Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.
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PMID:Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation. 874 44

alpha-SNAP [soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein] is required for fusion of transport vesicles with their target membrane. In this study, we have examined the membrane-binding properties of alpha-SNAP. We have found that in several tissues a much larger amount of alpha-SNAP per unit weight of protein is bound to membranes than is free in the cytosol. Biochemical analysis shows that a fraction of alpha-SNAP behaves in ways characteristic of hydrophobic, lipid-associated proteins. These findings suggest that membrane binding may be accounted for, at least in part, by interaction with membrane lipid. Consistent with this idea, binding of newly synthesized alpha-SNAP to brain membranes was found to be independent of functional SNAP receptors and could be accounted for by direct binding of alpha-SNAP to membrane lipid. Furthermore, membrane lipid enhanced the ability of alpha-SNAP to stimulate NSF-dependent ATPase activity.
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PMID:Evidence for interaction of the fusion protein alpha-SNAP with membrane lipid. 923 Jan 35

Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.
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PMID:Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. 926 32

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.
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PMID:A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network. 934 16

N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP were able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285), and alpha-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis.
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PMID:Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis. 936 6

The N-ethylmaleimide-sensitive factor (NSF) is required for multiple intracellular vesicle transport events. In vitro biochemical studies have demonstrated that NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors from a 20 S particle. This complex is disassembled by the ATPase activity of NSF. We have studied particle disassembly in a membrane environment by examining the binding of recombinant SNAPs and NSF to endosomal membranes. We present evidence that alpha-SNAP is released from the membranes in a temperature- and time-dependent manner and that this release is mediated by the ATPase activity of NSF. Our results indicate that NSF mutants in the first ATP binding domain completely abrogate alpha-SNAP release, whereas no inhibitory effect is observed with a mutant in the second ATP binding domain. Interestingly, neither beta-SNAP nor gamma-SNAP are released by the ATPase activity of NSF, indicating that these proteins are retained on the membranes by interactions that differ from those that retain alpha-SNAP. Although the small Rab GTPases are known to play a role in SNARE complex assembly, our results indicate that these GTPases do not regulate the NSF-dependent release of alpha-SNAP.
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PMID:N-ethylmaleimide-sensitive factor-dependent alpha-SNAP release, an early event in the docking/fusion process, is not regulated by Rab GTPases. 943 Jun 66

Vacuole fusion requires Sec18p (NSF), Sec17p (alpha-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (low Mr activity 1, a heterodimer of thioredoxin and I(B)2). LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p "priming" ATPase requires both Sec17p and LMA1. Either the sec18-1 mutation and deletion of I(B)2, or deletion of both I(B)2 and p13 (an I(B)2 homolog) causes a striking synthetic vacuole fragmentation phenotype. Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex. LMA1 is released from vacuoles in a phosphatase-regulated reaction. This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion.
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PMID:LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion. 965 46

The ATPase of the N-ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle-target membrane fusion. Complexes containing different combinations of NSF, alpha-SNAP, Vamp-2 (synaptobrevin 2), syntaxin 1, and SNAP-25 were reconstituted and then tested for their effect on the ATPase of NSF. While NSF interacts with all alpha-SNAP-containing complexes, only the alpha-SNAP/t-SNARE complex significantly stimulated ATPase activity. This stimulation was dependent on increasing SNAP/t-SNARE complex and was saturable. The apparent stimulation of ATPase activity is due to a 10-fold increase in initial hydrolysis rate. Complex containing both v- and t-SNAREs bound significantly more alpha-SNAP but did not stimulate the ATPase of NSF.
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PMID:The effects of SNAP/SNARE complexes on the ATPase of NSF. 976 11

Much recent work has focussed on the role of membrane-bound components in fusion. We show here that p97 and NSF are sufficient to mediate rapid membrane fusion. Fractionation of cytosol revealed that p97 and its co-factor, p47, constitutes the major fusion activity. This was confirmed by depleting p97 from the cytosol, which resulted in an 80% decrease in fusion. Using purified protein, p97 or NSF was found to be sufficient to mediate rapid fusion in an ATP-dependent manner. A regulatory role was observed for their corresponding co-factors, p47 and alpha-SNAP. When present at a molar ratio half of that of the ATPase, both co-factors increased fusion activity significantly. Intriguingly, at this ratio the ATPase activity of the complex measured in solution was at its lowest, suggesting that the co-factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the opposing membranes and contents of liposomes. We conclude from these data that p97, NSF and perhaps other related ATPases catalyse rapid and complete fusion between lipid bilayers on opposing membranes. This highlights a new role for p97 and NSF and prompts a re-evaluation of current fusion models.
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PMID:Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion. 1020 62

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.
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PMID:Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties. 1033 34


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