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PubMed1994

Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells.

Several proteins that are of importance for membrane trafficking in the nerve terminal have recently been characterized. We have used Western blot and immunohistochemistry to show that synaptotagmin, synaptobrevin/VAMP (vesicle-associated membrane protein), SNAP-25 (synaptosomal-associated protein of 25 kDa), and syntaxin proteins are present in cells of the islets of Langerhans in the endocrine pancreas. Synaptotagmin-like immunoreactivity (-LI) was localized to granules within the cytoplasm of a few endocrine cells located in the periphery of the islets, identified as somatostatin-containing cells, and in many nerve fibers within the islets. VAMP-LI was seen in granules of virtually all pancreatic islet cells and also in nerve fibers. SNAP-25-LI and syntaxin-LI were predominantly present in the plasma membrane of the endocrine cells, including insulin-producing beta cells. In situ hybridization, using isoform-specific oligonucleotide probes, detected VAMP-2, cellubrevin, SNAP-25, syntaxin 1A, 4, and 5, and munc-18 mRNAs in isolated pancreatic islets and in insulin-producing cells. The results show the presence of several synaptic proteins at protein and mRNA levels in pancreatic islet cells, suggesting that they may have specific roles in the molecular regulation of exocytosis also in insulin-secreting cells.

Animals↗

PubMed1994

n-Sec1: a neural-specific syntaxin-binding protein.

We have identified n-Sec1, a rat brain homolog of the yeast Sec1p protein that participates in the constitutive secretory pathway between the Golgi apparatus and the plasma membrane. The rat brain cDNA is predicted to encode a 68-kDa protein with 65% amino acid identity to Drosophila rop, 59% identity to Caenorhabditis elegans unc-18, and 27% identity to Saccharomyces cerevisiae Sec1p. By RNA blot analysis, n-Sec1 mRNA expression is neural-specific. An anti-peptide antiserum directed against the n-Sec1 carboxyl terminus detects a 68-kDa protein in rat brain cytosol and membranes, but not in peripheral tissues. In the presence of syntaxin 1a, a plasma membrane protein implicated in synaptic vesicle docking, n-Sec1 becomes membrane-associated. n-Sec1 binds to syntaxin 1a, 2, and 3 fusion proteins coupled to agarose beads, but not to syntaxin 4 fusion protein or beads coupled to a variety of other proteins. These findings indicate that n-Sec1 is a neural-specific, syntaxin-binding protein that may participate in the regulation of synaptic vesicle docking and fusion.

Amino Acid Sequence↗

PubMed1994

A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.

Amino Acid Sequence↗

PubMed1995

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

Amino Acid Sequence↗

PubMed1998

Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions.

Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl- currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant DeltaF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.

Animals↗

PubMed1998

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Amino Acid Sequence↗

PubMed1999

Neurosecretory control of aging in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, an insulin receptor signaling pathway regulates adult life span and developmental arrest at the dauer larval stage. Here we show that the unc-64 and unc-31 genes also function in this pathway. These two genes are involved in mediating Ca2+-regulated secretion. Mutations in unc-64 and unc-31 increase adult life span and cause constitutive dauer formation. Both phenotypes are suppressed by mutations in daf-16, which also suppresses other mutations in this pathway. We present evidence that the site of action of unc-64 is neuronal, suggesting that a neurosecretory signal regulates life span and dauer formation.

Aging↗

PubMed1995

Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins.

Botulinum neurotoxin serotype C (BoNT/C) is a 150-kDa protein produced by Clostridium botulinum, which causes animal botulism. In contrast to the other botulinum neurotoxins that contain one atom of zinc, highly purified preparations of BoNT/C bind two atoms of zinc per toxin molecule. BoNT/C is a zinc-endopeptidase that cleaves syntaxin 1A at the Lys253-Ala254 and syntaxin 1B at the Lys252-Ala253 peptide bonds, only when they are inserted into a lipid bilayer. The other Lys-Ala bond present within the carboxyl-terminal region is not hydrolyzed. Syntaxin isoforms 2 and 3 are also cleaved by BoNT/C, while syntaxin 4 is resistant. These data suggest that BoNT/C recognizes a specific spatial organization of syntaxin, adopted upon membrane insertion, which brings a selected Lys-Ala peptide bond of its carboxyl-terminal region to the active site of this novel metalloproteinase.

Alanine↗

PubMed1996

Evidence that syntaxin 1A is involved in storage in the secretory pathway.

Syntaxin 1A is a nervous system-specific protein thought to function during the late steps of the regulated secretory pathway by mediating the docking of secretory vesicles with the plasma membrane. We have examined the effects of transiently overexpressing syntaxin 1A on protein secretion in constitutively secreting cell lines that do not normally express the protein. Syntaxin 1A showed the constitutive release of marker proteins human growth hormone (hGH) and vesicular stomatitis virus glycoprotein from COS-1 cells, increasing the intracellular half-life of human growth hormone from 90 min to 18 h. A similar effect was observed in HEK 293 cells. Immunofluorescence microscopy revealed that these secretory proteins were concentrated in the periphery of the cell. The effect was specific for the full-length neuronal protein. Neither a syntaxin 1A variant which lacks a membrane attachment domain nor syntaxin 2 caused the cells to retain human growth hormone. The effect of syntaxin 1A was partially reversed by incubating the cells with botulinum type C1 neurotoxin, which specifically cleaves syntaxin 1A. Release of human growth hormone from syntaxin 1A-expressing cells was maintained during a blockade of protein synthesis, suggesting that the hormone was being released from a pool of stored vesicles which accumulated before the addition of cycloheximide. The existence of a post-Golgi storage compartment in syntaxin 1A-expressing cells was confirmed using brefeldin A to collapse the Golgi stacks in both HEK 293 and COS-1 cells. Brefeldin A rapidly blocked growth hormone release in control cultures while having no effect on release in cells expressing syntaxin 1A. Reducing the temperature to 19 degrees C, which inhibits transport from the trans-Golgi network, also inhibited hGH secretion from cells without syntaxin 1A but had little effect on hGH secretion from cells with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released.

Animals↗

PubMed1996

Synthesis of GDP-L-fucose by the human FX protein.

FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity. Its function has been unrecognized despite partial structural and genetic characterization. Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen. In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing. This sequence revealed a significant homology with many proteins from different organisms. Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E. coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose. Accordingly, a possible role of FX in this metabolism was investigated. The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells. Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.

Amino Acid Sequence↗

PubMed1997

Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4.

We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, -2, and -3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.

3T3 Cells↗

PubMed1998

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure.

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.

Animals↗

PubMed1998

Functional properties of the unc-64 gene encoding a Caenorhabditis elegans syntaxin.

Phenotypes of Caenorhabditis elegans unc-18 and unc-64 gene mutations are similar. While unc-18 is known to be essential for normal synaptic transmission (Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishiwaki, S., Miwa, J., Taketo, A., and Kodaira, K.-I. (1992) J. Neurochem. 58, 1517-1525), the function of unc-64 remains unclear. Here we describe the cloning, and the molecular and genetic characterization of the unc-64 gene, especially in relation to unc-18. unc-64 encodes a protein (C. elegans syntaxin) showing sequence and structural similarities to mammalian syntaxin 1A. From unc-64, at least three types of poly(A)+ RNA are transcribed, which encode two types of syntaxin that differ in the deduced transmembrane domain. In gene expression, unc-64 closely resembles unc-18, that is, both are expressed in neural cells, especially in motor neurons and neurons constituting head ganglions. C. elegans syntaxin binds to UNC-18 with high affinity. The unc-64 (e246) mutation producing a mild phenotype causes an Ala-->Val conversion in the conserved COOH-terminal region in mammalian syntaxin 1A or Drosophila syntaxin-1A whose site is included in three types of transcripts. The binding of the mutant C. elegans syntaxin to UNC-18 is greatly reduced, indicating the mutation site contributes to the binding.

Amino Acid Sequence↗

PubMed1998

Assembly of a ternary complex by the predicted minimal coiled-coil-forming domains of syntaxin, SNAP-25, and synaptobrevin. A circular dichroism study.

The assembly of target (t-SNARE) and vesicle-associated SNAP receptor (v-SNARE) proteins is a critical step for the docking of synaptic vesicles to the plasma membrane. Syntaxin-1A, SNAP-25, and synaptobrevin-2 (also known as vesicle-associated membrane protein, or VAMP-2) bind to each other with high affinity, and their binding regions are predicted to form a trimeric coiled-coil. Here, we have designed three peptides, which correspond to sequences located in the syntaxin-1A H3 domain, the C-terminal domain of SNAP-25, and a conserved central domain of synaptobrevin-2, that exhibit a high propensity to form a minimal trimeric coiled-coil. The peptides were synthesized by solid phase methods, and their interactions were studied by CD spectroscopy. In aqueous solution, the peptides were unstructured and showed no interactions with each other. In contrast, upon the addition of moderate amounts of trifluoroethanol (30%), the peptides adopted an alpha-helical structure and displayed both homomeric and heteromeric interactions. The interactions observed in ternary mixtures induce a stabilization of peptide structure that is greater than that predicted from individual binary interactions, suggesting the formation of a higher order structure compatible with the assembly of a trimeric coiled-coil.

Amino Acid Sequence↗

PubMed1998

Targeting of SNAP-23 and SNAP-25 in polarized epithelial cells.

SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.

Animals↗

PubMed1998

Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings.

The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.

Animals↗

PubMed1999

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.

Animals↗

PubMed1999

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.

Animals↗