Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca ²⁺, but Ca ²⁺-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca ²⁺ and restored Ca ²⁺-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca ²⁺-binding residues in C2A and C2B. Munc13-4 exhibited Ca ²⁺-stimulated SNARE interactions dependent on C2A and Ca ²⁺-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca ²⁺-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca ²⁺-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca ²⁺ sensor at ratelimiting priming steps in granule exocytosis.