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PubMed1995

Anti-syntaxin antibodies inhibit calcium-dependent catecholamine secretion from permeabilized chromaffin cells.

Adrenomedullary chromaffin cells release catecholamines in response to the intracellular calcium rise upon stimulation by different secretagogues. The presence of syntaxin 1, a protein presumably involved in docking of synaptic vesicles to presynaptic membranes, has been investigated in chromaffin cells. The study using two different monoclonal antibodies shows that syntaxin 1 is present in the chromaffin cell membrane fraction. Functional experiments demonstrate that anti-syntaxin antibodies inhibit calcium-dependent secretion in permeabilized cells. These results suggest that syntaxin 1 is an important component of the secretory machinery in chromaffin cells.

Adrenal Medulla↗

PubMed1999

Organization of the secretory machinery in the rodent brain: distribution of the t-SNAREs, SNAP-25 and SNAP-23.

Vesicular transport events appear to be facilitated by the VAMP/synaptobrevin family of membrane proteins in the vesicle (v-SNAREs) and a heterodimeric complex of syntaxin and SNAP-23/25 family members in the target membrane (t-SNAREs). In this manuscript we examine the tissue distribution and composition of the heterodimeric t-SNARE complexes in adult rodent brain. Analysis of protein extracts from brain regions shows that SNAP-25, syntaxin 1, and 4 are broadly distributed, while SNAP-23, syntaxin 3, and 7 show distinct patterns of expression. Further immunohistochemistry and fractionation studies show that while SNAP-25 is enriched in axons and nerve terminals, SNAP-23 is concentrated in cell bodies. Both SNAP-23 and SNAP-25 associate with the plasma membrane and can be metabolically labeled with [(3)H] palmitate in AtT-20 cells. Anti-SNAP-25 antibodies co-immunoprecipitate t-SNARE heterodimers from brain extracts that predominantly contain syntaxin 1 and 2. Contrary to results from in vitro binding assays, SNAP-23 was found predominantly associated with syntaxin 3. These observations suggest that t-SNARE, heterodimer composition is governed more by SNARE expression and localization than by simple protein-protein affinity.

Acylation↗

PubMed1998

Snare proteins essential for cyclic AMP-regulated exocytosis in salivary glands.

Rat parotid acinar cells secrete amylase through the stimulation of beta-adrenoceptors followed by accumulation of intracellular cAMP. However, it remains unclear at the molecular level how secretory granules fuse with the apical membranes. We have examined whether SNARE proteins are involved in exocytosis in the salivary glands, and have found that one of the SNARE proteins, VAMP-2, is localized at the secretory granule membrane of rat parotid acinar cells. Moreover, botulinum neurotoxin B, which has endoprotease activity that cleaves VAMP-2, inhibited cAMP-dependent amylase release but did not inhibit basal secretion in the absence of cAMP. These results suggest that VAMP-2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. In contrast, both neurotoxins A and C1 (endoproteases that cleave SNAP-25 and syntaxin 1 respectively) failed to inhibit cAMP-dependent amylase release. Therefore, neither SNAP-25 nor syntaxin 1 are involved in amylase secretion in the parotid glands. Clarification of the mechanism of secretion will require the identification of proteins that interact and function cooperatively with VAMP-2. This approach may also reveal details of the molecular mechanism by which the cAMP facilitates secretion in other systems, including neurotransmission.

Animals↗

PubMed1997

Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c.

SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc 18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.

Adipocytes↗

PubMed1996

A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina.

In contrast to conventional synapses, which release neurotransmitter transiently, ribbon synapses formed by photoreceptors and bipolar cells of the retina release neurotransmitter continuously and modulate the rate in response to light. Both modes of release are mediated by synaptic vesicles but probably differ in the regulation of docking and fusion of synaptic vesicles with the plasma membrane. We have found that syntaxin 1, an essential component of the core fusion complex in conventional synapses, is absent from ribbon synapses of the retina, raising the possibility that these synapses contain a different type of syntaxin or syntaxin-like protein. By immunoprecipitating syntaxin 1-depleted retina and brain extracts with a SNAP-25 antibody and microsequencing the precipitated proteins, syntaxin 3 was detected in retina complexed with SNAP-25, synaptobrevin, and complexin. Using an anti-syntaxin 3 antiserum, syntaxin 3 was demonstrated to be present at high levels in retina compared to brain. Immunofluorescent staining of rat retina sections confirmed that syntaxin 3 is expressed by photoreceptor and bipolar cells in the retina. Thus, in the retina, expression of syntaxin 3 is correlated with ribbon synapses and may play a role in the tonic release of neurotransmitter.

Adaptor Proteins, Vesicular Transport↗

PubMed1999

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP-25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP-23, an analogue of SNAP-25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP-23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t-SNAREs SNAP-23 and syntaxin 1 and the v-SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis.

Animals↗

PubMed1997

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Animals↗

PubMed1995

A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Amino Acid Sequence↗

PubMed1998

Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis.

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized omega conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.

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↗

PubMed1998

Synaptobrevin isoforms in secretory granules and synaptic-like microvesicles in anterior pituitary cells.

A set of synaptic proteins have been shown to be essential for the life cycle and exocytosis of synaptic vesicles at the nerve terminal. Recently, these proteins have also been identified in certain endocrine cells. Here we analysed the presence and location of some of these synaptic proteins in anterior pituitary cells. Immunoblotting data demonstrated that Rab3a, synaptotagmin, cellubrevin, synaptobrevin 2, syntaxin 1, SNAP-25 and synaptophysin were well represented in anterior pituitary cells as well as in the corticotroph cell line AtT-20. Cellubrevin was the most abundant synaptobrevin isoform present in pituitary cells. Moreover, both cellubrevin and synaptobrevin 2 took part of a protein complex involved in the fusion process in adenohypophyseal cells. Immunocytochemical and subcellular fractionation showed that cellubrevin, synaptobrevin 2, Rab3a and synaptotagmin were located in both secretory granules and synaptic-like microvesicles fractions. In contrast, SNAP-25 and syntaxin 1 were mainly associated with plasma membrane fractions. Therefore, these results suggest similar secretory mechanisms for synaptic vesicles and secretory organelles in both neuronal and endocrine cells.

Animals↗

PubMed1998

Dissociation between genes activated in long-term potentiation and in spatial learning in the rat.

We have compared changes in mRNA of three genes, zif268, raf B, and syntaxin 1 B, following the unilateral induction of long-term potentiation (LTP) in rats previously trained in a water maze, and in behaviourally naive animals. mRNA of all three genes was enhanced in the potentiated dentate gyrus of naive animals 3 h after the induction of LTP. Training did not affect expression of mRNA for zif268 or for syntaxin 1 B. Expression of raf B was enhanced by training, and in trained animals the LTP-associated increase in expression of raf B was occluded. These results suggest that LTP and spatial training engage a common pathway utilizing an increase in mRNA for raf B, and demonstrate a dissociation between LTP and spatial learning with respect to expression of zif268 and syntaxin 1B.

Animals↗

PubMed1999

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.

Amino Acid Sequence↗

PubMed1997

Human syntaxin 7: a Pep12p/Vps6p homologue implicated in vesicle trafficking to lysosomes.

The movement of hydrolases and other proteins to lysosomes is accomplished by vesicle trafficking. Specific vesicles are targeted from the trans-Golgi network via a prelysosomal compartment to lysosomes. The specificity of vesicle transport is thought to occur through the interaction of vesicle proteins with receptors on a particular target membrane. The syntaxins are a family of transmembrane proteins that have been implicated as vesicle receptors involved in vesicle docking and fusion. Syntaxins 1-4 are localized to the plasma membrane, and in particular, syntaxin 1a mediates synaptic vesicle docking in the nerve terminal. Syntaxins 5 and 6 have been localized to cis-Golgi and trans-Golgi network compartments, respectively. We now report the identification of syntaxin 7 from a human brain cDNA library. The syntaxin 7 gene is localized to human chromosome 6. By Northern analysis, the syntaxin RNA was found to be broadly distributed. Based on its homology to yeast and plant vacuolar syntaxins, we propose that syntaxin 7 has a role in vesicle trafficking between the Golgi complex and lysosomes. In vitro binding studies reveal that syntaxin 7 binds alphaSNAP, a key regulator of transport vesicle fusion at multiple stages of the secretory pathway.

Amino Acid Sequence↗

PubMed1999

Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms.

The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1 A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca2+- and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.

Animals↗

PubMed1999

A conformational switch in syntaxin during exocytosis: role of munc18.

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

Animals↗

PubMed1995

Cellular and subcellular localization of syntaxin-like immunoreactivity in the rat striatum and cortex.

Syntaxin is a synapse-specific protein previously localized to the plasma membrane of axon terminals. Biochemical and molecular biological studies indicate a prominent role for syntaxin 1A and 1B in synaptic vesicle docking and/or fusion, suggesting that these proteins are localized to active zone regions of most terminal varicosities in the central nervous system. We sought to test this hypothesis by examining the cellular and subcellular immunocytochemical localization of syntaxin 1 proteins in the striatum and frontal cortex of rats. Using either a polyclonal anti-syntaxin antibody, or a monoclonal antibody directed against the identical protein, HPC-1, immunoperoxidase reaction product was localized to preterminal axons and terminal varicosities that made almost exclusively Type I (asymmetric) synapses on dendritic spines or distal shafts. Immunoreactive terminals forming Type II (symmetric) synapses were observed rarely and only in tissue that was pretreated by rapid freeze-thaw to enhance antibody penetration. From a semi-quantitative analysis, it was estimated that at least 48-62% of all vesicle-filled varicosities and 67-69% of all terminals forming Type I synapses were immunoreactive for syntaxin or HPC-1, respectively. Using a pre-embedding immunogold-silver technique to provide a non-diffusible marker for subcellular localization, gold-silver particles for syntaxin or HPC-1 were localized to the cytoplasmic surface of non-synaptic portions of the plasma membrane of preterminal axons and terminal varicosities. Enrichment of presynaptic active zone regions was not observed with immunogold-silver staining. These findings suggest that syntaxin is primarily contained in a subpopulation of terminals that are associated with excitatory amino acid transmitters, but appears not to be ubiquitously expressed in all terminal classes. The results further indicate that syntaxin is localized to non-synaptic regions of axon and terminal membranes, but may not be enriched in presynaptic active zones. The apparent inconsistency between the subcellular localization of syntaxin and its proposed role in vesicle exocytosis is discussed in terms of possible technical limitations and alternative functions for syntaxin.

Animals↗

PubMed1999

Syntaxin 1A and 1B display distinct distribution patterns in the rat peripheral nervous system.

Syntaxin 1 has been shown to play an outstanding role in synaptic vesicle exocytosis. Two isoforms of this protein are expressed in neurons, syntaxin 1A and 1B. However, the physiological significance of the occurrence of such closely related isoforms is not still understood. Here, by means of isoform-specific immunocytochemistry, we show that syntaxin 1A and 1B display different patterns of expression in the rat peripheral nervous system. Nerve terminals of sensory neurons reaching the spinal cord were clearly enriched in immunoreactive syntaxin 1A. Both isoforms were detected in cell bodies of sensory neurons at the dorsal root ganglia, although specific immunolabelling displayed very different patterns at the cellular level. Motor endplates and muscle spindles were only immunostained for syntaxin 1B. Syntaxin 1A was mainly associated with nerve fibres reaching small blood vessels. In addition, nerve plexuses of the enteric nervous system showed immunostaining for both syntaxin isoforms. The different distribution pattern of the two neuronal syntaxin isoforms in the rat peripheral nervous system could be related to isoform-specific biochemical properties involved in the exocytotic process.

Animals↗