The goal of this work is to understand the molecular basis by which cell polarity is determined. Cells of the budding yeast, Saccharomyces cerevisiae, exhibit a characteristic pattern of budding which reflects genetic programming of the site of cell polarization. They respond to mating pheromones by reprogramming the site of cell polarization towards the mating partner. We have identified five genes -- BUDl-BUD5 -- necessary for the proper budding pattern of mitotically growing cells and three genes -- BEM1, PEA1, and TNY1 -- necessary for proper morphological response to the mating pheromones. We propose that the BUD proteins play key roles in a morphogenetic pathway that determines the site of budding by determining the positions of bud-formation proteins (CDC24, CDC42, and BEM1) at the site for bud growth. These proteins then organize the cytoskeleton, in particular, actin and tubulin.
Our specific aims concern tests of these hypotheses and include a goal of understanding the role of ras-related proteins and their regulators in this process. Our studies employ techniques of genetics, enzymology, molecular biology, and cell biology. (1) We shall determine the role of the BUD products in bud-site selection by determining their cellular location; test whether BUD proteins interact with bud-formation proteins; determine whether BUD2 protein is a GTPase activating protein and whether BUD5 is a guanine nucleotide exchange protein by in vitro and in vivo analyses, and whether their substrates are the ras-related proteins, BUD1 and CDC42; and seek to identify additional genes involved in bud-site selection by genetic methods. (2) We shall determine the molecular basis for the difference in bud-site selection exhibited by a/alpha cells vs. a and alpha cells by testing our hypothesis that synthesis or activity of BUD3 or BUD4 is negatively regulated in a/alpha cells and by other analyses. (3) We shall determine the molecular basis by which mating pheromones cause cells to reorient their cytoskeleton towards their mating partner by examining the effects of alpha-factor on BUD and other proteins and by studying the mutants that we have isolated.
