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J. Biol. Chem., Vol. 281, Issue 12, 7801-7808, March 24, 2006

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The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p^*

Rafal Dutkiewicz¹, Jaroslaw Marszalek², Brenda Schilke, Elizabeth A. Craig, Roland Lill^¶, and Ulrich Mühlenhoff^¶3

From the Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland, the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, and the ^¶Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch Strasse 6, 35033 Marburg, Germany

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.

Received for publication, December 14, 2005 , and in revised form, January 23, 2006.

^* This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (SFB 593 and Gottfried-Wilhelm Leibniz program), Fonds der Chemischen Industrie, Deutsches Humangenomprojekt, the Fritz-Thyssen-Stiftung, and by National Institutes of Health Grant RO1GM27870. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a Short Term EMBO Fellowship in 2003 and by a Fellowship from United Nation Education and Science Commission/Polish Academy of Sciences Cellular and Molecular Biology Network in 2004.

² Supported by Polish State Committee for Scientific Research Project 3 P04A 050 23.

³ To whom correspondence should be addressed. Tel.: 49-6421-286-4171; Fax: 49-6421-286-6414; E-mail: muehlenh{at}mailer.uni-marburg.de.

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