerevisiae may lead to the development of therapeutic agents against medically important fungi.
