Characterization of the stimulation of SNARE-mediated membrane fusion by the SM protein Munc18a

The mammalian neuronal Sec1p/Munc18 protein Muncl8a binds tightly to the t-SNARE Syntaxin1a in isolation, and the binary Muncl8a/Syntaxin1a interaction is thought to prevent formation of the Syntaxin1a/SNAP25b t-SNARE complex required for intracellular membrane fusion. However, both Sec1p and Munc18a are required factors for exocytosis and stimulate in vitro membrane fusion of the appropriate preassembled t-SNARE complex and v-SNARE. The experiments presented here show that Muncl8a stimulates the initial rate and final extent of membrane fusion driven by the neuronal t-SNAREs Syntaxin1a/SNAP25b and v-SNARE VAMP2 and elucidate the molecular mechanism underlying the increase in membrane fusion. Munc18a binds to the assembled neuronal t-SNARE complex in a functional manner much like its yeast homolog Sec1p. The Munc18a/t-SNARE complex interaction appears to occur through Syntaxin1a but not SNAP25b because Munc18a stimulates fusion of a Syntaxin1a/yeast Sec9c t-SNARE complex and fails to interact with yeast SNAREs. Munc18a displays a well-characterized interaction with the Syntaxin1a N-terminal regulatory domain (NRD), but also contacts the Syntaxin1a SNARE Core Complex (H3) domain to stimulate fusion because fusion of a yeast t-SNARE complex chimera in which the yeast Sso1p NRD was replaced with the Syntaxin1a NRD was not stimulated by Munc18a. Deletion of a flexible linker region between the Syntaxin1a NRD and H3 domain that permits movement of the NRD abolishes the ability of Munc18a to stimulate membrane fusion, suggesting that Munc1a may actively position the Syntaxin1a NRD to favor membrane fusion. In addition, Munc18a directly and functionally interacts with VAMP2. Taken together, these results suggest strongly that Munc18a acts as a scaffold that increases the efficiency of t-SNARE complex and v-SNARE interactions, resulting in accelerated membrane fusion. Furthermore, Munc18a may also function in combination with the calcium sensor Synaptotagmin I to enhance neurosecretion at the last stage of exocytosis.