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At least 19 recordsLinked to original sources
PubMed2002

Effects of selective endocrine or exocrine induction of AR42J on SNARE and Munc18 protein expression.

INTRODUCTION AND AIM: We used the amphicrine AR42J as an excellent model to study the differentiation of the secretory machinery of pancreatic endocrine and exocrine cells. Dexamethasone treatment induced the AR42J to differentiate towards the exocrine phenotype capable of secreting amylase in response to cholecystokinin. In contrast, activin A plus hepatocyte growth factor treatment of a subclone of AR42J, AR42J-B13, induced this cell to differentiate morphologically and functionally toward an insulin-containing and insulin-secreting endocrine phenotype. We took advantage of these unique properties of selective exocrine and endocrine induction of the AR42J to reveal which distinct combinations of exocytic SNARE complex proteins (syntaxin, SNAP-25 and VAMP) and associated Munc18 proteins were preferentially expressed to play a role in enzyme and insulin secretion. RESULTS AND CONCLUSION: To our surprise, both endocrine and exocrine induction of AR42J and AR42J-B13 caused very similar upregulation in the expression of the exocytic member isoforms of the syntaxin, SNAP-25, VAMP, and Munc18 families. We conclude that whereas the differentiation of the proximal components of the secretory machinery of the exocrine acinar and endocrine islet beta-cells is distinct, the differentiation of the distal components of exocytosis between these two cell types is very similar.

Activins↗
PubMed2006

Cyclin-dependent kinase 5, Munc18a and Munc18-interacting protein 1/X11alpha protein up-regulation in Alzheimer's disease.

Besides formation of neurofibrillary tangles and neuron loss, the Alzheimer's disease brain is characterized by neuritic plaques consisting of beta-amyloid peptide deposits and impaired neurotransmission. The proteins Munc18a, Munc18-interacting protein 1 and Munc18-interacting protein 2 mediate exocytosis and decrease beta-amyloid peptide formation. Cyclin-dependent kinase 5 and its activator p35 disrupt Munc18a-syntaxin 1 binding, thereby promoting synaptic vesicle fusion during exocytosis. We investigated protein levels of the signaling pathway: p35, cyclin-dependent kinase 5, Munc18a, syntaxin 1A and 1B, Munc18-interacting protein 1 and Munc18-interacting protein 2 in Alzheimer's disease cortex and found that this pathway was up-regulated in the Alzheimer's disease parietal and occipital cortex. In the cortex of transgenic Tg2576 mice over-expressing human beta-amyloid precursor protein with the Swedish mutation known to lead to familial Alzheimer's disease, which have substantial levels of beta-amyloid peptide but lack neurofibrillary tangles and neuron loss, no alterations of protein levels were detected. These data suggest that the pathway is enhanced in dying or surviving neurons and might serve a protective role by compensating for decreased neurotransmission and decreasing beta-amyloid peptide levels early during the progression of Alzheimer's disease.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins.

Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

Animals↗
PubMed1997

Mints, Munc18-interacting proteins in synaptic vesicle exocytosis.

Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis. We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1. Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane. In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion. The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takonawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177). Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint.Munc18-1 complex in conjunction with syntaxin 1.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Munc18 interacting proteins: ADP-ribosylation factor-dependent coat proteins that regulate the traffic of beta-Alzheimer's precursor protein.

Coat proteins cycle between soluble and membrane-bound locations at the time of vesicle biogenesis and act to regulate the assembly of the vesicle coat that determines the specificity in cargo selection and the destination of the vesicle. A transmembrane cargo protein, an Arf GTPase, and a coat protein (e.g. COPs, APs, or GGAs) are minimal components required for budding of vesicles. Munc18 interacting proteins (MINTs) are a family of three proteins implicated in the localization of receptors to the plasma membrane. We show that MINTs bind Arfs directly, co-localize with Arf and the Alzheimer's precursor protein (beta-APP) to regions of the Golgi/trans-Golgi network, and can co-immunoprecipitate clathrin. We demonstrate that MINTs bind Arfs through a region of the PTB domain and the PDZ2 domain, and Arf-MINT interaction is necessary for the increased cellular levels of beta-APP produced by MINT overexpression. Knockdown (small interference RNA) experiments implicate beta-APP as a transmembrane cargo protein that works together with MINTs. We propose that MINTs are a family of Arf-dependent, vesicle-coat proteins that can regulate the traffic of beta-APP.

ADP-Ribosylation Factors↗
PubMed2000

Synaptic assembly of the brain in the absence of neurotransmitter secretion.

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

Animals↗
PubMed1998

Genomic structure of MUNC18-1 protein, which is involved in docking and fusion of synaptic vesicles in brain.

MUNC18-1 (n-Sec1) is a brain-specific protein and is known to play a role in neurotransmitter release by mediating docking and fusion of synaptic vesicles to presynaptic membranes. The protein is also implicated in the cellular excretion process of hormones and other biological substances in other mammalian tissues and yeasts. We have studied the structure of mouse munc18-1 gene by sequencing the genomic munc18-1 gene and its 5'-flanking region. munc18-1 gene comprises 19 exons whose size ranges from 50 base pairs (2nd exon) to 1676 base pairs (19th exon) with a total gene size of approximately 56 kilobases. In the 5'-flanking region, there are several transcription factor binding sites such as for HSF2, Lyf-1, and Sp1 but no TATA or CAAT sequences. munc18-1 gene was mapped on mouse chromosome 2 between two anchor markers D2Mit152 and D2Mit242. Transfection experiments employing these and upstream sequences suggest the presence of a sequence(s) that negatively regulates the expression of munc18-1 gene.

Animals↗
PubMed2003

Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis.

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.

Animals↗
PubMed2004

Alcoholic chronic pancreatitis involves displacement of Munc18c from the pancreatic acinar basal membrane surface.

The minimal machinery for fusion of secretory vesicles with the cell membrane is a cognate set of v- and t-SNAREs on opposing membranes. Spontaneous SNARE complex assembly leading to unregulated membrane fusion is prevented by Munc18 proteins that bind membrane SNAREs syntaxins. Munc18 blocks syntaxin interactions with cognate SNARE proteins and thereby act as an inhibitor of exocytosis. The pancreatic acinar cell contains several sets of cognate SNAREs and Munc18 proteins that mediate the distinct exocytic events. We had reported that in the rat pancreas, Munc18c co-localizes with t-SNAREs syntaxin4 and SNAP23 on the acinar cell basolateral plasma membrane. Under conditions that induce pancreatitis in vivo, displacement of Munc18c from the basolateral plasma membrane relieved its blockade of SNARE-mediated membrane fusion in this region and thereby redirected apical exocytosis to the basal membrane surface. Here we show in a case of human mild alcoholic chronic pancreatitis that Munc18c is also displaced from the plasma membrane of intact acinar cells, which would render these cells receptive to pathologic basolateral exocytosis and further episodes of pancreatitis.

Carrier Proteins↗
PubMed2001

Physiological control of Xunc18 expression in neuroendocrine melanotrope cells of Xenopus laevis.

In mammals, the brain-specific protein munc18-1 regulates synaptic vesicle exocytosis at the synaptic junction, in a step before vesicle fusion. We hypothesize that the rate of biosynthesis of munc18-1 messenger RNA (mRNA) and the amount of munc18-1 present in neurons and neuroendocrine cells are related to the physiologically controlled state of activity. To test this hypothesis, the homolog of munc18-1 in the clawed toad Xenopus laevis, xunc18, was studied in the brain and in the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland, at both the mRNA and the protein level. In toads adapted to a black background, the melanotropes release the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces darkening of the skin, whereas in animals adapted to a white background the cells hardly release but store alpha-MSH, making the animal's skin look pale. The intermediate pituitary lobe of black-adapted animals revealed a strong hybridization reaction with the xunc18 mRNA probe, whereas a much weaker hybridization was observed in the intermediate lobe of white-adapted animals (optical density black: 3.4 +/- 0.2 vs. white: 0.8 +/- 0.1; P < 0.02). Immunocytochemically, Xenopus munc18-like protein has been detected throughout the brain, in identified neuronal perikarya as well as in axon tracts. Western blot analysis and immunocytochemistry further demonstrated the presence of xunc18 in the neural, intermediate and distal lobe of the pituitary gland. Xunc18 protein was furthermore determined in immunoblots of homogenates of melanotropes dissociated from the pituitary gland. In melanotropes of toads adapted to a black background, the integrated optical density of the xunc18 immunosignal was 2.7 +/- 0.5 times higher than in cells of white-adapted toads (P < 0.0001). It is concluded that, in Xenopus melanotrope cells, the amounts of both xunc18 mRNA and xunc18 protein are up-regulated in conjunction with the induction of exocytosis of alpha-MSH as a result of a physiological stimulation (environmental light condition). We propose that xunc18 is involved in physiologically controlled exocytotic secretion of neuroendocrine messengers.

Animals↗
PubMed2005

Expression and transcriptional regulation of Munc18 isoforms in mast cells.

Mast cells are specialized immune cells with a central pathophysiological role in allergic reactions and important roles in pathogen defense. Their main effector response is the exocytic release of preformed inflammatory mediators from secretory granules. Munc18 proteins are essential for exocytic function, so we analyzed the expression of Munc18 transcripts in RBL-2H3 mast cells and mouse bone marrow derived mast cells (BMMC). All three isoforms of Munc18 are expressed in both cell types, but Munc18-2 transcripts are most abundant. The proximal 181 bp region of the Munc18-2 gene promoter is conserved between mice and humans, and shows maximal promoter activity among a series of truncation mutants. Binding sites for Ets, E-box and CREB transcription factors that are known to be important for mast cell development are highly conserved and functionally active. Thus, mast cells upregulate an essential component of their exocytic machinery as they develop morphologic and functional features of the regulated secretory phenotype.

Animals↗
PubMed2004

Protease-activated receptor-1 activation of endothelial cells induces protein kinase Calpha-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression.

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Calpha activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Calpha activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.

Animals↗
PubMed2005

Arachidonic acid allows SNARE complex formation in the presence of Munc18.

SNARE complex formation underlies intracellular membrane fusion in eukaryotic organisms; however, the factors regulating the SNARE assembly are not well understood. The neuronal SNARE complex is composed of synaptobrevin2, SNAP-25, and syntaxin1, the latter being under tight control by the cytosolic protein Munc18. We found that the inhibition of syntaxin1 by Munc18 both in nerve terminals and in defined in vitro reactions can be overcome by specific detergents. This serendipitous finding led us to screen biologically relevant fatty acids, revealing that unsaturated arachidonic and linolenic acids can stimulate Munc18-regulated SNARE complex formation in a direct manner. The direct effect of arachidonic acid on the syntaxin1/Munc18 complex suggests a mechanism for the activation of the SNARE assembly pathway and provides a lead for the further investigation of fatty acids that may regulate SNARE-mediated membrane fusion in eukaryotes.

Animals↗
PubMed2005

The role of Munc18-1 in docking and exocytosis of peptide hormone vesicles in the anterior pituitary.

BACKGROUND INFORMATION: Many neurons secrete classical transmitters from synaptic vesicles as well as peptide transmitters from LDCVs (large dense-core vesicles). Little is known about the mechanistic differences between these two secretory pathways. The soluble protein Munc18-1 is essential for synaptic vesicle secretion [Verhage, Maia, Plomp, Brussaard, Heeroma, Vermeer, Toonen, Hammer, van den Berg, Missler, et al. (2000) Science 287, 864-869.]. RESULTS: In the present study, we tested if Munc18 genes are also involved in peptidergic secretion from LDCVs using the anterior pituitary as a model system. We show that Munc18-1 is the dominant isoform expressed in the anterior pituitary. In Munc18-1 null mutant mice, the anterior pituitary developed normally and the five major endocrine cell types had a normal distribution. However, circulating peptide hormone levels were decreased by up to 50-fold in the null mutant, whereas the intracellular levels were significantly higher than that in controls. Ultrastructural analysis using the tannic acid method revealed striking differences in the distribution of secretory vesicles: (i) the number of exocytotic figures was mostly decreased in the null mutants and (ii) the LDCVs accumulated near but not at their target membrane. This is in contrast with the apparently normal distribution of synaptic vesicles in developing synapses in the null mutant (Verhage et al., 2000). CONCLUSIONS: We conclude that Munc18-1 is involved in the secretion of peptide hormones and in the docking of LDCVs. These results unmask an apparent mechanistic difference between LDCVs and synaptic vesicles.

Animals↗
PubMed2005

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

Animals↗
PubMed2000

Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A.

In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies.

Amino Acid Transport Systems, Neutral↗
PubMed2003

Evidence for SNARE zippering during Ca2+-triggered exocytosis in PC12 cells.

SNAREs (soluble NSF attachment protein receptors) are membrane proteins that catalyze membrane fusion. SNAREs are defined by a characteristic 70 residue sequence called the SNARE motif. During synaptic vesicle fusion, the single SNARE motif of the synaptic vesicle SNARE protein synaptobrevin/VAMP associates into a four-helical bundle with SNARE motifs from the plasma membrane SNARE proteins syntaxin 1 and SNAP-25. The four SNARE motifs (one each from synaptobrevin and syntaxin, and two from SNAP-25) assume a parallel orientation in the complex, suggesting that formation of the complex initiates fusion by forcing the membranes containing the SNAREs into close proximity. It has been proposed that SNARE complexes assemble in an N- to C-terminal progression, a process referred to as zippering, but little direct evidence for zippering exists. Furthermore, the SM protein Munc18-1, which binds to syntaxin 1 and is essential for synaptic fusion, is thought to prepare SNAREs for complex formation by an unknown mechanism, possibly by nucleating zippering. We now show that fragments containing the N- and C-terminal regions of the SNARE motif from syntaxin 1A bind SNAP-25 similarly. However, in permeabilized PC12 cells which are used as a biochemical model system to study synaptic fusion, only fragments containing the N-terminal region are powerful inhibitors of fusion. Furthermore, mutations in the N-terminal part of the Syntaxin SNARE motif have only a moderate effect on SNAP-25 binding but abolish the inhibitory activity of the SNARE motif. Finally, larger fragments of syntaxin 1A that strongly bind to Munc18-1 but do not readily assemble into SNARE complexes had no effect on exocytosis in permeabilized PC12 cells. Together these results suggest that Munc18-1 acts before SNARE complex assembly, and is no longer required at the stage of fusion assayed in permeabilized PC12 cells. The selective effect of the N-terminal half of the syntaxin 1A SNARE motif on PC12 cell exocytosis shows that the SNARE motif is functionally polarized, and supports the notion that SNARE complexes assemble in an N- to C-terminal zippering reaction during fusion without a stable, partially assembled intermediate.

Animals↗