Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins (original) (raw)
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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.
SNARE proteins and membrane fusion
El Mednifico Journal, 2013
Diverse proteins catalyze membrane fusion reactions. These mediate recognition of the membranes for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. In the nervous system, membrane fusion is vital for neuroexocytosis, neuro transmitter release and chemical synaptic transmission. Three neuronal soluble NSF (N-ethyl-maleimide-sensitive fusion protein) attachment receptor (SNARE) proteins exist, namely: (i) vesicle associated membrane protein (VAMP-2), also called synaptobrevin; (ii) 25 kDa synaptosome-associated protein (SNAP-25); and (iii) syntaxin 1A (STX1). The SNAREs are involved in the neuronal membrane fusion process. (El Med J 2:1; 2014)
Compartmental specificity of cellular membrane fusion encoded in SNARE proteins
Nature, 2000
Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.
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...
Molecular Cell, 1999
Sacher et al., 1998). Additional general factors acting on SNAREs are NSF (NEM-sensitive factor; Block New York, New York 10021 † Department of Molecular Biophysics et al., 1988) and SNAPs (soluble NSF attachment proteins; Clary et al., 1990), which alter the conformation and Biochemistry Yale University of SNAREs and disassemble v-t SNARE complexes, thereby regenerating separate v-and t-SNAREs for re-New Haven, Connecticut 06520 peated use. Most SNARE proteins possess a single transmembrane domain at their extreme carboxy terminus and Summary are predicted to have a high propensity to form coiledcoil structures. Assembled cytosolic domains of SNARE The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to proteins form very stable structures in all cases that have been closely examined (Hayashi et al., 1994; Yang do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins et al., 1999), likely due to their coiled-coil nature. Furthermore, electron microscopic (Hanson et al., 1997b; Hohl and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline et al., 1998) and biophysical (Lin and Scheller, 1997; Poirier et al., 1998) analysis of assembled full-length residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, neuronal SNARE complexes revealed that the transmembrane domains of both the v-SNARE VAMP/synap-and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a tobrevin (Trimble et al., 1988; Baumert et al., 1989; Sü dhof et al., 1989) and the t-SNARE syntaxin 1A (Bennett flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately et al., 1992, 1993) emerge at the same end of the 7S or 20S particle establishing a parallel orientation of the affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. assembled SNARE proteins. These characteristics suggested that SNARE proteins are also directly responsible Together, these results rule out a requirement for helical continuity and suggest that distance is a critical for membrane fusion (Hanson et al., 1997a, 1997b; Lin and Scheller, 1997; Hohl et al., 1998), and this has now factor for membrane fusion. been directly demonstrated with artificial liposomes and isolated SNAREs (Weber et al., 1998) and confirmed with Introduction permeabilized cells (Chen et al., 1999). The recently obtained three-dimensional structure of Members of the SNARE (SNAP receptor) protein family the neuronal SNARE complex (Poirier et al., 1998; Sutton (Sö llner et al., 1993a, 1993b) are key components in et al., 1998) has provided additional information to guide the process of transport vesicle docking and fusion. structure-function studies aimed at clarifying the bio-Individual SNARE family members are maintained in disphysical mechanisms involved in fusion. The crystal crete locations throughout the secretory pathway prostructure confirms the previous biophysical predictions viding a roadmap of vesicle flow patterns (Hay and of coiled-coil structure and provides a structural frame-Scheller, 1997; Linial, 1997; Advani et al., 1998; Nichols work to explain the intrinsic stability of the assembled and Pelham, 1998; Steegmaier et al., 1998). The assem-SNARE complex. Another striking feature of the core bly of trans-SNARE complexes (SNAREpins) between SNARE complex structure is its overall similarity to the membranes is likely the underlying principle of lipid biproposed fusogenic cores of a variety of virally encoded layer fusion (Weber et al., 1998; Chen et al., 1999), and fusion proteins (Skehel and Wiley, 1998). This similarity as such, it must be highly regulated in many cell types.
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
The Journal of Cell Biology, 2002
e utilize structurally targeted peptides to identify a "t C fusion switch" inherent to the coil domains of the neuronal t-SNARE that pairs with the cognate v-SNARE. The t C fusion switch is located in the membrane-proximal portion of the t-SNARE and controls the rate at which the helical bundle that forms the SNAREpin can zip up to drive bilayer fusion. When the fusion switch is "off" (the intrinsic state of the t-SNARE), zippering of the helices from their membrane-distal ends is impeded and fusion is slow. When the t C fusion switch is "on," fusion is much faster. The t C fusion switch can be thrown by a W peptide that corresponds to the membrane-proximal half of the cognate v-SNARE, and binds reversibly to the cognate region of the t-SNARE. This structures the coil in the membrane-proximal domain of the t-SNARE and accelerates fusion, implying that the intrinsically unstable coil in that region is a natural impediment to the completion of zippering, and thus, fusion. Proteins that stabilize or destabilize one or the other state of the t C fusion switch would exert fine temporal control over the rate of fusion after SNAREs have already partly zippered up.
SNAREs Contribute to the Specificity of Membrane Fusion
Neuron, 2000
Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information