Delineation of functional regions within the subunits of the Saccharomycescerevisiae cell adhesion molecule a-agglutinin

a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an ancho rage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachme nt of Agap is mediated through two disulfide bonds to Aga1p (Cappellaro, C. , Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha -agglutinin, Aga2p co-expressed wi th a 149-residue fragment of Aga1p formed a disulfide-linked complex with s pecific activity 43-fold higher than Aga2p expressed alone. Circular dichro ism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alo ne had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragmen t was partially active in stabilization of Aga2p activity. Mutation of eith er or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA 2 identified only C-terminal residues of Aga2p as being essential for bindi ng activity. Aga2p residues 45-72 are similar to sequences in soybean Nod g enes, and include residues implicated in interactions with both Aga1p (incl uding Cys(68)) and alpha -agglutinin.