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Functional studies of the PreP peptidasome in Arabidopsis thaliana
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-7434ISBN: 978-91-7155-601-1 (print)OAI: oai:DiVA.org:su-7434DiVA: diva2:198269
Public defence
2008-04-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-03-19 Created: 2008-03-19Bibliographically approved
List of papers
1. NMR Solution Structure of the Mitochondrial F1β Presequence from Nicotiana plumbaginifolia
Open this publication in new window or tab >>NMR Solution Structure of the Mitochondrial F1β Presequence from Nicotiana plumbaginifolia
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2004 In: Journal of molecular biology, ISSN 0022-2836, Vol. 336, no 5, 1129-1140 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24759 (URN)
Note
Part of urn:nbn:se:su:diva-7434Available from: 2008-03-19 Created: 2008-03-19Bibliographically approved
2. Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, AtPreP2
Open this publication in new window or tab >>Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, AtPreP2
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2005 In: Plant & cell physiology, ISSN 0032-0781, Vol. 46, no 6, 985-996 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24760 (URN)
Note
Part of urn:nbn:se:su:diva-7434Available from: 2008-03-19 Created: 2008-03-19Bibliographically approved
3. Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different
Open this publication in new window or tab >>Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different
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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.

Keyword
targeting, protease, degradation, mitochondria, chloroplast
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-24761 (URN)
Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2015-04-21Bibliographically approved
4. Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
Open this publication in new window or tab >>Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
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2009 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 71, no 4-5, 497-508 p.Article in journal (Refereed) Published
Abstract [en]

A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of ~40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.

Keyword
PreP, Protease, Knockout, Targeting peptide, Mitochondria, Chloroplast
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24762 (URN)10.1007/s11103-009-9534-6 (DOI)
Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2014-04-01Bibliographically approved

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