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)

Membrane trafficking has heretofore been studied with intact organelles. Here, fusion-competent proteoliposomes were reconstituted from a yeast vacuole detergent extract. Homotypic vacuole fusion requires many membrane proteins, including the Ypt7p guanosine triphosphatase and a "SNARE complex" with Vam3p and Nyv1p. Proteoliposomes from extracts immunodepleted of either Vam3p or Ypt7p could not fuse, but vesicles reconstituted from a mixture of these depleted extracts had restored fusion activity. Purified preassembled vacuolar SNARE complex, when reconstituted with a SNARE-depleted extract, was fully functional for fusion. Thus, solubilized integral membrane components can be reconstituted for priming, docking, and fusion steps of organelle trafficking.
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PMID:Functional reconstitution of ypt7p GTPase and a purified vacuole SNARE complex. 968 64

Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell. They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs. Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion. NSF is an ATPase which will disrupt complexes composed of different SNAREs. However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane. The essential role of NSF may be to prime SNAREs for a direct role during fusion. The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi. The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar. Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes. Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles. In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue. There are only eight members of the syntaxin family in yeast. Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex. They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi. We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles.
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PMID:SNAREs and membrane fusion in the Golgi apparatus. 971 10

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

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 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

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

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

Vacuole fusion occurs in three stages: priming, in which Sec18p mediates Sec17p release, LMA1 (low M(r) activity 1) relocation, and cis-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex disassembly; docking, mediated by Ypt7p and trans-SNARE association; and fusion of docked vacuoles. Ca(2+) and calmodulin regulate late stages of the reaction. We now show that the vacuole proton gradient, generated by the vacuolar proton ATPase, is needed for trans-SNARE complex formation during docking and hence for the subsequent LMA1 release. Though neither the vacuolar Pmc1p Ca(2+)-ATPase nor the Vcx1p Ca(2+)/H(+) exchanger are needed for the fusion reaction, they participate in Ca(2+) and Delta mu(H)(+) homeostasis. Fusion itself does not require the maintenance of trans-SNARE pairs.
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PMID:Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion. 1050 Jan 53


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