Date of Award

Summer 8-19-2022

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Joshua B. Kelley

Second Committee Member

Melissa Maginnis

Third Committee Member

Samuel T. Hess

Additional Committee Members

Dustin Updike

Kristy L. Townsend

Abstract

G-protein coupled receptors (GPCRs) are the target of approximately 35% of all FDA-approved pharmaceuticals. This is because GPCRs regulate different cellular signals including control of cell polarity. The yeast, Saccharomyces cerevisiae, use a GPCR for mating which induces chemotropic growth and morphogenesis toward a mate. In mammalian cells, signaling downstream of the receptor is primarily conducted by the Gα subunit of the large G-protein. In the yeast pheromone response little is known about Gα’s contribution to signaling and the Gβγ subunit is the primary known contributor. This begs the question, what is the contribution of Gα to the yeast pheromone response? I have found two novel mechanisms in the yeast pheromone response that implicate Gα signaling in yeast mating. First, I have identified a previously unknown role of the regulator of G-protein signaling (RGS), which facilitates hydrolysis of GTP to GDP on Gα to turn off downstream signaling. I found that RGS is phosphorylated early in the pheromone response to facilitate the transition from mitosis to pheromone-induced polarization. Importantly, I also found that the phosphorylation state of the RGS dictates the distribution of the downstream Fus3-MAPK response. The Fus3-MAPK binds directly to Gα-GTP to enhance signal output. In cells with phosphomimetic RGS, the distribution of Fus3-MAPK, is broadly localized across the polar cap while unphosphorylatable RGS mutants were similar to controls. Therefore, the phosphorylation state of RGS controls the distribution of active Gα and drives localization of the Fus3-MAPK signal. iii Previous work indicated that cells lacking RGS-activity do not generate mating projections and have aberrant septin distribution. Septins are cytoskeletal filaments that function as diffusion barriers and protein scaffolds during cell division, but their roles in the pheromone response are not well understood. I found that septin distribution is driven by epsin-mediated endocytosis of Gα via its ubiquitination domain. Additionally, the septin chaperone Gic1 and the Cdc42 GTPase activating protein (Cdc42-GAP) Bem3 were differentially involved in pheromone-induced septin deposition when compared to other septin chaperones and Cdc42-GAPs respectively. These results provide new evidence for Gα as a signaling contributor to the yeast pheromone response.

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