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Josep Rizo - One of the best experts on this subject based on the ideXlab platform.
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synaptotagmin 1 munc18 1 and munc13 1 dependent liposome fusion with a few neuronal snares
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Karolina P Stepien, Josep RizoAbstract:Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs Syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and Syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the Syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low Syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.
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re examining how munc13 1 facilitates opening of Syntaxin 1
Protein Science, 2020Co-Authors: Magdalena Magdziarek, Karolina P Stepien, Agnieszka A Bolembach, Bradley Quade, Xiaoxia Liu, Josep RizoAbstract:Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by Syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with Syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of Syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-Syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of Syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the Syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening Syntaxin-1 via interactions with the linker.
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mechanism of neurotransmitter release coming into focus
Protein Science, 2018Co-Authors: Josep RizoAbstract:Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca2+ -triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca2+ -dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) Syntaxin-1, SNAP-25, and synaptobrevin-2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N-ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18-1 and Munc13-1 orchestrate SNARE complex formation in an NSF-SNAP-resistant manner by a mechanism whereby Munc18-1 binds to synaptobrevin and to a self-inhibited "closed" conformation of Syntaxin-1, thus forming a template to assemble the SNARE complex, and Munc13-1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of Syntaxin-1. Synaptotagmin-1 functions as the major Ca2+ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.
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Reconstitution of the Vital Functions of Munc18 and Munc13 in Neurotransmitter Release
2016Co-Authors: Alpay B. Seven, Josep RizoAbstract:and the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) Syntaxin-1, synaptobrevin, and SNAP-25. In vitro reconstitutions have shown that Syntaxin-1–SNAP-25 liposomes fuse efficiently with synaptobrevin liposomes in the presence of synaptotagmin-1–Ca2+, but neurotransmitter release also requires Munc18-1 and Munc13 in vivo. We found that Munc18-1 could displace SNAP-25 from Syntaxin-1 and that fusion of Syntaxin-1–Munc18-1 liposomes with synaptobrevin liposomes required Munc13, in addition to SNAP-25 and synaptotagmin-1-Ca2+. Moreover, when starting with Syntaxin-1–SNAP-25 liposomes, NSF–a-SNAP disassembled the Syntaxin-1–SNAP-25 heterodimers and abrogated fusion, which then required Munc18-1 and Munc13. We propose that fusion does not proceed through Syntaxin-1–SNAP-25 heterodimers but starts with the Syntaxin-1–Munc18-1 complex; Munc18-1 and Munc13 then orchestrate membrane fusion together with the SNAREs and synaptotagmin-1-Ca2+ in an NSF- and SNAP-resistant manner. Neurotransmitter release by Ca 2+-triggered synaptic vesicle fusion is crucial for neu-ral function. Key components of the re
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Syntaxin opening by the mun domain underlies the function of munc13 in synaptic vesicle priming
Nature Structural & Molecular Biology, 2015Co-Authors: Xiaoyu Yang, Josep Rizo, Shen Wang, Yi Sheng, Mingshu Zhang, Wenjuan Zou, Lijun Kang, Rongguang ZhangAbstract:Crystallographic and functional studies reveal the arch-shaped architecture of the Munc13 MUN domain and show the molecular basis for Munc13's role in synaptic-vesicle priming by mediating Syntaxin-1 opening and SNARE-complex assembly.
Thomas C. Südhof - One of the best experts on this subject based on the ideXlab platform.
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microsecond dissection of neurotransmitter release snare complex assembly dictates speed and ca2 sensitivity
Neuron, 2014Co-Authors: Claudio Acuna, Jianyuan Sun, Qingchen Guo, Jacqueline Burre, Manu Sharma, Thomas C. SüdhofAbstract:Summary SNARE-complex assembly mediates synaptic vesicle fusion during neurotransmitter release and requires that the target-SNARE protein Syntaxin-1 switches from a closed to an open conformation. Although many SNARE proteins are available per vesicle, only one to three SNARE complexes are minimally needed for a fusion reaction. Here, we use high-resolution measurements of synaptic transmission in the calyx-of-Held synapse from mutant mice in which Syntaxin-1 is rendered constitutively open and SNARE-complex assembly is enhanced to examine the relation between SNARE-complex assembly and neurotransmitter release. We show that enhancing SNARE-complex assembly dramatically increases the speed of evoked release, potentiates the Ca 2+ -affinity of release, and accelerates fusion-pore expansion during individual vesicle fusion events. Our data indicate that the number of assembled SNARE complexes per vesicle during fusion determines the presynaptic release probability and fusion kinetics and suggest a mechanism whereby proteins (Munc13 or RIM) may control presynaptic plasticity by regulating SNARE-complex assembly.
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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, Taulant Bacaj, Zhiping P. Pang, Xiaofei Yang, Yingsha Zhang, Christian Rosenmund, 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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the membrane fusion enigma snares sec1 munc18 proteins and their accomplices guilty as charged
Annual Review of Cell and Developmental Biology, 2012Co-Authors: Josep Rizo, Thomas C. SüdhofAbstract:Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins Syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of Syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca2+ and RI...
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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, Josep Rizo, Irina Dulubova, 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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Munc18-1 stabilizes Syntaxin 1, but is not essential for Syntaxin 1 targeting and SNARE complex formation
Journal of neurochemistry, 2005Co-Authors: Ruud F. Toonen, Thomas C. Südhof, Klaas Jan De Vries, Robbert Zalm, Matthijs VerhageAbstract:Munc18–1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18–1 binds tightly to the SNARE protein Syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that Syntaxin 1 levels are reduced by 70% in munc18–1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18–1 directly promotes the stability of Syntaxin 1, consistent with a chaperone function. However, the residual Syntaxin 1 in munc18–1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18–1 is not required for Syntaxin 1 function as such. These data demonstrate that the Munc18–1 interaction with Syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18–1 knockout mice indicates that Munc18–1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs
Scientific Reports, 2017Co-Authors: Torben-tobias Kliesch, Reinhard Jahn, Jörn Dietz, Laura Turco, Partho Halder, Elena Polo, Marco Tarantola, Andreas JanshoffAbstract:The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of Syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing Syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m^−1, at which the number of fusion events is increased substantially.
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calcium promotes the formation of Syntaxin 1 mesoscale domains through phosphatidylinositol 4 5 bisphosphate
Journal of Biological Chemistry, 2016Co-Authors: Dragomir Milovanovic, Ulf Diederichsen, Reinhard Jahn, Mitja Platen, Meike Junius, Iwan A T Schaap, Alf Honigmann, Geert Van Den BogaartAbstract:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and Syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote Syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple Syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.
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Munc18-bound Syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.
PLoS biology, 2006Co-Authors: Felipe E. Zilly, Jakob B Sorensen, Reinhard Jahn, Thorsten LangAbstract:Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE Syntaxin 1, in which Syntaxin is stabilized in a closed conformation. Since closed Syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to Syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with Syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound Syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not Syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between Syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.
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single molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of Syntaxin 1
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Martin Margittai, Dirk Fasshauer, Jerker Widengren, E Schweinberger, Gunnar F Schroder, Suren Felekyan, E Haustein, Marcelle Konig, Helmut Grubmuller, Reinhard JahnAbstract:Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of Syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of Syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free Syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.
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axonal transport and targeting of the t snares snap 25 and Syntaxin 1 in the peripheral nervous system
European Journal of Cell Biology, 1996Co-Authors: Reinhard Jahn, A DahlstromAbstract:Axonal transport and targeting of the t-SNAREs SNAP-25 and Syntaxin 1 were investigated in the rat peripheral nervous system using a stop-flow (crush) technique. In crush-operated sciatic nerves, accumulations of SNAP-25 and Syntaxin 1 immunoreactivities were detected as early as 1 h after operation, indicating fast axonal transport. The amounts increased on the proximal side of the crush with time after crushing. Distal accumulations of SNAP-25, representing recycling to the cell body, were less than 10% of the proximal accumulations, but 40% for Syntaxin 1, 50% for synaptobrevin II and 70% for synaptophysin. Immunoelectron microscopic studies demonstrated that SNAP-25 and Syntaxin 1 are present on pleiotropic membranes within a diameter of 50 to 100 nm in axons proximal to a crush. Distal to the crush, labeling for Syntaxin 1 and SNAP-25 were sparse and barely detectable, respectively. In addition, the two proteins were found in the axolemma. In nerve terminals of the spinal cord, both proteins were concentrated around small synaptic vesicles (about 50 nm in diameter), whereas only very few gold particles were observed near the presynaptic membrane or the active zones.
Xiaofei Yang - One of the best experts on this subject based on the ideXlab platform.
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munc13 activates the munc18 1 Syntaxin 1 complex and enables munc18 1 to prime snare assembly
The EMBO Journal, 2020Co-Authors: Xianping Wang, Xiaoyu Yang, Shen Wang, Jihong Gong, Le Zhu, Xiaofei YangAbstract:Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/Syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of Syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/Syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of Syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of Syntaxin-1. This interaction ensures Munc18-1 to persistently associate with Syntaxin-1 during the conformational change of Syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/Syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.
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Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly
Nature Publishing Group, 2019Co-Authors: Shen Wang, Xiaofei Yang, Jihong Gong, Rongguang ZhangAbstract:Synaptic exocytosis depends on formation of the SNARE complex but its assembly mechanism is still under debate. Here, the authors identify an interaction between Munc13-1 and synaptobrevin-2 that is critical for the transition of the Munc18-1/Syntaxin-1 complex to the SNARE complex
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conformational change of Syntaxin linker region induced by munc13s initiates snare complex formation in synaptic exocytosis
The EMBO Journal, 2017Co-Authors: Shen Wang, Xiaofei Yang, Xiaoyu Yang, Jihong Gong, Ucheor B Choi, Yun Li, Austin L Wang, Axel T BrungerAbstract:Abstract The soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) protein Syntaxin‐1 adopts a closed conformation when bound to Munc18‐1, preventing binding to synaptobrevin‐2 and SNAP‐25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13‐1 catalyzes the transition from the Munc18‐1/Syntaxin‐1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the Syntaxin‐1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18‐1/Syntaxin‐1/MUN complex, in which Syntaxin‐1 still adopts a closed conformation tightly bound to Munc18‐1, whereas the Syntaxin‐1 linker region changes its conformation, similar to that of the LE mutant of Syntaxin‐1 when bound to Munc18‐1. We suggest that the conformational change of the Syntaxin‐1 linker region induced by Munc13‐1 initiates ternary SNARE complex formation in the neuronal system.
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Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release
Neuron, 2013Co-Authors: Peng Zhou, Taulant Bacaj, Xiaofei Yang, Zhiping P. PangAbstract:Synaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of Syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored Syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca2+-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion.
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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, Taulant Bacaj, Zhiping P. Pang, Xiaofei Yang, Yingsha Zhang, Christian Rosenmund, 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.
Thorsten Lang - One of the best experts on this subject based on the ideXlab platform.
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Munc18-bound Syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.
PLoS biology, 2006Co-Authors: Felipe E. Zilly, Jakob B Sorensen, Reinhard Jahn, Thorsten LangAbstract:Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE Syntaxin 1, in which Syntaxin is stabilized in a closed conformation. Since closed Syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to Syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with Syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound Syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not Syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between Syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.
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The SNARE motif is essential for the formation of Syntaxin clusters in the plasma membrane.
Biophysical journal, 2006Co-Authors: Jochen J. Sieber, Katrin I. Willig, Rainer Heintzmann, Stefan W. Hell, Thorsten LangAbstract:In the plasma membrane, Syntaxin 1 and Syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct Syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the Syntaxin 1 concentration. For Syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types Syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of Syntaxin 1 and 4 mediate specific Syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, Syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions.
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the r snare motif of tomosyn forms snare core complexes with Syntaxin 1 and snap 25 and down regulates exocytosis
Journal of Biological Chemistry, 2003Co-Authors: Kiyotaka Hatsuzawa, Dirk Fasshauer, Thorsten Lang, Dieter BrunsAbstract:Tomosyn is a 130-kDa Syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs Syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfoldingrefolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either Syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous Syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor -SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.
