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)

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity. Exchanging DnaJ homologs between the S. cerevisiae and E. coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved. In contrast, GrpE further stimulated only DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.
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PMID:Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators. 763 93

Ydj1p, a cytosolic DnaJ homolog from Saccharomyces cerevisiae, is demonstrated to function as a molecular chaperone. Purified Ydj1p formed complexes with non-native polypeptides and suppressed protein aggregation. Ydj1p cooperated with Ssa Hsp70 proteins in the prevention of protein aggregation, but not with the Ssb Hsp70 proteins. Cooperation between these different molecular chaperones was only observed in the presence of hydrolyzable ATP and correlated with the ability of Ydj1p to stimulate the ATPase activity of the Hsp70 homolog with which it was paired. The regulatory and chaperone activities of a eukarytic DnaJ homolog thus act together to assist Hsp70 in modulating the conformation of proteins.
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PMID:Cooperation of the molecular chaperone Ydj1 with specific Hsp70 homologs to suppress protein aggregation. 786 84

A DnaK homolog (T.DnaK) has been purified as a stable complex with a DnaJ homolog (T.DnaJ) from a thermophilic bacterium, Thermus thermophilus. This complex has an approximate molecular size of 300 kDa and appears to contain three copies of each of T.DnaK and T.DnaJ molecules. Consistently, trigonal ring structures with a diameter (trigonal apex-to-apex) of about 11 nm were observed with electron microscopy. The complex has no endogenously bound AT(D)P and is stable in the presence of Mg-AT(D)P. It possesses a weak ATPase activity and retains about 3 mol of ADP/mole of the complex when incubated with Mg-ATP. This complex is able to interact with the reduced carboxymethylated alpha-lactalbumin which we used as a model unfolded protein.
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PMID:Isolation of the stable hexameric DnaK.DnaJ complex from Thermus thermophilus. 792 50

In Saccharomyces cerevisiae Ydj1p, a DnaJ homolog, is localized to the cytosol with the Ssa and Ssb Hsp70 proteins. Ydj1p helps facilitate polypeptide translocation across mitochondrial and endoplasmic reticulum membranes (Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992) Cell 71, 1143-1155) and can directly interact with Ssa1p to regulate chaperone activity (Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931). In this study, the role of Ydj1p in modulating ATP-dependent reactions catalyzed by Ssa and Ssb Hsp70 proteins has been examined using purified components and compared with that of other Hsp70 homologs BiP and DnaK. Ssa1p, Ssa2p, and Ssb1/2p all formed stable complexes with the mitochondrial presequence peptide, F1 beta(1-51). ATP alone had only modest effects on polypeptide complex formation with Ssa1p and Ssa2p, but prevented the majority of polypeptide binding to BiP and DnaK. ATP by itself also reduced polypeptide binding to Ssb1/2p to a level that was intermediate between that observed for the Ssa Hsp70 proteins tested and BiP and DnaK. ATP hydrolysis by Ssa1p, Ssa2p, and Ssb1/2p occurred at similar rates. Ydj1p was a potent modulator of the both the ATPase and polypeptide binding activities of Ssa1p and Ssa2p. In contrast, Ydj1p had little effect on the ATPase and polypeptide binding activity of Ssb1/2p. Therefore the chaperone-related activities of Ssa and Ssb Hsp70 proteins exhibit significant differences in sensitivity to ATP and YDJ1p. These data indicate that regulation of Hsp70 activity by DnaJ homologs can be specific. The specificity of interactions between Ydj1p and the Ssa and Ssb Hsp70 proteins observed could contribute in determining the functional specificity of these chaperones in the cytosol. In related experiments, F1 beta(1-51) was found to reduce the extent to which Ydj1p stimulated Ssa1p ATPase activity. This effect correlated with the formation of F1 beta(1-51).Ssa1p complexes. We propose that intramolecular communication between the polypeptide binding, ATPase and DnaJ regulatory domains on Ssa1p plays a role in the regulation of chaperone activity.
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PMID:Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1. 814 72

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.
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PMID:Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. 870 58

We have studied the direct interaction of the constitutive isoform of Hsp70 (Hsc70) with the DnaJ homolog, auxilin, a cofactor that binds to clathrin-coated vesicles and is required for their uncoating by Hsc70. Auxilin caused a 5-fold increase in Hsc70 ATPase activity and a corresponding increase in steady-state levels of bound ADP; the dissociation constant for this effect was 0.6 microM. Auxilin also induced polymerization of Hsc70 and bound to the resulting polymer at a 1:1 molar ratio; here too the dissociation constant was 0.6 microM. Both this binding and polymerization required ATP; the Hsc70 depolymerized with a 4-min half-life when ATP was completely hydrolyzed to ADP. Although auxilin induces polymerization stoichiometrically and other DnaJ homologs induce polymerization catalytically, these data show that auxilin is similar to other DnaJ homologs in its ability to activate the Hsc70 ATPase activity, to polymerize Hsc70, and in the nucleotide dependence of this polymerization. Furthermore, the 70-amino acid J-domain of auxilin polymerized Hsc70 with the same nucleotide dependence as intact auxilin. Therefore, although only auxilin and not other DnaJ homologs support uncoating, our data suggest that various DnaJ homologs share a common mechanism of interaction with Hsc70, perhaps because their J-domains interact similarly with Hsc70.
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PMID:Interaction of auxilin with the molecular chaperone, Hsc70. 904 25

We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaJ homolog which is required for Hsc70 to uncoat clathrin baskets. In the present study we investigated the effect of auxilin on the interaction of Hsc70 with pure clathrin baskets at pH 6, where no uncoating occurs. In a reaction which required auxilin, the baskets activated the Hsc70 ATPase activity more than 100-fold with an apparent dissociation constant of about 0.2 microM. Maximal ATPase activity occurred at a 1 to 1 molar ratio of auxilin to clathrin triskelion independent of the Hsc70 concentration suggesting that auxilin is primarily complexed with the clathrin baskets. The binding of Hsc70 to baskets also required auxilin, but less auxilin was needed for maximum binding than for maximum ATPase activity showing that auxilin can catalytically induce binding of Hsc70. The binding also required ATP; Hsc70 dissociated from baskets with a 6 min half-life when ATP was hydrolyzed to ADP. In contrast to auxilin, the assembly proteins, AP-2 and AP180, did not support activation of the Hsc70 ATPase activity by clathrin baskets nor did soluble clathrin triskelions at pH 7 significantly activate the ATPase activity with auxilin present. Therefore, the interaction of auxilin, clathrin baskets, and Hsc70-ATP is highly specific with auxilin first binding to a clathrin triskelion in the baskets and then Hsc70-ATP strongly binding to the auxilin-clathrin complex; the auxilin can then migrate to another clathrin triskelion before the ATPase cycle is complete.
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PMID:Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets. 910 26

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.
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PMID:Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli. 932 54

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.
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PMID:N-Ethylmaleimide-modified Hsp70 inhibits protein folding. 1046 52

Calf thymus (ct) Hsc70 has been shown previously to reactivate heat-inactivated prokaryotic and eukaryotic enzymes, while DnaK was able to reactivate solely prokaryotic enzymes. Here, we report on isolation from calf thymus of a DnaJ homolog, ctHsc40, and on testing of its cooperative function in three different assays: (i) reactivation of heat-inactivated DNA polymerases, (ii) stimulation of the ATPase activity of ctHsc70 chaperone, and (iii) replication of bacteriophage lambda DNA. Surprisingly, ctHsc70/ctHsc40 chaperones were found to reactivate the denatured prokaryotic and eukaryotic enzymes but not to promote bacteriophage lambda DNA replication, suggesting species specificity in DNA replication.
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PMID:Calf thymus Hsc70 and Hsc40 can substitute for DnaK and DnaJ function in protein renaturation but not in bacteriophage DNA replication. 1168 50


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