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.     

Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex

Sec1/Munc18 proteins play a fundamental role in multiple steps of intracellular membrane trafficking. Dual functions have been attributed to Munc18-1: it can act as a chaperone when it interacts with monomeric syntaxin 1A, and it can activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion when it binds to SNARE complexes. Although both modes of binding involve the central cavity of Munc18-1, their precise molecular mechanisms of action are not fully understood. In this paper, we describe a novel Munc18-1 mutant in the central cavity that showed a reduced interaction with syntaxin 1A and impaired chaperone function, but still bound to assembled SNARE complexes and promoted liposome fusion and secretion in neuroendocrine cells. Soluble syntaxin 1A H3 domain partially blocks Munc18-1 activation of liposome fusion by occupying the Munc18-1 central cavity. Our findings lead us to propose a transition model between the two distinct binding modes by which Munc18 can control and assist in SNARE-complex assembly during neurotransmitter release.     

Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation

Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.     

An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Complex Assembly

Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended α-helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.     

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.     

Syntaxin-1蛋白的多重功能(英文)

Syntaxin-1是特异性地分布在神经细胞突触前质膜上的蛋白。它早期被作为分子量为35 kD的synaptotagmin-1结合蛋白,但很快就被认识到是细胞质膜融合的关键蛋白。Syntaxin-1通过与SNAP25和Synaptobrevin/VAMP蛋白聚合,进而形成被认为是神经突触囊泡融合必要因子的SNARE核心复合体。作为一个多结构域的蛋白,syntaxin-1与多个突触蛋白相互作用,其作用远超出了仅作为SNARE核心复合体中一个蛋白质成员的作用。本文着重介绍了有关syntaxin-1与其它SNARE组份蛋白、munc18蛋白和钙离子通道的相互作用及其功能的最新研究进展。全面揭示syntaxin-1作为SNARE核心复合体成员的功能以及超越这一功能的作用,还有待于对其结构以及与其它突触蛋白相互作用特性的进一步深刻理解。     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Binding of the Munc13-1 MUN domain to membrane-anchored SNARE complexes

The core of the membrane fusion machinery that governs neurotransmitter release includes the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin, which form a tight "SNARE complex", and Munc18-1, which binds to the SNARE complex and to syntaxin-1 folded into a closed conformation. Release is also controlled by specialized proteins such as complexins, which also bind to the SNARE complex, and unc13/Munc13s, which are crucial for synaptic vesicle priming and were proposed to open syntaxin-1, promoting SNARE complex assembly. However, the biochemical basis for unc13/Munc13 function and its relationship to other SNARE interactions are unclear. To address this question, we have analyzed interactions of the MUN domain of Munc13-1, which is key for this priming function, using solution binding assays and cofloatation experiments with SNARE-containing proteoliposomes. Our results indicate that the Munc13-1 MUN domain binds to membrane-anchored SNARE complexes, even though binding is barely detectable in solution. The MUN domain appears to compete with Munc18-1 but not with complexin-1 for SNARE complex binding, although more quantitative assays will be required to verify these conclusions. Moreover, our data also uncover interactions of membrane-anchored syntaxin-1/SNAP-25 heterodimers with the MUN domain, Munc18-1 and complexin-1. The interaction with complexin-1 is surprising, as it was not observed in previous solution studies. Our results emphasize the importance of studying interactions within the neurotransmitter release machinery in a native membrane environment, and suggest that unc13/Munc13s may provide a template to assemble syntaxin-1/SNAP-25 heterodimers, leading to an acceptor complex for synaptobrevin.     

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Munc13‐1 is crucial for neurotransmitter release and, together with Munc18‐1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin‐1, SNAP‐25, and synaptobrevin. Assembly starts with syntaxin‐1 folded into a self‐inhibited closed conformation that binds to Munc18‐1. Munc13‐1 is believed to catalyze the opening of syntaxin‐1 to facilitate SNARE complex formation. However, different types of Munc13‐1‐syntaxin‐1 interactions have been reported to underlie this activity, and the critical nature of Munc13‐1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13‐1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin‐1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13‐1 fragments, even though binding of this linker region to Munc13‐1 is barely detectable. Conversely, the syntaxin‐1 SNARE motif clearly binds to Munc13‐1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13‐1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13‐1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin‐1 via interactions with the linker.     

An InCytes from MBC Selection: Yeast Sec1p Functions before and after Vesicle Docking

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.     

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

WNK1 is a novel regulator of Munc18c-syntaxin 4 complex formation in soluble NSF attachment protein receptor (SNARE)-mediated vesicle exocytosis

Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159-491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.     

NMR analysis of the closed conformation of syntaxin-1

The Sec1/Munc18 (SM) protein Munc18-1 and the SNAREs syntaxin-1, SNAP-25 and synaptobrevin form the core of the membrane fusion machinery that triggers neurotransmitter release. Munc18-1 binds to syntaxin-1 folded into a closed conformation and to the SNARE complex formed by the three SNAREs, which involves an open syntaxin-1 conformation. The former interaction is likely specialized for neurotransmitter release, whereas SM protein/SNARE complex interactions are likely key for all types of intracellular membrane fusion. It is currently unclear whether the closed conformation is highly or only marginally populated in isolated syntaxin-1, and whether Munc18-1 stabilizes the close conformation or helps to open it to facilitate SNARE complex formation. A detailed NMR analysis now suggests that the closed conformation is almost quantitatively populated in isolated syntaxin-1 in the absence of oligomerization, and indicates that its structure is very similar to that observed previously in the crystal structure of the Munc18-1/syntaxin-1 complex. Moreover, we demonstrate that Munc18-1 binding prevents opening of the syntaxin-1 closed conformation. These results support a model whereby the closed conformation constitutes a key intrinsic property of isolated syntaxin-1 and Munc18-1 binding stabilizes this conformation; in this model, Munc18-1 plays in addition an active role in downstream events after another factor(s) helps to open the syntaxin-1 conformation.     

Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet β-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and β-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ～20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in β-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.     

Selective activation of cognate SNAREpins by Sec1/Munc18 proteins

Sec1/Munc18 (SM) proteins are required for every step of intracellular membrane fusion, but their molecular mechanism of action has been unclear. In this work, we demonstrate a fundamental role of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery. In a reconstituted system, mammalian SNARE pairs assemble between bilayers to drive a basal fusion reaction. Munc18-1/nSec1, a synaptic SM protein required for neurotransmitter release, strongly accelerates this reaction through direct contact with both t- and v-SNAREs. Munc18-1 accelerates fusion only for the cognate SNAREs for exocytosis, therefore enhancing fusion specificity.     

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP‐25

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE‐mediated membrane fusion. Recently, a new protein–protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature‐sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18‐1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP‐25b, respectively. Incubation of SNAP‐25b with the Munc18‐1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild‐type Munc18‐1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p–SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP‐25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.      

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

A conserved regulatory mode in exocytic membrane fusion revealed by Mso1p membrane interactions

Sec1/Munc18 family proteins are important components of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex–mediated membrane fusion processes. However, the molecular interactions and the mechanisms involved in Sec1p/Munc18 control and SNARE complex assembly are not well understood. We provide evidence that Mso1p, a Sec1p- and Sec4p-binding protein, interacts with membranes to regulate membrane fusion. We identify two membrane-binding sites on Mso1p. The N-terminal region inserts into the lipid bilayer and appears to interact with the plasma membrane, whereas the C-terminal region of the protein binds phospholipids mainly through electrostatic interactions and may associate with secretory vesicles. The Mso1p membrane interactions are essential for correct subcellular localization of Mso1p–Sec1p complexes and for membrane fusion in Saccharomyces cerevisiae. These characteristics are conserved in the phosphotyrosine-binding (PTB) domain of β-amyloid precursor protein–binding Mint1, the mammalian homologue of Mso1p. Both Mint1 PTB domain and Mso1p induce vesicle aggregation/clustering in vitro, supporting a role in a membrane-associated process. The results identify Mso1p as a novel lipid-interacting protein in the SNARE complex assembly machinery. Furthermore, our data suggest that a general mode of interaction, consisting of a lipid-binding protein, a Rab family GTPase, and a Sec1/Munc18 family protein, is important in all SNARE-mediated membrane fusion events.