The Experts below are selected from a list of 11817 Experts worldwide ranked by ideXlab platform
Paul A. Roche - One of the best experts on this subject based on the ideXlab platform.
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SNAP-29 Is a Promiscuous Syntaxin-Binding SNARE
Biochemical and biophysical research communications, 2001Co-Authors: Anita C. Hohenstein, Paul A. RocheAbstract:SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific Syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular Syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized Syntaxins, SNAP-29 binds to both plasma membrane and intracellular Syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of Syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to Syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways.
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Cloning and identification of human Syntaxin 5 as a synaptobrevin/VAMP binding protein
Journal of molecular neuroscience : MN, 1997Co-Authors: Veerasamy Ravichandran, Paul A. RocheAbstract:Syntaxins are transmembrane proteins that function in regulating transport vesicle docking and fusion with target membranes in neuronal and nonneuronal cells. Vesicle docking is thought to be regulated in part by the specific interactions of Syntaxin with a vesicle-associated membrane protein termed synaptobrevin/VAMP. We have cloned a 1557-bp cDNA that encodes the human Syntaxin 5 isoform, using a combination of PCR and colony-screening methods. The deduced 301 amino-acid sequence of human Syntaxin 5 shares 96% identity with rat Syntaxin 5. Like rat Syntaxin 1A, human Syntaxin 5 binds to synaptobrevin/VAMP in vitro. The identification of human Syntaxin 5 as a synaptobrevin/VAMP-binding protein supports the hypothesis that Syntaxin 5 regulates protein transport by binding to vesicle-associated membrane proteins.
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Identification of a Novel Syntaxin- and Synaptobrevin/VAMP-binding Protein, SNAP-23, Expressed in Non-neuronal Tissues
The Journal of biological chemistry, 1996Co-Authors: Veerasamy Ravichandran, Ashish Chawla, Paul A. RocheAbstract:Abstract The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed Syntaxin. These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery. Synaptobrevin/VAMP and Syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells. However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues. Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple Syntaxins and synaptobrevins/VAMPs in vitro. SNAP-23 is 59% identical with SNAP-25. Unlike SNAP-25, SNAP-23 was expressed in all tissues examined. These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.
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identification of a novel Syntaxin and synaptobrevin vamp binding protein snap 23 expressed in non neuronal tissues
Journal of Biological Chemistry, 1996Co-Authors: Veerasamy Ravichandran, Ashish Chawla, Paul A. RocheAbstract:Abstract The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed Syntaxin. These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery. Synaptobrevin/VAMP and Syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells. However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues. Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple Syntaxins and synaptobrevins/VAMPs in vitro. SNAP-23 is 59% identical with SNAP-25. Unlike SNAP-25, SNAP-23 was expressed in all tissues examined. These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.
Josep Rizo - One of the best experts on this subject based on the ideXlab platform.
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Conformational Switch of Syntaxin-1 Controls Synaptic Vesicle Fusion
Science, 2008Co-Authors: Stefan H. Gerber, Jong-cheol Rah, Sang-won Min, Xinran Liu, Heidi De Wit, Irina Dulubova, Alexander C. Meyer, Josep Rizo, Marife Arancillo, Robert E. HammerAbstract:During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein Syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open Syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed Syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open Syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to Syntaxin-1 was impaired in Syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of Syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.
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dual modes of munc18 1 snare interactions are coupled by functionally critical binding to Syntaxin 1 n terminus
The Journal of Neuroscience, 2007Co-Authors: Mikhail Khvotchev, Irina Dulubova, Josep Rizo, Jianyuan Sun, Han Dai, Thomas C. SüdhofAbstract:The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins Syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated Syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing Syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme Syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated Syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated Syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal Syntaxin/Munc18 interaction is essential. Introduction of an N-terminal Syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the Syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.
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How Tlg2p/Syntaxin 16 ‘snares’ Vps45
The EMBO journal, 2002Co-Authors: Irina Dulubova, Sang-won Min, Thomas C. Südhof, Tomohiro Yamaguchi, Yan Gao, Iryna Huryeva, Josep RizoAbstract:Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the Syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal Syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of Syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian Syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the Syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between Syntaxins and SM proteins.
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how tlg2p Syntaxin 16 snares vps45
The EMBO Journal, 2002Co-Authors: Irina Dulubova, Sang-won Min, Thomas C. Südhof, Tomohiro Yamaguchi, Yan Gao, Iryna Huryeva, Josep RizoAbstract:Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the Syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal Syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of Syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian Syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the Syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between Syntaxins and SM proteins.
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Vam3p structure reveals conserved and divergent properties of Syntaxins.
Nature structural biology, 2001Co-Authors: Irina Dulubova, Thomas C. Südhof, Tomohiro Yamaguchi, Yun Wang, Josep RizoAbstract:Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All Syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal Syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of Syntaxins by NMR analysis of Vam3p, a yeast Syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with Syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by Syntaxins to govern different types of membrane fusion.
Thomas C. Südhof - One of the best experts on this subject based on the ideXlab platform.
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Syntaxin-1 N-peptide and Habc-domain perform distinct essential functions in synaptic vesicle fusion
The EMBO Journal, 2012Co-Authors: Peng Zhou, Zhiping P. Pang, Xiaofei Yang, Yingsha Zhang, Christian Rosenmund, Taulant Bacaj, Thomas C. SüdhofAbstract:Among SNARE proteins mediating synaptic vesicle fusion, Syntaxin-1 uniquely includes an N-terminal peptide (‘Npeptide’) that binds to Munc18-1, and a large, conserved Habc-domain that also binds to Munc18-1. Previous in vitro studies suggested that the Syntaxin-1 N-peptide is functionally important, whereas the Syntaxin-1 Habc-domain is not, but limited information is available about the in vivo functions of these Syntaxin-1 domains. Using rescue experiments in cultured Syntaxin-deficient neurons, we now show that the N-peptide and the Habc-domain of Syntaxin1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the Habc-domain regulates this fusion, in part by forming the closed Syntaxin-1 conformation. Moreover, we observed that deletion of the Habc-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the Habc-domain stabilizes Munc18-1. Thus, the N-terminal Syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes.
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dual modes of munc18 1 snare interactions are coupled by functionally critical binding to Syntaxin 1 n terminus
The Journal of Neuroscience, 2007Co-Authors: Mikhail Khvotchev, Irina Dulubova, Josep Rizo, Jianyuan Sun, Han Dai, Thomas C. SüdhofAbstract:The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins Syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated Syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing Syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme Syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated Syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated Syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal Syntaxin/Munc18 interaction is essential. Introduction of an N-terminal Syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the Syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.
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How Tlg2p/Syntaxin 16 ‘snares’ Vps45
The EMBO journal, 2002Co-Authors: Irina Dulubova, Sang-won Min, Thomas C. Südhof, Tomohiro Yamaguchi, Yan Gao, Iryna Huryeva, Josep RizoAbstract:Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the Syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal Syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of Syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian Syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the Syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between Syntaxins and SM proteins.
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how tlg2p Syntaxin 16 snares vps45
The EMBO Journal, 2002Co-Authors: Irina Dulubova, Sang-won Min, Thomas C. Südhof, Tomohiro Yamaguchi, Yan Gao, Iryna Huryeva, Josep RizoAbstract:Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the Syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal Syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of Syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian Syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the Syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between Syntaxins and SM proteins.
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Vam3p structure reveals conserved and divergent properties of Syntaxins.
Nature structural biology, 2001Co-Authors: Irina Dulubova, Thomas C. Südhof, Tomohiro Yamaguchi, Yun Wang, Josep RizoAbstract:Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All Syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal Syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of Syntaxins by NMR analysis of Vam3p, a yeast Syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with Syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by Syntaxins to govern different types of membrane fusion.
Richard H. Scheller - One of the best experts on this subject based on the ideXlab platform.
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SNARE-mediated membrane fusion
Nature Reviews Molecular Cell Biology, 2001Co-Authors: Yu A. Chen, Richard H. SchellerAbstract:SNARE proteins have been proposed to mediate all intracellular membrane fusion events. There are over 30 SNARE family members in mammalian cells and each is found in a distinct subcellular compartment. It is likely that SNAREs encode aspects of membrane transport specificity but the mechanism by which this specificity is achieved remains controversial. Functional studies have provided exciting insights into how SNARE proteins interact with each other to generate the driving force needed to fuse lipid bilayers. Membrane fusion is important for various biological processes, including maintenance of the basic eukaryotic cellular organization. A vesicle fusion event involves many coordinated steps, such as targeting, tethering, priming and finally the triggering of the fusion event. More than a hundred SNARE proteins have been found, and most of them can be assigned to three protein families ? the Syntaxins, the VAMPs and the SNAP-25 family. The hallmark of all SNARE proteins is their coiled-coil domains. SNARE proteins were initially thought to confer docking specificity. However, more recent functional data have shown that they are probably involved in fusion, rather than docking. It is likely that both SNARE-mediated fusion specificity and small GTPase Rab-mediated docking specificity ensure the fidelity of intracellular membrane transport. SNAREs bind to each other to form a very stable four-stranded coiled-coil core complex. Neuronal core complexes are formed by one coil each from Syntaxin and VAMP, and two coils from SNAP-25. The regulation of the core complex formation is still largely unknown. Syntaxins have a large amino-terminal domain that interacts with its coil domain in the presence of the chaperone n-Sec1. After a conformational change that is triggered by unknown mechanisms, Syntaxin opens up to allow the coil domain to assemble into the core complex, thus promoting fusion. SNAREs on two membranes probably interact to form a partial and reversible complex before the final fusion trigger arrives to promote the full assembly of the core complex and membrane fusion. The emerging model for membrane fusion is that vesicles dock with the help of Rab proteins and/or other factors, bringing SNAREs into proximity. The assembly of the SNARE core complex then directs the two membranes towards each other and creates membrane curvature and tension. Once the membranes are close enough, hemifusion occurs followed by fusion pore opening and expansion, leading to complete membrane fusion. SNARE proteins provide the driving force and stabilize the transition state in this reaction.
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three novel proteins of the Syntaxin snap 25 family
Journal of Biological Chemistry, 1998Co-Authors: Martin Steegmaier, Bin Yang, Jin-san Yoo, Betty Huang, Mary Shen, Ying Luo, Richard H. SchellerAbstract:Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the Syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different Syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the Syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of Syntaxins.
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Three Novel Proteins of the Syntaxin/SNAP-25 Family
The Journal of biological chemistry, 1998Co-Authors: Martin Steegmaier, Bin Yang, Jin-san Yoo, Betty Huang, Mary Shen, Ying Luo, Richard H. SchellerAbstract:Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the Syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different Syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the Syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of Syntaxins.
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The Syntaxin family of vesicular transport receptors
Cell, 1993Co-Authors: Mark K. Bennett, Josée. Garcia-arrarás, Lisa A. Elferink, Karen Peterson, Anne M. Fleming, Christopher D. Hazuka, Richard H. SchellerAbstract:Syntaxins A and B are nervous system-specific proteins implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. A family of Syntaxin-related proteins from rat has been identified that shares 23%-84% amino acid identity. Each of the six Syntaxins terminate with a carboxy-terminal hydrophobic domain that anchors the protein on the cytoplasmic surface of cellular membranes. The Syntaxins display a broad tissue distribution and, when expressed in COS cells, are targeted to different subcellular compartments. Microinjection studies suggest that the nervous system-specific Syntaxin 1A is important for calcium-regulated secretion from neuro-endocrine PC12 cells. These results indicate that the Syntaxins are a family of receptors for intracellular transport vesicles and that each target membrane may be identified by a specific member of the Syntaxin family.
Kimio Akagawa - One of the best experts on this subject based on the ideXlab platform.
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activation of Syntaxin 1c an alternative splice variant of hpc 1 Syntaxin 1a by phorbol 12 myristate 13 acetate pma suppresses glucose transport into astroglioma cells via the glucose transporter 1 glut 1
Journal of Biological Chemistry, 2004Co-Authors: Takahiro Nakayama, Katsuhiko Mikoshiba, Tetsuo Yamamori, Kimio AkagawaAbstract:Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/Syntaxin 1A. We found previously that Syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and Syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of Syntaxin 1C is not known. In this study, we examined the relationship between Syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous Syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane Syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous Syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in Syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed Syntaxin 1C. Based on these results, we postulate that Syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a Syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.
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Roles of the cytoplasmic and transmembrane domains of Syntaxins in intracellular localization and trafficking.
Journal of Cell Science, 2001Co-Authors: Kazuo Kasai, Kimio AkagawaAbstract:Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many Syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of Syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of Syntaxin chimeras, each containing a Syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different Syntaxin. We show that the cytoplasmic domains of Syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of Syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type Syntaxin 5. Additionally, we find that Syntaxins 6, 7 and 8, but not Syntaxin 5, are present at the plasma membrane, and that these Syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of Syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of Syntaxins.
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HPC-1/Syntaxin 1A suppresses exocytosis of PC12 cells.
Journal of biochemistry, 1999Co-Authors: Takuya Watanabe, Tomonori Fujiwara, Shinji Komazaki, Kazuhiko Yamaguchi, Osamu Tajima, Kimio AkagawaAbstract:The membrane protein Syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of Syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins. To elucidate the function of Syntaxin 1A in exocytosis, HPC-1/Syntaxin 1A-reduced PC12h cells (PC12h/Deltasyx) that were stably transfected with a plasmid for antisense Syntaxin 1A expression were constructed. Depolarizing stimulation of PC12h/Deltasyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between Syntaxin 1A protein expression and enhancement of dopamine release. Reduction of Syntaxin 1A had no effect on increase of the cytoplasmic free Ca2+ concentration by depolarized stimulation. Moreover, PC12h/Deltasyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that Syntaxin 1A has more than one function in exocytosis.
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Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic β Cells: Syntaxin 1A, BUT NOT 1B, PLAYS A NEGATIVE ROLE IN REGULATORY INSULIN RELEASE PATHWAY
The Journal of biological chemistry, 1996Co-Authors: Shinya Nagamatsu, Takashi Watanabe, Tomonori Fujiwara, Yoko Nakamichi, Hiroshi Katahira, Hiroki Sawa, Kimio AkagawaAbstract:Abstract Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated Syntaxin 1 expression in pancreatic β cells and the functional role of Syntaxin 1 in the insulin release mechanism. Expression of Syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat Syntaxin 1/HPC-1 antibody demonstrated Syntaxin 1A protein with an apparent molecular mass of ∼35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that Syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of Syntaxin 1 in pancreatic β-cells, rat Syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either Syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by Syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by Syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed Syntaxin 1A and used them to evaluate the effect of Syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in Syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by Syntaxin 1A overexpression, indicating a specific role of Syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that Syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of Syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that Syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.
