Mechanism and function of ubiquitin binding by conserved endocytic protein Rvs167

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DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis is a fundamental process that is conserved in all eukaryotic cells, and is required for numerous important functions including nutrient uptake, membrane remodeling, synaptic vesicle recycling and down-regulation of activated signaling receptors. Rvs167, a BAR domain protein homologous to the mammalian endocytic factors endophilin and amphiphysin, is required for growth control and endocytosis in yeast. Recently, Rvs167 was demonstrated to bind ubiquitin, and preliminary studies suggest that ubiquitin binding is required for the function of Rvs167. This application proposes to define the mechanism and function of ubiquitin binding by Rvs167 through the following aims: 1) Determine the molecular basis for the novel interaction between Rvs167 and ubiquitin by conducting binding assays with different fragments or mutants of recombinant Rvs167. 2) Define the role of ubiquitin binding in cell growth, actin organization and endocytosis. Ubiquitin binding-defective cells will be analyzed for growth arrest in response to nutrient deprivation, actin filament structure, and endocytosis of both bulk-phase and receptordependent markers. Additionally, real-time fluorescent microscopy will be used to monitor the localization and dynamics of wild-type and ubiquitin binding-defective fluorescent protein fusions of Rvs167 in strains co-expressing other endocytic proteins with alternate fluorescent tags. 3) Identify and characterize protein(s) that interact with Rvs167 via ubiquitin binding. Interacting proteins will be identified by a whole genome yeast-2-hybrid screen, GST fusion protein affinity chromatography, or by coimmunoprecipitation from yeast lysates. Once identified, deletion and Rvs167-binding defective mutants of interacting proteins will be subjected to assays described in aim 2. These studies are anticipated to contribute to understanding of the molecular mechanisms responsible for endocytosis and cell growth control, and to provide a foundation to understand how defects in endocytosis contribute to human disease. Endocytosis is a fundamental cellular process that is conserved from yeast to humans. Rvs167 is a close relative of two human endocytic proteins and is required for growth control and endocytosis in yeast. These studies on Rvs167 are anticipated to contribute to understanding the basic molecular mechanisms responsible for endocytosis and to provide a foundation for understanding how defects in endocytosis contribute to human disease.