Inducible orthogonal aminoacylation demonstrates that charging is required for mitochondrial tRNA import in Trypanosoma brucei

Abstract

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have emerged as powerful means of site-specifically introducing non-standard amino acids into proteins in vivo . Using amino acids with crosslinking moieties this method allows the identification of transient protein-protein interactions. Here we have introduced a previously characterized evolved tyrosyl-tRNA synthetase/suppressor tRNA Tyr pair from E. coli into the parasitic protozoan Trypanosoma brucei . Upon addition of a suitable non-standard amino acid the suppressor tRNA Tyr was charged and allowed translation of a green fluorescent protein whose gene contained a nonsense mutation. - T. brucei is unusual in that its mitochondrion lacks tRNA genes indicating that all its organellar tRNAs are imported from the cytosol. Expression of the bacterial tyrosyl-tRNA synthetase in our system is tetracycline-inducible. We have therefore used it to demonstrate that cytosolic aminoacylation of the suppressor tRNA Tyr induces its import into the mitochondrion.

The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria

“…Since elongation factors only recognize aminoacylated tRNAs (40) this implies that only charged tRNAs can be imported. This was independently confirmed in a recent publication showing that, while a mutant E. coli tRNA Tyr expressed in T. brucei could not be imported into mitochondria, because it was not recognized by the trypanosomal tyrosyl-tRNA synthetase, its import could be induced by co-expression of the E. coli tyrosyl-tRNA synthetase (12). These two examples illustrate that in T. brucei only aminoacylated tRNAs are imported into mitochondria in vivo.…”

“…As in other systems a connection to mitochondrial protein import has emerged, even though in contrast to what has been proposed in yeast, trypanosomal tRNAs are not co-imported with proteins (9). Moreover, tRNAs appear to be imported as fully processed molecules (11,12). The exclusive reliance on mitochondrial tRNA import has initiated a more general interest in trypanosomal tRNA biology (1).…”