Positively charged amino acids at the N terminus of select mitochondrial proteins mediate early recognition by import proteins αβ′-NAC and Sam37

A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence [MTS]) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone nascent polypeptide–associated complex (NAC), which in yeast is represented as the two different heterodimers αβ-NAC and αβ′-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αβ′-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αβ′-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αβ′-NAC-dependent Oxa1 and αβ′-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αβ′-NAC. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αβ′-NAC and Sam37.