Gic2p May Link Activated Cdc42p to Components Involved in Actin Polarization, Including Bni1p and Bud6p (Aip3p)

Abstract

Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p^W23A) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.

ACKNOWLEDGMENTS

We thank Charlie Boone, Danny Lew, Michael Snyder, David Amberg, Anne-Christine Butty, Pamela Silver, Rosine Hagenauer-Tsapis, and Mike Tyers for kind gifts of strains, plasmids, and antibodies and Miranda Sanders and Phil Crews (UCSC) for the synthesis of LatA; work in their laboratory is supported by the NIH. We are grateful to members of the laboratory for helpful discussions, Audrey Petit for help with the SMART system, Nathalie Perrinjaquet for excellent technical assistance, and Bruno Amati and Richard Iggo for critical reading of the manuscript.

This work was supported by grants from the Swiss National Science Foundation, the Swiss Cancer League, and a Helmut Horten Incentive Award.