Journal of Biological Chemistry
Volume 284, Issue 8, 20 February 2009, Pages 4865-4872
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Membrane Transport, Structure, Function, and Biogenesis
Interaction of Tim23 with Tim50 Is Essential for Protein Translocation by the Mitochondrial TIM23 Complex*

https://doi.org/10.1074/jbc.M807041200Get rights and content
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The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

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*

This work was supported by German-Israeli Foundation for Scientific Research and Development Grant 753/181 and German-Israeli Project Cooperation Grant F5.1. 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 Figs. S1–S4.

1

These authors contributed equally to this work.