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J. Biol. Chem., Vol. 280, Issue 32, 28966-28972, August 12, 2005

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Compensation for a Defective Interaction of the Hsp70 Ssq1 with the Mitochondrial Fe-S Cluster Scaffold Isu^*

Helena Knieszner, Brenda Schilke¶, Rafal Dutkiewicz, Patrick D'Silva¶, Sara Cheng¶, Maikke Ohlson¶||, Elizabeth A. Craig¶**, and Jaroslaw Marszalek¶

From the Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland and ^¶Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Ssq1, a specialized yeast mitochondrial Hsp70, plays a critical role in the biogenesis of proteins containing Fe-S clusters through its interaction with Isu, the scaffold on which clusters are built. Two substitutions within the Ssq1 substrate binding cleft, both of which severely reduced affinity for Isu, had very different effects in vivo. Cells expressing Ssq1(F462S), which had no detectable affinity for Isu, are indistinguishable from ssq1 cells, underscoring the importance of the Ssq1-Isu1 interaction in vivo. In contrast, cells expressing Ssq1(V472F), whose affinity for Isu is at least 10-fold lower than that of wild-type Ssq1, had only moderately reduced Fe-S enzyme activities and increased iron levels and grew similarly to wild-type cells. Consistent with the reduced affinity for Isu, the ATPase activity of Ssq1(V472F) was stimulated less well than that of Ssq1 upon addition of Isu and Jac1, the J-protein partner of Ssq1. However, higher concentrations of Jac1 or Isu1, which form a stable complex, could compensate for this defect in stimulation of Ssq1(V472F). Expression of Isu1 was up-regulated 10-fold in ssq1(V472F) compared with wild-type cells, suggesting that formation of a Jac1-Isu1 complex can overcome a lowered affinity of Ssq1 for Isu in vivo as well as in vitro.

Received for publication, March 18, 2005 , and in revised form, June 14, 2005.

^* This work was supported by the Polish State Committee for Scientific Research Project 3 P04A 050 23 (to J. M.), National Institutes of Health (NIH) Grant R01GM27870 (to E. A. C.), and American Heart Association Fellowship 04200492 (to P. D.). CD data were obtained at the University of Wisconsin-Madison Biophysics Instrumentation Facility, which is supported by the University of Wisconsin-Madison, by National Science Foundation Grant BIR-9512577, and NIH Grant S10 RR13790. 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.

Both authors contributed equally to this work.

^|| Present address: Dept. of Microbiology, University of Washington, Seattle, WA 98195.

^** To whom correspondence should be addressed: Dept. of Biochemistry, 441E Biochemistry Addition, 433 Babcock Dr., University of Wisconsin, Madison WI 53706-1544. Tel.: 608-263-7105; Fax: 608-262-3453; E-mail: ecraig{at}wisc.edu.

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