Abstract
Mitochondrial protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17.
Highlights
The parasitic protozoan Trypanosoma brucei is the causative agent of the devastating human sleeping sickness and of nagana in cattle [1]
In the present study we focus on the small Tim family of intermembrane space (IMS) localized chaperones, which is conserved in all mitochondria-containing eukaryotes including trypanosomes [11, 12]
It has been shown that all trypanosomal small Tims are associated with the single trypanosomal the inner membrane (TIM) complex, irrespectively of whether it is engaged in Mitochondrial carrier proteins (MCPs) import or in import of presequence containing proteins [10]
Summary
The parasitic protozoan Trypanosoma brucei is the causative agent of the devastating human sleeping sickness and of nagana in cattle [1]. Trypanosomes are a member of the supergroup of the Excavates and are phylogenetically very distant to Opisthokonts [7] Due to this position in the eukaryotic evolutionary tree and its experimental accessibility, T. brucei is excellently suited to investigate which features of mitochondrial protein import are conserved and which ones are not [3, 4]. Two subunits of the ATOM complex and one integral membrane subunit of the single trypanosomal TIM complex are orthologous to TOM and TIM complex subunits of any other eukaryote [3, 4] This is surprising since protein import is considered to be one of the first, if not the first, mitochondria-specific trait to evolve, which suggests that the machineries mediating the process would be conserved
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