The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting process 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 S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has identified a large set of genes that control the sorting of Gap1. These genes include factors directly involved in the membrane trafficking of Gap1, such as proteins required to modify Gap1 with poly-ubiquitin, a tag found to be required for the proper sorting between Golgi and endosomal compartments. In addition, genes required to generate the proper regulatory signals for Gap1 sorting were also identified. Building on these findings the proposal is to: (i) determine how Gap1 is ubiquitinated and to identify the cellular components required for recognition of the ubiquitin tag, (ii) use a combination of genetic and biochemical methods to characterize the gene products that control Gap1 sorting and to determine their site(s) of action in the secretory and endocytic paths, and (iii) determine how nitrogen-derived signals are generated and determine how the membrane trafficking machinery responsible for sorting Gap1 decodes these signals.
