Identification of glycoprotein components of alpha-agglutinin, a cell adhesion protein from Saccharomyces cerevisiae

Abstract

Several glycoproteins which inhibit the agglutinability of Saccharomyces cerevisiae mating type a cells were partially purified from extracts of mating type a cells. These proteins, called a-agglutinin, were labeled with 1251-Bolton-Hunter reagent. The labeled oi-agglutinin showed specific binding to a cells. Such specific binding approached saturation with respect to agglutinin or cells and was inhibited in the presence of excess unlabeled a-agglutinin. Nonspecific binding was similar in a and d cells, was neither saturable nor competable, and was three-to fourfold less than the specific binding to a cells at maximum tested agglutinin concentrations. The major a-specific binding species had a low electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and had an apparent molecular weight of 155,000 by rate zonal centrifugation. Endo-N-acetylglucosaminidase H digestion of the purified glycoprotein complex converted the low-mobility material to four maijor and several minor bands which were resolved by polyacrylamide gel electrophoresis. All but two minor peptides bound specifically to a cells. Analyses of agglutinin from mnn mutants confirmed the deglycosylation results in suggesting that the N-linked carbohydrate portion of a-agglutinin was not necessary for activity. Gl (8,27), and a pronouhced change in cell morphology (3, 7).Sexual agglutination provides an excellent model system for the study of eucaryotic cell interactions and their regulation at the molecular level. The agglutination reaction has been characterized in yeasts of the genus Hansenula and found to be the result of the interaction of a pair of complementary glycoproteins, one from each mating type (5,23,24). More recently, similar molecules have been isolated from Saccharomyces kluyveri (18) and Saccharomyces cerevisiae (12, 17, 26, 28, 29, 31). These studies revealed certain common elements in sexual agglutination in yeasts. Ih each species except S. cerevisiae the agglutination molecule (agglutinin) from the cells corresponding to the a mating type has been fdund to be relatively heat stable and sensitive to reducing agents and to contain a higher proportion of carbohydrate than the corresponding molecule from the opposite mating type. By contrast, the agglutinin from mating types analogous to a cells is heat labile and stable to reducing agents and contains a smaller proportion of carbohydrate (4). Most of the studies of agglutinins in S. cerevisiae, however, have used autoclaving to solubilize the molecules. Therefore the reported a-agglutinin glycoprotein must be heat stable

Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

“…Determination of the sites of glycosylation of domain III indicates that the area of the putative binding site contains little carbohydrate whereas a face on the opposite side of domain III is highly glycosylated (Figure 2). These results are consistent with experiments that indicate that carbohydrate does not affect binding activity (Terrance et al, 1987;Hauser and Tanner, 1989;Cappellaro et al, 1991).…”

Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

“…Binding between these complementary cell wall proteins mediates aggregation of cells during mating (for reviews of agglutinin structure and function, see references 26 and 50). The agglutinins facilitate mating under conditions that do not promote cell-cell contact (28,35,38 (5,16,40,44).Both agglutinins are transported to the cell surface through the secretory pathway. Consistent with this finding, all three agglutinin structural genes, AGao1, AGA1, and AGA2, initiate with prototypical signal sequences for proteins that are transported through the secretory pathway (5, 16,28,35,42,43 (14).…”

“…Binding between these complementary cell wall proteins mediates aggregation of cells during mating (for reviews of agglutinin structure and function, see references 26 and 50). The agglutinins facilitate mating under conditions that do not promote cell-cell contact (28,35,38 (5,16,40,44).…”

“…For cell extracts, the cells were pelleted and resuspended in 50 ml of 100 mM sodium acetate (pH 5.5)-0.03% Triton X-100-1 mM p-chloromercuribenzoate. The resuspended cells were disrupted with glass beads as described previously (40), using a Bead Beater. The cell mixture was centrifuged at 28,000 x g for 10 min, and the supernatant (crude cell extract) was harvested.…”

Developmental accumulation of lectin in Rhizoctonia solani: a potential role as a storage protein

“…Others have postulated that fungal lectins are involved in adhesion and recognition phenomena. In Saccharomyces cerevisiae the mating interaction is mediated by agglutinins (Terrance et al, 1987) whereas in lichens, fungal lectins are involved in the relationship between the fungal and algal components (Lockhart et al, 1978). Adhesion by lectins may also be the initial step in certain infection processes.…”