Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts

Abstract

Both authors contributed equally to this work. SummaryWe have recently isolated and identi®ed a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate speci®city and intracellular localization. Mutagenesis studies of the residues involved in metal binding identi®ed the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate speci®city studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria ± degradation of targeting peptides.

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

“…Consistent with this result, A. thaliana PreP (AtPreP) was shown to have a dual localization in mitochondrial matrix and chloroplastic stroma (18,20). In addition to targeting peptides generated by processing, AtPreP1 degrades a wide range of unstructured peptides ranging from 10 from 65 aa (18). The crystal structure of AtPreP1 revealed that the enzyme consists of two bowl-shaped halves connected by a hinge region, forming a catalytic chamber of 10,000 Å 3 , which is big enough to accommodate unstructured peptides up to 65 aa long; however, small folded proteins cannot be degraded.…”

“…2C), having the same intracellular localization as the dual-localized presequence protease, AtPreP ( Fig. 2B) (18,36). According to TargetP ( Fig.…”

“…PreP initially was identified in plant mitochondria as the protease responsible for the degradation of the presequence of the ATPase beta subunit (pF 1 β) and the chloroplastic transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (18,19). Consistent with this result, A. thaliana PreP (AtPreP) was shown to have a dual localization in mitochondrial matrix and chloroplastic stroma (18,20). In addition to targeting peptides generated by processing, AtPreP1 degrades a wide range of unstructured peptides ranging from 10 from 65 aa (18).…”

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

“…For proteins with noncleavable sequences detected only by LC, we cannot rule out the possibility that removed presequences are stable and have been detected by MS. But presequence turnover is considered to be very rapid in mitochondria due to the activity of PreP (Moberg et al, 2003), so we consider this unlikely.…”

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

“…Presequence protease from Arabidopsis thaliana, AtPreP, is a protease that degrades targeting sequences and other unstructured peptides of 10-65 amino acids in length in both mitochondria and chloroplasts [11][12][13][14]. AtPreP is a 110 kDa zinc metallooligopeptidase that belongs to the pitrilysin M16C family of peptidases (MEROPS database) [15].…”