Mast cell exocytosis, which includes compound degranulation and vesicle-associated piecemeal degranulation, requires multiple Q- and R- SNAREs. It is not clear how these SNAREs pair to form functional trans-SNARE complexes and how these trans-SNARE complexes are selectively regulated for fusion. Here we undertake a comprehensive examination of the capacity of two Q-SNARE subcomplexes (syntaxin3/SNAP-23 and syntaxin4/SNAP-23) to form fusogenic trans-SNARE complexes with each of the four granule-borne R-SNAREs (VAMP2, 3, 7, 8). We report the identification of at least six distinct trans-SNARE complexes under enhanced tethering conditions: i) VAMP2/syntaxin3/SNAP-23, ii) VAMP2/syntaxin4/SNAP-23, iii) VAMP3/syntaxin3/SNAP-23, iv) VAMP3/syntaxin4/SNAP-23, v) VAMP8/syntaxin3/SNAP-23, and vi) VAMP8/syntaxin4/SNAP-23. We show for the first time that Munc18a operates synergistically with SNAP-23-based non-neuronal SNARE complexes (i to iv) in lipid mixing, in contrast to Munc18b and c, which exhibit no positive effect on any SNARE combination tested. Pre-incubation with Munc18a renders the SNARE-dependent fusion reactions insensitive to the otherwise inhibitory R-SNARE cytoplasmic domains, suggesting a protective role of Munc18a for its cognate SNAREs. Our findings substantiate the recently discovered but unexpected requirement for Munc18a in mast cell exocytosis, and implicate post-translational modifications in Munc18b/c activation.

Sec1-Munc18 (SM) proteins cooperate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.