Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos

Abstract

In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1Brandner K. Rehling P. Truscott K.N. The carboxyl-terminal third of the dicarboxylate carrier is crucial for productive association with the inner membrane twin-pore translocase.J. Biol. Chem. 2005; 280: 6215-6221Crossref PubMed Scopus (24) Google Scholar, 2Brix J. Ziegler G.A. Dietmeier K. Schneider-Mergener J. Schulz G.E. Pfanner N. The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins.J. Mol. Biol. 2000; 303: 479-488Crossref PubMed Scopus (68) Google Scholar, 3Pfanner N. Hoeben P. Tropschug M. Neupert W. The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria.J. Biol. Chem. 1987; 262: 14851-14854PubMed Google Scholar]. 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Bersch B. et al.Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.Cell. 2018; 175: 1365-1379Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. Tim22 represents the pore-forming core unit of the complex [13Kovermann P. Truscott K.N. Guiard B. Rehling P. Sepuri N.B. Müller H. Jensen R.E. Wagner R. Pfanner N. Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.Mol. Cell. 2002; 9: 363-373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Rehling P. Model K. Brandner K. Kovermann P. Sickmann A. Meyer H.E. Kühlbrandt W. Wagner R. Truscott K.N. Pfanner N. Protein insertion into the mitochondrial inner membrane by a twin-pore translocase.Science. 2003; 299: 1747-1751Crossref PubMed Scopus (224) Google Scholar]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.

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