Eukaryotic-type elongator tRNA^Met of Trypanosoma brucei becomes formylated after import into mitochondria

Abstract

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA^Met, whereas the only other trypanosomal tRNA^Met, the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs^Met carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA^Met is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA^Met becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA^Met is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA^Met formyltransferases, it has about twice the mass of any of these proteins.

• mitochondrial translation‖initiator tRNAMet‖formylation‖mitochondrial tRNA import‖methionyl-tRNAMet formyltransferase