The mating-specific G sub( alpha ) protein of Saccharomyces cerevisiae downregulates the mating signal by a mechanism that is dependent on pheromone and independent of G sub( beta gamma ) sequestration

It has been inferred from compelling genetic evidence that the pheromone-responsive G sub( alpha ) protein of Saccharomyces cerevisiae, Gpa1, directly inhibits the mating signal by binding to its own beta gamma subunit. Gpa1 has also been implicated in a distinct but as yet uncharacterized negative regulatory mechanism. We have used three mutant alleles of GPA1, each of which confers resistance to otherwise lethal doses of pheromone, to explore this possibility. Our results indicate that although the G322E allele of GPA1 completely blocks the pheromone response, the E364K allele promotes recovery from pheromone treatment rather than insensitivity to it. This observation suggests that Gpa1, like other G sub( alpha ), proteins, interacts with an effector molecule and stimulates a positive signal-in this case, an adaptive signal. Moreover, the Gpa1-mediated adaptive signal is itself induced by pheromone, is delayed relative to the mating signal, and does not involve sequestration of G sub( beta gamma ). The behavior of N388D, a mutant form of Gpa1 predicted to be activated, strongly supports these conclusions. Although N388D cannot sequester beta gamma , as evidenced by two-hybrid analysis and its inability to complement a gpa1 null allele under normal growth conditions, it can stimulate adaptation and rescue a gpa1 Delta strain when cells are exposed to pheromone. Considered as a whole, our data suggest that the pheromone-responsive heterotrimeric G protein of S. cerevisiae has a self-regulatory signaling function. Upon activation, the heterotrimer dissociates into its two subunits, one of which stimulates the pheromone response, while the other slowly induces a negative regulatory mechanism that ultimately shuts off the mating signal downstream of the receptor.