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J. Biol. Chem., Vol. 278, Issue 40, 38505-38513, October 3, 2003

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Juxtaposition of the Two Distal CX3C Motifs via Intrachain Disulfide Bonding Is Essential for the Folding of Tim10^*

Scott Allen  , Hui Lu , David Thornton  and Kostas Tokatlidis  ¶ ||

From the School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom and the ^¶Department of Chemistry, University of Crete and the Institute of Molecular Biology and Biotechnology Foundation for Research and Technology, P. O. Box 1527, GR-711 10, Heraklion, Greece

The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays). As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10. The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry. The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys⁴⁴ with Cys⁶¹ and an outer pair between Cys⁴⁰ and Cys⁶⁵. These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects. Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process.

Received for publication, June 9, 2003 , and in revised form, July 25, 2003.

^* This work was supported by the United Kingdom Medical Research Council, the Leverhulme Trust, the United Kingdom Biotechnology and Biological Sciences Research Council, and a Medical Research Council co-operative group grant. 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.

Supported by a Biotechnology and Biological Sciences Research Council Studentship.

^|| Lister Institute Research Fellow. To whom correspondence should be addressed: School of Biological Sciences, 2.205 Stopford Bldg., University of Manchester, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-5687; Fax: 44-161-275-5082; E-mail: tokatlidis{at}man.ac.uk.

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