The Role of ATP in the Functional Cycle of the DnaK Chaperone System

McCarty, John S.; Buchberger, Alexander; Reinstein, Jochen; Bukau, Bernd

doi:10.1006/jmbi.1995.0284

Cited by 373 publications

(372 citation statements)

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“…The maximal increase observed, -4-fold at 25"C, is less than the maximal stimulation of DnaK intrinsic ATPase activity by DnaJ which is -10-fold (Jordan & McMacken, 1995;McCarty et al, 1995). Because of the higher intrinsic activity of Hsc66 compared to DnaK, however, the maximal Hsc20-stimulated ATPase activity of Hsc66 (-0.5 min-I at 25°C) is in the same range as the maximal Dnd-stimulated ATPase activity of DnaK.…”

Section: Discussionmentioning

confidence: 81%

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

1997

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The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc2O in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc2O are constitutively expressed at levels corresponding to cell concentrations -20 pM and -10 pM, respectively. The levels do not change appreciably following heat shock (44"C), but a small increase in Hsc2O is observed following a shift to 10°C. Purified Hsc66 exhibits a low intrinsic ATPase activity (-0.6 min" at 37"C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration -5 pM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/Dnd and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions. Keywords: ATPase activity; chaperone; co-chaperone; constitutive expression; hsp70; hsp40; purification The hsp70, or stress-70, proteins comprise a large and widespread family of proteins -7O-kDa, which function as ATP-dependent molecular chaperones (reviewed in Gething

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Section: Discussionmentioning

confidence: 81%

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

1997

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“…The most extensively studied cochaperones that modulate Hsp70 function are DnaJ and GrpE in the E. coli DnaK system. DnaJ stimulates the ATP hydrolysis step in the DnaK ATPase cycle (50), which yields the high affinity ADP state for substrate binding. To release the bound substrate and allow the cycle to begin anew, GrpE, which functions as a nucleotide exchange factor, is needed in a next step (51,52).…”

Section: Resultsmentioning

confidence: 99%

Cns1 Is an Activator of the Ssa1 ATPase Activity

Hainzl¹,

Wegele²,

Richter

et al. 2004

Journal of Biological Chemistry

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Hsp90 is a key mediator in the folding process of a growing number of client proteins. The molecular chaperone cooperates with many co-chaperones and partner proteins to fulfill its task. In Saccharomyces cerevisiae, several co-chaperones of Hsp90 interact with Hsp90 via a tetratricopeptide repeat (TPR) domain. Here we show that one of these proteins, Cns1, binds both to Hsp90 and to the yeast Hsp70 protein Ssa1 with comparable affinities. This is reminiscent of Sti1, another TPR-containing co-chaperone. Unlike Sti1, Cns1 exhibits no influence on the ATPase of Hsp90. However, it activates the ATPase of Ssa1 up to 30-fold by accelerating the ratelimiting ATP hydrolysis step. This stimulating effect is mediated by the N-terminal TPR-containing part of Cns1, whereas the C-terminal part showed no effect. Competition experiments allow the conclusion that Hsp90 and Ssa1 compete for binding to the single TPR domain of Cns1. Taken together, Cns1 is a potent cochaperone of Ssa1. Our findings highlight the importance of the regulation of Hsp70 function in the context of the Hsp90 chaperone cycle.

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“…The unfolded protein remains bound to DnaK-ADP until a favourable physiological state is regained. GrpE, in turn, functions as a nucleotide-exchange factor, and promotes dissociation of ADP from DnaK for ATP (Harrison, 2003;Liberek et al, 1991;McCarty et al, 1995;Winter & Jakob, 2004). Thus, all three proteins are essential for the DnaK system to be functional.…”

Section: Discussionmentioning

confidence: 99%