The composition and function of the plasma membrane is maintained by a complex intracellular traffic moving cell surface glycoproteins between organelles. This requires the recognition and sorting of different classes of proteins, not only during biosynthesis, but also during redistributive processes, such as the internalization and recycling of receptors during receptor-mediated endocytosis, transcytosis, or during the down-regulation of hormone receptors. Disease can result from the failure of any one of these processes, or from its subversion for use by a pathogen such as a virus. The focus of the work described in this application is to determine the molecular details of post-Golgi sorting events. By establishing the details of sorting events for a few, well- understood proteins such as the influenza virus hemagglutinin (HA), in a few cell lines, we intend to provide the basis for a comparison of the sorting events that occur in other cell types involving other proteins. Once the relationship between the sorting of different proteins in different cell types is understood, it may be possible to use this information for diagnostic purposes. Studies in vitro of cell types easily isolated from human patients might be used to learn about disease processes in organs for which biopsy material is limiting, difficult to isolate, or the cells difficult to maintain in culture. Recent observations suggest that there should be families of cytosolic proteins that control many intracellular sorting events by binding transiently to specific peptide sequences in the cytosolic domains of transmembrane proteins. The experiments proposed in this application will test that hypothesis by identifying those proteins, cloning their genes, and determining if sequence relationships exist. Specifically, experiments are proposed: (1) to identify cellular factors that interact with the sorting signals by identifying proteins that physically interact with fusion proteins containing sorting signals using both the yeast-two hybrid system and glutathione-S-transferase fusions; (2) to identify novel cytosolic sequences that influence the intracellular traffic of HA reporter proteins and to identify the proteins that bind to novel sorting signals: (3) to precisely define by site-directed mutagenesis the cytosolic sequence of HA+8 responsible for sorting that protein to lysosomes. (4) To determine the role of the transmembrane domain of HA for its intracellular transport in polarized and nonpolarized cells and for its incorporation into influenza virus virions.
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