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Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2005 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 4, 847-860 p.Article in journal (Refereed) Published
Abstract [en]

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P1P′1

position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10–16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10–23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

Place, publisher, year, edition, pages
2005. Vol. 349, no 4, 847-860 p.
Keyword [en]
targeting, protease, degradation, mitochondria, chloroplast
National Category
Biological Sciences Chemical Sciences
URN: urn:nbn:se:su:diva-24761OAI: diva2:198267
Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2015-04-21Bibliographically approved
In thesis
1. Functional studies of the PreP peptidasome in Arabidopsis thaliana
Open this publication in new window or tab >>Functional studies of the PreP peptidasome in Arabidopsis thaliana
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Two independent endosymbiotic events gave rise to mitochondria and chloroplasts. Despite the fact that both organelles have their own small genome the majority of organellar proteins are encoded in the nucleus, synthesized in the cytosol and imported into the organelles. The targeting information for most organellar proteins is located in an N-terminal extension called a targeting peptide. Targeting peptides are cleaved off after import by organellar processing peptidases. The cleaved targeting peptides are toxic to organellar functions and are degraded by the PreP peptidasome, the metalloendopeptidase which is the main topic of this thesis.

We have overexpressed, purified and determined the first structure of a plant mitochondrial targeting peptide, the F1β presequence from Nicotiana plumbaginifolia, by NMR in a membrane mimetic environment. The structure showed that the targeting peptide formed two helices separated by an unstructured domain. The N-terminal helix being amphipatic. The F1β targeting peptide has been used as a model substrate for the mitochondrial and chloroplast PreP peptidasome. In Arabidopsis thaliana the PreP peptidasome is present as two isoforms, AtPreP1 and AtPreP2. We have shown that both forms are expressed and dually targeted to mitochondria and chloroplasts. Both AtPreP1 and AtPreP2 degrade targeting peptides and other non-related unstructured peptides up to 65 amino acid residues. Substrate specificity studies showed that both PreP isoforms have a preference for positively charged amino acid residues in the P1′ position and small uncharged residues in the P1 position. Mapping of cleavage sites revealed unique cleavage sites for both isoforms. We have generated and characterized both single and double AtPreP1 and AtPreP2 knockouts in A. thaliana. AtPreP1 was shown to be the major isoform. The double knockout exhibited a chlorotic phenotype with altered mitochondrial and chloroplast morphology. Furthermore,mitochondria were partially uncoupled. Throughout the development there was a slower growth rate and 40% lower biomass production. These results show that the PreP peptidasome is important for efficient organellar functions and normal plant development.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 63 p.
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:su:diva-7434 (URN)978-91-7155-601-1 (ISBN)
Public defence
2008-04-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Available from: 2008-03-19 Created: 2008-03-19Bibliographically approved

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