Saccharomyces cerevisiae will be used to study cell polarization in response to an external signal. Cell polarization is a fundamental biological phenomenon. underlying process such as asymmetric cell division, chemotaxis, and neurite outgrowth. In S. cerevisiae, the haploid a and alpha cells polarize and grow toward the source of mating pheromone in preparation for the mating event. The polarity establishment proteins are localized to the site of new growth and orient the cytoskeleton toward this site. How the polarity establishment proteins are recruited to the site of growth and the mechanism by which they affect the cytoskeleton is unknown. I will study the localization of these proteins and how they orient the cytoskeleton by initially focusing on BEM1, a polarity establishment gene. A BEM1-Green Fluorescent Protein (GFP) fusion will be used to determine BEM1's localization kinetics. Structure-function analysis of BEM1 will define protein domains important for its localization. Two approaches will be used to identify components important for BEM1 function: 1) The two-hybrid approach will identify proteins that physically interact with BEM1; these can act either upstream or downstream of BEM1. 2) A genetic screen will be done to identify loci involved in localizing the polarity establishment genes. Mutants in these loci will be unable to mate to bem1 (based on Chenevert. et al. [(1994) Genetics, 136: 1287-1297]) and unable to properly localize BEM1-GFP.
