Targeting of tRNA into yeast and human mitochondria: the role of anticodon nucleotides

In vivo, yeast mitochondria import a single cytoplasmic tRNA, tRNA(CUU)Lys, while human mitochondria do not import any cytoplasmic tRNA. We have previously demonstrated that both yeast and human isolated mitochondria can specifically internalize tRNA(CUU)Lys, several of its mutant versions and some mutant versions of yeast cytosolic tRNA(UUU)Lys (not imported in vivo). Aminoacylation of tRNA(CUU)Lys by the cytoplasmic lysyl-tRNA synthetase was a prerequisite for its import. Here we are studying the influence of one-base replacements in the anticodon of tRNAs(Lys) on their aminoacylation, on binding to the precursor of the mitochondrial lysyl-tRNA synthetase (carrier protein directing the import), and on the efficiency of import into isolated yeast and human mitochondria. We show that the base U35 is the main identity element for the yeast cytoplasmic lysyl-tRNA synthetase. The single replacement that abolished import was C34G, while all the others only modulated the import efficiency. The need of aminoacylation for import and for interaction with the carrier protein was shown only for a subset of mutant versions, while the others could be recognized and internalized without aminoacylation or in misacylated forms.