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

Abstract

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptidedegrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N-and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å 3 . The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…Organellar oligopeptidase (OOP) is another zinc metalloprotease that participates in targeting peptide degradation. OOP degrades peptides with lengths of eight to 23 amino acid residues but fails to act on folded proteins, consistent with its catalytic cavity size, as in the case of PreP1/2 (Kmiec et al, 2013(Kmiec et al, , 2014. The OOP-null mutant phenotype is wild type like, but genetic interaction with the prep1 prep2 double mutant is observed, indicating complementary functions for OOP and PreP1/2 (Kmiec et al, 2013).…”

“…Transit peptides are 26 to 146 amino acids long (Zybailov et al, 2008). Structural and biochemical studies have reported that PreP1 degrades various peptide substrates of 10 to 65 amino acids (Moberg et al, 2003;Ståhl et al, 2005), whereas OOP cleaves peptide fragments ranging from eight to 23 amino acids (Kmiec et al, 2013). Consequently, both proteases are able to function in transit peptide degradation, with OOP acting in parallel or downstream to PreP.…”

“…FtsH11, Lon4, PrePs, and OOP are targeted to both mitochondria and chloroplasts (Moberg et al, 2003;Kmiec et al, 2013). Given that the distribution of a dual-targeted protein is altered by organellar stress and dysfunction (Nargund et al, 2012), allocation of these protease and peptidase machineries between the chloroplast and mitochondrion could be regulated at the protein import step, depending on the status of organellar protein homeostasis.…”

Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

“…Out of the various peptidases quantified, only the OOP peptidase was significantly affected (nearly 2-fold reduced levels) in clpf-1 ( Figure 3C; Supplemental Data Set 3). This M3 peptidase is thought to degrade cleavage products from upstream proteases, such as the Clp system or Prep1,2 (Kmiec et al, 2013;Nishimura and van Wijk, 2015).…”

INTERMEDIATE CLEAVAGE PEPTIDASE55 Modifies Enzyme Amino Termini and Alters Protein Stability in Arabidopsis Mitochondria

“…Most mitochondrial proteins responsible for the machinery of respiration and metabolism are synthesized in the cytosol and imported into mitochondria. After import, N-terminal presequences containing targeting signals are cleaved from many proteins by the mitochondrial processing peptidase (MPP; Sjoling and Glaser, 1998;Zhang and Glaser, 2002), and the mitochondrial presequences themselves are then degraded by presequence peptidases (Ståhl et al, 2002;Moberg et al, 2003;Bhushan et al, 2005) and oligopeptidase (Kmiec et al, 2013). The specificity of the MPP cutting sites has been analyzed by comparison of the experimentally determined N-terminal sequences of mature proteins with the amino acid sequences of the precursor proteins.…”