MUTATION OF RGA1, WHICH ENCODES A PUTATIVE GTPASE-ACTIVATING PROTEIN FOR THE POLARITY-ESTABLISHMENT PROTEIN CDC42P, ACTIVATES THE PHEROMONE-RESPONSE PATHWAY IN THE YEAST SACCHAROMYCES-CEREVISIAE

We have selected yeast mutants that exhibit a constitutively active ph eromone-response pathway in the absence of the beta subunit of the tri meric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which con tributed to the constitutive phenotype. One mutation identifies the RG A1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is suffi cient to activate the pathway in strains lacking the G beta subunit. M oreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signal ing, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the origin al mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbse mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This fin ding coupled with recent experiments with mammalian and yeast cells in dicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest tha t Rga1p controls the activity of Cdc42p, which in turn controls the ma gnitude of signaling in the pheromone pathway via Ste20p.