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Motor-free mitochondrial presequence translocase drives membrane integration of preproteins


The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions1. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane2,3,4,5,6,7,8,9. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix8,10,11. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.

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Figure 1: The motor form of the mitochondrial presequence translocase (TIM23CORE) is disrupted in the tim23-76 mutant, whereas the sorting form (TIM23SORT) remains intact.
Figure 2: Reconstitution of the TIM23SORT complex into proteoliposomes.
Figure 3: Reconstituted TIM23SORT forms a presequence-sensitive channel.
Figure 4: Protein insertion by the reconstituted TIM23SORT complex.
Figure 5: Membrane insertion of cytochrome c1 precursor does not depend on the presence of respiratory chain complexes.


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We are grateful to A. Chacinska and C. Meisinger for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereiche 388, 431 and 746, an EMBO long-term fellowship (M.v.d.L.), Gottfried Wilhelm Leibniz Program, Max Planck Research Award, Alexander von Humboldt Foundation, Bundesministerium für Bildung und Forschung and the Fonds der Chemischen Industrie.

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Authors and Affiliations



M.v.d.L. established the reconstituted import system, performed and analysed import experiments. M.M. performed and analysed reconstitution/electrophysiological studies. J.D. and M.v.d.L. characterized the mutant TIM23 allele. D.P.H. performed and analysed coimmunoprecipitation experiments. M.L. performed and analysed experiments relating to Tim23 assembly in tim21 mutant mitochondria. I.P. purified the presequence translocase complex. B.G. generated conditional yeast mutant strains. R.W. supervised electrophysiological analysis and participated in data analysis. N.P. and P.R. led the project and participated in data analysis. All authors participated in the preparation of the manuscipt.

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Correspondence to Richard Wagner or Nikolaus Pfanner.

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van der Laan, M., Meinecke, M., Dudek, J. et al. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat Cell Biol 9, 1152–1159 (2007).

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