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

---

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

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.
...
PMID:Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion. 1020 62

The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide-sensitive fusion protein), and both proteins encode cytoplasmic ATPases which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.
...
PMID:Biochemical analysis of the Saccharomyces cerevisiae SEC18 gene product: implications for the molecular mechanism of membrane fusion. 1038 16

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.
...
PMID:Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. 1045 12

Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins.
...
PMID:The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding. 1054 42

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.
...
PMID:Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins. 1071 14

SNARE (SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.
...
PMID:SNAREpins are functionally resistant to disruption by NSF and alphaSNAP. 1083 10

Stimulation of parietal cells causes fusion of intracellular tubulovesicles with the canalicular plasma membrane thereby increasing the apical membrane area up to tenfold. The presence of the SNARE proteins synaptobrevin, syntaxin1, and SNAP25 in parietal cells and their intracellular redistribution after stimulation suggest a SNARE-mediated mechanism. Here we show that NSF and alpha, beta-SNAPs which are involved in the dissociation of the SNARE complex in neurons also occur in parietal cells exhibiting subcellular distributions similar to the ones obtained for SNARE proteins and for the H+, K(+)-ATPase. More importantly proteolytic cleavage of synaptobrevin by tetanus neurotoxin completely inhibits the cAMP-dependent increase of acid secretion further supporting the crucial role SNARE proteins play in parietal cells.
...
PMID:Acid secretion of parietal cells is paralleled by a redistribution of NSF and alpha, beta-SNAPs and inhibited by tetanus toxin. 1115 8

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.
...
PMID:Structure of the AAA ATPase p97. 1116 19

NSF is a cytosolic ATPase implicated in a variety of cellular functions including synaptic vesicle exocytosis. Here we report a lethal mutation in the Drosophila homolog of NSF (dNSF1). Lethality staging and rescue experiments with the wild type dNSF1 transgene show that NSF1 is critically required during early larval stages and during late pupariation. Lethality in larval stages is associated with defects in neurogenesis as evidenced by an overall reduction in synapse size and synapse branching. Moreover, escaper adults, though showing abnormal seizure-like paralytic behavior, are normal in terms of synaptic transmission as assayed by electroretinograms. Taken together, these data indicate a role for NSF in neural growth and branching in addition to its documented role in synaptic transmission.
...
PMID:Lethal comatose mutation in Drosophila reveals possible role for NSF in neurogenesis. 1138 12

The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events. The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation. This suggests that Vtc3p might act close to or at fusion pore opening. We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans-complex formation and/or control fusion pore opening.
...
PMID:The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation. 1182 19


<< Previous 1 2 3 4 5 6 7 Next >>

---