PHOSPHORYLATION OF THE PHEROMONE-RESPONSIVE G(BETA) PROTEIN OF SACCHAROMYCES-CEREVISIAE DOES NOT AFFECT ITS MATING-SPECIFIC SIGNALING FUNCTION

The pheromone-responsive G(beta) subunit of Saccharomyces cerevisiae ( encoded by STE4) is rapidly phosphorylated at multiple sites when yeas t cells are exposed to mating pheromone. It has been shown that a muta nt form of Ste4 lacking residues 310-346, ste4 Delta 310-346, cannot b e phosphorylated: and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was propo sed that phosphorylation of Ste4, is associated with an adaptive respo nse to marine pheromone. In this study we used site-directed mutagenes is to create two phosphorylation null (Pho(-)) alleles of STE4: ste4-T 320A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4, remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on e ither signaling or adaptation. In addition, disruption of the FUS3 gen e, which encodes a pheromone-specific MAP kinase, leads to partial los s of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis sugge sts that the ste4 Delta 310-346 deletion mutant is impaired in its int eraction with Gpa1, the pheromone-responsive G(alpha) of yeast, wherea s the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken toge ther, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhib ited by the 310-346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.