The T-Stem Determines the Cytosolic or Mitochondrial Localization of Trypanosomal tRNAsMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNAMet, all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNAMet is closely related to the imported elongator tRNAMet. Thus, the distinct localization of the two tRNAsMet must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAsMet. Furthermore, it was shown that the tRNAMet T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNAMet indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAsMet into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNAMet.