Regulation of SNARE-mediated membrane fusion by Sec1/Munc18 (SM) proteins
Scott, Brenton L.
McNew, James A.
Doctor of Philosophy
The intricate temporal and spatial regulation of membrane fusion is critical for all living organisms. Fusion of two opposing membranes occurs in a wide range of processes. These include intracellular transportation, cell-to-cell fusion and viral fusion. In all known cases, the SNARE proteins (Soluble NSF attachment protein receptors) (Sollner et al., 1993; Whiteheart et al., 1993) have been shown to be required for vesicular membrane fusion within cells and sufficient to drive membrane fusion in vitro (Nickel et al., 1999; Parlati et al., 1999; Weber et al., 1998). While SNAREs combine in specific combinations to drive highly specific membrane fusion, it is clear that SNARE proteins do not act independently to regulate the entire fusion process. Many regulatory proteins from different families have been identified that interact with individual SNARE proteins and SNARE complexes, yet the precise role of many of these remains unclear. One such group of regulatory proteins is the Sec1/Munc18 (SM) family. Sec1 proteins are likely to be critical players in membrane trafficking. My work has focused on the role of the yeast Sec1p in post-Golgi secretion in Saccharomyces cerevisiae. To analyze Sec1p function in vitro, I have utilized a well-characterized SNARE-mediated membrane fusion assay. For this application, conditions were optimized to allow for specific protein-protein interactions to be tested. Conditions for expression and purification of the previously elusive recombinant Sec1p are documented. In addition, an overexpressing Sec1p yeast strain was generated. Sec1p interactions with SNARE proteins that mediate post-Golgi secretion were then tested. I found that recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p;Sec9c) as well as to the fully assembled ternary-SNARE complex (Sso1p;Sec9c/Snc2p), and also weakly to free Sso1p. I tested the ability of Sec1p to regulate fusion in the fusion assay. Concentration dependent stimulation of membrane fusion is observed when Sec1p is associated with the SNARE proteins. The binding and fusion data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion. With this new information, specific binding modes of neuronal-Sec1 are currently being investigated further in yeast, Drosophila and mammalian SNARE systems.