Interaction of the dual targeting peptide of Thr‐tRNA synthetase with the chloroplastic receptor Toc34 inArabidopsis thaliana

Abstract

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl‐tRNA synthetase inArabidopsis thaliana (AtThrRS‐dTP) interacts with the mitochondrial receptorAtTom20 mainly through its N‐terminal part. Here we report on the interaction ofAtThrRS‐dTP with the chloroplastic receptorAtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in15N HSQC spectra ofAtThrRS‐dTP as a function ofAtToc34 concentration. Line broadening shows that the interaction withAtToc34 involves residues along the entire sequence, which is not the case forAtTom20. The N‐terminal φχχφφ motif, which plays an important role inAtTom20 recognition, shows no specificity forAtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments ofAtThrRS‐dTP onin vitro import of organelle specific proteins was examined. This demonstrates that the N‐terminal A2‐Y29 segment ofAtThrRS‐dTP is essential for import into both organelles, while the C‐terminal L30‐P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide ofAtThr‐tRNA synthetase is different for the mitochondrial and chloroplastic receptors.

Keywords

• Dual targeting
• Chloroplasts and mitochondria
• Protein import
• NMR
• Toc34 receptor