Mitochondrial tRNA import in Toxoplasma gondii

Apicomplexan parasites have the smallest known mitochondrial genome. It consists of a repeated element of similar to6-7 kb in length and encodes three mitochondrial proteins, a number of rRNA fragments, but no tRNAs. It has therefore been postulated that in apicomplexans all tRNAs required for mitochondrial translation are imported from the cytosol. To provide direct evidence for this process we have established a cell fractionation procedure allowing the isolation of defined organellar RNA fractions from the apicomplexan Toxoplasma gondii. Analysis of T. gondii total and organellar RNA by Northern hybridization showed that except for the cytosol-specific initiator tRNA(Met) all nucleus-encoded tRNAs tested were present in the cytosol and in the mitochondrion but not in the plastid. Thus, these results provide the first experimental evidence for mitochondrial tRNA import in apicomplexans. The only other taxon that imports the whole set of mitochondrial tRNAs are the trypanosomatids. Interestingly, the initiator tRNA(Met) is the only cytosol-specific tRNA in trypanosomatids, indicating that the import specificity is identical in both groups. In agreement with this, the T. gondii initiator tRNA(Met) remained in the cytosol when expressed in Trypanosoma brucei. However, in contrast to trypanosomatids, no thio- modifications were detected in the tRNA(Gln) of T. gondii indicating that, unlike what is suggested in Leishmania, they are not involved in regulating import.