Presequence Recognition by the Tom40 Channel Contributes to Precursor Translocation into the Mitochondrial Matrix

Abstract

More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined. Using a photoaffinity-labeling approach with modified presequence peptides, we identified Tom40 as a presequence interactor of the TOM complex. Utilizing mass spectrometry, we mapped Tom40's presequence-interacting regions to both sides of the β-barrel. Analysis of a phosphorylation site within one of the presequence-interacting regions revealed altered translocation kinetics along the presequence pathway. Our analyses assess the relation between the identified presequence-binding region of Tom40 and the intermembrane space domain of Tom22. The identified presequence-interacting region of Tom40 is capable of functioning independently of the established trans-acting TOM presequence-binding domain during matrix import.

ACKNOWLEDGMENTS

We are indebted to O. Lytovchenko for helpful discussion. We thank Klaus Neifer and Lars van Werven for expert technical support.

This work was supported by the Deutsche Forschungsgemeinschaft, SFB860, the Göttingen Graduate School for Neurosciences and Molecular Biosciences, and the Max Planck Society (P.R.). J.M. is a doctoral student of the Ph.D. program Molecular Biology—International Max Planck Research School and the Göttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB) (DFG grant GSC 226/1) at the Georg August University Göttingen. The research of L.W. and A.C. was supported by the Foundation for Polish Science-Welcome Program cofunded by the EU within the European Regional Development Fund.

We declare that we have no conflicts of interest.