NMR investigations of the dual targeting peptide of Thr‐tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana

Abstract

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mito-chondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting pep-tide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form a-helical structure in one region, S6-F27, and a very weak b-strand propensity for Q34-Q38. The a-helical structured region has an amphiphilic character and a uvvuu motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. Database The backbone chemical shift assignments for ThrRS-dTP(2-60) have been deposited with the Biological Magnetic Resonance Bank (BMRB) under the accession code 18248 Structured digital abstract ThrRS-dTP and Tom20-4 bind by nuclear magnetic resonance (View interaction) Abbreviations aaRS, aminoacyl-tRNA synthetase; AOX, alternative oxidase; CLEANEX, clean chemical exchange; cTP, chloroplastic targeting peptide; dTP, dually targeting peptide; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum coherence; mTP, mitochondrial targeting peptide; SSP, secondary structure propensity; ThrRS-dTP, threonyl tRNA synthetase dual targeting peptide; 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.

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

“…6). We do indeed see that AtThrRS-dTP(1-29) possesses high helical propensity, whereas AtThrRS-dTP(30-60) and the truncated peptides AtThrRS-dTP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and AtThrRS-dTP(20-29) do not. Taken together, our results imply that the mitochondrial receptor Tom20 interacts mostly with the N-terminal portion of the AtThrRS-dTP(2-60) peptide, whereas the chloroplastic receptor Toc34 interacts with both the N-and C-terminal segments of the dual targeting peptide.…”

“…The import into both organelles was strongly inhibited (73% for mitochondria and 88% for chloroplasts) by the N-terminal amphiphilic helical AtThrRS-dTP(1-29) peptide (lanes 4 and 5). The truncated versions of the AtThrRS-dTP(1-29) peptide, AtThrRS-dTP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and AtThrRS-dTP (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), exhibited only a weak inhibitory effect (622%) on both organelles (lanes 6-9). Interestingly, the C-terminal unstructured AtThrRS-dTP(30-60) part of the dual targeting peptide revealed very strong inhibition (90%) of the chloroplast import and affected the mitochondrial import to much lower extent (25%) (lanes 10 and 11).…”

Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis

“…These data indicate that the interactions of these nucleus-encoded proteins with AtPAP2 are not the sole determining factors of their targeting to these two organelles. Instead, other receptors on TOC (Toc34, Toc64, and Toc159) and TOM (Tom20 and OM64) complexes are responsible for cargo specificity (Ye et al, 2012(Ye et al, , 2015.…”

“…For both chloroplasts and mitochondria, over 1,000 different proteins are required to be specifically imported into each organelle. Furthermore, while most proteins are imported specifically into one organelle, a significant number of proteins are dually targeted to both via ambiguous targeting signals (Carrie et al, 2009a;Ye et al, 2012Ye et al, , 2015. The sorting of proteins to chloroplast and mitochondria is achieved through the inclusion of targeting signals in newly synthesized proteins that act in combination with receptor domains present in outer membrane multisubunit protein complexes to specifically direct proteins to their destination compartments (Jarvis, 2008;Shi and Theg, 2013;Murcha et al, 2014).…”

Translation in Mitochondria and Other Organelles

“…Rice paralogous proteins were found for most dual-targeted Arabidopsis aaRSs. Several nonexclusive possibilities exist such as the presence of alternative translation or transcription sites, existence of "ambiguous" targeting sequences, environmental conditions or differential mRNA sorting at the surface of the organelles Pujol et al 2007;Michaud et al 2010;Ye et al 2012). In algae, although no extensive experimental studies have been performed, in silico analyses strongly suggest that most aaRSs are at least dual localized (http://p lantrna.ibmp.cnrs.fr/), (Cognat et al 2013).…”