The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery

Abstract

The septin cytoskeleton plays a key role in the morphogenesis of the yeast mating projection, forming structures at the base of the projection. The yeast mating response uses the G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. Desensitization of the G, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. We hypothesized that Gpa1 would utilize known septin regulators to control septin organization. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the endocytic adaptor proteins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive G. We hypothesized that hyperactive G might increase the rate of endocytosis of a pheromone-responsive cargo, thereby altering where septins are localized. Mathematical modeling predicted that changes in endocytosis could explain the septin organizations we find in WT and mutant cells. Our results show that Gpa1-induced disorganization of septins requires clathrin-mediated endocytosis. Both the GPCR and the G 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, deleting the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the G delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.