Characterization of the potential Cdc42 regulators Dop1 and Rdi1 in budding yeast

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The small GTPase Cdc42 is a key regulator of cell polarity. In the budding yeast Saccharomyces cerevisiae it is required at different stages of the cell cycle during vegetative growth but also during mating and filamentous growth. Cdc42 interacts in the active state with numerous effectors which mediate various functions such a bud growth, exit from mitosis and cytokinesis. It seems very likely that this specific activation of Cdc42 effectors is controlled by Cdc42 regulators. Among these regulators are the GEF Cdc42, the GAPs Bem3, Rga1 and Rga2 and the GDI Rdi1. Research has mainly focused on Cdc24, whereas less is known about the other proteins. The conserved polarity protein Dop1 was identified as an interactor of Bem3 but has not been further analyzed to date. We plan to characterize the proteins Dop1 and Rdi1 and examine how they contribute to the regulation of Cdc42 activity and cell polarity in general. This includes the analysis of genetic interactions between cell polarity proteins and Dop1 and Rdi1, respectively. Protein interactions will be examined using the yeast two-hybrid system, co-immunoprecipitations and in vitro binding assays. Furthermore, the phenotypes of overexpression and inactivation of Dop1 and Rdi1 will be analyzed in detail. Assays for Cdc42 activity will be used to examine the relationship between other cell polarity proteins and Dop1 and Rdi1, respectively. Finally, we will screen for novel factors that regulate Cdc42 and characterize these interactors in a similar way.