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

Abstract

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 tRNAMetis closely related to the imported elongator tRNAMet. Thus, the distinct localization of the two tRNAsMetmust 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 tRNAMetT-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNAMetindicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAsMetinto isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNAMet.