My long-term objective is to use the genetically tractable eukaryote Saccharomyces cerevisiae to determine how Cdc42p, an evolutionarily conserved GTPase, controls the organization of the actin cytoskeleton and of the septins. Recent work suggests that Cdc42p controls the actin organization by two parallel pathways: one involving Msb3p, Msb4p, and Bnilp; the other involving Gic1p, Gic2p, and Bem4p. In the present studies, these pathways will be explored further, focusing initially on the roles of Msb3p and Msb4p. Msb3p and Msb4p, a pair of highly homologous proteins in yeast, play a redundant role in linking Cdc42p to the actin cytoskeleton. They belong to a family of proteins that includes the Drosophila cell adhesion molecule, Pollux, and the human oncoprotein, Tre17. Thus, studying the function of Msb3p and Msb4p will shed significant light on the function of this family of proteins. Specifically, research in this proposal will address the following questions: What proteins link Msb3p and Msb4p to the upstream Cdc42p, and to the downstream actin cytoskeleton? How are the proteins in the Msb-mediated pathways organized at the molecular level? What are the functions of the putative domains in Msb3p and Msb4p (the highly conserved PTM domain, the membrane-spanning domains, and a lipid modification site)? How are the localization of Msb3p and Msb4p regulated in the cell cycle? These questions will be answered with a combination of genetic, cytological, and biochemical approaches. To exapnd our hypothesis, additional genes that are involved in regulating Cdc42p activity or a specific effector pathway will be identified by genetic screens, and affinity chromatography coupled with tandem mass spectrometry. Homologs of Cdc42p are involved in diverse cellular processes, such as cell polarity, cell migration, and cell growth control. In addition, deregulation of Cdc42p activity in mammals is associated with serious diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast represents an important bridge between basic and clinical sciences.