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J. Biol. Chem., Vol. 279, Issue 43, 44376-44383, October 22, 2004

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Successive and Synergistic Action of the Hsp70 and Hsp100 Chaperones in Protein Disaggregation^*

Szymon Zitkiewicz, Joanna Krzewska, and Krzysztof Liberek

From the Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.

Received for publication, March 3, 2004 , and in revised form, July 26, 2004.

^* This work was supported by Polish State Committee for Scientific Research Grant 3P04A02924. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel./Fax: 48-58-301-92-22; E-mail: liberek{at}biotech.univ.gda.pl.

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