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)

Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.
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PMID:The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development. 900 87

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPgammaS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide-sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.
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PMID:Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte. 908 39

Rab7 has been shown to localize to late endosomes and to mediate transport from early to late endosome/lysosome in mammalian cells and in yeast. We developed a novel assay to quantify transport from early to late endosomes using the Xenopus oocyte. Oocytes were pulsed with avidin after which the oocytes were incubated to allow avidin transport to a late compartment. The oocytes were then allowed to internalize biotin-horseradish peroxidase (HRP). The oocytes were then injected with test proteins and incubated further to allow transport of biotin-HRP from early endosomes to late endosomal/lysosomal compartments. Transport was quantified by assessing the formation of HRP-biotin-avidin complexes. Injection of Rab7:wild-type (WT) and Rab7:Q67L, a GTPase defective mutant, stimulated transport. Rab5:WT had no effect. Rab7:WT-stimulated transport was inhibited by nocodazole, suggesting a role for intact microtubules. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked Rab7:WT-stimulated transport, but Rab7:Q67L-stimulated transport was unaffected by the drug. Rab7:Q67L is constitutively activated and may not require phosphatidylinositol 3-kinase activity for activation. Rab7-stimulated transport requires N-ethylmaleimide-sensitive factor (NSF) activity as transport was blocked by N-ethylmaleimide and ATPase defective NSF mutants. Our results indicate that sequentially acting endocytic Rab GTPases utilize similar factors although their modes of action may be different.
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PMID:Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes. 914 16

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.
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PMID:Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles. 917 94

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.
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PMID:Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells. 918 93

We characterized the in vitro fusion of endosomal compartments from Dictyostelium discoideum. Fusion activity was restricted to early compartments, was dependent on cytosolic proteins, and was activated by GTP and guanosine 5'-O(3-thio)triphosphate (GTPgammaS). This stimulation suggests the involvement of a small G protein, which we propose to be Rab7 on the basis of the strong inhibitory effect of anti-Rab7 antibodies. It is noteworthy that in the presence of GTPgammaS, the concentration of ATP-Mg2+ could be reduced to less than 1 nM without loss of fusion activity. Under these conditions, competing residual ATP with adenosine 5'-O-(3-thio)triphosphate-Mg2+ also failed to inhibit endosome fusion. The presence of an ATP-depleting system alone blocked fusion probably because endogenous GTP was removed by coupling through NDP kinase. Moreover, whether ATP was present or not, GTPgammaS-activated fusion was equally sensitive to anti-Rab7 antibodies or N-ethylmaleimide and was restricted to early compartments. These results show that soluble ATP-Mg2+ is not needed for endosome fusion. Since homotypic fusion of endosomes in D. discoideum has been shown to depend on the ATPase N-ethylmaleimide-sensitive factor (Lenhard, J. M., Mayorga, L. , and Stahl, P. D. (1992) J. Biol. Chem. 267, 1896-1903), the nucleotide exchange on the N-ethylmaleimide sensitive factor must take place before GTPgammaS activation in this system.
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PMID:In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7 but proceeds in the absence of ATP-Mg2+ from the bulk solution. 942 33

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

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase which primes and/or dissociates SNARE complexes involved in intracellular fusion events. Each NSF protomer contains three domains: an N-terminal domain required for SNARE binding and two ATPase domains, termed D1 and D2, with D2 being required for oligomerization. We have determined the 1.9 A crystal structure of the D2 domain of NSF complexed with ATP using multi-wavelength anomalous dispersion phasing. D2 consists of a nucleotide binding subdomain with a Rossmann fold and a C-terminal subdomain, which is structurally unique among nucleotide binding proteins. There are interactions between the ATP moiety and both the neighboring D2 protomer and the C-terminal subdomain that may be important for ATP-dependent oligomerization. Of particular importance are three well-ordered and conserved lysine residues that form ionic interactions with the beta- and gamma-phosphates, one of which likely contributes to the low hydrolytic activity of D2.
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PMID:Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. 973 75

The highly conserved ATPase p97, a member of the AAA-ATPases, is found in a complex with its co-factor p47 in rat liver cytosol. Previously it had been shown that p97-mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47 which mediates the binding of p97 to a Golgi t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment factor receptor), syntaxin 5. Here we show that it also suppresses the ATPase activity of p97 by up to 85% in a dose-dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle.
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PMID:The p47 co-factor regulates the ATPase activity of the membrane fusion protein, p97. 982 2

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly.
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PMID:NSF N-terminal domain crystal structure: models of NSF function. 1044 31


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