Description: Publication date: 2015 Source: FEBS Open Bio, Volume 5 Author(s): Weihua Ye, Erika Spånning, Elzbieta Glaser, Lena Mäler 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 in Arabidopsis thaliana ( At ThrRS-dTP) interacts with the mitochondrial receptor At Tom20 mainly through its N-terminal part. Here we report on the interaction of At ThrRS-dTP with the chloroplastic receptor At Toc34, 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 in 15 N HSQC spectra of At ThrRS-dTP as a function of At Toc34 concentration. Line broadening shows that the interaction with At Toc34 involves residues along the entire sequence, which is not the case for At Tom20. The N-terminal φχχφφ motif, which plays an important role in At Tom20 recognition, shows no specificity for At Toc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of At ThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of At ThrRS-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 of At Thr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors. Graphical abstract