title: Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis 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 in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, 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 AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal φχχφφ motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-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 AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden.
authors.forename: Weihua
authors.initials: W
authors.lastname: Ye
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden.
authors.forename: Erika
authors.initials: E
authors.lastname: Spånning
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden.
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Mäler
keywords: Chloroplasts and mitochondria; Dual targeting; HSQC, heteronuclear single-quantum coherence; NMR; Protein import; TIC, translocase of the inner envelope membrane of chloroplasts; TIM, translocase of the inner mitochondrial membrane; TOC, translocase of the outer envelope membrane of chloroplasts; TOM, translocase of the outer mitochondrial membrane; ThrRS, threonyl tRNA synthetase; Toc34 receptor; aaRS, amino acyl-tRNA synthetase; cTP, chloroplastic targeting peptide; dTP, dual targeting peptide; mTP, mitochondrial targeting peptide