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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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VDAC34 and VDAC36 sequences. (A) Schematic representation of VDAC34 (dark gray) and of VDAC36 (light gray). The three segments set up for the design of the different mutants are indicated. Subsequently, for segment I, two more divisions were done. The number of amino acids that differ between the two VDAC is indicated for each segment. (B) Sequence alignment of VDAC34 and VDAC36. Amino acids that differ between the two VDAC sequences are depicted on a gray background. Positions delimiting the different segments used for design of the different VDAC34 mutants are indicated. Amino acids of the first 50 residues predicted to potentially interact with RNA by BindN software are indicated in italics bold.
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Figure 3: VDAC34 and VDAC36 sequences. (A) Schematic representation of VDAC34 (dark gray) and of VDAC36 (light gray). The three segments set up for the design of the different mutants are indicated. Subsequently, for segment I, two more divisions were done. The number of amino acids that differ between the two VDAC is indicated for each segment. (B) Sequence alignment of VDAC34 and VDAC36. Amino acids that differ between the two VDAC sequences are depicted on a gray background. Positions delimiting the different segments used for design of the different VDAC34 mutants are indicated. Amino acids of the first 50 residues predicted to potentially interact with RNA by BindN software are indicated in italics bold.

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)

VDAC34 and VDAC36 sequences. (A) Schematic representation of VDAC34 (dark gray) and of VDAC36 (light gray). The three segments set up for the design of the different mutants are indicated. Subsequently, for segment I, two more divisions were done. The number of amino acids that differ between the two VDAC is indicated for each segment. (B) Sequence alignment of VDAC34 and VDAC36. Amino acids that differ between the two VDAC sequences are depicted on a gray background. Positions delimiting the different segments used for design of the different VDAC34 mutants are indicated. Amino acids of the first 50 residues predicted to potentially interact with RNA by BindN software are indicated in italics bold.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: VDAC34 and VDAC36 sequences. (A) Schematic representation of VDAC34 (dark gray) and of VDAC36 (light gray). The three segments set up for the design of the different mutants are indicated. Subsequently, for segment I, two more divisions were done. The number of amino acids that differ between the two VDAC is indicated for each segment. (B) Sequence alignment of VDAC34 and VDAC36. Amino acids that differ between the two VDAC sequences are depicted on a gray background. Positions delimiting the different segments used for design of the different VDAC34 mutants are indicated. Amino acids of the first 50 residues predicted to potentially interact with RNA by BindN software are indicated in italics bold.
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.

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