Transport vesicle docking and fusion with target membranes appears to be mediated by strong, oligomeric protein complexes between vesicle and target membrane SNAP receptors (SNARES). SNARE mechanisms in mammalian ER to Golgi transport are not well known. Understanding ER/Golgi SNARE protein interactions and their functional roles will advance our general knowledge about how cells handle any membrane trafficking event. It will also provide specific knowledge and reagents that can potentially be used to manipulate ER to Golgi transport. In some instances it may be medically beneficial to block or reduce secretion, for example during viral infection and secretion of autostimulatory agents by tumors. The proposed studies will characterize protein interactions among the mammalian ER/Golgi SNARES syntaxin 5, GOS-28, membrin, rsec22b, rbet1 and others. Binding assays employing soluble, purified recombinant SNAREs will be used to define all of the direct binding events among this set of SNARES. Structural determinants for each interaction will be mapped. The pattern of binding cooperativity among these SNARES will suggest which subsets of proteins assemble simultaneously into higher-order functional complexes that direct fusion events. The proposed work will characterize the subunit composition and stoichiometry of ER/Golgi SNARE complex(es). The work also seeks to determine the functional role(s) of ER/Golgi SNARE interactions. Reagents will be developed that inhibit or disrupt particular sets of SNARE interactions. The physiological consequences of disrupting the interactions will be explored using an in vitro ER to Golgi transport reconstitution. The stage in transport at which different SNARE interactions are required will be characterized by light microscopy and subcellular fractionation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Shapiro, Bert I
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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