Project Summary 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 (α-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.