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Dual Targeting of Proteins to Mitochondria and Chloroplasts
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
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: urn:nbn:se:su:diva-31048ISBN: 978-91-7155-971-5 (print)OAI: oai:DiVA.org:su-31048DiVA: diva2:275228
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
List of papers
1. The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting
Open this publication in new window or tab >>The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting
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2006 (English)In: FEBS Letters, ISSN 0014-5793, Vol. 580, no 16, 3966-3972 p.Article in journal (Refereed) Published
Abstract [en]

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.

Keyword
Chloroplast; Mitochondria; Targeting peptide; Protein import; Miss-sorting
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-8750 (URN)10.1016/j.febslet.2006.06.018 (DOI)16806197 (PubMedID)
Available from: 2007-12-21 Created: 2007-12-21 Last updated: 2009-11-04Bibliographically approved
2. Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide
Open this publication in new window or tab >>Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide
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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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-31102 (URN)10.1093/mp/ssp048 (DOI)000272182500013 ()
Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2014-10-31Bibliographically approved
3. Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts
Open this publication in new window or tab >>Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts
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.

Keyword
dual targeting; amino acyl-tRNA synthetase; mitochondria; chloroplast; targeting peptide
Identifiers
urn:nbn:se:su:diva-31103 (URN)10.1016/j.jmb.2009.08.072 (DOI)000271528300003 ()
Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2009-11-04Bibliographically approved
4. Matrix localized AtPreP complements intermembrane space located homologue, MOP112, in Saccaromyces cerevisiae
Open this publication in new window or tab >>Matrix localized AtPreP complements intermembrane space located homologue, MOP112, in Saccaromyces cerevisiae
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-31105 (URN)
Available from: 2009-11-04 Created: 2009-11-04 Last updated: 2017-02-22Bibliographically approved

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