CHARACTERIZING CONDITIONAL MUTANTS OF BOI1/2 IN SACCHAROMYCES CEREVISIAE

Abstract

Cell polarity is exhibited in Saccharomyces cerevisiae through the asymmetric budding of a new cell and segregation of organelles prior to cell division. This process occurs, in part, through the activation of a small Rho-GTPase, Cdc42, which helps direct assembly of polarized actin cables, allowing the class-V myosin, Myo2, to carry organelles and vesicles into the bud for cell growth. These vesicles contain cell wall components and are decorated with proteins that help with direction, membrane tethering, and fusion. The redundant yeast proteins, Boi1 and Boi2, were identified by their ability to bind Bem1, that binds an effector of Cdc42. Loss of both paralogs is lethal, indicating that they provide an essential function. Boi1/2 also have a secondary function in the absence of Bem1 that has never been studied before. This work aims to explore these two functions of Boi1/2 through the generation and characterization of conditional mutants of BOI1 in a boi2∆ background. Two mutations within the PH domain were found to abrogate the protein’s essential function. Characterization of the mutants show significant defects in budding patterns, actin patches, and abrupt cell lysis during growth at the restrictive temperature. In parallel, mutations in BEM1 were constructed to explore the over-lapping function of Boi1 and Bem1. These experiments illustrate that Boi1’s SH3 domain is necessary for cell survival in cells lacking Bem1 (bem1∆) and that this domain influences not only Boi1 localization at the bud neck, but also Bem1 localization, highlighting a novel role for this region of Boi1/2.