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Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs.

Salinas T, El Farouk-Ameqrane S, Ubrig E, Sauter C, Duchêne AM, Maréchal-Drouard L - Nucleic Acids Res. (2014)

Bottom Line: To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern.This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding.Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes, UPR 2357 CNRS, associated with Strasbourg University, 12 rue du Général Zimmer 67084 Strasbourg cedex, France laurence.drouard@ibmp-cnrs.unistra.fr.

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Solanum tuberosum mitochondrial VDACs differentially interact with RNA and DNA in vitro. Coomassie blue-stained profile (St), Northwestern (NW) and Southwestern analyses (SW) of His-tagged purified VDAC34 (34) and VDAC36 (36) proteins. For NW, a radiolabeled random 75 nt long RNA transcript was used. For SW, membranes were incubated with either a radiolabeled double-stranded DNA (dsDNA) substrate corresponding to the Arabidopsis thaliana cytosolic tRNAAla PCR product or with a radiolabeled single-stranded DNA substrate (ssDNA) corresponding to the A. thaliana cytosolic tRNAAla oligonucleotide sequence. NW and SW experiments were performed with increasing amounts of VDAC34 or VDAC36 and in presence of 0.2 nM of radiolabeled nucleic acids. The histograms show the percentages of interaction observed for each VDAC amount. For each nucleic acid substrate the interaction with the highest amount of VDAC34 was arbitrary taken as a reference (100%).
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Figure 2: Solanum tuberosum mitochondrial VDACs differentially interact with RNA and DNA in vitro. Coomassie blue-stained profile (St), Northwestern (NW) and Southwestern analyses (SW) of His-tagged purified VDAC34 (34) and VDAC36 (36) proteins. For NW, a radiolabeled random 75 nt long RNA transcript was used. For SW, membranes were incubated with either a radiolabeled double-stranded DNA (dsDNA) substrate corresponding to the Arabidopsis thaliana cytosolic tRNAAla PCR product or with a radiolabeled single-stranded DNA substrate (ssDNA) corresponding to the A. thaliana cytosolic tRNAAla oligonucleotide sequence. NW and SW experiments were performed with increasing amounts of VDAC34 or VDAC36 and in presence of 0.2 nM of radiolabeled nucleic acids. The histograms show the percentages of interaction observed for each VDAC amount. For each nucleic acid substrate the interaction with the highest amount of VDAC34 was arbitrary taken as a reference (100%).

Mentions: Plant tRNA mitochondrial import is highly specific, but we previously demonstrated that plant VDACs are not responsible for this selectivity (12). Furthermore, VDACs are involved not only in tRNA import but also in DNA uptake into mitochondria (6). We thus wondered whether the differential interaction observed with tRNA is transposable to other substrates. To answer this question, three radiolabeled substrates including a random RNA transcript, a single and a double stranded DNA fragment were used for northwestern or southwestern experiments. As shown in Figure 2, for each of these nucleic acid substrates, the interaction with VDAC34 increases continuously as a function of protein amounts. By contrast, although an increased signal was observed with VDAC36, it was about 6× weaker on average than with VDAC34 (Figure 2). As a whole, the data indicate that potato VDAC34 and VDAC36 differentially interact with all types of nucleic acids.


Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs.

Salinas T, El Farouk-Ameqrane S, Ubrig E, Sauter C, Duchêne AM, Maréchal-Drouard L - Nucleic Acids Res. (2014)

Solanum tuberosum mitochondrial VDACs differentially interact with RNA and DNA in vitro. Coomassie blue-stained profile (St), Northwestern (NW) and Southwestern analyses (SW) of His-tagged purified VDAC34 (34) and VDAC36 (36) proteins. For NW, a radiolabeled random 75 nt long RNA transcript was used. For SW, membranes were incubated with either a radiolabeled double-stranded DNA (dsDNA) substrate corresponding to the Arabidopsis thaliana cytosolic tRNAAla PCR product or with a radiolabeled single-stranded DNA substrate (ssDNA) corresponding to the A. thaliana cytosolic tRNAAla oligonucleotide sequence. NW and SW experiments were performed with increasing amounts of VDAC34 or VDAC36 and in presence of 0.2 nM of radiolabeled nucleic acids. The histograms show the percentages of interaction observed for each VDAC amount. For each nucleic acid substrate the interaction with the highest amount of VDAC34 was arbitrary taken as a reference (100%).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Solanum tuberosum mitochondrial VDACs differentially interact with RNA and DNA in vitro. Coomassie blue-stained profile (St), Northwestern (NW) and Southwestern analyses (SW) of His-tagged purified VDAC34 (34) and VDAC36 (36) proteins. For NW, a radiolabeled random 75 nt long RNA transcript was used. For SW, membranes were incubated with either a radiolabeled double-stranded DNA (dsDNA) substrate corresponding to the Arabidopsis thaliana cytosolic tRNAAla PCR product or with a radiolabeled single-stranded DNA substrate (ssDNA) corresponding to the A. thaliana cytosolic tRNAAla oligonucleotide sequence. NW and SW experiments were performed with increasing amounts of VDAC34 or VDAC36 and in presence of 0.2 nM of radiolabeled nucleic acids. The histograms show the percentages of interaction observed for each VDAC amount. For each nucleic acid substrate the interaction with the highest amount of VDAC34 was arbitrary taken as a reference (100%).
Mentions: Plant tRNA mitochondrial import is highly specific, but we previously demonstrated that plant VDACs are not responsible for this selectivity (12). Furthermore, VDACs are involved not only in tRNA import but also in DNA uptake into mitochondria (6). We thus wondered whether the differential interaction observed with tRNA is transposable to other substrates. To answer this question, three radiolabeled substrates including a random RNA transcript, a single and a double stranded DNA fragment were used for northwestern or southwestern experiments. As shown in Figure 2, for each of these nucleic acid substrates, the interaction with VDAC34 increases continuously as a function of protein amounts. By contrast, although an increased signal was observed with VDAC36, it was about 6× weaker on average than with VDAC34 (Figure 2). As a whole, the data indicate that potato VDAC34 and VDAC36 differentially interact with all types of nucleic acids.

Bottom Line: To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern.This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding.Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes, UPR 2357 CNRS, associated with Strasbourg University, 12 rue du Général Zimmer 67084 Strasbourg cedex, France laurence.drouard@ibmp-cnrs.unistra.fr.

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