Dual Targeting to Mitochondria and Chloroplasts: Characterization of Thr–tRNA Synthetase Targeting Peptide

Abstract

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase (ThrRS-dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS-dTP(2-60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP(2-60) inhibited import of pF1beta into mitochondria and of pSSU into chloroplasts at microM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP(2-60) indicated that the peptide has the propensity for forming alpha-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

Organelle import of proteins with dual targeting properties into mitochondria and chloroplasts takes place by the general import pathways

“…These experiments fit well to earlier observations [10][11][12] and reconfirm the conclusion that proteins with dual targeting properties do not use distinct import pathways that were specifically developed for this purpose. This observation can be taken as further hint for the hypothesis that dual targeting is an evolutionary remnant in the development of transit peptides of both endosymbiotic organelles.…”

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

“…2 and 3a). 27 It appears, however, that the deleted constructs can bind to both organelles, as shown by the presence of precursor in the absence of PK in the in vitro studies ( Fig. 2a and b, lane 2).…”

“…6 However, recent studies have not found the overall hydrophobicity to be increased in dTPs, even though phenylalanines and leucines are overrepresented. [25][26][27] It was also shown that the Nterminal portion of dTPs has significantly more serines than mTPs, and that the amount of arginines is similar as in mTPs, but almost twice as high as in cTPs. 27 An intriguing question is how the targeting information for the respective organelle is organised within the dTP.…”

“…[25][26][27] It was also shown that the Nterminal portion of dTPs has significantly more serines than mTPs, and that the amount of arginines is similar as in mTPs, but almost twice as high as in cTPs. 27 An intriguing question is how the targeting information for the respective organelle is organised within the dTP. Since both import machineries have evolved to separate its own set of proteins from all other cellular proteins, one hypothesis is that targeting determinants are located in different domains within dTPs.…”

The Aconitase C-Terminal Domain Is an Independent Dual Targeting Element

“…[1][2][3][4][5] Yeast aconitase [citrate (isocitrate) hydroxylase] is encoded by a single nuclear gene, ACO1 (778 amino acids), 6 which produces a single translation product that is nevertheless distributed between the mitochondria and the cytosol. 3,7 Aconitase employs a [ 4 Fe-4 S] cluster, 8 and the resolved crystal structure of aconitase isolated from porcine heart mitochondria revealed that it folds into four domains.…”