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Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
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 Analytical Chemistry.
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2009 (English)In: Molecular Plant, ISSN 1674-2052, Vol. 6, no 2, 1298-1309 p.Article in journal (Refereed) Published
Abstract [en]

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr–tRNA synthetase (ThrRS–dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS–dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS–dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS–dTP(2–60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS–dTP(2–60) inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS–dTP(2–60) indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

Place, publisher, year, edition, pages
2009. Vol. 6, no 2, 1298-1309 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-31102DOI: 10.1093/mp/ssp048ISI: 000272182500013OAI: oai:DiVA.org:su-31102DiVA: diva2:275226
Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2014-10-31Bibliographically approved
In thesis
1. Dual Targeting of Proteins to Mitochondria and Chloroplasts
Open this publication in new window or tab >>Dual Targeting of Proteins to Mitochondria and Chloroplasts
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The vast majority of mitochondrial and chloroplastic proteins are nuclear encoded, synthesized in the cytosol and imported into the respective organelle using an N-terminal extension, the targeting peptide (TP). After import into the organelle, the TP is cleaved off and degraded by the Presequence protease (PreP). The import process is thought to be highly specific, however there is a group of proteins that are localised to both mitochondria and chloroplasts, using an ambiguous, dual targeting peptide (dTP). The aim of this thesis was to investigate targeting properties of dTPs. Analysis of the amino acid content of all currently known dually targeted proteins revealed that the dTPs are enriched in hydroxylated, hydrophobic and positively charged residues, lacking acidic residues, whereas the content of serine, arginine and proline is intermediary in comparison to the mitochondrial and chloroplastic TPs. dTPs do not form amphiphilic a-helices, characteristic of the mitochondrial TPs, but the helical structure can be induced in membrane mimetic environment, as revealed by spectroscopic studies of a dTP of an aminoacyl- tRNA-synthetase (aaRS). In vitro and in vivo import experiments of fusion constructs containing N-terminal truncations of seven aaRS-dTPs coupled to green fluorescent protein (GFP) demonstrated different organisation of targeting determinants showing that the N-terminal portion of dTPs was crucial for import into both organelles or at least one organelle for different constructs. In addition, studies of targeting capacity of the TPs of PreP homologues from plant, mammal and yeast (AtPreP, hPreP and Mop112) showed species dependent intra-mitochondrial localisation of the coupled GFP and demonstrated functional complementation of an intermembrane space located Mop112 with a matrix located AtPreP. The studies presented here contribute to understanding of the intracellular and intra-mitochondrial sorting process of proteins in the eukaryotic cell.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 74 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-31048 (URN)978-91-7155-971-5 (ISBN)
Public defence
2009-12-17, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.Available from: 2009-11-25 Created: 2009-11-02 Last updated: 2009-11-04Bibliographically approved
2. Dual targeting of proteins to mitochondria and chloroplasts: Characterization of dual targeting peptides and their interaction with organellar receptors
Open this publication in new window or tab >>Dual targeting of proteins to mitochondria and chloroplasts: Characterization of dual targeting peptides and their interaction with organellar receptors
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins with an N-terminal targeting peptide (TP), which directs them to the correct organelle. There is also a group of proteins that are dual targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). The aim of this thesis was to characterize dTPs with respect to physicochemical features, sequence patterns, structural properties and interaction with the mitochondrial and chloroplastic receptors.

We have used different statistical methods, including a multivariate data analysis (MVDA) to analyse all available dTPs and compare them to organelle-specific TPs of proteome-identified mitochondrial and chloroplastic proteins from Arabidopsis thaliana. The overall amino acid sequence patterns of dTPs were intermediate between mitochondrial targeting peptides (mTPs) and chloroplastic targeting peptides (cTPs) but the greatest differences in amino acid composition were found within the very N-terminal region of dTPs, where especially arginines are highly overrepresented in relation to cTPs. Interestingly, introducing arginines to the dTPs showed clustering towards the mTPs in silico and resulted in inhibition of chloroplast import in vitro, suggesting that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for chloroplast import.

Studies with the dTP of threonyl-tRNA synthetase (ThrRS-dTP) revealed that 60 amino acids were required to confer dual targeting. The purified ThrRS-dTP(2-60) inhibited import of organelle-specific proteins, providing evidence that dual and organelle-specific proteins use the same organellar import pathways. CD spectra indicated that ThrRS-dTP(2-60) has the propensity to form a-helical structure in membrane mimetic environments. Further, NMR investigations of interaction profiles of ThrRS-dTP(2-60) with the mitochondrial Tom20 and the chloroplastic Toc34 receptor demonstrated that the mode of the recognition of a dual targeting peptide by mitochondrial and chloroplastic receptors is different. Our studies provide thorough characterization of dTPs and present for the first time dTP-organellar receptor interactions on the molecular level.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 76 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-108471 (URN)978-91-7649-027-3 (ISBN)
Public defence
2014-12-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.

Available from: 2014-11-20 Created: 2014-10-28 Last updated: 2014-12-17Bibliographically approved

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Berglund, Anna-KarinSpånning, ErikaBiverståhl, HenrikMaddalo, GianlucaMäler, LenaGlaser, Elzbieta
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