The Slp4-a linker domain controls exocytosis through interaction with Munc18-1.syntaxin-1a complex

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

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

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

Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane

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.     

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.     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

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.     

Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells

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.     

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.     

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.     

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.     

Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1

Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion.     

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.     

Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.     

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.     

Dissecting docking and tethering of secretory vesicles at the target membrane

Secretory vesicles dock at their target in preparation for fusion. Using single-vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18-1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking-deficient munc18-1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t-SNARE and Munc18-1 binding partner syntaxin, but not the v-SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18-1/syntaxin-dependent, strongly tethered, fusion-competent state.     

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.     

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Roles of Munc18-3 in amylase release from rat parotid acinar cells

Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.     

Munc13-1 is required for the sustained release of insulin from pancreatic beta cells

Munc13-1 is a presynaptic protein that is essential for synaptic vesicle priming. Deletion of Munc13-1/unc13 causes total arrest of synaptic transmission due to a complete loss of fusion-competent synaptic vesicles. The requirement of Munc13-1 for large dense-core vesicles (LDCVs), however, has not been established. In the present study, we use Munc13-1 knockout (KO) and diacylglycerol (DAG) binding-deficient Munc13-1^H567K mutant knockin (KI) mice to determine the role of Munc13-1 in the secretion of insulin-containing LDCVs from primary cultured pancreatic β cells. We show that Munc13-1 is required for the sustained insulin release upon prolonged stimulation. The sustained release involves signaling of DAG second messenger, since it is also reduced in KI mice. Insulin secretion in response to glucose stimulation is characterized by a biphasic time course. Our data show that Munc13-1 plays an essential role in the development of the second phase of insulin secretion by priming insulin-containing LDCVs.     

The role of syntaxins in the specificity of vesicle targeting in polarized epithelial cells

In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes.