DESCRIPTION (provided by applicant): The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting processes can produce rapid changes in the rate that a transporter is delivered to the plasma membrane in response to an intracellular signal and thus provide a way for a cell to alter its capacity to take up small molecules from the extracellular environment in response to regulatory cues. For example, the GLUT4 glucose transporter is delivered to the plasma membrane of fat and muscle cells in response to insulin, and defects in this regulated trafficking are thought to be a root cause of non insulin-dependent diabetes. Many aspects of the intracellular sorting of GLUT4 remain poorly understood at this time. Dr. Kaiser's research group proposes to continue to study regulated sorting of general amino acid permease (Gap1) in response to the nitrogen source in the growth medium. By studying amino acid permease sorting in the model organism S. cerevisiae it will be possible to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has shown that Gap 1 p sorting is controlled by the rate at which Gap1 p protein can recycle from the endosome to the plasma membrane and that this sorting step is controlled by the abundance of intracellular amino acids. A genetic screen for mutants defective in Gap1 p recycling identified a GTPase containing complex (GSE) that interacts with Gap 1 p so as to suggest that the GSE complex may form part of a vesicle coat for Gap 1 p trafficking. Building on these findings the proposal is to: (i) identify additional components of the GSE complex with the ultimate aim of reconstituting activity in vitro, (ii) determine the structure and function of the GTPase component of the GSE complex, including functional studies of the mammalian orthologs, and (iii) determine how the nitrogen source controls Gap1 p recycling. We have found that Gap1 p modification by ubiquitin is necessary for sorting from the Golgi to endosome. A final aim of the project is (iv) to understand how ubiquitination is regulated and to identify the cellular components required for recognition of the ubiquitin tag.