Identification and analysis of novel genes involved in cellular morphogenesis in Saccharomyces cerevisiae.

Abstract

The cell cycle of the budding yeast Saccharomyces cerevisiae involves a variety of morphogenetic events, and given its amenability to both classical and molecular genetic techniques, offers an ideal system to study the mechanisms of cell polarity. In this dissertation I report the analysis of two mutants that display temperature-sensitive defects in polarity establishment. In each mutant, the original phenotype is dependent on the simultaneous presence of two mutations. I have shown that in each case, one of the two mutations is in BEM1, a gene that appears to be required for polarity establishment both during budding and during mating. In one mutant, the second mutation is in BUD5, a nonessential gene that is required for normal bud-site selection. In the other mutant, the second mutation is a temperature-sensitive-lethal mutation in a newly identified gene ABD1. The ABD1 sequence predicts a polypeptide of $\sim$50 kD that shows no similarity to other known proteins. Disruption of ABD1 is lethal; the germinated spores display no obvious terminal phenotype. In this work, I have also shown that the predicted sequence of the BEM1 protein (Bem1p) contains two copies of a domain (denoted SH3) that has been found in many proteins associated with the cellular cortical cytoskeleton. Complete deletion of BEM1 leads to the defect in polarization of vegetative cells observed for the temperature-sensitive bem1 mutants. Antibodies specific for Bem1p were generated and used to demonstrate that this protein localizes to the presumptive bud site in unbudded cells and to the bud tip of cells with small buds. These antibodies were also used to explore further the timing and dependency relationships of early events in polarity establishment. Results from these experiments suggest that assembly of Bem1p at the bud-site prior to bud emergence is independent of the assembly of actin and of the assembly of the neck-filament-associated proteins, yet dependent on the function of CDC24. The close association between actin and Bem1p at the presumptive bud site suggests that these two proteins interact to establish a polarized cellular axis during bud formation.