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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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Mitochondrial VDAC proteins from Solanum tuberosum differentially interact with tRNA. (A) VDAC34 interacts with labeled tRNAs. (St) corresponds to the Coomassie blue staining of total mitochondrial outer membrane proteins fractionated on SDS/PAGE and electroblotted onto Immobilon-P membrane. (W) corresponds to the western blot analysis with antibodies raised against S. tuberosum VDAC proteins. The position of the two major VDACs, VDAC34 and VDAC36, is indicated. (NW) corresponds to the northwestern blot analysis. The proteins fixed on the membrane were renatured and incubated with radiolabeled cytosolic Arabidopsis thaliana tRNAAla transcript. (B) Differential interaction was confirmed with the His-tagged purified proteins. (St) corresponds to the Coomassie blue staining of the purified VDAC34 (34) and VDAC36 (36) proteins fractionated by SDS/PAGE and transferred onto nylon membrane. (NW) corresponds to the Northwestern blot analysis with labeled tRNAAla. The histogram shows the percentage of interaction between VDAC36 and tRNA as compared to VDAC34 interaction (100%). It is the average of 11 independent experiments and standard error is indicated for VDAC36. (C) Gel shift assay with 20 to 200 nM of purified VDAC34 or VDAC36 proteins in presence of an excess of labeled tRNAAla (1 nM). Unbound tRNA probe and tRNA probe bound to the VDAC protein are indicated by the letters U and B respectively. The histogram shows the tRNA–VDAC complex signal intensities. The signal intensity observed in presence of 200 nM of VDAC34 was arbitrary taken as 100%. (D) Constant dissociation (Kd) values for VDAC34 and VDAC36. The values are the average of seven and five independent experiments for VDAC34 and VDAC36 respectively and standard errors are indicated. Examples of gel-shift assays and binding curve graphics are shown in supplemental information (Supplemental Figure S3). (E) Gel shift competition assay with 200 nM of VDAC34 and an excess of labeled tRNAAla (1 nM) in presence of increasing amounts of unlabeled tRNAAla or increasing amounts of ATP.
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Figure 1: Mitochondrial VDAC proteins from Solanum tuberosum differentially interact with tRNA. (A) VDAC34 interacts with labeled tRNAs. (St) corresponds to the Coomassie blue staining of total mitochondrial outer membrane proteins fractionated on SDS/PAGE and electroblotted onto Immobilon-P membrane. (W) corresponds to the western blot analysis with antibodies raised against S. tuberosum VDAC proteins. The position of the two major VDACs, VDAC34 and VDAC36, is indicated. (NW) corresponds to the northwestern blot analysis. The proteins fixed on the membrane were renatured and incubated with radiolabeled cytosolic Arabidopsis thaliana tRNAAla transcript. (B) Differential interaction was confirmed with the His-tagged purified proteins. (St) corresponds to the Coomassie blue staining of the purified VDAC34 (34) and VDAC36 (36) proteins fractionated by SDS/PAGE and transferred onto nylon membrane. (NW) corresponds to the Northwestern blot analysis with labeled tRNAAla. The histogram shows the percentage of interaction between VDAC36 and tRNA as compared to VDAC34 interaction (100%). It is the average of 11 independent experiments and standard error is indicated for VDAC36. (C) Gel shift assay with 20 to 200 nM of purified VDAC34 or VDAC36 proteins in presence of an excess of labeled tRNAAla (1 nM). Unbound tRNA probe and tRNA probe bound to the VDAC protein are indicated by the letters U and B respectively. The histogram shows the tRNA–VDAC complex signal intensities. The signal intensity observed in presence of 200 nM of VDAC34 was arbitrary taken as 100%. (D) Constant dissociation (Kd) values for VDAC34 and VDAC36. The values are the average of seven and five independent experiments for VDAC34 and VDAC36 respectively and standard errors are indicated. Examples of gel-shift assays and binding curve graphics are shown in supplemental information (Supplemental Figure S3). (E) Gel shift competition assay with 200 nM of VDAC34 and an excess of labeled tRNAAla (1 nM) in presence of increasing amounts of unlabeled tRNAAla or increasing amounts of ATP.

Mentions: We previously showed that a 34 kDa potato VDAC can interact with tRNA molecules (12). However, in S. tuberosum two major VDACs are present in the OMM, namely VDAC34 and VDAC36 (13). Indeed, while northwestern experiments performed on total OMM proteins in the presence of radiolabeled plant cytosolic tRNAAla gave only one signal corresponding in size to VDAC34 (Figure 1A), a western blot experiment performed with antibodies recognizing the two VDAC isoforms showed that both proteins were present in equal amount in the OMM (Figure 1A). This suggested that the two VDAC isoforms differentially interact with tRNA. In order to confirm this observation, the two VDACs were overexpressed and His-Tag purified. Northwestern experiments with the purified recombinant proteins confirmed that VDAC34 interacts about 7.5× fold more with labeled tRNAAla transcript than VDAC36 (Figure 1B). This differential interaction, although of less magnitude, was further supported by gel shift assay with labeled tRNAAla transcript and increasing amounts of overexpressed VDAC proteins (Figure 1C), as 2× more tRNA–VDAC complex on an average was formed with VDAC34 than with VDAC36. In order to rule out a defect of refolding and/or of stability of VDAC36, we analyzed VDAC hydrodynamic properties by dynamic light scattering and confirmed that both VDACs are stable and behave as monomers in solution (Supplemental Figure S2). Gel-shift quantitative analyses allowed calculating the apparent constant dissociation (Kd) values for both VDAC proteins (Figure 1D and Supplemental Figure S3). These analyses showed that the Kd values were in the 0.1 μM range and that VDAC36 Kd value was on average 1.4× higher than that of VDAC34. It is worth to note that the Kd for VDAC34 yielded highly reproducible values whereas the VDAC36 Kd was more variable (Figure 1D). Furthermore, the interaction between radiolabeled tRNAAla transcript and VDAC34 remained unchanged when increasing amounts of ATP, one of the major metabolites transported through VDAC, were added to gel shift assays. By contrast, this interaction was almost completely competed out in the presence of an excess of unlabeled tRNA transcript (Figure 1E). This indicates that the binding site(s) of ATP molecules on VDAC34 is likely different to those required for tRNA interaction.


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)

Mitochondrial VDAC proteins from Solanum tuberosum differentially interact with tRNA. (A) VDAC34 interacts with labeled tRNAs. (St) corresponds to the Coomassie blue staining of total mitochondrial outer membrane proteins fractionated on SDS/PAGE and electroblotted onto Immobilon-P membrane. (W) corresponds to the western blot analysis with antibodies raised against S. tuberosum VDAC proteins. The position of the two major VDACs, VDAC34 and VDAC36, is indicated. (NW) corresponds to the northwestern blot analysis. The proteins fixed on the membrane were renatured and incubated with radiolabeled cytosolic Arabidopsis thaliana tRNAAla transcript. (B) Differential interaction was confirmed with the His-tagged purified proteins. (St) corresponds to the Coomassie blue staining of the purified VDAC34 (34) and VDAC36 (36) proteins fractionated by SDS/PAGE and transferred onto nylon membrane. (NW) corresponds to the Northwestern blot analysis with labeled tRNAAla. The histogram shows the percentage of interaction between VDAC36 and tRNA as compared to VDAC34 interaction (100%). It is the average of 11 independent experiments and standard error is indicated for VDAC36. (C) Gel shift assay with 20 to 200 nM of purified VDAC34 or VDAC36 proteins in presence of an excess of labeled tRNAAla (1 nM). Unbound tRNA probe and tRNA probe bound to the VDAC protein are indicated by the letters U and B respectively. The histogram shows the tRNA–VDAC complex signal intensities. The signal intensity observed in presence of 200 nM of VDAC34 was arbitrary taken as 100%. (D) Constant dissociation (Kd) values for VDAC34 and VDAC36. The values are the average of seven and five independent experiments for VDAC34 and VDAC36 respectively and standard errors are indicated. Examples of gel-shift assays and binding curve graphics are shown in supplemental information (Supplemental Figure S3). (E) Gel shift competition assay with 200 nM of VDAC34 and an excess of labeled tRNAAla (1 nM) in presence of increasing amounts of unlabeled tRNAAla or increasing amounts of ATP.
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Figure 1: Mitochondrial VDAC proteins from Solanum tuberosum differentially interact with tRNA. (A) VDAC34 interacts with labeled tRNAs. (St) corresponds to the Coomassie blue staining of total mitochondrial outer membrane proteins fractionated on SDS/PAGE and electroblotted onto Immobilon-P membrane. (W) corresponds to the western blot analysis with antibodies raised against S. tuberosum VDAC proteins. The position of the two major VDACs, VDAC34 and VDAC36, is indicated. (NW) corresponds to the northwestern blot analysis. The proteins fixed on the membrane were renatured and incubated with radiolabeled cytosolic Arabidopsis thaliana tRNAAla transcript. (B) Differential interaction was confirmed with the His-tagged purified proteins. (St) corresponds to the Coomassie blue staining of the purified VDAC34 (34) and VDAC36 (36) proteins fractionated by SDS/PAGE and transferred onto nylon membrane. (NW) corresponds to the Northwestern blot analysis with labeled tRNAAla. The histogram shows the percentage of interaction between VDAC36 and tRNA as compared to VDAC34 interaction (100%). It is the average of 11 independent experiments and standard error is indicated for VDAC36. (C) Gel shift assay with 20 to 200 nM of purified VDAC34 or VDAC36 proteins in presence of an excess of labeled tRNAAla (1 nM). Unbound tRNA probe and tRNA probe bound to the VDAC protein are indicated by the letters U and B respectively. The histogram shows the tRNA–VDAC complex signal intensities. The signal intensity observed in presence of 200 nM of VDAC34 was arbitrary taken as 100%. (D) Constant dissociation (Kd) values for VDAC34 and VDAC36. The values are the average of seven and five independent experiments for VDAC34 and VDAC36 respectively and standard errors are indicated. Examples of gel-shift assays and binding curve graphics are shown in supplemental information (Supplemental Figure S3). (E) Gel shift competition assay with 200 nM of VDAC34 and an excess of labeled tRNAAla (1 nM) in presence of increasing amounts of unlabeled tRNAAla or increasing amounts of ATP.
Mentions: We previously showed that a 34 kDa potato VDAC can interact with tRNA molecules (12). However, in S. tuberosum two major VDACs are present in the OMM, namely VDAC34 and VDAC36 (13). Indeed, while northwestern experiments performed on total OMM proteins in the presence of radiolabeled plant cytosolic tRNAAla gave only one signal corresponding in size to VDAC34 (Figure 1A), a western blot experiment performed with antibodies recognizing the two VDAC isoforms showed that both proteins were present in equal amount in the OMM (Figure 1A). This suggested that the two VDAC isoforms differentially interact with tRNA. In order to confirm this observation, the two VDACs were overexpressed and His-Tag purified. Northwestern experiments with the purified recombinant proteins confirmed that VDAC34 interacts about 7.5× fold more with labeled tRNAAla transcript than VDAC36 (Figure 1B). This differential interaction, although of less magnitude, was further supported by gel shift assay with labeled tRNAAla transcript and increasing amounts of overexpressed VDAC proteins (Figure 1C), as 2× more tRNA–VDAC complex on an average was formed with VDAC34 than with VDAC36. In order to rule out a defect of refolding and/or of stability of VDAC36, we analyzed VDAC hydrodynamic properties by dynamic light scattering and confirmed that both VDACs are stable and behave as monomers in solution (Supplemental Figure S2). Gel-shift quantitative analyses allowed calculating the apparent constant dissociation (Kd) values for both VDAC proteins (Figure 1D and Supplemental Figure S3). These analyses showed that the Kd values were in the 0.1 μM range and that VDAC36 Kd value was on average 1.4× higher than that of VDAC34. It is worth to note that the Kd for VDAC34 yielded highly reproducible values whereas the VDAC36 Kd was more variable (Figure 1D). Furthermore, the interaction between radiolabeled tRNAAla transcript and VDAC34 remained unchanged when increasing amounts of ATP, one of the major metabolites transported through VDAC, were added to gel shift assays. By contrast, this interaction was almost completely competed out in the presence of an excess of unlabeled tRNA transcript (Figure 1E). This indicates that the binding site(s) of ATP molecules on VDAC34 is likely different to those required for tRNA interaction.

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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Related in: MedlinePlus