Epithelial cells exhibit structural and functional polarity crucial to their role as mediators between two often very different environments. The apical and the basolateral domains of their plasma membranes have different protein compositions enabling epithelia to perform vectorial tasks. The long term goals of this project are to understand how epithelial cells assemble and maintain unique plasma membrane domains and how this is related to the development of polarized cells during embryogenesis. In the work proposed here, the transport of a model apical membrane protein, the influenza virus hemagglutinin, will be examined in viral-infected MDCK cells, a polarized epithelial cell line, first by a unique cell culture approach and later in a reconstituted cell-free system.
The specific aims of this proposal are 1) to determine if the route of the hemagglutinin to the apical plasma membrane goes via the basolateral domain using cells grown on permeable supports so that both domains may be independently assayed, 2) to identify the intracellular organelles traversed by the hemagglutinin between the Golgi complex and plasma membrane by immunocytochemistry, 3) to establish the metabolic and some biochemical requirements for transport and sorting using a pharmacological approach, 4) to isolate and characterize the distal intracellular transport compartments by cell fractionation procedures, 5) to identify possible transport carrier or receptor proteins by immunprecipitation, protein-blotting and chemical cross-linking, and to investigate the role of the hemagglutinin cytoplasmic sequence using a specific antibody, and 6) to devise an in vitro exocytosis assay based upon trypsin proteolysis of the hemagglutinin and the removal of sialic acid by the viral neuraminidase.
