The STE2 gene product is the ligand-binding component of the α-factor receptor of Saccharomyces cerevisiae

The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for α-factor in MATa haploids. Methods were devised to prepare membrane fractions from MATa cells that retained high levels of α-factor binding activity, consistent with the view that the α-factor receptor resides in the plasma membrane. To demonstrate that the membrane constituent responsible for α-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis. Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000. Affinity labeling of yeast membrane preparations by chemical cross-linking to ³⁵S-α-factor indicated that a molecule of 49,000 molecular weight was the major α-factor-binding species. This α-factor-binding species was shown to be the product of the STE2 gene in three ways. First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced α-factor binding activity about 15-fold. Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of α-factor (equivalent to the level displayed by MATα haploids) and possessed no species that could be cross-linked to ³⁵S-α-factor. Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to ³⁵S-α-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide. These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast α-factor receptor.