Membrane traffic is among the most ancient innovations of eukaryotic cells. It is central to neurotransmission, immune signaling, and normal development, and disrupted in a wide array of infectious and degenerative diseases. The fusion of transport vesicles with target membranes is central to many trafficking processes and employs a core and conserved machinery: three or four SNARE proteins, the disassembly chaperones Sec17 (?-SNAP) and Sec18 (NSF), and proteins of the SM (Sec1/Munc18) family. The mechanism of SM protein function is not understood, though several SM proteins are associated with human infectious disease, neurodegeneration, neutropenia, and diabetes. The overall goal of this Project is to scrutinize the emerging hypothesis that the biochemical basis of SM function entails not only SM?SNARE interactions but an elegant network of physical and functional interactions among SNAREs, SMs, Sec17, and Sec18, operating in tightly coupled assembly and disassembly reactions. Much of the recent work on SM proteins and Sec17 has been done in vitro. This Project combines powerful in vitro biochemical approaches with state-of-the-art in vivo structure?function analyses that are currently feasible only in Saccharomyces cerevisiae.
In Aim 1, the mechanisms of SM protein activation and activity are assessed in vivo and in vitro.
In Aim 2, Sec17 interactions with SNAREs, Sec18, and specific SMs are assessed.
In Aim 3, biophysical techniques are applied to elucidate the architecture of SM assembly with SNAREs and with Sec17. These studies will test general and deep hypotheses about the conserved mechanisms of SNARE-mediated membrane fusion, with particular emphasis on comparisons between two different transport steps, and between in vitro results and stringent and quantitative in vivo structure? function analyses.
The specific proteins studied in this proposal are central to the organization of the endomembrane system. For example, Vps33 is one of two human proteins most critical for Ebola and Marburg virus infection, and is also centrally implicated in nutrient sensing and in one variant of the Charcot-Marie-Tooth neurodegenerative disease. Our experiments will clarify how Vps33 and related proteins function, and will illuminate fundamental mechanisms that contribute to infectious disease, immunity, neurotransmission, neurodegeneration, and endocrine function and dysfunction.
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