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)

Whether hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone and, to day, the involvement of ATP hydrolysis in hsp90 function is still a controversial subject. ATPase activities may be detected in partially purified hsp90's preparations from rabbit muscle. We demonstrate that the major contaminant associated with hsp90 is the p97 fusion protein and that these oligomeric structures are copurifying together with the 20S proteasome and its PA28 activator. Improving the purification procedure permits to separate hsp90 and p97 to homogeneity. Then, our attempts failed to detect any significant ATPase activity in the hsp90 fraction. Thus, p97 would be principally responsible for the ATPase activity detected in partially purified hsp90 preparations from rabbit muscle.
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PMID:20S proteasome, hsp90, p97 fusion protein, PA28 activator copurifying oligomers and ATPase activities. 1020 83

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

The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation. p97/Cdc48p contains two conserved consensus p34cdc2 kinase phosphorylation sites within its second ATP binding domain. This domain is likely to play a role in stabilising the hexameric form of the protein. We therefore investigated whether p97 could be phosphorylated by p34cdc2 kinase in vitro, and whether phosphorylation might influence the oligomeric status of p97. Monomeric, but not hexameric, p97 was phosphorylated by p34cdc2 kinase, as was the p97-associated protein p47. However, phosphorylation by p34cdc2 kinase did not impair subsequent re-hexamerisation of p97, implying that the phosphorylated residue(s) are not critical for interaction between p97 monomers. Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is not regulated during mitosis by influencing the extent of oligomerisation.
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PMID:Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase. 1035 Feb 10

Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.
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PMID:Structure of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum as studied by electron tomography. 1035 78

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.
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PMID:The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding. 1054 42

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.
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PMID:Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein. 1055 5

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.
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PMID:A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. 1081 9

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
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PMID:Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum. 1093 Apr 51

The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.
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PMID:Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro. 1108 17

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.
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PMID:Structure of the AAA ATPase p97. 1116 19


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