We are studying bud emergence in the yeast S. cerevisiae to elucidate basic mechanisms governing cell polarity development. Bud emergence is regulated by Cdc42, which is a conserved member of the Rho family of GTPases. In yeast and animals, Cdc42 controls cortical actin organization and the activation of MAP kinase pathways, and the actions of Cdc42 are linked to those of other Rho-type GTPases. We would like to know how Cdc42 becomes activated and associated with its targets at the appropriate places and times, what structures are formed in response to Cdc42, and how the actions of Cdc42 are coordinated with those of Rho3, a Rho-type GTPase that is implicated in bud growth. The activation of Cdc42 requires the guanine nucleotide exchange factor Cdc24. From a variety of genetic and two-hybrid screens, we have identified a set of proteins whose functions are linked to Cdc42 and Cdc24. These include Bem1, which is an SH3 (Src homology 3) domain-bearing protein that binds to Cdc24 and displays a two-hybrid interaction with Cdc42; Boi1 and Boi2, which are functionally and structurally similar SH3 and PH (Pleckstrin homology) domain-containing proteins that bind to Bem1, display two-hybrid interactions with Cdc42, and interact genetically with both Cdc42 and Rho3; Bem4, which displays genetic and two-hybrid interactions with Cdc42; and Akr1, which contains six ankyrin-like repeats, displays a synthetic-lethal interaction with Rho3, and (like Cdc24, Cdc42, and Bem1) is involved in pheromone signaling. To help elucidate the roles of Cdc24, Boi1, Bem1, Bem4, and Akr1 in the regulation and function of Cdc42, we will: I. Investigate how Boi1, Bem1, Bem4, and Akr1 affect the regulation and action of Cdc42. II. Characterize associations among Cdc42, Cdc24, Boi1, Bem1, Bem4, and Akr1. III. Use affinity techniques to purify other proteins associated with Boi1, Bem1, and Bem4. IV. Immunolocalize Boi1, Bem4, and Rho3, and investigate the mechanisms by which Bem1, Boi1, and Rho3 become properly localized. V. Screen for genes whose products link the actions of Boi1/Boi2 and Rho3.
