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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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The N-terminal part of VDAC34 is essential for an efficient interaction with tRNA. Two kinds of mutants were designed: (A) deleted versions of VDAC34 (D1–D7) and (B) chimeric proteins (C1–C9) in which segments of VDAC34 were replaced by segments of VDAC36. Numbers indicate position within VDAC sequences. The variants overexpressed in Escherichia coli were purified and used to perform NW experiments in the presence of Arabidopsis thaliana cytosolic tRNAAla in order to quantify percentage of interaction between mutant proteins and tRNA. Obtained values are represented on a diagram and are given in a summary table. They are the average of 2–9 independent experiments and correspond to the percentage of interaction of the mutant protein with tRNA compared to the wild-type VDAC34 interaction. Standard error is indicated for each value. Examples of NW experiments allowing the quantification of interactions are given as supplemental information (Supplemental Figure S4).
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Figure 4: The N-terminal part of VDAC34 is essential for an efficient interaction with tRNA. Two kinds of mutants were designed: (A) deleted versions of VDAC34 (D1–D7) and (B) chimeric proteins (C1–C9) in which segments of VDAC34 were replaced by segments of VDAC36. Numbers indicate position within VDAC sequences. The variants overexpressed in Escherichia coli were purified and used to perform NW experiments in the presence of Arabidopsis thaliana cytosolic tRNAAla in order to quantify percentage of interaction between mutant proteins and tRNA. Obtained values are represented on a diagram and are given in a summary table. They are the average of 2–9 independent experiments and correspond to the percentage of interaction of the mutant protein with tRNA compared to the wild-type VDAC34 interaction. Standard error is indicated for each value. Examples of NW experiments allowing the quantification of interactions are given as supplemental information (Supplemental Figure S4).

Mentions: In order to determine which regions are important for the interaction with tRNA, different mutant versions of VDAC34 were constructed. To do so, VDAC proteins were arbitrarily divided into three segments: I (position 1–90), II (position 91–171) and III (position 172–276) (Figure 3A). In total, seven deletion mutants (D1–D7) and nine chimeric (C1–C9) were designed (Figure 4). Northwestern analysis on deletion mutants showed that none of the VDAC34 segments with the exception of the D3 mutant are able to interact efficiently with tRNAs (Figure 4A). In addition, analysis on chimeric mutants indicated that the gradual replacement of VDAC34 sequence by VDAC36 sequence from the C-terminal side (C1-C2-C3-C4) or from the N-terminal side (C5-C6-C7-C8-C9) leads to decreased interaction with tRNA (Figure 4B). These two observations are in agreement with the fact that the VDAC34–tRNA interaction does not involve a specific RNA binding domain but rather a set of amino acids distributed all along the sequence (Figure 3B). Furthermore, analyzing deletion mutants highlighted the importance of preserving a complete VDAC34 sequence since short deletions of 25 amino acids at the N-terminus (D2) or 30 amino acids at the C-terminus (D4) induced an important decrease of interaction up to 70%. Even the deletion of 10 amino acids (D3) decreased the interaction level by 10%. Indeed, the comparison of deleted and corresponding chimeric structures such as D5 with C2, D6 with C1 and D2 with C6, showed that deleted proteins could not interact as efficiently as full-length proteins.


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)

The N-terminal part of VDAC34 is essential for an efficient interaction with tRNA. Two kinds of mutants were designed: (A) deleted versions of VDAC34 (D1–D7) and (B) chimeric proteins (C1–C9) in which segments of VDAC34 were replaced by segments of VDAC36. Numbers indicate position within VDAC sequences. The variants overexpressed in Escherichia coli were purified and used to perform NW experiments in the presence of Arabidopsis thaliana cytosolic tRNAAla in order to quantify percentage of interaction between mutant proteins and tRNA. Obtained values are represented on a diagram and are given in a summary table. They are the average of 2–9 independent experiments and correspond to the percentage of interaction of the mutant protein with tRNA compared to the wild-type VDAC34 interaction. Standard error is indicated for each value. Examples of NW experiments allowing the quantification of interactions are given as supplemental information (Supplemental Figure S4).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150812&req=5

Figure 4: The N-terminal part of VDAC34 is essential for an efficient interaction with tRNA. Two kinds of mutants were designed: (A) deleted versions of VDAC34 (D1–D7) and (B) chimeric proteins (C1–C9) in which segments of VDAC34 were replaced by segments of VDAC36. Numbers indicate position within VDAC sequences. The variants overexpressed in Escherichia coli were purified and used to perform NW experiments in the presence of Arabidopsis thaliana cytosolic tRNAAla in order to quantify percentage of interaction between mutant proteins and tRNA. Obtained values are represented on a diagram and are given in a summary table. They are the average of 2–9 independent experiments and correspond to the percentage of interaction of the mutant protein with tRNA compared to the wild-type VDAC34 interaction. Standard error is indicated for each value. Examples of NW experiments allowing the quantification of interactions are given as supplemental information (Supplemental Figure S4).
Mentions: In order to determine which regions are important for the interaction with tRNA, different mutant versions of VDAC34 were constructed. To do so, VDAC proteins were arbitrarily divided into three segments: I (position 1–90), II (position 91–171) and III (position 172–276) (Figure 3A). In total, seven deletion mutants (D1–D7) and nine chimeric (C1–C9) were designed (Figure 4). Northwestern analysis on deletion mutants showed that none of the VDAC34 segments with the exception of the D3 mutant are able to interact efficiently with tRNAs (Figure 4A). In addition, analysis on chimeric mutants indicated that the gradual replacement of VDAC34 sequence by VDAC36 sequence from the C-terminal side (C1-C2-C3-C4) or from the N-terminal side (C5-C6-C7-C8-C9) leads to decreased interaction with tRNA (Figure 4B). These two observations are in agreement with the fact that the VDAC34–tRNA interaction does not involve a specific RNA binding domain but rather a set of amino acids distributed all along the sequence (Figure 3B). Furthermore, analyzing deletion mutants highlighted the importance of preserving a complete VDAC34 sequence since short deletions of 25 amino acids at the N-terminus (D2) or 30 amino acids at the C-terminus (D4) induced an important decrease of interaction up to 70%. Even the deletion of 10 amino acids (D3) decreased the interaction level by 10%. Indeed, the comparison of deleted and corresponding chimeric structures such as D5 with C2, D6 with C1 and D2 with C6, showed that deleted proteins could not interact as efficiently as full-length proteins.

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.

Show MeSH
Related in: MedlinePlus