Endocytosis is required for the normal growth and development of all eukaryotes. It is an important route by which cells take up many nutrients and regulatory molecules and down-regulate membrane proteins in response to their ligands and environmental signals. A major pathway for receptor-mediated endocytosis involves the coat protein clathrin along with a large number of accessory proteins that facilitate the capture of cargo and formation of endocytic vesicles. Also, in the last few years a close relationship between the actin cytoskeleton and clathrin-mediated uptake has emerged. Studies in the applicant's laboratory have taken advantage of the powerful molecular genetic methods of the yeast system to analyze the function of clathrin. In yeast the actin cytoskeleton is central to endocytosis, but recent work indicates that clathrin pIays a role in this process through interaction with components of the cortical actin cytoskeleton. Furthermore, many of these actin-associated proteins have counterparts in animal cells that are involved in clathrin-mediated internalization, indicating that yeast can serve as an important model for studying clathrin and endocytosis. This project focuses extensively on a protein, Scd5p, that was identified as a suppressor of clathrin deficiency in yeast. Work from the investigator's laboratory has shown that Scd5p regulates actin organization and endocytosis, most likely as a targeting subunit for type 1 protein phosphatase (PP1). In addition Scd5p may be regulated by a new family of actin-regulating kinases (ARKs). These kinases appear to promote the disassembly of cortical actin complexes, and substrates include proteins that interact with clathrin. Studies proposed here will: (1) investigate how Scd5p function is regulated, particularly by phosphorylation of the central triple repeat motif, which is a potential target for ARK phosphorylation, and by the C-terminal region of the protein; (2) examine whether PP1 regulates Scd5p itself and/or Scd5p targets PP1 to other targets for regulation of actin organization, and endocytosis. Scd5p/PPl substrates will also be sought; and (3) generate and analyze new clathrin mutants that affect clathrin association with cortical actin components. These studies should provide new information on the role of clathrin and actin in endocytosis. Furthermore, novel insight into the importance of phosphorylation in regulation of these dynamic processes will be gained.