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 neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.
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PMID:Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly. 972 21

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

N-Ethylmaleimide-sensitive factor (NSF) plays a key role in vesicular traffic by disassembling and priming SNARE proteins for their function in docking and fusion. We demonstrate that the ATPase activity of NSF is activated by alpha-soluble NSF attachment protein (alpha-SNAP) in a complex with syntaxin 1A. In addition, we show that a construct consisting of the H3 domain of syntaxin IA (GST-synt(195-263), which does not support NSF disassembly in the presence of MgATP gave a larger stimulation. NSF ATPase activation was specific and did not occur using mutant alpha-SNAPs unable to bind GST-synt or with mutated C-termini. We suggest that activation of NSF ATPase activity in the SNARE complex may be essential to allow SNARE priming.
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PMID:Stimulation of NSF ATPase activity during t-SNARE priming. 977 83

SNAP-25 belongs to a family of evolutionarily conserved proteins whose members are essential for exocytosis. Neurons and neuroendocrine cells differentially express two SNAP-25 isoforms in a developmentally regulated manner, and related homologues have been detected in most eukaryotic cells. SNAP-25 is localised on the cytoplasmic face of the plasma membrane and on secretory vesicles. It forms a stable ternary complex with two other exocytotic proteins: syntaxin and the synaptic vesicle protein synaptobrevin. A cytosolic ATPase dissociates this complex during priming of the exocytotic apparatus. Subsequent reassembly is promoted by SNAP-25 and may drive Ca(2+)-triggered vesicle-plasma membrane fusion. A mutant mouse that lacks the SNAP-25 gene is defective in neuronal dopamine signalling and exhibits similar behaviour as sufferers from hyperactivity disorders. Use of this animal model thus provides a promising avenue for the development of therapeutic treatments. Additionally, SNAP-25-based peptides that mimic the effect of botulinum neurotoxin A may be used for the treatment of involuntary muscle spasms.
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PMID:SNAP-25. 978 71

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

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.
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PMID:Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. 1045 12

The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion. In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H(+)-ATPase to and from the apical membrane. We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells. The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H(+)-ATPase in IMCD cells during exocytosis and to determine the effects of clostridial toxins on SNARE-mediated trafficking of H(+)-ATPase. Cell acidification induced a marked increment of H(+)-ATPase in the apical membrane. However, pretreating cells with clostridial toxins blocked the cellular translocation of the 31-kDa subunit. Immunoprecipitation of IMCD cell homogenate, using antibodies against either the 31-kDa subunit of H(+)-ATPase or vesicle-associated membrane protein-2, co-immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF attachment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2. Pretreatment with clostridial toxin resulted in reduced co-immunoprecipitation of H(+)-ATPase and syntaxin. These experiments document, for the first time, a putative docking fusion complex in IMCD cells and a physical association of the H(+)-ATPase with the complex. The sensitivity to the action of clostridial toxin indicates the docking-fusion complex is a part of the exocytotic mechanism of the proton pump.
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PMID:SNARE proteins regulate H(+)-ATPase redistribution to the apical membrane in rat renal inner medullary collecting duct cells. 1047 13

SNARE proteins - rab3A - parietal cells - H+/K+-ATPase When stimulated by histamine, acetylcholine, or gastrin the luminal compartments of oxyntic parietal cells display conspicuous morphological changes. The luminal plasma membrane surface becomes greatly expanded, while the cytoplasmic tubulovesicles are decreased in parallel. Due to these membrane rearrangements the H+/K(+)-ATPase obtains access to the luminal surface, where proton secretion occurs. The stimulation-induced translocation of H+/K(+)-ATPase involves a fusion process. Exocytotic membrane fusion in neurons is achieved by the highly regulated interaction of mainly three proteins, the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 (synaptosomal-associated protein of 25 kDa), also referred to as SNARE proteins. Using immunofluorescence microscopy we analysed the subcellular distribution of neuronal synaptic proteins and rab3A in resting and stimulated parietal cells from pig and rat. In resting cells all synaptic proteins colocalized with the H+/ K(+)-ATPase trapped in the tubulovesicular compartment. After stimulation, translocated H+/K(+)-ATPase showed a typical canalicular distribution. Syntaxin, synaptobrevin, SNAP25 and rab3A underwent a similar redistribution in stimulated cells and consequently localized to the canalicular compartment. Using immunoprecipitation we found that the SNARE complex consisting of synaptobrevin, syntaxin and SNAP25, which is a prerequisite for membrane fusion in neurons, is also assembled in parietal cells. In addition the parietal cell-derived synaptobrevin could be proteolytically cleaved by tetanus toxin light chain. These data may provide evidence that SNARE proteins and rab3A are functionally involved in the stimulation-induced translocation of the H+/K(+)-ATPase.
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PMID:SNARE proteins and rab3A contribute to canalicular formation in parietal cells. 1060 54

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.
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PMID:Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins. 1071 14

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.
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PMID:Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells. 1124 93


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