Interaction of Tim23 with Tim50 Is Essential for Protein Translocation by the Mitochondrial TIM23 Complex

Lada Gevorkyan-Airapetov,Keren Zohary,Dušan Popov-Čeleketić,Koyeli Mapa,Kai Hell,Walter Neupert,Abdussalam Azem,Dejana Mokranjac

doi:10.1074/jbc.m807041200

Abstract

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.

Highlights

Maintenance and growth of mitochondria depend on the constitutive import of newly synthesized proteins that are encoded by nuclear DNA, translated on cytoplasmic ribosomes as precursors and eventually taken up from the cytosol
The TIM23 complex can be distinguished into two functionally defined parts: a core complex comprised of integral membrane proteins whose subunits are firmly associated with the membrane and the import motor
Tim50 is anchored in the inner membrane with one transmembrane helix and comprises a large C-terminal hydrophilic domain (ϳ350 amino acid residues) that is exposed to the intermembrane space (IMS)

Summary

Introduction

Maintenance and growth of mitochondria depend on the constitutive import of newly synthesized proteins that are encoded by nuclear DNA, translated on cytoplasmic ribosomes as precursors and eventually taken up from the cytosol. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. The TIM23 (translocase of the inner mitochondrial membrane) complex handles the import of precursor proteins that

Results

Conclusion