Regulation of Yeast Gβγ Interactions and Chemotropism by Gα-Targeted Phosphorylation

Mating in the yeast Saccharomyces cerevisiae is chemotropic, cells respond by polarizing their growth in response to pheromone. Pheromone gradients are interpreted by surface displayed seven transmembrane receptors that are coupled to a heterotrimer of G-proteins. Receptor-pheromone binding induces the dissociation of the G-protein heterotrimer into the Gα and Gβγ subunits. We have previously shown that pheromone induces Gβ phosphorylation and that it is important for chemotropism. In this study, we mapped the sites of Gβ phosphorylation and determined that they lie in MAPK and casein kinase recognition motifs. Pull-down experiments using a phospho-site mutant of Gβ (GβP-) suggest that Gβ phosphorylation regulates the interaction between Gβγ and effectors such as Far1, the MAPK Fus3, and casein kinases Yck1/Yck2. These data suggest that Gβ is likely phosphorylated by the Fus3 and Yck1/Yck2 kinases and that Gβ phosphorylation contributes to chemotropism by regulating Gβγ-effector interactions. Previous evidence also suggests that the interaction between Gα and Fus3 contributes to chemotropism and the phosphorylation of Gβ. Does Gα recruit Fus3 to phosphorylate other proteins important for mating? Using mass spectrometry, we identified fourteen potential Gα-Fus3 targets that were enriched for Gene Ontology terms related to polarization and cortical localization. Pull-down experiments with a phospho-site mutant form of Spa2 (Spa2P-), suggest that phosphorylation of Spa2 on specific residues regulates Spa2’s interaction with polarisome components and actin regulating proteins. Fluorescence microscopy of vegetative cells, expressing fluorescently tagged Spa2 and Spa2P-, revealed striking cell morphology and Spa2 localization phenotypes that were 100% penetrant. Further, pheromone-responding Spa2P- mutants were unable to form mating projections, and were more likely to lyse upon cell fusion. These data suggest Spa2 phosphorylation affects its protein interactions and its localization. We discuss the possibility that Gα-Fus3 is a spatiotemporal hub that coordinately regulates multiple cortical MAPK targets during yeast mating. This work presents many new putative targets of Gα-Fus3, each of which could lead to significant insights about the yeast mating process.