Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

20Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, 21 but their mechanism(s) of action remain controversial. Using single-molecule force 22 spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of 23 three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. 24 Catalysis requires formation of an intermediate template complex in whichMunc18-1 25 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while 26 keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs 27to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. 28 Munc18-1 mutations modulate the stability of the template complex in a manner consistent 29with their effects on membrane fusion, indicating that chaperoned SNARE assembly is 30 essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone 31 SNARE assembly via a template complex, suggesting that SM protein mechanism is 32 conserved. 33 34 lymphocytes to kill cancerous or infected cells (Cote et al., 2009) and for glucose uptake (Bryant 43 and Gould, 2011), respectively. Consequently, dysfunctions of SM proteins are associated with 44 neurological and immunological disorders, cancers, diabetes, and other diseases (Bryant and 45

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depolarization to synchronous neurotransmitter release. Syt1 is a vesiculartethered protein, with two homologous C2A and C2B domains attached through a juxtamembrane linker. Upon Ca 2þ influx, loops on the two domains bind Ca 2þ and insert into charged membrane. The C2B domain contains other regions capable of membrane interaction, including the lysine rich polybasic face. Syt1 can interact with either bilayer surface, binding cis-to the SV membrane, or trans-to the PM. Cis-binding likely plays an inhibitory role by back-binding both domains to the SV preventing membrane fusion. Trans-binding then permits fusion, docking the SV to the PM surface. These membrane interactions are lipid specific in order to drive membrane bridging. On the synaptic vesicle surface, only phosphatidylserine (PS) contributes negative charge, while on the PM both PS and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) contribute to the charge density. Through various EPR techniques, we explored cis-and trans-binding under various physiologically relevant lipid, salt, and ionic compositions, to determine if Syt1 may act as a distance regulator between the SV and PM. A series of methods were developed to determine and differentiate membrane insertion of the domains in the full-length protein. With this, we characterized an ATP and PIP 2 competition to the polybasic face which blocks or promotes transbinding. We also characterized the juxtamembrane linker and the preferential binding Synaptic vesicles fuse with the plasma membrane to release neurotransmitter both synchronously and asynchronously following an action potential. How many vesicles can fuse at a single active zone, and where these vesicles fuse within the active zone, is not well understood, particularly in the context of these two phases. To capture synaptic vesicle exocytosis during synchronous and asynchronous release at cultured mouse hippocampal synapses, we induced single action potentials (AP) by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release (<5 ms after AP) multiple vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing the extracellular calcium concentration. Synchronous fusions are distributed throughout the active zone, whereas asynchronous fusions (5-11 ms after AP) are biased toward the center of the active zone. As exocytosis proceeds, new vesicles are recruited to the active zone and fully replenish the docked pool, but docking of these vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that multivesicular release occurs even at low-release-probability synapses and suggest a spatial organization underlying synchronous and asynchronous release. Synapsin (Syn), a family of phosphoproteins are found abundantly in neurons of both vertebrate and invertebrate systems. Syn is located in the nervous terminals and regulates the availability of synaptic vesicles by reversible associ...