Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion

Abstract

Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion.

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

“…Munc18c selectively activated the fusion reactions reconstituted with its cognate SNARE isoforms, in agreement with the pathway-specific activities of SM proteins observed in vivo (16). +1 +2 +3 +4 +5 +6 +7 +8 39 42 46 49 53 56 60 63 67 70 74 77 81 84 Munc18c recognition (12,26,27,30,31). However, it was unclear whether this stimulatory function represents a conserved mechanism of SM proteins.…”

“…Commonly used solution binding assays (e.g., ITC measurements) only examine the binding of SM proteins to the cis-SNARE complex, and thus cannot reflect the dynamic SM/ SNARE interactions en route to fusion. The binding of SM proteins to the trans-SNARE involves multiple motifs on each of the distinct subunits in the context of the SNARE complex (12,25,26,30). Therefore, measurements of SM binding to a single SNARE subunit in isolation may not be representative of the interaction in the content of the trans-SNARE complex, which likely underlies the observed heterogeneity in the SM/SNARE binding modes.…”

“…Munc18-1 has been shown to play dual roles in synaptic vesicle fusion. First, Munc18-1 positively regulates the SNARE-dependent fusion reaction by interacting with the trans-SNARE complex and accelerating the fusion kinetics (12,(25)(26)(27)(28)(29)(30)(31)(32)(33). Second, Munc18-1 binds to syntaxin-1 monomer and locks the latter into a "closed" configuration that prevents SNARE complex formation (34-36).…”

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

“…2). This interaction has been demonstrated using both pull-down experiments and liposomal fusion assays (Table 1) (Dá ak et al, 2009;Latham et al, 2006;Rodkey et al, 2008;Shen et al, 2007). Chemical cross-linking and two-dimensional NMR spectroscopy experiments have confirmed this interaction of Munc18a with an assembled SNARE complex (Dulubova et al, 2007;Shen et al, 2007).…”

“…On the other hand, soluble Sx1a (1-262) that has a C-terminus free in solution was unable to form a SNARE complex in the presence of Munc18a (Burkhardt et al, 2008) (Table 1). Similarly, when Sx1a is anchored via its C-terminal TMD onto liposomes, Munc18a promotes vesicle fusion in liposomal fusion assays Rodkey et al, 2008;Shen et al, 2010Shen et al, , 2007Tareste et al, 2008) (Table 1).…”