Positively charged amino acids at the N terminus of select mitochondrial proteins mediate early recognition by import proteins αß'-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 αß-NAC and αß'-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 αß'-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 αß'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αß'-NAC-dependent Oxa1 and αß'-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 αß'-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 αß'-NAC and Sam37.

From cytosol to mitochondria: the beginning of a protein journey.

Mitochondrial protein import is one of the key processes during mitochondrial biogenesis that involves a series of events necessary for recognition and delivery of nucleus-encoded/cytosol-synthesized mitochondrial proteins into the organelle. The past research efforts have mainly unraveled how membrane translocases ensure the correct protein sorting within the different mitochondrial subcompartments. However, early steps of recognition and delivery remain relatively uncharacterized. In this review, we discuss our current understanding about the signals on mitochondrial proteins, as well as in the mRNAs encoding them, which with the help of cytosolic chaperones and membrane receptors support protein targeting to the organelle in order to avoid improper localization. In addition, we discuss recent findings that illustrate how mistargeting of mitochondrial proteins triggers stress responses, aiming to restore cellular homeostasis.

αß'-NAC cooperates with Sam37 to mediate early stages of mitochondrial protein import.

The mitochondrial proteome is mostly composed of nuclear-encoded proteins. Such polypeptides are synthesized with signals that guide their intracellular transport to the surface of the organelle and later within the different mitochondrial subcompartments until they reach their functional destination. It has been suggested that the nascent-polypeptide associated complex (NAC) - a cytosolic chaperone that recognizes nascent chains on translationally active ribosomes - has a role in the import of nuclear-encoded mitochondrial proteins. However, the molecular mechanisms that regulate the NAC-mediated cotranslational import are still not clear. Here, we show that a particular NAC heterodimer formed by subunits α and ß' in Saccharomyces cerevisiae is specifically involved in the process of mitochondrial import and functionally cooperates with Sam37, an outer membrane protein subunit of the sorting and assembly machinery complex. Mutants in both components display growth defects, incorrectly accumulate precursor forms of mitochondrial proteins in the cytosol, and have an altered mitochondrial protein content. We propose that αß'-NAC and Sam37 are members of the system that recognizes mitochondrial proteins at early stages of their synthesis, escorting them to the import machinery of mitochondria.