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1.  WNK1 Is a Novel Regulator of Munc18c-Syntaxin 4 Complex Formation in Soluble NSF Attachment Protein Receptor (SNARE)-mediated Vesicle Exocytosis* 
The Journal of biological chemistry  2007;282(45):32613-32622.
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.
doi:10.1074/jbc.M706591200
PMCID: PMC2423411  PMID: 17848561
2.  Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells 
Molecular Biology of the Cell  2009;20(23):4962-4975.
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.
doi:10.1091/mbc.E09-08-0712
PMCID: PMC2785739  PMID: 19812250
3.  Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding 
PLoS Computational Biology  2011;7(3):e1001097.
The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.
Author Summary
Protein phosphorylation plays a significant regulatory role in multi-component systems engaged in signal transduction or coordination of cellular processes, by activating or deactivating proteins. The potential of phosphorylation to induce substantial conformational changes in proteins, thereby changing their affinity to target proteins, has already been shown but the dynamics of the process is not fully elucidated. In the present study, we investigated, by molecular dynamics simulations, the dynamic conformational changes in munc18a, a protein that is crucial for neurotransmitter release and interacts tightly with the SNARE syntaxin-1. We further investigated the conformational changes that occur in munc18a when it is phosphorylated, reducing its affinity to syntaxin-1a. The results of the simulations suggest that there is a conformational flexibility of the syntaxin-unbounded munc18a that allows changes in the shape of the syntaxin-1a binding cavity. In silico insertion of phosphomimetic mutations into munc18a led to a reduction in the flexibility and closure of the syntaxin-binding site. We suggest that the reduced affinity of phosphorylated munc18a to syntaxin-1a stems from the difficulty of syntaxin-1a to bind to the munc18a closed-cavity conformation, induced by the PKC phosphorylation of munc18a.
doi:10.1371/journal.pcbi.1001097
PMCID: PMC3048386  PMID: 21390273
4.  Munc18c Depletion Selectively Impairs the Sustained Phase of Insulin Release 
Diabetes  2009;58(5):1165-1174.
OBJECTIVE
The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4–mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mechanism(s) using the islet β-cell as a model system.
RESEARCH DESIGN AND METHODS
Perifusion analyses of isolated Munc18c- (−/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization.
RESULTS
Munc18c (−/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (−/+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2–Syntaxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion.
CONCLUSIONS
Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion.
doi:10.2337/db08-1059
PMCID: PMC2671048  PMID: 19188424
5.  Munc18/SNARE proteins’ regulation of exocytosis in guinea pig duodenal Brunner’s gland acini 
AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell.
METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy.
RESULTS: One and 6 μmol/L carbachol evoked a dose-dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact.
CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.
doi:10.3748/wjg.14.2314
PMCID: PMC2705084  PMID: 18416456
Apical exocytosis; Brunner’s gland acini; Munc18c; Syntaxin-4; Carbachol
6.  A Mechanism of Munc18b-Syntaxin 3-SANP25 complex assembly in regulated epithelial secretion 
FEBS letters  2007;581(22):4318-4324.
Syntaxin and Munc18 are essential for regulated exocytosis in all eukaryotes. It was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by CDK5 phosphorylation, indicating that structural change disrupts the syntaxin-Munc18 interaction. Here we show that this phosphorylation promotes the assembly of Munc18b-Syntaxin 3-SNAP25 tripartite complex and membrane fusion machinery SNARE. Using siRNAs to screen for genes required for regulated epithelial secretion, we identified the requirements of CDK5 and Munc18b in cAMP-dependent gastric acid secretion. Biochemical characterization revealed that Munc18b bears a syntaxin 3-selective binding site located at its most C-terminal 53 amino acids. Significantly, the phosphorylation of Thr572 by CDK5 attenuates Munc18b-Syntaxin 3 interaction and promotes formation of Munc18b-Syntaxin 3-SNAP25 tripartite complex, leading to an assembly of functional Munc18b-Syntaxin 3-SNAP25-VAMP2 membrane fusion machinery. Thus, our studies suggest a novel regulatory mechanism in which phosphorylation of Munc18b operates vesicle docking and fusion in regulated exocytosis.
doi:10.1016/j.febslet.2007.07.083
PMCID: PMC3690314  PMID: 17716669
SNARE; syntaxin 3; Munc18b; CDK5; exocytosis
7.  Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice. 
Journal of Clinical Investigation  1997;100(12):3044-3052.
Rab3D, a member of the ras-related GTP-binding protein Rab family, is localized to secretory granules of various exocrine tissues such as acinar cells of the pancreas, chief cells of the stomach, and parotid and lacrimal secretory cells. To elucidate the function of Rab3D in exocytosis, we have generated transgenic mice that over-express Rab3D specifically in pancreatic acinar cells. Hemagglutinin-tagged Rab3D was localized to zymogen granules by immunohistochemistry, and was shown to be present on zymogen granule membranes by Western blotting; both results are similar to previous studies of endogenous Rab3D. Secretion measurements in isolated acinar preparations showed that overexpression of Rab3D enhanced amylase release. Amylase secretion from intact acini of transgenic mice 5 min after 10 pM cholecystokinin octapeptide (CCK) stimulation was enhanced by 160% of control. In streptolysin-O-permeabilized acini of transgenic mice, amylase secretion induced by 100 microM GTP-gamma-S was enhanced by 150%, and 10 microM Ca2+-stimulated amylase secretion was augmented by 206% of that of the control. To further elucidate Rab3D involvement in stimulus-secretion coupling, we examined the effect of CCK on the rate of GTP binding to Rab3D. Stimulation of permeabilized acini with 10 pM CCK increased the incorporation of radiolabeled GTP into HA-tagged Rab3D. These results indicate that overexpression of Rab3D enhances secretagogue-stimulated amylase secretion through both calcium and GTP pathways. We conclude that Rab3D protein on zymogen granules plays a stimulatory role in regulated amylase secretion from pancreatic acini.
PMCID: PMC508517  PMID: 9399951
8.  Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites* 
The Journal of Biological Chemistry  2012;288(7):5102-5113.
Background: The molecular architecture of the secretory machinery is undefined.
Results: Munc18 moves between membrane depots distinct from vesicle docking sites.
Conclusion: Munc18 is not a docking factor, and the membrane environment is likely to determine fusion likelihood.
Significance: It is now possible to test directly previous models of the molecular mechanisms of secretion.
Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.
doi:10.1074/jbc.M112.407585
PMCID: PMC3576115  PMID: 23223447
Membrane Biophysics; Membrane Function; Membrane Proteins; Membrane Trafficking; Vesicles
9.  Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex 
Molecular Biology of the Cell  2011;22(21):4150-4160.
The Munc18 central cavity plays a major role in trafficking syntaxin 1 (Stx 1) to the plasma membrane and in activating SNARE-mediated membrane fusion. This paper provides critical insight into the mechanisms of how the Stx1A H3 domain can compete with the SNARE complex for binding the Munc18 central cavity, first inhibiting, and later assisting, SNARE-complex assembly.
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.
doi:10.1091/mbc.E11-02-0150
PMCID: PMC3204075  PMID: 21900493
10.  Munc18b Is a Major Mediator of Insulin Exocytosis in Rat Pancreatic β-Cells 
Diabetes  2013;62(7):2416-2428.
Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic β-cells to exocytose insulin becomes compromised in diabetes. β-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted β-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6–8 SGs) not normally (2–3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of β-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.
doi:10.2337/db12-1380
PMCID: PMC3712044  PMID: 23423569
11.  Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization 
Journal of Clinical Investigation  2005;115(2):291-301.
Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c−/− mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c−/− cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c−/− cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.
doi:10.1172/JCI200522681
PMCID: PMC546422  PMID: 15690082
12.  Doc2b Is a Key Effector of Insulin Secretion and Skeletal Muscle Insulin Sensitivity 
Diabetes  2012;61(10):2424-2432.
Exocytosis of intracellular vesicles, such as insulin granules, is carried out by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. An additional regulatory protein, Doc2b (double C2 domain), has recently been implicated in exocytosis from clonal β-cells and 3T3-L1 adipocytes. Here, we investigated the role of Doc2b in insulin secretion, insulin sensitivity, and the maintenance of whole-body glucose homeostasis. Doc2b heterozygous (Doc2b+/−) and homozygous (Doc2b−/−) knockout mice exhibited significant whole-body glucose intolerance and peripheral insulin resistance, compared with wild-type littermates. Correspondingly, Doc2b+/− and Doc2b−/− mice exhibited decreased responsiveness of pancreatic islets to glucose in vivo, with significant attenuation of both phases of insulin secretion ex vivo. Peripheral insulin resistance correlated with ablated insulin-stimulated glucose uptake and GLUT4 vesicle translocation in skeletal muscle from Doc2b-deficient mice, which was coupled to impairments in Munc18c-syntaxin 4 dissociation and in SNARE complex assembly. Hence, Doc2b is a key positive regulator of Munc18c-syntaxin 4–mediated insulin secretion as well as of insulin responsiveness in skeletal muscle, and thus a key effector for glucose homeostasis in vivo. Doc2b’s actions in glucose homeostasis may be related to its ability to bind Munc18c and/or directly promote fusion of insulin granules and GLUT4 vesicles in a stimulus-dependent manner.
doi:10.2337/db11-1525
PMCID: PMC3447898  PMID: 22698913
13.  Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen 
The Journal of Cell Biology  1996;133(2):257-268.
We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142- 19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium- binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.
PMCID: PMC2120788  PMID: 8609160
14.  Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4 
BMC Biochemistry  2011;12:19.
Background
Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4.
Results
We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro. Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c (Y521E).
Conclusion
Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic.
doi:10.1186/1471-2091-12-19
PMCID: PMC3103433  PMID: 21548926
15.  Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion 
Molecular Biology of the Cell  2008;19(12):5422-5434.
Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion.
doi:10.1091/mbc.E08-05-0538
PMCID: PMC2592651  PMID: 18829865
16.  Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes  
PLoS Biology  2006;4(10):e330.
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.
The authors take a fresh look at the role of Munc-18, an SM protein, in vesicle fusion and find that it facilitates the formation of fusion-competent SNARE complexes by binding to syntaxin.
doi:10.1371/journal.pbio.0040330
PMCID: PMC1570500  PMID: 17002520
17.  Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane 
Molecular Biology of the Cell  2011;22(21):4134-4149.
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.
Munc18-1 plays pleiotropic roles in neurosecretion by acting as 1) a molecular chaperone of syntaxin-1, 2) a mediator of dense-core vesicle docking, and 3) a priming factor for soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion. However, how these functions are executed and whether they are correlated remains unclear. Here we analyzed the role of the domain-1 cleft of Munc18-1 by measuring the abilities of various mutants (D34N, D34N/M38V, K46E, E59K, K46E/E59K, K63E, and E66A) to bind and chaperone syntaxin-1 and to restore the docking and secretion of dense-core vesicles in Munc18-1/-2 double-knockdown cells. We identified striking correlations between the abilities of these mutants to bind and chaperone syntaxin-1 with their ability to restore vesicle docking and secretion. These results suggest that the domain-1 cleft of Munc18-1 is essential for binding to syntaxin-1 and thereby critical for its chaperoning, docking, and secretory functions. Our results demonstrate that the effect of the alleged priming mutants (E59K, D34N/M38V) on exocytosis can largely be explained by their reduced syntaxin-1–chaperoning functions. Finally, our data suggest that the intracellular expression and distribution of syntaxin-1 determines the level of dense-core vesicle docking.
doi:10.1091/mbc.E11-02-0135
PMCID: PMC3204074  PMID: 21900502
18.  Role of tissue transglutaminase type 2 in calbindin-D28k interaction with ataxin-1 
Neuroscience letters  2007;420(1):53-57.
Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeats within the disease protein, ataxin-1. The mutant ataxin-1 precipitates as large intranuclear aggregates in the affected neurons. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding protein calbindin-D28k (CaB) associated with SCA1 pathogenesis is recruited to ataxin-1 aggregates in Purkinje cells of SCA1 mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in cross-linking and aggregation of ataxin-1, the present study was initiated to determine if TG2 has any role in CaB-ataxin-1 interaction. The guinea pig TG2 covalently cross-linked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-1 was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-1 with normal and expanded polyglutamine repeats (Q2, Q30 and Q82). The reaction products were analyzed by Western blots using anti- polyglutamine, CaB or GFP antibodies. CaB interacted with ataxin-1 independent of TG2 as the protein-protein cross-linker DSS stabilized CaB-ataxin-1 complex. TG2 cross-linked CaB preferentially with Q82 ataxin-1. The cross-linking was inhibited with EGTA or TG2 inhibitor cystamine. The present data indicate that CaB may be a TG2 substrate. In addition, aggregates of mutant ataxin-1 may recruit CaB via TG2 mediated covalent cross-linking, further supporting the argument that ataxin-1 aggregates may be toxic to neurons.
doi:10.1016/j.neulet.2007.04.005
PMCID: PMC1949022  PMID: 17442486
SCA1; Transglutaminase; Ataxin-1; Cerebellum
19.  Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell 
Journal of cell science  2006;119(Pt 9):1833-1842.
Summary
Newly synthesized secretory granule content proteins are delivered via the Golgi complex for storage within mature granules, whereas constitutive secretory proteins are not stored. Most soluble proteins traveling anterograde through the trans-Golgi network are not excluded from entering immature secretory granules, whether or not they have granule-targeting signals. However, the ‘sorting-for-entry’ hypothesis suggests that soluble lumenal proteins lacking signals enter transport intermediates for the constitutive secretory pathway. We aimed to investigate how these constitutive secretory proteins are sorted. In a pancreatic β-cell line, we stably expressed two lumenal proteins whose normal sorting information has been deleted: alkaline phosphatase, truncated to eliminate its glycosylphosphatidylinositol membrane anchor (SEAP); and Cab45361, a Golgi lumenal resident, truncated to eliminate its intracellular retention (Cab308Myc). Both truncated proteins are efficiently secreted, but whereas SEAP enters secretory granules, Cab308Myc behaves as a true constitutive marker excluded from granules. Interestingly, upon permeabilization of organelle membranes with saponin, SEAP is extracted as a soluble protein whereas Cab308Myc remains associated with the membrane. These are among the first data to support a model in which association with the lumenal aspect of Golgi and/or post-Golgi membranes can serve as a means for selective sorting of constitutive secretory proteins.
doi:10.1242/JCS.02905
PMCID: PMC2547412  PMID: 16608874
Trans-Golgi network; Granule maturation; Constitutive; like secretory pathway
20.  Munc18-2 and Syntaxin 3 control distinct essential steps in mast cell degranulation1 
Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)6 and mammalian unc18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. Here we found that siRNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE binding partner Syntaxin 3 (STX3) also markedly inhibited degranulation, while combined knock-down produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.
doi:10.4049/jimmunol.1301277
PMCID: PMC3905451  PMID: 24323579
21.  Munc18/Syntaxin Interaction Kinetics Control Secretory Vesicle Dynamics* 
The Journal of Biological Chemistry  2009;285(6):3965-3972.
In neuronal and hormonal release, regulated exocytosis requires an essential set of proteins: the soluble N-ethylmaleimide sensitive-factor attachment receptor proteins (SNAREs) syntaxin 1, SNAP-25, VAMP, and their regulator, Munc18. Recently, it was found that Munc18-1 can interact with syntaxin 1 through distinct mechanisms: an inhibitory mode enveloping syntaxin (mode 1), sequestering it from SNARE protein interactions, and direct binding to an evolutionarily conserved N-terminal peptide of syntaxin (mode 2/3). The latter interaction has been proposed to control “priming” of the fusion reaction, defined using electrophysiology, but it is unknown how this interaction is regulated, and any dynamic effect at the molecular or vesicular level in cells remains undiscovered. We now show that a phosphorylation site in syntaxin 1 (Ser14) regulates the N-terminal interaction with Munc18-1. Probing syntaxin 1 association with Munc18-1, in real-time and in living cells, we found that modification of Ser14 modulated the dynamics of this interaction, specifically at the plasma membrane. Destabilization of this dynamic interaction enhanced vesicle immobilization at the plasma membrane with a resulting inhibition of exocytosis.
doi:10.1074/jbc.M109.040402
PMCID: PMC2823538  PMID: 19748891
22.  The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis* 
The Journal of biological chemistry  2006;281(26):17624-17634.
Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [32P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.
doi:10.1074/jbc.M601581200
PMCID: PMC2396333  PMID: 16638745
23.  Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion 
The Journal of Cell Biology  2012;197(2):301-312.
Munc13-4 is a Ca2+-dependent membrane- and SNARE-binding protein that promotes 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 Ca2+, but Ca2+-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 Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-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 Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis.
doi:10.1083/jcb.201109132
PMCID: PMC3328385  PMID: 22508512
24.  SYNTAXIN 3B IS A T-SNARE SPECIFIC FOR RIBBON SYNAPSES OF THE RETINA 
Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in non-neuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxin 3A, 3B, 3C and 3D. We demonstrate here using analysis of EST databases, RT-PCR, in situ hybridization and Northern blot analysis that cells in the mouse retina only express syntaxin 3B. In contrast non-neuronal tissues, such as kidney express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP-25 compared to that of syntaxin1A. Using an in vitro fusion assay we could demonstrate that vesicles containing syntaxin 3B and SNAP-25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.
doi:10.1002/cne.21806
PMCID: PMC2893228  PMID: 18683220
vesicle fusion; SNAP-25; Synaptobrevin; VAMP; splicing; isoform
25.  Structure-Function Study of Mammalian Munc18-1 and C. elegans UNC-18 Implicates Domain 3b in the Regulation of Exocytosis 
PLoS ONE  2011;6(3):e17999.
Munc18-1 is an essential synaptic protein functioning during multiple stages of the exocytotic process including vesicle recruitment, docking and fusion. These functions require a number of distinct syntaxin-dependent interactions; however, Munc18-1 also regulates vesicle fusion via syntaxin-independent interactions with other exocytotic proteins. Although the structural regions of the Munc18-1 protein involved in closed-conformation syntaxin binding have been thoroughly examined, regions of the protein involved in other interactions are poorly characterised. To investigate this we performed a random transposon mutagenesis, identifying domain 3b of Munc18-1 as a functionally important region of the protein. Transposon insertion in an exposed loop within this domain specifically disrupted Mint1 binding despite leaving affinity for closed conformation syntaxin and binding to the SNARE complex unaffected. The insertion mutation significantly reduced total amounts of exocytosis as measured by carbon fiber amperometry in chromaffin cells. Introduction of the equivalent mutation in UNC-18 in Caenorhabditis elegans also reduced neurotransmitter release as assessed by aldicarb sensitivity. Correlation between the two experimental methods for recording changes in the number of exocytotic events was verified using a previously identified gain of function Munc18-1 mutation E466K (increased exocytosis in chromaffin cells and aldicarb hypersensitivity of C. elegans). These data implicate a novel role for an exposed loop in domain 3b of Munc18-1 in transducing regulation of vesicle fusion independent of closed-conformation syntaxin binding.
doi:10.1371/journal.pone.0017999
PMCID: PMC3061876  PMID: 21445306

Results 1-25 (747993)