Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences

The biogenesis of mitochondria depends on the import of hundreds of preproteins. N-terminal matrix-targeting signals (MTSs) direct preproteins to the surface receptors Tom20, Tom22, and Tom70. In this study, we show that many preproteins contain additional internal MTS-like signals (iMTS-Ls) in their mature region that share the characteristic properties of presequences. These features allow the in silico prediction of iMTS-Ls. Using Atp1 as model substrate, we show that iMTS-Ls mediate the binding to Tom70 and have the potential to target the protein to mitochondria if they are presented at its N terminus. The import of preproteins with high iMTS-L content is significantly impaired in the absence of Tom70, whereas preproteins with low iMTS-L scores are less dependent on Tom70. We propose a stepping stone model according to which the Tom70-mediated interaction with internal binding sites improves the import competence of preproteins and increases the efficiency of their translocation into the mitochondrial matrix.