Hob3 is required for SAPK pathway dependent Gef1 plasma membrane recruitment

Abstract

Cell polarity is a fundamental feature of cells that refers to the asymmetry observed in cellular shape, structure, and function. The fission yeast Schizosaccharomyces pombe is a valuable model to study core polarity regulation pathways owing to its simple genome and polarized pattern of growth during the cell cycle. In S. pombe, activation of the highly conserved polarity regulator Cdc42 is controlled via two guanine nucleotide exchange factors, Scd1 and Gef1. Normally Scd1 is tip localized, while Gef1 is cytosolic. The Sawin lab have found that upon activation of the stressactivated protein kinase (SAPK) pathway, Scd1 is dispersed from cell tips while Gef1 is recruited to the plasma membrane. Additionally, they have shown that active Cdc42 is no longer maintained at the plasma membrane in stressed gef1Δ mutants. Unlike Scd1, Gef1 does not possess a pleckstrin homology domain for plasma membrane association; however, Gef1 binds to Hob3, a plasma membraneassociated N-BAR protein. This study aimed to investigate whether Hob3 has a role in the mechanisms regulating Gef1 plasma membrane recruitment upon Sty1 activation.

The findings presented in this thesis establish a role for Hob3 in stress induced Gef1 plasma membrane recruitment. Here I describe experiments which show that Hob3 is necessary for the establishment and maintenance of Gef1 plasma membrane patches. Additionally, I show that Hob3 puncta do not co-localize with Gef1, instead non-puncta associated Hob3 is essential for recruitment of Gef1 to the plasma membrane. Furthermore, I show that SAPK activation does not lead to changes in Gef1-Hob3 interaction. This thesis also focuses on co-immunoprecipitation experiments coupled with mass spectrometry-based proteomics in the search to identify Hob3 interactors which change upon Sty1 activation, facilitating Gef1 plasma membrane recruitment. I identify several novel Hob3 interactors pre and post Sty1 activation. However, further investigation of these interactors did not reveal insights into the mechanisms involved in Gef1 plasma membrane recruitment.