TRANSFER-RNAS ARE IMPORTED INTO MITOCHONDRIA OF TRYPANOSOMA-BRUCEI INDEPENDENTLY OF THEIR GENOMIC CONTEXT AND GENETIC-ORIGIN

The mitochondrial genome of Trypanosoma brucei does not encode any ide ntifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the n ucleus and subsequently imported into mitochondria. In order to analys e the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episom ally and their import into mitochondria assessed by purification and n uclease treatment of the mitochondrial fraction, Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA (Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosoli c tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5'-flanking sequence of the trypano somal tRNA(Tyr) gene, In all cases efficient import of each of the tRN As was observed, We independently confirmed the mitochondrial import o f the yeast tRNA(His), since in organello [alpha-P-32]ATP-labelling of the 3'-end of the tRNA was inhibited by carboxyatractyloside, a highl y specific inhibitor of the mitochondrial adenine nucleotide transloca tor, Import of heterologous tRNAs in their own genomic contexts suppor ts the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T.brucei, but rather that the tRNA s tructure itself is sufficient to specify import.