Abstract 1081 Autophagy leads to desensitization of the Saccharomyces cerevisiae mating pathway through the ligand-independent endocytosis of the mating GPCR Ste2

GPCRs are conserved sensors of extracellular cues which coordinate signaling networks to produce an adaptive response. In humans, prolonged GPCR signaling can lead to maladaptive consequences such as heart failure, tumorigenesis, and cell death. Various negative regulators of GPCR signaling desensitize cells to certain stimuli, preventing the deleterious effects of prolonged signaling. Ste2 is a pheromone responsive GPCR in budding yeast that directs the transcriptional and morphological changes required for mating. Yeast cells face a metabolic gamble upon detection of pheromone, whereby significant quantities of energy and resources are consumed while attempting to mate. Therefore, cells must ensure they are supplied with sufficient nutrients to respond to mating pheromone. The objective of this study is to examine whether autophagy signaling impacts the pheromone receptor in Saccharomyces cerevisiae. Using live-cell fluorescence microscopy, we observed that Ste2 is removed from the plasma membrane under nutrient limitation and reappears following nutrient repletion. Nutrient-directed control of Ste2 localization points toward autophagy as a mechanism to prevent cells from responding to mating pheromone while starving. Autophagy is a cytoprotective stress response that promotes survival through the recycling of cytoplasmic regions to provide metabolites to the cell. This response is primarily controlled by the Target of Rapamycin Complex 1 (TORC1), whose inhibition via nutrient limitation or rapamycin treatment results in autophagy induction. To test if the change in Ste2 localization was due to autophagy signaling, cells were treated with rapamycin in an unsaturated culture for 2 hours and imaged. Rapamycin treatment decreased peripheral Ste2 levels, suggesting autophagy is the signaling pathway directing the internalization of the GPCR. To test the specificity of this effect on all yeast GPCRs, the mating receptor of the opposite mating type Ste3, the sugar sensing GPCR Gpr1, and the proton pump Pma1 (as a negative control), were each tagged and imaged following rapamycin treatment. Rapamycin treatment resulted in a decrease of peripheral Ste3, while causing an increase in Gpr1, suggesting that autophagy signaling can differentially control yeast GPCR levels at the plasma membrane. Pma1 levels did not significantly change in response to rapamycin, indicating that autophagy-induced internalization is specific to GPCRs. The reduction in both mating receptors further supports the idea that autophagy may be used to prevent the cell from mating while starving. To test this, we used a transcriptional reporter assay to measure the pheromone-induced expression of mating genes in cells treated with rapamycin. Cells treated with rapamycin showed a decrease in the maximal output of mating gene expression. In conclusion, autophagy signaling suppresses mating when nutrients are unavailable through the control of surface mating receptor presentation, revealing a novel mechanism of GPCR desensitization through the induction of autophagy. This research was supported by NIH grants R15GM140409 and P20GM144265-8804.