Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Dual targeting of proteins to mitochondria and chloroplasts: Characterization of dual targeting peptides and their interaction with organellar receptors
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
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: urn:nbn:se:su:diva-108471ISBN: 978-91-7649-027-3 (print)OAI: oai:DiVA.org:su-108471DiVA: diva2:759220
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
List of papers
1. 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
Show others...
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
2. Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana
Open this publication in new window or tab >>Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana
2014 (English)In: Molecular Plant, ISSN 1674-2052, Vol. 7, no 1, 121-136 p.Article in journal (Refereed) Published
Abstract [en]

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide (TP) directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis (MVDA) with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed. dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for the chloroplast import.

Keyword
dual targeting, ambiguous targeting signal, mitochondria, chloroplast, protein import, partial least square discriminant analysis, Arabidopsis thaliana
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-100099 (URN)10.1093/mp/sst148 (DOI)000329254200010 ()
Note

AuthorCount:4;

Available from: 2014-01-30 Created: 2014-01-27 Last updated: 2014-10-29Bibliographically approved
3. Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species
Open this publication in new window or tab >>Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species
Show others...
2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 410, no 3, 400-410 p.Article in journal (Refereed) Published
Abstract [en]

Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.

Keyword
mitochondrial import, presequence, PreP, Cym1/Mop112, peptide degradation
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:su:diva-66595 (URN)10.1016/j.jmb.2011.05.009 (DOI)000292718400003 ()
Note
authorCount :9Available from: 2011-12-20 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved
4. NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana
Open this publication in new window or tab >>NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana
Show others...
2012 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 19, 3738-3748 p.Article in journal (Refereed) Published
Abstract [en]

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form a-helical structure in one region, S6F27, and a very weak beta-strand propensity for Q34Q38. The a-helical structured region has an amphiphilic character and a f??ff motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9V28, overlaps with the amphiphilic region, and the other comprises residues L30Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. Database The backbone chemical shift assignments for ThrRS-dTP(260) have been deposited with the Biological Magnetic Resonance Bank (BMRB) under the accession code 18248 Structured digital abstract ThrRS-dTP and Tom20-4 bind by nuclear magnetic resonance (View interaction)

Keyword
dual targeting peptide, mitochondria, NMR, protein import, structure
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biophysics
Identifiers
urn:nbn:se:su:diva-82129 (URN)10.1111/j.1742-4658.2012.08735.x (DOI)000308928900019 ()
Funder
Swedish Research Council, 621-2011-5964
Note

AuthorCount:6;

Available from: 2012-11-09 Created: 2012-11-08 Last updated: 2017-12-07Bibliographically approved
5. Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana
Open this publication in new window or tab >>Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana
2015 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 5, 405-412 p.Article in journal (Refereed) Published
Abstract [en]

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in (15)(N) HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal phi chi chi phi phi motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.

Keyword
Dual targeting, Chloroplasts and mitochondria, Protein import, NMR, Toc34 receptor
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biophysics
Identifiers
urn:nbn:se:su:diva-108469 (URN)10.1016/j.fob.2015.04.014 (DOI)000366999300050 ()
Available from: 2014-10-28 Created: 2014-10-28 Last updated: 2017-12-05Bibliographically approved

Open Access in DiVA

Erika Spånning Dissertation(3062 kB)121 downloads
File information
File name FULLTEXT01.pdfFile size 3062 kBChecksum SHA-512
d625dcbc1011f6ca4b3951f0440993f9c354107085d76150fd2089fe0f98623d981628904b2bf9db9a0187cf37c72a4901f4dee4d1b131381c267837c9f5c80d
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Spånning, Erika
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 121 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 329 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf