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 firstname.lastname@example.org.Show MeSH
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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.
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.