Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs.
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.
Affiliation: Institut de Biologie Moléculaire des Plantes, UPR 2357 CNRS, associated with Strasbourg University, 12 rue du Général Zimmer 67084 Strasbourg cedex, France email@example.com.Show MeSH
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.
Affiliation: Institut de Biologie Moléculaire des Plantes, UPR 2357 CNRS, associated with Strasbourg University, 12 rue du Général Zimmer 67084 Strasbourg cedex, France firstname.lastname@example.org.