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Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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pi 4 5 p2 dependent regulation of exocytosis by amisyn the vertebrate specific competitor of Synaptobrevin 2
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Ilona Kondratiuk, Shrutee Jakhanwal, Jialin Jin, Udhayabhaskar Sathyanarayanan, Benjamin Kroppen, Ajaybabu V Pobbati, Anita Krisko, Uri Ashery, Michael Meinecke, Reinhard JahnAbstract:The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with Synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2. However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.
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Dynamic structure of lipid-bound Synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Jeffrey F. Ellena, Reinhard Jahn, Alexander Stein, Binyong Liang, David S. Cafiso, Maciej Wiktor, Lukas K. TammAbstract:The synaptic vesicle protein Synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of Synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length Synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most N-terminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.
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The synaptophysin-Synaptobrevin complex: a hallmark of synaptic vesicle maturation.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999Co-Authors: Anja Becher, Reinhard Jahn, Anne Drenckhahn, Ingrid Pahner, Martin Margittai, Gudrun Ahnert-hilgerAbstract:Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein Synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that Synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound Synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-Synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and Synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of Synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to Synaptobrevin changes in a similar manner during development. Recombinant Synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-Synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-Synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of Synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.
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A stable interaction between syntaxin 1a and Synaptobrevin 2 mediated by their transmembrane domains
FEBS Letters, 1999Co-Authors: Martin Margittai, Henning Otto, Reinhard JahnAbstract:Abstract The proteins Synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and Synaptobrevin are not required for association. Here we report a novel interaction between Synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of Synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.
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Axonal transport and distribution of Synaptobrevin I and II in the rat peripheral nervous system.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996Co-Authors: Lisa Edelmann, Reinhard Jahn, Annica DahlströmAbstract:Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, Synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for Synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, Synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for Synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, Synaptobrevin I and II were colocalized in many terminals. However, labeling for Synaptobrevin I was more intense whereas labeling for Synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only Synaptobrevin I. In the sciatic nerve, Synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that Synaptobrevin I is present predominantly in motor neurons whereas Synaptobrevin II is present in adrenergic and sensory neurons. We conclude that Synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons.
Thomas C Sudhof - One of the best experts on this subject based on the ideXlab platform.
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Ablation of All Synaptobrevin vSNAREs Blocks Evoked But Not Spontaneous Neurotransmitter Release at Neuromuscular Synapses.
The Journal of Neuroscience, 2019Co-Authors: Yoshie Sugiura, Thomas C SudhofAbstract:Synaptic transmission occurs when an action potential triggers neurotransmitter release via the fusion of synaptic vesicles with the pre-synaptic membrane, driven by the formation of SNARE complexes composed of the vesicular (v)-SNARE Synaptobrevin and the target (t)-SNAREs Snap-25 and syntaxin-1. Neurotransmitters are also released spontaneously, independent of an action potential, through the fusion of synaptic vesicles with the pre-synaptic membrane. The major neuronal vSNAREs, Synaptobrevin-1 and Synaptobrevin-2, are expressed at the developing neuromuscular junction (NMJ) in mice, but their specific roles in NMJ formation and function remain unclear. Here, we examine the NMJs in mutant mouse embryos lacking either Synaptobrevin 1 ( Syb1 lew/lew ) or Synaptobrevin 2 ( Syb2 —/— ), and those lacking both ( Syb1 lew/lew Syb2 —/— ). We found that compared with controls: (1) the number and size of NMJs was markedly increased in Syb2 —/— and Syb1 lew/lew Syb2 —/— mice, but not in Syb1 lew/lew mice; (2) synaptic vesicle density was markedly reduced in Syb1 lew/lew Syb2 —/— NMJs, and (3) evoked neurotransmission was markedly reduced in Syb2 —/— NMJs and completely abolished in Syb1 lew/lew Syb2 —/— NMJs. Surprisingly, however, spontaneous neurotransmission persists in the absence of both Syb1 and Syb2. Furthermore, spontaneous neurotransmission remains constant in Syb1 lew/lew Syb2 —/— NMJs despite changing Ca 2+ levels. These findings reveal an overlapping role for Syb1 and Syb2 (with Syb2 being dominant) in developing NMJs in mice. Moreover, because spontaneous release becomes Ca 2+ insensitive in Syb1 lew/lew Syb2 —/— NMJs, our findings suggest that Synaptobrevin-based SNARE complexes play a critical role in conferring Ca 2+ sensitivity during spontaneous release. SIGNIFICANCE STATEMENT Neurotransmitters can be released at synapses with (evoked) or without (spontaneous) the influence of action potentials. Whereas evoked neurotransmission requires Ca 2+ influx, those underlying the spontaneous neurotransmission may occur with or without Ca 2+ . Our findings show that, in the absence neuronal vSNARE Synaptobrevin-1 and Synaptobrevin-2, evoked neurotransmission is completely abolished; however, spontaneous synaptic transmission not only persists but even increased. Furthermore, spontaneous synaptic transmission that is normally highly Ca 2+ -sensitive became Ca 2+ -independent upon deletion of vSNARE Synaptobrevin-1 and Synaptobrevin-2. These findings reveal distinct mechanisms for evoked and spontaneous neurotransmitter release. Moreover, these findings suggest that Synaptobrevin-based SNARE complexes play critical roles in conferring Ca 2+ sensitivity during spontaneous neurotransmission at developing neuromuscular synapses in mice.
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structural determinants of Synaptobrevin 2 function in synaptic vesicle fusion
The Journal of Neuroscience, 2006Co-Authors: Ferenc Deak, Thomas C Sudhof, Ok Ho Shin, Ege T KavalaliAbstract:Deletion of Synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in Synaptobrevin-deficient neurons but that deletion of both cellubrevin and Synaptobrevin does not cause a more severe decrease in exocytosis than deletion of Synaptobrevin alone. We then examined the structural requirements for Synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair Synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of Synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, Synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in Synaptobrevin-deficient neurons. Our data suggest that Synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.
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Stimulus-dependent dynamic homo- and heteromultimerization of Synaptobrevin/VAMP and synaptophysin.
Biochemistry, 2004Co-Authors: Mikhail Khvotchev, Thomas C SudhofAbstract:Synaptophysin and Synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K+ solution, the excitatory neurotoxin α-latrotoxin, or the Ca2+-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and Synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total Synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of Synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobre...
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stimulus dependent dynamic homo and heteromultimerization of Synaptobrevin vamp and synaptophysin
Biochemistry, 2004Co-Authors: Mikhail Khvotchev, Thomas C SudhofAbstract:Synaptophysin and Synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K+ solution, the excitatory neurotoxin α-latrotoxin, or the Ca2+-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and Synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total Synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of Synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobre...
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Synaptobrevin is essential for fast synaptic-vesicle endocytosis.
Nature cell biology, 2004Co-Authors: Ferenc Deak, Thomas C Sudhof, Susanne Schoch, Xinran Liu, Ege T KavalaliAbstract:Synaptobrevin-2 (VAMP-2), the major SNARE protein of synaptic vesicles, is required for fast calcium-triggered synaptic-vesicle exocytosis. Here we show that Synaptobrevin-2 is also essential for fast synaptic-vesicle endocytosis. We demonstrate that after depletion of the readily releasable vesicle pool, replenishment of the pool is delayed by knockout of Synaptobrevin. This delay was not from a loss of vesicles, because the total number of pre-synaptic vesicles, docked vesicles and actively recycling vesicles was unaffected. However, altered shape and size of the vesicles in Synaptobrevin-deficient synapses suggests a defect in endocytosis. Consistent with such a defect, the stimulus-dependent endocytosis of horseradish peroxidase and fluorescent FM1-43 were delayed, indicating that fast vesicle endocytosis may normally be nucleated by a SNARE-dependent coat. Thus, Synaptobrevin is essential for two fast synapse-specific membrane trafficking reactions: fast exocytosis for neurotransmitter release and fast endocytosis that mediates rapid reuse of synaptic vesicles.
Gudrun Ahnert-hilger - One of the best experts on this subject based on the ideXlab platform.
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The C-terminal transmembrane region of Synaptobrevin binds synaptophysin from adult synaptic vesicles.
European journal of cell biology, 2005Co-Authors: Sowmya V. Yelamanchili, Stefan Sikorra, Thomas Binz, Anja Becher, Clemens Reisinger, Hans Bigalke, Gudrun Ahnert-hilgerAbstract:Synaptophysin and Synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/Synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that Synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged Synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal Synaptobrevin cleavage product. In addition, the His-tagged C-terminal Synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of Synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding Synaptobrevin, synaptophysin may positively modulate neurotransmission.
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The synaptophysin/Synaptobrevin complex dissociates independently of neuroexocytosis
Journal of neurochemistry, 2004Co-Authors: Clemens Reisinger, Anja Becher, Britta Hinz, Diana Mitter, Sowmya V. Yelamanchili, Hans Bigalke, Gudrun Ahnert-hilgerAbstract:Synaptophysin is one of the most abundant membrane proteins of small synaptic vesicles. In mature nerve terminals it forms a complex with the vesicular membrane protein Synaptobrevin, which appears to modulate Synaptobrevin's interaction with the plasma membrane-associated proteins syntaxin and SNAP25 to form the SNARE complex as a prerequisite for membrane fusion. Here we show that Synaptobrevin is preferentially cleaved by tetanus toxin while bound to synaptophysin or when existing as a homodimer. The synaptophysin/Synaptobrevin complex is, however, not affected when neuronal secretion is blocked by botulinum A toxin which cleaves SNAP25. Excessive stimulation with alpha-latrotoxin or Ca(2+)-ionophores dissociates the synaptophysin/Synaptobrevin complex and increases the interaction of the other SNARE proteins. The stimulation-induced dissociation of the synaptophysin/Synaptobrevin complex is not inhibited by pre-incubating neurones with botulinum A toxin, but depends on extracellular calcium. However, the synaptophysin/Synaptobrevin complex cannot be directly dissociated by calcium alone or in combination with magnesium. The dissociation of Synaptobrevin from synaptophysin appears to precede its interaction with the other SNARE proteins and does not depend on the final fusion event. This finding further supports the modulatory role the synaptophysin/Synaptobrevin complex may play in mature neurones.
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The synaptophysin/Synaptobrevin interaction critically depends on the cholesterol content
Journal of neurochemistry, 2002Co-Authors: Diana Mitter, Britta Hinz, Clemens Reisinger, Susanne Hollmann, Sowmya V. Yelamanchili, Stephanie Treiber-held, Thomas G. Ohm, Andreas Herrmann, Gudrun Ahnert-hilgerAbstract:Synaptophysin interacts with Synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/Synaptobrevin complex promotes Synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on Synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/Synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/Synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/Synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/Synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/Synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.
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Activity-dependent changes of the presynaptic synaptophysin-Synaptobrevin complex in adult rat brain.
European journal of cell biology, 2001Co-Authors: Britta Hinz, Anja Becher, Diana Mitter, Katrin Schulze, Uwe Heinemann, Andreas Draguhn, Gudrun Ahnert-hilgerAbstract:Summary The vesicular protein Synaptobrevin contributes to two mutually exclusive complexes in mature synapses. Synaptobrevin tightly interacts with the plasma membrane proteins syntaxin and SNAP 25 forming the SNARE complex as a prerequisite for exocytotic membrane fusion. Alternatively, Synaptobrevin binds to the vesicular protein synaptophysin. It is unclear whether SNARE complex formation is diminished or facilitated when Synaptobrevin is bound to synaptophysin. Here we show that the synaptophysin-Synaptobrevin complex is increased in adult rat brain after repeated synaptic hyperactivity in the kindling model of epilepsy. Two days after the last kindling-induced stage V seizure the relative amount of synaptophysin-Synaptobrevin complex obtained by co-immunoprecipitation from cortical and hippocampal membranes was increased twofold compared to controls. By contrast the relative amounts of various synaptic proteins as well as that of the SNARE complex did not change in membrane preparations from kindled rats compared to controls. The increased amount of synaptophysin-Synaptobrevin complex in kindled rats supports the idea that this complex represents a reserve pool for Synaptobrevin enabling synaptic vesicles to adjust to an increased demand for synaptic efficiency. We conclude that the synaptophysin-Synaptobrevin interaction is involved in activity-dependent plastic changes in adult rat brain.
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Synaptobrevin 2 is palmitoylated in synaptic vesicles prepared from adult, but not from embryonic brain.
Molecular and cellular neurosciences, 2000Co-Authors: Michael Veit, Anja Becher, Gudrun Ahnert-hilgerAbstract:Neuronal SNARE-proteins such as Synaptobrevin, SNAP 25, and synaptotagmin are key players during neurosecretion. So far palmitoylation of SNAP-25 and synaptotagmin 1 have been described in vivo. Here we have analyzed palmitoylation of the SNARE-proteins Synaptobrevin 2 and synaptotagmin in vitro using synaptosomal and synaptic vesicle preparations from rat brain. Labeling of synaptic vesicles prepared from adult brain with [3H]palmitate revealed Synaptobrevin 2 besides synaptotagmin 1 as major palmitoylated proteins. [3H]Palmitoylation of Synaptobrevin 2 was resistant to chloroform/methanol extraction, but sensitive to reducing agents indicating a covalent fatty acid bond to cysteine residues. Palmitoylation of Synaptobrevin 2 was also confirmed using endogenous Synaptobrevin 2 present in PC-12 cells and Synaptobrevin 2 expressed with a vacciniavirus system in Cos cells. In contrast to the situation seen with membrane preparations obtained from adult brain, synaptic vesicles prepared from embryonic rat brain did not support [3H]palmitoylation of Synaptobrevin and synaptotagmin. These results suggest, that both Synaptobrevin 2 and synaptotagmin were efficiently palmitoylated from mature synaptic vesicles. However, at least one component of the palmitoylation machinery is developmentally upregulated.
Thomas Binz - One of the best experts on this subject based on the ideXlab platform.
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Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins
Nature Neuroscience, 2011Co-Authors: Régis Nouvian, Anna V. Bulankina, Ellen Reisinger, Tina Pangršič, Stefan Sikorra, Jakob Neef, Thomas Binz, Nils Brose, Thomas Frank, Tobias MoserAbstract:SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and Synaptobrevin-1 or Synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.
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substrate recognition mechanism of vamp Synaptobrevin cleaving clostridial neurotoxins
Journal of Biological Chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/Synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/Synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/Synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/Synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4′) do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/Synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.
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Substrate recognition mechanism of VAMP/Synaptobrevin-cleaving clostridial neurotoxins.
The Journal of biological chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/Synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/Synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/Synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/Synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/Synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.
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Substrate recognition mechanism of VAMP/Synaptobrevin cleaving clostridial neurotoxins.
Journal of Biological Chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle associated membrane protein (VAMP)/Synaptobrevin. TeNT and BoNT/B, D, F and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/Synaptobrevin. Except for BoNT/B and TeNT they cleave unique peptide bonds and prior work suggested that different substrate segments are required for the interaction of each toxin. Whereas the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/Synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, F, and TeNT by means of systematic mutagenesis of VAMP/Synaptobrevin. For each of the toxins three or more residues clustered at an N terminal site remote to the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number, however, the importance of individual positions at the cleavage sites varied for each toxin. The data evidence that similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/Synaptobrevin specific CNTs also initiate substrate interaction employing an exosite located N terminal of the scissile peptide bond.
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The C-terminal transmembrane region of Synaptobrevin binds synaptophysin from adult synaptic vesicles.
European journal of cell biology, 2005Co-Authors: Sowmya V. Yelamanchili, Stefan Sikorra, Thomas Binz, Anja Becher, Clemens Reisinger, Hans Bigalke, Gudrun Ahnert-hilgerAbstract:Synaptophysin and Synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/Synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that Synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged Synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal Synaptobrevin cleavage product. In addition, the His-tagged C-terminal Synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of Synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding Synaptobrevin, synaptophysin may positively modulate neurotransmission.
Manfred Gratzl - One of the best experts on this subject based on the ideXlab platform.
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Functional Importance of Synaptobrevin and SNAP-25 during Exocytosis of Histamine by Rat Gastric Enterochromaffin-Like Cells
Endocrinology, 1997Co-Authors: Barbara Höhne-zell, Manfred Gratzl, Angela Galler, Wolfgang Schepp, Christian PrinzAbstract:Gastric enterochromaffin-like (ECL) cells release histamine upon stimulation with gastrin in a calcium-dependent manner. The intracellular mechanisms and proteins mediating exocytosis of histamine-containing vesicles in ECL cells have not been determined yet. We used immunocytochemistry to show the localization of SNAP-25 (synaptosome-associated protein of 25 kDa) and Synaptobrevin VAMP (vesicle-associated membrane protein) in ECL cells of the rat gastric mucosa and in isolated, highly enriched ECL cells, which were identified with an antibody directed against the marker enzyme histidine decarboxylase. Immunoblots of isolated ECL cells demonstrated the presence of SNAP-25, Synaptobrevin, synaptophysin, synaptotagmin, and syntaxin. Histamine release from isolated ECL cells permeabilized with 8 microM digitonin (2 min) was stimulated approximately 2.5-fold upon exposure to calcium (30 microM; 10-min incubation). Preincubation with 1 microM tetanus toxin light chain for 15 min attenuated calcium-induced histamine release by 40-50% and almost completely cleaved Synaptobrevin. Botulinum neurotoxin A (100 nM) totally blocked calcium-induced histamine release and cleaved SNAP-25. We conclude that Synaptobrevin, synaptophysin, synaptotagmin, SNAP-25, and syntaxin are present in gastric ECL cells. Inhibition of histamine secretion by clostridial neurotoxins associated with the cleavage of Synaptobrevin and SNAP-25 implicates the functional importance of these proteins in the docking and fusion of histamine vesicles.
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Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells
FEBS Letters, 1994Co-Authors: Barbara Höhne-zell, Axel Ecker, Ulrich Weller, Manfred GratzlAbstract:Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular Synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that Synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of Synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of Synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by Synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves Synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells.
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Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells
FEBS Letters, 1993Co-Authors: Barbara Höhne-zell, Brigitte Stecher, Manfred GratzlAbstract:The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of Synaptobrevin cleavage nor peptides spanning the site of Synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that Synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells.
