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 central to the life of a cell. To accomplish this, experiments have been designed to identify cells with mutations in four different sorting events: (1)the internalization phase of endocytosis, in which receptors for nutrients and hormones are selected from other proteins of the plasma membrane for incorporation into coated pits; (2)the recycling phase of endocytosis, in which receptors that constitutively internalize nutrients are separated from proteins destined for lysosomes; (3)the sorting of plasma membrane glycoproteins during exocytosis in epithelial cells; (4)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.
For specific aim 3 and 4, polarized cell line expressing either a mutant influenza virus hemagglutinin or the human LDL receptor have been constructed and will be used to investigate the pathway by which the mutant protein reaches the basolateral plasma membrane.
