Glycoprotein-glycoprotein interactions play essential roles in viral-cell, cell-cell, and cell-extracellular matrix interactions in higher eukaryotes.
The aim of the proposed research is to use a system in which genetic and molecular approaches are possible, i.e. Saccharomyces cerevisiae and other yeasts, to study glycoprotein-glycoprotein mediated cell-cell interactions. Haploid cells of opposite mating types (a and alpha) of S. cerevisiae mate with one another to form the a/alpha diploid. Cell-surface glycoproteins, called a- and alpha-agglutinins, mediate aggregation between a and alpha cells. Mutants that show a- or alpha-specific agglutination defects have been isolated and used to clone and characterize the alpha-agglutinin structural gene and the structural gene for one of two a-agglutinin subunits, the core or cell-surface attachment domain. Other mutants will be used to isolate the second a-agglutinin structural gene, which encodes the binding fragment. The primary function of the agglutinins is to interact with one another to mediate cell-cell interactions. One of our primary goals, therefore, is to identify and characterize the regions of the agglutinins that are involved in this interaction. Using truncation and deletion mutants, we are in progress of localizing the binding domain of alpha-agglutinin; similar experiments will be used for the a-agglutinin. After determination of the smallest region of the agglutinins sufficient for binding to the opposite agglutinin, mutagenesis of these regions will be done to determine particular amino acids and features critical for binding. Further investigation of the interactions between the two agglutinins will involve the isolation of suppressor mutants, i.e. mutations in one agglutinin that allow an interaction with a mutant agglutinin of the opposite type. A feature of both the alpha-agglutinin and a-agglutinin core structural genes suggests that glycosyl phosphatidylinositol anchors may be involved in cell-surface localization and attachment. Experiments will be done to test this hypothesis. Experiments aimed at investigating the species-specificity of agglutinin interactions will involve the isolation of agglutinin genes from other yeasts, comparison of these genes to the S. cerevisiae genes, and construction and analysis of hybrid genes. These studies will provide a detailed characterization of glycoprotein-glycoprotein interactions that mediate interactions between particular cell types.
