Atp23 – An unconventional substrate of the Mia40 import pathway

Most proteins of the mitochondrial intermembrane space (IMS) lack mitochondrial targeting sequences. Many of these proteins contain characteristic patterns of cysteine residues (twin Cx3C or twin Cx9C) that are essential for their import. During import of these components, the IMS-located oxidoreductase Mia40 directly binds to these proteins and converts them into oxidized, stably folded structures. Reoxidation of Mia40 is achieved by the sulfhydryl oxidase Erv1. All Mia40 substrates that have been analyzed so far are folded into helix-loop-helix structures containing two parallel disulfide bonds. It was suggested that this consistent architecture of all substrates reflects the specific property of the oxidation machinery of the IMS and a lack of an isomerase activity that would be required to reshuffle disulfides in more complex substrates. Here we present data on the biogenesis of the IMS-located protease Atp23. This protein is conserved from yeast to human and contains 10 cysteine residues. Alkylation experiments suggest that the cysteine residues in Atp23 are oxidized. We show that the import of Atp23 depends on Mia40 and Erv1. A direct interaction between Atp23 and Mia40 could be shown by means of coimmunoprecipitation. Moreover, the import of Atp23 strongly depends on the presence of glutathione which presumably is critical for disulfide reshuffling. Our data show that the Mia40-driven import system is not limited to simple substrate proteins and allows significant insights into the mechanisms by which proteins are imported into the IMS of mitochondria.