Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex (original) (raw)
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Traffic, 2004
Membrane fusion depends on the formation of a complex of four SNARE motifs, three that bear a central glutamine and are localized in the target membrane (t-SNARE) and one that bears an arginine and is localized in the donor vesicle (v-SNARE). We have characterized the arginine 56 to proline mutant (R56P) of synaptobrevin-2 (Sb). SbR56P was blocked at the plasma membrane in association with the endogenous plasma membrane t-SNARE due to an inhibition of SNARE complex dissociation, suggesting that the plasma membrane is its first target. Cell surface blockade of SbR56P could be rescued by coexpression of synaptophysin, a partner of Sb. Sb was blocked at the plasma membrane but SNARE complexes were unaffected in cells expressing defective dynamin, indicating that the phenotype of SbR56P was not due to an internalization defect. When expressed in neurons, SbR56P localized both to axonal and dendritic plasma membranes, showing that both domains are initial targets of Sb. The R56P mutation affects a highly conserved position in v-SNAREs, and might thus provide a general tool for identifying their first target membranes.
α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery
Journal of Biological Chemistry, 2014
Background: Soluble N-ethylmaleimide-sensitive factor attachment protein ␣ (␣-SNAP) regulates the pre-fusion step as well as SNARE disassembly. Results: ␣-SNAP on its own interferes with SNARE zippering and inhibits chromaffin granule fusion, but not synaptic vesicle fusion. Conclusion: Retardation of SNARE zippering by ␣-SNAP results in the partial SNARE zippering. Significance: This is the first direct evidence showing the partial SNARE zippering in the physiological context. Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor ␣-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that ␣-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled transcomplex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of ␣-SNAP interference with SNARE zippering. We suggest that binding of ␣-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. Neurotransmitters are stored in synaptic vesicles and secretory granules and are released by Ca 2ϩ-dependent exocytosis upon stimulation. Fusion between vesicles and the plasma membrane are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. These include the transmembrane synaptobrevin-2 residing in the vesicle membrane, and SNAP 3-25A and syntaxin-1A resid
A Novel Site of Action for -SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion
Molecular Biology of the Cell, 2007
sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of ␣-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of ␣-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of ␣-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of ␣-SNAP potently inhibits Ca 2؉ -dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an ␣-SNAP mutant defective in NSF activation is used. We conclude that ␣-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for ␣-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
Proceedings of the …, 1998
SNARE [soluble NSF (N-ethylmaleimidesensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four ␣-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.
A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion
Molecular biology of the cell, 2008
Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocy...
Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins
… et Biophysica Acta (BBA …, 2003
SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains. Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade. This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast. Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function. D
Structural determinants for SNARE-mediated neurosecretion
2010
14 synaptobrevin2) and the synaptic plasma membrane proteins syntaxin1A and SNAP-25 [44-47]. A molecular model of SNARE-mediated vesicle exocytosis has emerged within the last 30 years [2, 48] (Figure 4). This model of regulated exocytosis consists of a series of transition steps that are controlled by additional late regulatory proteins including synaptotagmin, complexin, tomosyn, Munc-13, syntaphilin and snapin [49-55]. The process begins when syntaxin1A and SNAP-25, which are organized in clusters at the plasma membrane [56, 57], assemble together to form a binary complex called acceptor complex [58-60]. The acceptor complex provides a binding interface for the vesicular SNARE VAMP2, thus forming a ternary complex. Figure 4. Model for SNARE-mediated neuronal exocytosis. The neuronal t-SNAREs SNAP-25 and syntaxin1A (labelled in green and red, respectively), assemble together to form the acceptor complex followed by binding of the v-SNARE partner VAMP2 (in blue). The three SNARE proteins form the trans-SNARE complex that brings opposing membranes into close proximity awaiting a Ca 2+ signal. Additional proteins such as synaptotagmin (purple) and complexin (pink) bind to this trans-SNARE complex with possibly distinct outcomes. Ca 2+ entry triggers membrane fusion, followed by the generation of cis-SNARE complexes, which are disassembled by NSF and α-SNAP upon ATP-hydrolysis This ternary complex proceeds from a loose state (in which only the Nterminal part is assembled) as studied in vivo [61-63] and in vitro [64] to a tight Ca + Calcium influx Membrane fusion Synaptobrevin/Vamp