Role of the Novel Metallopeptidase MoP112 and Saccharolysin for the Complete Degradation of Proteins Residing in Different Subcompartments of Mitochondria

Abstract

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m-and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.Mitochondria are essential organelles with central anabolic and catabolic functions. To maintain their homeostasis and thereby avoid cell damage, a precise control of the steady state levels of mitochondrial proteins is required. First, evidence for the presence of an independent proteolytic system within mitochondria came from early studies that revealed different turnover rates of proteins residing in different mitochondrial subcompartments (1, 2). Many components of this system, often highly conserved throughout evolution, have been identified since then and found to exert crucial functions within mitochondria (3, 4). They control distinct steps in the biogenesis of mitochondria and selectively degrade misfolded and nonassembled polypeptides accumulating in the organelle. These could be non-assembled proteins, which accumulate in case of an imperfect coordination of nuclear and mitochondrial gene expression, or oxidatively damaged proteins progressively generated in aging cells. Quantitative measurements of mitochondrial protein turnover in logarithmically growing yeast cells suggested the degradation of up to 10% of the mitochondrial proteome per hour, most likely reflecting to a large extent misfolded or damaged proteins (5).Central c...

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

“…In total, of 12 peptides degraded by OOP, 10 also were substrates for PreP. The apparent overlap in the substrate pool between PreP and OOP and the same subcellular localization suggests that these two peptidases might cooperate in vivo for peptide degradation in endosymbiotic organelles, a possibility supported by previous studies in yeast (27).…”

“…Previous reports suggested that M3A peptidases may be involved in the degradation of targeting peptides and even may cooperate with M16C peptidases (as Cym1 or PreP) in this function (27). We approached this idea by testing the activity of the mitochondria-and chloroplast-resident OOP against the Nicotiana plumbaginifolia pF 1 β, a well-characterized substrate of AtPreP.…”

“…Mitochondria-localized peptidases of M3A family were found in yeast (PRD1) and mammals (neurolysin) (25,26). Their function in mitochondria remains unknown, although it has been suggested that PRD1 cooperates with Cym1 (the PreP ortholog in yeast) in the degradation of mitochondrial presequences (27).…”

“…For these reasons, the turnover of peptides generated in mitochondria and chloroplasts must be regulated, either by active export or local degradation. In yeast, peptides up to 20 aa long can be exported out of mitochondria by an ABC-type transporter termed "Mdl1" (27,34), but a similar system has not been identified in plants.…”

Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP

“…AtPreP1 and AtPreP2 are dually targeted to both the mitochondrial matrix and chloroplast stroma [14]. The yeast homolog of AtPreP, called MOP112 or CYM1p, appears to be located in the mitochondrial intermembrane space [17] whereas the human homolog (hPreP or MP1) is localized to the mitochondrial matrix and is also known to degrade amyloidbeta peptide (Ab) [18], the toxic agent that accumulates in mitochondria in Alzheimer's disease [19].…”