ISOLATION OF CELLS DEFECTIVE IN SORTING GLYCOPROTEINS

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A central feature of all eukaryotic cells is the ability to maintain and control a complex traffic of proteins moving between cellular organelles. The mechanisms by which this control is achieved are currently unknown. The goal of the research proposed in this application is to identify the molecular mechanisms responsible for several of the sorting events integral to the life of a cell. To accomplish this, experiments have been designed to identify cells with temperature-conditional mutations in three different sorting events: (1) the internalization phase of endocytosis' the event by which receptors for nutrients and hormones are selected from other proteins of the membrane for incorporation into coated pits; (2) the sorting of plasma membrane glycoproteins during exocytosis in kidney epithelial cells, the event that defines and maintains the surface polarity of these cells that is a fundamental requirement for their function; (3) the sorting of cell surface receptors after endocytosis in kidney epithelial cells that serves to recycle those receptors to the serosal surface, and thus maintains the protein composition of that membrane domain. Once cells with the desired phenotype have been identified, the mutants will be subjected to complementation analysis, immunocytochemical and biochemical assays to group mutants with common defects. Revertants of interesting mutants will be isolated after transfection with complementing, wild-type DNA. Through serial DNA transfer techniques, genes capable of reverting mutations in each of the sorting events will be isolated and the gene products characterized.