Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptide

Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.     

SNARE assembly and membrane fusion, a kinetic analysis

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) assembly may promote intracellular membrane fusion, an essential process for vesicular transport in cells. Core complex formation between vesicle-associated SNARE and target membrane SNARE perhaps drives the merging of two membranes into a single bilayer. Using spin-labeling EPR, trans-SNARE complex formation was monitored "locally" at four different core locations of recombinant yeast SNAREs, which are individually reconstituted into phospholipid vesicles. The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the "zipper" model. The EPR data were then compared with the kinetics of the lipid mixing measured with the fluorescence assay. The analysis suggests that SNARE core assembly occurs on a much faster time scale than the lipid mixing, providing a new insight into the timing of individual events in SNARE-induced membrane fusion.     

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H_abc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H_abc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H_abc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q_bc configuration. We found that neither the linker nor the Q_bc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.     

Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.     

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H_abc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.     

Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.     

Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca²⁺-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1–SNARE–complexin macromolecular assembly that is poised for Ca²⁺ triggering of fusion.     

Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

How the Sec1/Munc18-syntaxin complex might transition to form the SNARE core complex remains unclear. Toward this, Munc18c tyrosine phosphorylation has been correlated with its dissociation from syntaxin 4. Using 3T3-L1 adipocytes subjected to small interfering ribonucleic acid reduction of Munc18c as a model of impaired insulin-stimulated GLUT4 vesicle exocytosis, we found that coordinate expression of Munc18c-wild type or select phosphomimetic Munc18c mutants, but not phosphodefective mutants, restored GLUT4 vesicle exocytosis, suggesting a requirement for Munc18c tyrosine phosphorylation at Tyr219 and Tyr521. Surprisingly, the insulin receptor (IR) tyrosine kinase was found to target Munc18c at Tyr521 in vitro, rapidly binding and phosphorylating endogenous Munc18c within adipocytes and skeletal muscle. IR, but not phosphatidylinositol 3-kinase, activation was required. Altogether, we identify IR as the first known tyrosine kinase for Munc18c as part of a new insulin-signaling step in GLUT4 vesicle exocytosis, exemplifying a new model for the coordination of SNARE assembly and vesicle mobilization events in response to a single extracellular stimulus.     

Sec1/Munc18 protein stabilizes fusion-competent syntaxin for membrane fusion in Arabidopsis cytokinesis

Intracellular membrane fusion requires complexes of syntaxins with other SNARE proteins and regulatory Sec1/Munc18 (SM) proteins. In membrane fusion mediating, e.g., neurotransmitter release or glucose-stimulated insulin secretion in mammals, SM proteins preferentially interact with the inactive closed, rather than the active open, conformation of syntaxin or with the assembled SNARE complex. Other membrane fusion processes such as vacuolar fusion in yeast involve like membranes carrying cis-SNARE complexes, and the role of SM protein is unknown. We investigated syntaxin-SM protein interaction in membrane fusion of Arabidopsis cytokinesis, which involves cytokinesis-specific syntaxin KNOLLE and SM protein KEULE. KEULE interacted with an open conformation of KNOLLE that complemented both knolle and keule mutants. This interaction occurred at the cell division plane and required the KNOLLE linker sequence between helix Hc and SNARE domain. Our results suggest that in cytokinesis, SM protein stabilizes the fusion-competent open form of syntaxin, thereby promoting trans-SNARE complex formation.     

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca²⁺ sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca²⁺-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.     

Regulation of neuronal SNARE assembly by the membrane

In the neuron, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) assembly acts centrally in driving membrane fusion, a required process for neurotransmitter release. In the cytoplasm, vesicular SNARE VAMP-2 (vesicle-associated membrane protein-2) engages with two plasma membrane SNAREs, syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa), to form the core complex that bridges two membranes. Although various factors regulate SNARE assembly, the membrane also aids in regulation by trapping VAMP-2 in the membrane. Fluorescence and EPR analyses revealed that the insertion of seven C-terminal core-forming residues into the membrane controls complex formation of the entire core region, even though the preceding 54 core-forming residues are fully exposed and freely moving. When two interfacial tryptophan residues in this region were replaced with hydrophilic serine residues, the mutation supported rapid complex formation. The results suggest that the membrane-proximal region of VAMP-2 is a regulatory module for SNARE assembly, providing new insights into calcium-triggered membrane fusion.     

Munc18-1 Controls SNARE Protein Complex Assembly during Human Sperm Acrosomal Exocytosis

The spermatozoon is a very specialized cell capable of carrying out a limited set of functions with high efficiency. Sperm are then excellent model cells to dissect fundamental processes such as regulated exocytosis. The secretion of the single dense-core granule of mammalian spermatozoa relies on the same highly conserved molecules and goes through the same stages as exocytosis in other types of cells. In this study, we describe the presence of Munc18-1 in human sperm and show that this protein has an essential role in acrosomal exocytosis. We observed that inactivation of endogenous Munc18-1 with a specific antibody precluded the stabilization of trans-SNARE complexes and inhibited acrosomal exocytosis. Addition of recombinant Munc18-1 blocked secretion by sequestering monomeric syntaxin, an effect that was rescued by α-soluble NSF attachment protein. By electron microscopy, we observed that both the anti-Munc18-1 antibody and recombinant Munc18-1 inhibited the docking of the acrosome to the plasma membrane. In conclusion, our results indicate that Munc18-1 plays a key role in the dynamics of trans-SNARE complex assembly and/or stabilization, a process that is necessary for the docking of the outer acrosomal membrane to the plasma membrane and subsequent fusion pore opening.     

Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

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 rate-limiting priming steps in granule exocytosis.     

Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

During exocytosis a four-helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C-terminal end affected fusion triggering in vivo and led to two-step unfolding of the SNARE complex in vitro, indicating that the C-terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C-terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N-terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N- to C-terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.