Munc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly

Abstract

Synaptic vesicle exocytosis in neurons requires SNARE complex formation during which VAMP2 from vesicles zippers with t-SNAREs (SNAP25 and Syntaxin1a) on the plasma membrane. Munc13-1 acts as a chaperone for this process by both tethering vesicles to PIP2-microdomains on the plasma membrane containing Syntaxin1a and by activating Syntaxin1a. How membrane-anchored SNAP25 enters this reaction is unknown. Here we report a novel function of Munc13 in the recruitment of SNAP25 to initiate the SNARE priming process. Using biochemical analyses, including quantitative binding studies, we show the MUN domain of Munc13 directly binds the SNAP25 loop region (connecting the two helices). Indeed, replacing the SNAP25 loop with the loop region of closely-related SNAP23 or with a non-specific GGGGS repeat sequence abrogated the Munc13 interaction. Interestingly, the Munc13 mutant NF->AA (1128/1131) that has been shown to be incapable of activating Syntaxin1a, still bound SNAP25 suggesting the binding sites on Munc13 for Syntaxin1a and SNAP25 are different. Furthermore, total internal reflection fluorescence (TIRF) microscopy analysis revealed that Munc13 co-clusters with SNAP25 on a supported lipid bilayer setup and these clusters typically contain 5-6 molecules each of Munc13 and SNAP25. This co-clustering was absent in the GGGGS mutant. Overall, our data suggest Munc13 recruits SNAP25 to the site of vesicle docking via direct MUN-SNAP25 loop interaction and by virtue of increasing its local concentration, it facilitates the initiation of the SNARE complex formation.