Budding yeast display polarity in the site of bud emergence. Haploid cells bud in an axial fashion, in which the new bud emerges close to the site of the old bud, while diploid cells bud in a bipolar pattern. This site selection requires the BUD1, 2 and 5 gene products encoding a ras-like G protein and the GTPase activating and nucleotide exchange proteins, respectively. This GTPase module interacts with Bem1 and the Cdc42 and 24 proteins, a second G-protein set that is presumed to interact with the cytoskeleton to fix polarity. In addition to the above proteins, axial budding requires the BUD3 and 4 and AXL1 gene products; mutations in any one of the three leads to bipolar budding. AXL1 is repressed in diploid cells accounting for their bipolar, non- axial budding pattern. Mating yeast cells bud towards the source of pheromone, and this over-riding of normal cell polarity requires the FAR1 gene product. These ten proteins represent the known factors, discovered over the past several years. Dr. Herskowitz proposes in this application to continuate the characterization of cell polarity determination. During the present funding period, the PI has demonstrated a number of pairwise interactions between various combinations of these ten proteins using in vitro studies with bacterial expressed proteins and the two-hybrid system. These interactions provide the basis for a model of a Bud1, 2, and 5, Cdc24 and 42, Bem1 complex-pathway which will be tested in the proposed experiments. The in vitro binding assays will be combined with competition studies and in vivo two-hybrid studies as well as mutational analyses to determine which of the predicted interactions are correct. Also, immunolocalization studies will be carried out to test the prediction that Bud2 anchors the complex to the bud emergence site. A variety of mutant alleles that disrupt specific interactions but allow others will be used to test whether the conclusions reached from these studies are relevant to native interactions. One interesting aspect of polarity determination is that the BUD gene products are not essential; their absence leads only to random budding. Therefore, there must be a backup system which the PI will seek using synthetic lethal hunts. The role of Bud3 and 4 and Axl1 in determining axial budding will be explored. The hypothesis that these proteins are localized to the bud neck and interact with septins will be tested using the two-hybrid system and immunolocalization studies. The Far1- dependent alteration in polarity induced by mating pheromone will be studied. The cellular localization of Far1 and its interaction with the polarity-determining proteins will be investigated. Additional gene products will be sought through extended mutant hunts.
