Abstract 2021 The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and endocytic machinery

The yeast mating response uses a G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. The septin cytoskeleton forms structures at the base of the mating projection and promoting morphogenesis. Desensitization of the Gα, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. In cells where the Gα is hyperactive, septins are mislocalized to the site of polarity, and the cells are unable to track a pheromone gradient. We set out to identify the proteins that mediate Gα control of septins during the Saccharomyces cerevisiae mating response by making mutations to rescue septin localization in cells expressing the hyperactive Gα mutant gpa1G302S. We tested this using fluorescence microscopy in microfluidic devices, computational image analysis, and mathematical modeling. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the epsins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive Gα. Ent1 and Ent2 are endocytic adaptor proteins, which lead us to consider the potential for endocytosis to be involved in septin organization. We examined Ent1-GFP during the pheromone response and found that it routinely colocalizes with septins. We created an agent-based model of vesicle trafficking that predicts that changes in endocytic cargo licensing alter localization of endocytosis in a way that mirrors the septin localization we see experimentally. We hypothesized that hyperactive Gα may increase the rate of endocytosis of a pheromone-responsive cargo, thereby altering where septins are localized. Both the GPCR and the Gα are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deletion of the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Disruption of all endocytosis through deletion of END3 also lead to rescue of septin organization to the base of the mating projection. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the Gα delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity. This work was funded by NIH Grant R15 GM140409 to Joshua Kelley.