The Mating-Specific G_α Protein of Saccharomyces cerevisiae Downregulates the Mating Signal by a Mechanism That Is Dependent on Pheromone and Independent of G_βγ Sequestration

Abstract

It has been inferred from compelling genetic evidence that the pheromone-responsive G ␣ protein of Saccharomyces cerevisiae, Gpa1, directly inhibits the mating signal by binding to its own ␤␥ 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 ␣ 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 ␤␥ . The behavior of N388D, a mutant form of Gpa1 predicted to be activated, strongly supports these conclusions. Although N388D cannot sequester ␤␥, 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⌬ 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.In the life cycle of the budding yeast Saccharomyces cerevisiae, two haploid cells of opposite mating type, MATa and MAT␣, can conjugate to form a MATa/␣ diploid cell. Each mating type constitutively secretes a peptide mating pheromone that activates surface receptors on cells of the opposite type. Binding of pheromone to receptor triggers a genetic program that transforms vegetatively growing cells into gametes. In preparation for cell and nuclear fusion, cells stimulated by pheromone induce mating-specific genes, arrest in the G 1 phase of the cell cycle, reorganize their cytoskeletons, and elongate toward their mating partners. The molecular mechanisms by which pheromone elicits these responses have been studied by numerous laboratories, and the yeast pheromone response is currently one of the best-understood eukaryotic signaling pathways (for reviews, see references 27 and 44). Moreover, it has gradually become apparent that the pheromone signal of S. cerevisiae is propagated by many of the same types of signaling elements found in higher eukaryotes. The pheromone receptors, for example, belong to the seven-transmembrane-domain class of receptors and, as such, are coupled to a heterotrimeric G protein. When occupied by ligand, these receptors are thought to activate the pheromone-responsive protein G ␣ (encoded...

Effect of the Pheromone-Responsive Gα and Phosphatase Proteins of Saccharomyces cerevisiae on the Subcellular Localization of the Fus3 Mitogen-Activated Protein Kinase

“…Strip-purified antiserum raised against Gpa1 (62) and crude antiserum raised against Kap104 (generously provided by John Aitchison) were used as the primary antibodies.…”

“…In all cases, transformants resulting from single-copy integration were identified by Southern blot. To construct strain TBY163, the msg5⌬ ptp3⌬ strain (HKY161) was switched to MAT␣ by induction of a GAL1-HO plasmid and crossed to strain DSY253 (62), which contains two copies of GAL1…”

“…We are studying adaptive mechanisms that are stimulated by the pheromone-responsive G ␣ protein, Gpa1 (36,62,69). Recently, we have shown that Gpa1 binds directly to the matingspecific MAPK, Fus3 (44).…”

Sequence elements that contribute to the degradation of yeast Gα

“…G␣ has several roles related to pheromone response including activation of the signalling pathway, control of pheromone sensitivity and adaptation from prolonged pheromone exposure (Sprague & Thorner 1992;Stratton et al 1996). To understand the significance of G␣ degradation, we examined the stability of well-defined mutants, and the effects of various in vivo conditions.…”

“…Sst2 is important for adaptation and is predicted to function as a GTPaseactivating protein (GAP) (Chan & Otte 1982;Dohlman et al 1996). In addition, Sst2 may regulate the ability of G␣ to stimulate adaptation in a G␤␥-independent manner (Dohlman et al 1996;Stratton et al 1996).…”