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Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2009. Vol. 71, no 4-5, 497-508 p.
Keyword [en]
PreP, Protease, Knockout, Targeting peptide, Mitochondria, Chloroplast
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-24762DOI: 10.1007/s11103-009-9534-6OAI: oai:DiVA.org:su-24762DiVA: diva2:198268
Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2017-12-13Bibliographically 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
Biochemistry
Identifiers
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
Opponent
Supervisors
Available from: 2008-03-19 Created: 2008-03-19Bibliographically approved
2. Functional and structural studies of the Presequence protease, PreP
Open this publication in new window or tab >>Functional and structural studies of the Presequence protease, PreP
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2. 

We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP.

In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP.

We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 37 p.
Keyword
Presequence Protease, PreP, AtPreP, hPreP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102310 (URN)
Presentation
2014-04-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 15:00 (English)
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Supervisors
Available from: 2014-04-01 Created: 2014-03-31 Last updated: 2014-04-01Bibliographically approved

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