Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes

Abstract

SummaryFrataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxindepleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.

Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis

“…After the addition of tetracycline, which triggers RNAi, growth of the Tb-frataxin and Tb-IscS knock-down controls virtually stopped at day 5 postinduction (SI Appendix, Figs. S16 and S17), as, in the absence of these proteins, the cells cease dividing and eventually die (29,30). However, the procyclic trypanosomes in which RNAi-ablated Tb-frataxin was replaced by Bh-frataxin in trans retained almost the same growth as their noninduced counterparts (SI Appendix, Fig.…”

“…S16 and S17) indicates that Bh-frataxin and Bh-IscS plus Pt-Isd11 can partially replace the function of Tb-frataxin and Tb-IscS in the Tb mitochondrion. As previous studies have shown that the down-regulation of Tb-frataxin and Tb-IscS causes a disruption of Fe/S cluster assembly (29,36), it is likely that this function is provided in trans by the Blastocystis homologue. To verify this assumption, we measured activities of several previously described Fe/S cluster-containing enzymes with mitochondrial and/ or cytosolic localization (29,37).…”

“…As previous studies have shown that the down-regulation of Tb-frataxin and Tb-IscS causes a disruption of Fe/S cluster assembly (29,36), it is likely that this function is provided in trans by the Blastocystis homologue. To verify this assumption, we measured activities of several previously described Fe/S cluster-containing enzymes with mitochondrial and/ or cytosolic localization (29,37). Activity of the mitochondrial Fe/S cluster-containing aconitase dropped to less than 20% of the WT activity in the RNAi-induced Tb-frataxin (SI Appendix, Fig.…”

“…Western blot analyses were used to determine the specificity of antibodies raised against IscS of Trichomonas vaginalis (36), IscU of Giardia intestinalis (40), Frataxin of Tb (29), and SufC of Erwinia chrysanthemi (41) in Blastocystis protein extracts as previously described (42). To determine the cellular localization of these proteins, we used confocal immunofluorescence analyses, and, additionally, in the case of Bh-SufCB, immunogold transmission EM.…”

“…After linearization, the vectors were transfected into Tb knockdown strains. The rescued strains were tested for complementation by measuring the activities of mitochondrial and cytosolic Fe/S enzymes as demonstrated earlier (29). In the case of Bh-SufCB, functional characterization of the protein was performed to determine the FADH 2 binding affinities of the protein, the presence of an ATPase activity, and its capacity to interact with a cysteine desulfurase as described for E. coli homologues (26).…”