Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Slalp is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom 1) domain. It does not, however, contain either a gamma-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Slalp and with Las17p, two regulators of actin dynamics. The site of interaction with Slalp involves the Slal HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of ArD with the endocytic machinery in yeast.