This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. IcsA, an autotransported outer membrane protein is targeted to the old pole of the bacterium; the same pole where actin filaments are assembled for intracellular swimming and intercellular dissemination in host colonic epithelial cells. Several lines of evidence suggests that IcsA is targeted to the inner face of the cytoplasmic membrane before secretion and that IcsA must be targeted to the old pole before it is secreted. During the summer we have exploited these findings in a screen to identify a putative polar target for IcsA. We are also interested in the targeting of four proteins in yeast--Bud8p, Bud9p, Rax1p and Rax2p that are all involved in bipolar budding in diploid yeast cells. Rax1p and Rax2p are both targeted to both ends of diploid cells and are both involved in the targeting of Bud8p and Bud9p to these poles. Interestingly, the Rax proteins target Bud8p and Bud9p to the distal and proximal poles (with respect to the most recent bud), respectively. Deletions in Rax1p and/ or Rax2p lead to aberrant targeting of the Bud proteins such that an axial distribution of budding is observed as opposed to the oscillating bipolar distribution of buds characteristic of wild type diploid cells. We are interested in the molecular mechanism for targeting of the Rax1p/Rax2p complex and the role that these proteins play in the differential targeting of Bud8p and Bud9p. The immediate goal for August, 2005 is to define the function of Rax1p and Rax2p in oscillating, bipolar budding. To this end we are making deletions in the corresponding genes for use in a Synthetic Genomic Array screen. Later, we will apply the SGA screen to the Bud8p, Bud9p proteins to determine the function of these proteins in budding and also apply the Yeast 2-Hybrid screen to define proteins with which Bud8p, Bud9p, Rax1p and Rax2p interact.
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