Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import

Author summaryMitochondria play a central role in eukaryotic cells and defects in their biogenesis are implicated in many serious human diseases. Most mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and directed to their final destination by N-terminal extensions, called mitochondrial targeting sequences (MTS). A good efficiency of this import mechanism is critical for mitochondrial function and for the success of gene therapies involving the replacement in the nucleus of a defective mitochondrial gene by a functional non-mutated version. Through a systematic analysis of amino acid preferences in yeasts, we observed a strong and specific overrepresentation of hydrophobic residues at position 2 of MTSs that are known to avoid cleavage of the hydrophobic initiator methionine and to define potential substrates of the N-terminal acetyltransferase NatC. Therefore, most MTSs have two consecutive hydrophobic residues at position 1 and 2, a feature that is conserved throughout evolution. Using CRISPR/Cas9 technology, we showed that mutation of mitochondrial proteins at position 2 reduced their import efficiency. We thus demonstrated that the residue at position 2 of MTS is an important determinant of mitochondrial import under strong selective pressure. These findings may have valuable implications for improving the therapy of human mitochondrial diseases. N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.