Mitochondrial Lysyl-tRNA Synthetase Independent Import of tRNA Lysine into Yeast Mitochondria

Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

This work was supported by the Leverhulme Trust (Grant number RL2012-025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1 , a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3 , a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1 , to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.Publisher PDFPeer reviewe

Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei

Mitochondrial tRNA import is widespread in eukaryotes. Yet, the mechanism that determines its specificity is unknown. Previous in vivo experiments using the tRNAsMet, tRNAIle and tRNALys have suggested that the T-stem nucleotide pair 51:63 is the main localization determinant of tRNAs in Trypanosoma brucei. In the cytosol-specific initiator tRNAMet, this nucleotide pair is identical to the main antideterminant that prevents interaction with cytosolic elongation factor (eEF1a). Here we show that ablation of cytosolic eEF1a, but not of initiation factor 2, inhibits mitochondrial import of newly synthesized tRNAs well before translation or growth is affected. tRNASec is the only other cytosol-specific tRNA in T. brucei. It has its own elongation factor and does not bind eEF1a. However, a mutant of the tRNASec expected to bind to eEF1a is imported into mitochondria. This import requires eEF1a and aminoacylation of the tRNA. Thus, for a tRNA to be imported into the mitochondrion of T. brucei, it needs to bind eEF1a, and it is this interaction that mediates the import specificity