RESUMEN
The mating-specific yeast Gα controls pheromone signaling by sequestering Gßγ and by regulating the Fus3 MAP kinase. Disrupting Gα-Fus3 interaction leads to severe defects in chemotropism. Because Gα concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Gα-Fus3 interaction. Using a mutant form of Gα severely defective in Fus3-binding, GαDSD, and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Gα-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated GαDSD cells. Six sites were mutagenized to create the Spa26XSA mutant protein. Spa26XSA exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa26XSA cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Gα promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the Gα-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. SIGNIFICANCE: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis.