Interactions among Proteins Involved in Bud-site Selection and Bud-site Assembly in Saccharomyces cerevisiae

Abstract

Bud formation in yeast involves the actions of the Ras-type GTPase Rsr1, which is required for the proper selection of the bud site, and the Rho-type GTPase Cdc42, which is necessary for the assembly of cytoskeletal structures at that site. The Cdc24 protein is required both for proper bud-site selection and bud-site assembly and has been recently shown to display guanine-nucleotide-exchange factor (GEF) activity toward Cdc42. Here, we demonstrate, using recombinant proteins, that Cdc24 can also bind directly to Rsr1. This binding has no effect on the ability of Rsr1 to undergo intrinsic or GEF-stimulated GDP-GTP exchange. However, Cdc24 can inhibit both the intrinsic and GTPase-activating protein-stimulated GTPase activity of Rsr1 and thereby acts as a GTPase-inhibitor protein for Rsr1. Cdc24 thus appears to bind preferentially to the activated form of Rsr1. The SH3 domain-containing bud-site assembly protein Bem1 also binds directly to Cdc24, and we show here that this interaction is inhibited by Ca2+. Neither Bem1 nor Cdc42 affects the GTPase-inhibitor protein activity of Cdc24 toward Rsr1, and neither Bem1 nor Rsr1 affects the GEF activity of Cdc24 toward Cdc42. Taken together, these results suggest that Cdc24 enables the direct convergence of a Ras-like protein (Rsr1) and a Rho-like protein (Cdc42) with the SH3-domain-containing protein (Bem1) and that independent domains of Cdc24 are responsible for these different interactions. These results also suggest that rather than directly controlling the GEF activity of Cdc24, the primary roles of Rsr1 and Bem1 might be to control the positioning of Cdc24 within the cell.