A protein-specific priority code in presequences determines the efficiency of mitochondrial protein import.

The biogenesis of mitochondria relies on the import of hundreds of different precursor proteins from the cytosol. Most of these proteins are synthesized with N-terminal presequences which serve as mitochondrial targeting signals. Presequences consistently form amphipathic helices, but they considerably differ with respect to their primary structure and length. Here we show that presequences can be classified into seven different groups based on their specific features. Using a test set of different presequences, we observed that group A presequences endow precursor proteins with improved in vitro import characteristics. We developed IQ-Compete (for Import and de-Quenching Competition assay), a novel assay based on fluorescence de-quenching, to monitor the import efficiencies of mitochondrial precursors in vivo. With this assay, we confirmed the increased import competence of group A presequences. Using mass spectrometry, we found that the presequence of the group A protein Oxa1 specifically recruits the tetratricopeptide repeat (TPR)-containing protein TOMM34 to the cytosolic precursor protein. TOMM34, and the structurally related yeast co-chaperone Cns1, apparently serve as presequence-specific targeting factors which increases the import efficiency of a specific subset of mitochondrial precursor proteins. Our results suggest that presequences contain a protein-specific priority code that encrypts the targeting mechanism of individual mitochondrial precursor proteins.