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One key to two doors: Dual targeting peptides and membrane mimetics
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an α-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a φχχφφ motif, where φ represents a hydrophobic/aromatic residue and χ represents any amino acid residue. In contrast, neither the amphiphilic helix nor φχχφφ motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction.

In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form α-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2015. , 69 p.
Keyword [en]
: Dual targeting peptides, protein import, mitochondria and chloroplasts, bicelles, galactolipids, NMR spectroscopy
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-116817ISBN: 978-91-7649-159-1 (print)OAI: oai:DiVA.org:su-116817DiVA: diva2:808519
Public defence
2015-05-29, Magnéli hall, Arrhenius Laboratory, 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 2: In press.

Available from: 2015-05-07 Created: 2015-04-28 Last updated: 2015-06-29Bibliographically approved
List of papers
1. 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
2. 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
3. Characterization of the Morphology of Fast-Tumbling Bicelles with Varying Composition
Open this publication in new window or tab >>Characterization of the Morphology of Fast-Tumbling Bicelles with Varying Composition
2014 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 30, no 19, 5488-5496 p.Article in journal (Refereed) Published
Abstract [en]

Small, fast-tumbling bicelles are frequently used in solution NMR studies of protein lipid interactions. For this purpose it is critical to have information about the organization of the lipids within the bicelle structure. We have studied the morphology of small, fast-tumbling bicelles containing DMPC and DHPC as a function of temperature, lipid concentration, and the relative ratio (q value) of lipid (DMPC) to detergent (DHPC) amounts. Dynamic light scattering and cryo-transmission electron microscopy techniques were used to measure the size of the bicelles and to monitor the shape and dispersity of the particles in the samples. The stability and size of DMPC-containing bicelle mixtures were found to be highly dependent on temperature and the total lipid concentration for mixtures with q = 1 and q = 1.5. Stable DMPC/DHPC bicelles are only formed at low q values (0.5). Bicelle mixtures with q > 0.5 appear to be multidisperse containing more than one component, one with r(H) around 2.5 nm and one with r(H) of 6-8 nm. This is interpreted as a coexistence of small (possibly mixed micelles) bicelles and much larger bicelles. Incubating the sample at 37 degrees C increases the phase separation. Moreover, low total amphiphile concentrations and low q values lead to the formation of a temperature-independent morphology, interpreted as the formation of small particles in which the DHPC and DMPC are more mixed. On the basis of these results, we propose the existence of a critical bicelle concentration, a parameter that determines the existence of bilayered bicelles, which varies with q value. This polymorphism was not observed at any concentrations for q = 0.5 bicelles, for which a small but detectable temperature dependence was observed at high concentrations. The results demonstrate that q = 0.5 mixtures predominantly form classical bicelles, but that caution is needed when using fast-tumbling mixtures with q values higher than 0.5.

National Category
Chemical Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-105194 (URN)10.1021/la500231z (DOI)000336414500019 ()
Funder
Swedish Research Council
Note

AuthorCount:4;

Available from: 2014-07-04 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
4. New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles
Open this publication in new window or tab >>New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 4, 1044-1050 p.Article in journal (Refereed) Published
Abstract [en]

Galactolipids are the main structural component of plant chloroplastic (thylakoid) membranes and of blue-green algae cell membranes. The predominant lipids in this class are monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG). We here present a method for the preparation of bicelles that contain these galactolipids together with a characterization of the bicelles, and the lipids within the bicelles. NMR diffusion data show that up to 3096 of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a q = 0.5 DMPC/DHPC lipid matrix can be replaced with either monogalactosyl-diacylglycerol or digalactosyl-diacylglycerol and that these lipids incorporate into the bicelles. No evidence for phase separation is observed. Bicelles made with monogalactosyl-diacylglycerol are significantly larger than bicelles containing only DMPC, already with only 1096 of the DMPC replaced with the galactolipid. The effect of digalactosyl-diacylglycerol on bicelle size is much smaller. These observations are likely to be correlated with the different bilayer-forming properties of the lipids. Monogalactosyl-diacylglycerol is a non-bilayer-forming lipid, while digalactosyl-diacylglycerol is a bilayer-forming lipid. Both galactolipids display extensive local motion within the bilayer, as evidenced by natural abundance carbon-13 relaxation of the lipid molecules. The sugar headgroup regions are motionally restricted and cannot be described by a model that does not take into account anisotropic reorientation of the sugar units. No significant effect of the galactolipids on DMPC dynamics was observed. Our results indicate that these bicelles may become useful as model membrane mimetic media for studies of galactolipid-protein interactions.

National Category
Physical Chemistry
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-88249 (URN)10.1021/jp311093p (DOI)000314492300009 ()
Funder
Swedish Research Council, 621-2011-5964
Note

AuthorCount:3;

Available from: 2013-03-14 Created: 2013-03-12 Last updated: 2017-09-15Bibliographically approved

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