Abstract
Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.
Highlights
The mitochondrion is the home of several redox processes that are confined into distinct subcompartments
Distinct redox processes located in distinct mitochondrial subcompartments can generate superoxide radicals by the monoelectronic reduction of O2, which can be dismutated into H2O2 and O2
The leakage of electrons in the respiratory chain is the best-studied process of mitochondrial ROS4 generation, but it is currently clear that the superoxide radicals and H2O2 are generated by other processes in the mitochondria [4, 5]
Summary
Trx, and Prx are synthesized as precursor proteins on cytosolic ribosomes and subsequently imported into the mitochondria [16, 35]. Similar to the ␣-KGD protein profile, Trr, Trx, and Prx were protected from the digestion in the mitochondria and mitoplasts, indicating their matrix compartment localization (Fig. 1B, lanes 1– 4). Prx was detected in the mitoplast-derived supernatant (Fig. 1D, lanes 5 and 7, and supplemental Fig. S1A, lane 7), which provided direct evidence for the dual mitochondrial distribution of this protein (matrix and the intermembrane space) This finding will be further analyzed below. Based on a global analysis of the mature mitochondrial N termini in yeast, the Trr and Trx proteins are not cleaved by Oct1 [27], and as expected, these proteins did not show differences in the molecular sizes between the WT and ⌬OCT1 strains (Fig. 2C). As yeast Prx, the levels of human Prx in the ⌬OCT1 strain were the same as those in the ⌬OCT1 ϩ OCT1 strain (Fig. 5E)
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