GENETIC IDENTIFICATION OF RESIDUES INVOLVED IN ASSOCIATION OF ALPHA AND BETA G-PROTEIN SUBUNITS

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma submits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W 136G, W136R, and Delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signal ing pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be de fective in association with the Gpa1 protein. A mutation at position E 307 of the Gpa1 protein both suppresses the constitutive signaling phe notype of some mutant Ste4 proteins and allows the mutant alpha subuni t to physically associate with a specific mutant G beta subunit. The m utation in the Gpa1 protein is adjacent to the hinge, or switch, regio n that is required for the conformational change which triggers subuni t dissociation, but the mutation does not affect the interaction of th e alpha subunit with the wild-type beta subunit. Yeast cells construct ed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromo ne response pathway. Because the ability of the modified G alpha subun it to suppress the Ste4 mutations is allele specific, it is likely tha t the residues defined by this analysis play a direct role in G-protei n subunit association.