Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/ E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins initiate membrane fusion through the formation of the SNARE complex, a process tightly regulated by Sec1/Munc18-1 (SM) proteins. The emerging trend is that SM proteins promote SNARE-mediated membrane fusion by binding to a Syntaxin N-terminal motif. Here we report that mutations in the hydrophobic pocket of Munc18-1 (F115E and E132A), predicted to disrupt the N-terminal Sx1a interaction have a modest effect on binding to Sx1a in its free state, but abolish binding to the SNARE complex. Overexpression of the Munc18-1 mutant in PC12 cells lacking Munc18-1 rescues both neuroexocytosis and the plasma membrane localization of Syntaxin. However, total internal reflection fluorescence microscopy analysis reveals that expression of a Munc18-1 double mutant reduces the rate of vesicle fusion, an effect only detectable at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore critical for SNARE complex binding. However, mutations abrogating this interaction have a limited impact on Ca 2؉ -dependent exocytosis in PC12 cells.Following stimulation of neurons, a number of well orchestrated protein/protein (1) and protein/lipid (2) interactions underpin the fusion of secretory vesicles with the presynaptic plasma membrane. In this sequence of interactions, vesicles approach the plasma membrane (tethering and docking), undergo priming and, upon Ca 2ϩ influx, fuse with the plasma membrane, thereby releasing neurotransmitter into the synaptic cleft (1). Vesicular exocytosis relies on the function of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) 2 proteins as demonstrated by the blockade of neuroexocytosis following SNARE protein cleavage by clostridial neurotoxins (3). One of the key players in SNARE regulation is the cytosolic regulatory protein, Munc18-1 (Munc18a, nsec-1) (4 -7). Although the function of SNARE proteins in mediating exocytosis is well established (2, 8), the precise role of Munc18-1 in exocytosis is still a subject of heated debate (6, 7, 9, 10).Munc18-1 belongs to the Sec1/Munc18 (SM) family of proteins that are involved in mediating membrane trafficking events (11-13). Mutations in these proteins have recently been associated with infantile epileptic encephalopathy (14). Although the function of Munc18-1 and its interaction with SNAREs have been studied for over 10 years, the molecular mechanism of Munc18-1 regulation of membrane fusion is still not clear. Munc18-1 was originally characterized as a negative regulator of exocytosis as it binds to the target membrane SNARE, Syntaxin 1a (Sx1a) (5) in a conformation that sequesters the Sx1a SNARE helix and inhibits SNARE complex formation (7, 15). Other SM proteins have been shown to bind to their cognate syntaxins via an N-terminal motif (16 -19), allowing interactions that are associated with a posit...

Striking correlations exist between the abilities of domain-1 cleft mutants of Munc18-1 to bind and chaperone syntaxin-1 and their ability to restore vesicle docking and secretion.

J. Neurochem. (2010) 115, 1–10. Abstract Munc18‐1 plays essential roles in neurosecretion by interacting with syntaxin‐1 and controlling the formation of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18‐1 have been proposed: (i) molecular chaperone of syntaxin‐1 for appropriate localization and expression of syntaxin‐1, (ii) priming/stimulation of the SNARE‐mediated membrane fusion, and (iii) docking of large dense‐core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18‐1 and syntaxin‐1: (i) binary binding to a ‘closed’ conformation of syntaxin‐1, and (ii) binding to the N‐terminal peptide of syntaxin‐1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18‐1 and closed syntaxin‐1. Recent structural analyses have identified critical Munc18‐1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18‐1 null neurons, chromaffin cells and Munc18‐1/‐2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18‐1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18‐1 in neuroexocytosis.

Phosphorylation of the DYD motif of myosin VI small insert isoform controls the sustainability of exocytosis by tethering secretory granules to the cortical actin network.

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SummaryMunc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1 D317-333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1 D317-333to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1 D317-333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1 D317-333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.