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Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2009 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 393, no 4, 803-814 p.Article in journal (Refereed) Published
Abstract [en]

Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26–51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.

Place, publisher, year, edition, pages
2009. Vol. 393, no 4, 803-814 p.
Keyword [en]
dual targeting; amino acyl-tRNA synthetase; mitochondria; chloroplast; targeting peptide
URN: urn:nbn:se:su:diva-31103DOI: 10.1016/j.jmb.2009.08.072ISI: 000271528300003OAI: diva2:275225
Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2009-11-04Bibliographically 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
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)
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

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