Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Abstract

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA Met , all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA Met present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA Met carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA Met . In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA Met . Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA Met , suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knockdown of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyltRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.

Pentatricopeptide Repeat Proteins Stimulate mRNA Adenylation/Uridylation to Activate Mitochondrial Translation in Trypanosomes

“…S1). Translation factors were distributed among ribosomal subunits as expected: initiation factor 2 (IF2) (Charriere et al, 2005) was detected exclusively in the small subunit, while elongation factor Tu (EF-Tu) was found in both small and large subunits (Table S1). The discrete sets of PPRs, including two repeat-containing proteins originally identified in the KPAP1 complex (Tb11.02.3180 and Tb927.8.3170, (Etheridge et al, 2008)), were enriched in either small or large ribosomal subunits (Tables S1 and S3).…”

The enzymes of the 10-formyl-tetrahydrofolate synthetic pathway are found exclusively in the cytosol of the trypanosomatid parasite Leishmania major

“…While not studied in Leishmania , methionyl-tRNA Met-e formylation appears essential for mitochondrial protein biosynthesis in the related trypanosomatid T. brucei [24]. Since both DHCH1 and FTL are located in the cytosol of L. major , and previous studies in T. brucei showed that the initiator methionyl-tRNA Met-e is formylated in the mitochondrial matrix [23], this implies that 10-CHO-THF can be transported across the mitochondrial membrane in trypanosomatids.…”

“…Secondly, following import into the mitochondrion, the cytosolic elongator tRNA Met-e acts as either an elongator tRNA Met , or is formylated and used as an initiator tRNA Met ; remarkably, the tRNA Met-i was not used in the trypanosome mitochondrion [23]. Lastly, methionyl-tRNA formylation is required for binding of the fMet-tRNA Met to initiation factor 2, and appears to be essential in trypanosomes [24]. In contrast, the requirement for initiator tRNA Met formylation appears highly variable in evolution; while initially considered essential in bacteria, later data suggested otherwise [25, 26].…”

Folate metabolic pathways in Leishmania

“…There is considerable debate about the requirement for initiator Met-tRNA Met formylation, with genetic studies in both prokaryotes and eukaryotes yielding divergent conclusions – often within the same species [51]. RNAi (RNA interference) studies in trypanosomes showed little phenotype when targeting solely FMT , but strong growth inhibition when both FMT and IF2 were targeted simultaneously [52]. …”