SYNTHESIS & DISTRIBUTION OF PROTEINS IN MEMBRANES

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The overall goal of this proposal is to elucidate the sorting mechanisms that serve to establish the distinct protein compositions of the two plasma membrane domains of polarized epithelial cells. We wish to determine: i) how newly synthesized membrane proteins emerging from the trans Golgi network (TGN) are incorporated into specific vesicles, and, ii) how these vesicles are directed to one aspect of the cell surface. We propose to test a model in which certain proteins (Class I) sort by themselves by interaction in the TGN with adaptor molecules that recognize their cytoplasmic segments and lead to the incorporation of the proteins into vesicles directed to the plasma membrane. Other proteins, such as the HA of influenza and G of VSV, would be recognized by sorting receptors, which are Class I molecules that interact with the luminal domains of the proteins to be sorted and transport them to the cell surface. Polarized cell monolayers, perforated in one or the other surface, and a cell-free system, will be employed to identify, and ultimately purify, cellular components, including adaptor proteins and GTP-binding proteins and their cognate docking proteins, that participate in the formation and targeting of the transport vesicles. Complexes between the viral glycoproteins and their putative sorting receptors and the corresponding adaptors will be searched for in total cell extracts and subcellular fractions, including purified post Golgi vesicles. To investigate the role in sorting of the cytoplasmic segments of proteins that sort by themselves, peptides corresponding to the cytoplasmic tails of various receptors will be used in permeabilized cells and in the cell-free system, in attempts to specifically inhibit transport to one or the other aspect of the cell surface. The specific association of the cytoplasmic segments with adaptor-like molecules will be directly investigated by cross-linking and label-transfer techniques and by attempts to purify the adaptors using the cytoplasmic segments as affinity ligands. The perforated cell system will be used to examine the requirements for the formation of endocytic vesicles from each surface and for the transport of these vesicles within the cell that leads to their fusion with endosomes. The role of specific cytoskeletal elements, such as actin and fodrin, in endocytosis at each surface will be examined.