RESUMO
Saccharomyces cerevisiae respond to mating pheromone through the GPCRs Ste2 and Ste3, which promote growth of a mating projection in response to ligand binding. This commitment to mating is nutritionally and energetically taxing, and so we hypothesized that the cell may suppress mating signaling during starvation. We set out to investigate negative regulators of the mating pathway in nutritionally depleted environments. Here, we report that nutrient deprivation led to loss of Ste2 from the plasma membrane. Recapitulating this effect with nitrogen starvation led us to hypothesize that it was due to TORC1 signaling. Rapamycin inhibition of TORC1 impacted membrane levels of all yeast GPCRs. Inhibition of TORC1 also dampened mating pathway output. Deletion analysis revealed that TORC1 repression leads to α-arrestin-directed CME through TORC2-Ypk1 signaling. We then set out to determine whether major downstream effectors of the TOR complexes also downregulate pathway output during mating. We found that autophagy contributes to pathway downregulation through analysis of strains lacking ATG8 . We also show that Ypk1 significantly reduced pathway output. Thus, both autophagy machinery and TORC2-Ypk1 signaling serve as attenuators of pheromone signaling during mating. Altogether, we demonstrate that the stress-responsive TOR complexes coordinate GPCR endocytosis and reduce the magnitude of pheromone signaling, in ligand-independent and ligand-dependent contexts. One Sentence Summary: TOR signaling regulates the localization of all Saccharomyces cerevisiae GPCRs during starvation and suppress the mating pathway in the presence and absence of ligand.
RESUMO
In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.