Three-Dimensional Structure of the Complexin/SNARE Complex

Chen, Xiaocheng; Tomchick, Diana R.; Kovrigin, Evguenii; Araç, Demet; Machius, Mischa; Südhof, Thomas C.; Rizo, Josep

doi:10.1016/s0896-6273(02)00583-4

Cited by 419 publications

(636 citation statements)

References 47 publications

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“…The triple mutation had no detectable effect on the synaptobrevin/t-SNARE interaction. Complexin, a soluble protein that specifically interacts with the fully folded ternary SNARE complex (Chen et al, 2002), exhibited comparable binding of the SNARE complexes made with SNAP-25, carrying the triple mutation or not ( Figure 5B). Although this experiment provided strong evidence that folding of the SNARE helices is not affected by the mutation, it was essential to test the ability of the mutated SNAP-25 to form the SDS-resistant SNARE complex implicated in the fusion process (Sutton et al, 1998).…”

Section: The Syt Binding Epitope On Snap-25 Is Essential For Regulatementioning

confidence: 99%

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Rickman

Jiménez

Graham

et al. 2006

MBoC

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The regulated release of hormones and neurotransmitters is a fundamental process throughout the animal kingdom. The short time scale for the calcium triggering of vesicle fusion in regulated secretion suggests that the calcium sensor synaptotagmin and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) membrane fusion machinery are well ordered before the calcium signal. To gain insight into the organization of the prefusion protein assembly in regulated exocytosis, we undertook a structural/functional study of the vesicular synaptotagmin1 and the plasma membrane SNARE proteins, which copurify from the brain in the absence of calcium. Based on an evolutionary analysis, mutagenesis screens, and a computational protein docking approach, we now provide the first testable description of the supramolecular prefusion assembly. Perturbing the determined synaptotagmin/SNARE-interacting interface in several models of regulated exocytosis altered the secretion of hormones and neurotransmitters. These mutations also disrupted the constitutive synaptotagmin/SNARE link in full agreement with our model. We conclude that the interaction of synaptotagmin with preassembled plasma membrane SNARE proteins, before the action of calcium, can provide a precisely organized "tethering" scaffold that underlies regulated secretion throughout evolution. INTRODUCTIONIn regulated secretion, fusion of docked secretory vesicles with the plasma membrane is triggered by the rapid elevation of the intracellular calcium concentration (Katz and Miledi, 1967). The membrane fusion itself is brought about by the action of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, whereas the vesicular protein synaptotagmin (SYT) acts as the calcium sensor (Sollner et al., 1993;Sudhof and Scheller, 2001;Bonifacino and Glick, 2004). In neuroendocrine cells, the principal SNAREs are syntaxin1 and synaptosome-associated protein of 25 kDa (SNAP-25) on the plasma membrane (so-called target SNAREs or t-SNAREs), and vesicular synaptobrevin, also known as VAMP. Interaction between synaptobrevin and the two t-SNAREs leads to the formation of the ternary SNARE complex, a twisted parallel fourhelical bundle that probably drives membrane fusion (Sutton et al., 1998). With all the major players involved in vesicle fusion identified, it is becoming possible to tackle a central problem of this cellular process-how does calcium trigger vesicle fusion? Arguably, to understand the action of calcium it is essential to know 1) the extent of the assembly of the SNARE fusion proteins and 2) their organization in relation to the calcium sensor, SYT, before calcium-triggered events.It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al., 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two...

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Section: The Syt Binding Epitope On Snap-25 Is Essential For Regulatementioning

confidence: 99%

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Rickman

Jiménez

Graham

et al. 2006

MBoC

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“…Complexins are a set of small (18 -21 kDa), highly charged, cytosolic proteins that bind to the fully formed exocytotic core complex at a late step in synaptic vesicle release (McMahon et al, 1995;Reim et al, 2001;Chen et al, 2002;Pabst et al, 2002) to regulate the Ca 2ϩ -dependent triggering of transmitter exocytosis (Reim et al, 2001;Archer et al, 2002). It is thought that complexins do so by binding and stabilizing the open conformation of syntaxin in the SNARE complex (Pabst et al, 2002;Chen et al, 2002;Archer et al, 2002).…”

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confidence: 99%

“…It is thought that complexins do so by binding and stabilizing the open conformation of syntaxin in the SNARE complex (Pabst et al, 2002;Chen et al, 2002;Archer et al, 2002). Synapsins are a family of abundant synaptic vesicle-associated proteins, involved in the calcium-dependent recruitment of synaptic vesicles .…”

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confidence: 99%

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina

Hirano

Brandstätter

Brecha

2005

J of Comparative Neurology

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The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A-and B-type horizontal cells, of ␥-aminobutyric acid (GABA)-and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca 2ϩ -dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells.

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“…On the other hand, we reported that knock-down of complexin II decreases exocytotic release in mast cells. 10) Complexin I binds to SNARE complex in nerve terminal and make it stable, 23) therefore it is reasonable that SNARE complex become unstable in mast cells when complexin II is knocked down. We think that synaptotagmin 2 fails to bind to unstable SNARE complex, resulting in decrease of Ca 2+ sensitivity and exocytotic release.…”

Section: Discussionmentioning

confidence: 99%

“…Complexin I binds to neuronal SNARE complexes, and Arg59 residue of complexin I contributes to this crucial interaction. 23) Mutant (R59H) complexin I exhibits decreased binding to neuronal SNARE complexes. 24) Mutant (R59H) complexin II also exhibits reduced binding to SNARE complexes in mast cells.…”

Section: Discussionmentioning

confidence: 99%

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins

Tadokoro

Hirashima

Utsunomiya‐Tate

2016

Biological & Pharmaceutical Bulletin

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Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.Key words mast cell; exocytosis; membrane fusion; soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE); degranulation Mast cells are involved in allergic reactions. Cross-linking of FcεRI, the high-affinity receptor for the Fc region of immunoglobulin E, by multivalent antigens increases intracellular Ca 2+ concentration, causing exocytotic inflammatory mediator release from mast cells. 1) Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this process. 2-9) SNARE-binding proteins also regulate exocytotic release in mast cells. 9,10) Syntaxin-3, -4 and synaptosomal-associated protein 23 kDa (SNAP-23) are SNARE proteins on the plasma membrane (t-SNARE) in mast cells. 2-4) Vesicle associated membrane protein (VAMP)-2, -7, and -8 are SNARE proteins on the secretory vesicles membrane (v-SNARE). 3,4) These t-SNARE and v-SNARE interact with each other and form SNARE complex which induce exocytotic release. [4][5][6] Final stage of exocytosis is membrane fusion process between secretory granule membrane and plasma membrane. SNAP-23/syntaxin-3 or SANP23/syntaxin-4 incorporated liposomes fuse with VAMP-8 but not VAMP-7 incorporated liposomes. 11)Complexins possess an α-helix in their central region that binds to and regulates SNAREs. 12) Complexins I and II are expressed in neuronal cells and may be involved in neurotransmitter release. 13,14) Complexin affects neuronal SNARE-mediated membrane fusion between liposomes. 15,16) Complexin II, but not complexin I, is expressed in mast cells, and exocytotic release after antigen stimulation is suppressed in mast cells where complexin II has been knocked down. 10) To elucidate the molecular mechanism of SNARE-dependent exocytosis in mast cells, we examined effects of complexin II on mast cell SNARE-dependent liposomal membrane fusion that is a simple assay system without other factors derived from cells. 11,[17][18][19] MATERIALS AND METHODS Protein Expression and PurificationFull-length rat syntaxin-3, SNAP-23, VAMP-2 or -8, and complexin II were expressed in Escherichia coli (E....

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Three-Dimensional Structure of the Complexin/SNARE Complex

Cited by 419 publications

References 47 publications

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins

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