A main goal of this proposal is an elucidation of the biogenetic mechanisms which operate in polarized epithelial cells to ensure the segregation of specific plasma membrane proteins to either the luminal or basolateral domains of the cell surface. Using model systems of cultured kidney epithelial cells (MDCK) infected with enveloped viruses which bud exclusively from one or the other plasma membrane domain, or transfected with suitable expression vectors containing genes for natural or modified viral envelope proteins, we will determine the subcellular route followed by viral glycoproteins of different polarity from the Golgi apparatus to each plasma membrane domain, identify sorting signals within the membrane proteins and characterize cellular elements of the sorting apparatus. Aspects of the biosynthesis and structure of the Na+,K+ ATPase, a cellular enzyme located in the basolateral surface of epithelial cells, will be studied in cultured MDCK cells and in rat brain, using biochemical, cell fractionation and recombinant DNA methods. We will also study the determinants of polarized secretion in epithelial cells employing expression vectors containing genes encoding natural or modified endocrine or exocrine proteins and truncated fragments of membrane proteins which are normally segregated to one plasma membrane domain. The extent of convergence of the secretory, membrane biogenetic and endocytic pathways will be investigated. The major protein components of tight junctions will be isolated and characterized with respect to their transmembrane disposition, and the process of their assembly into junctions. cDNA clones for the junction proteins(s) will be obtained and used to determine if differences in junction permeability of different tissues result from variations in the organization or nature of junctional components.
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