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.
Trypanosoma brucei and its relatives are prominent pathogens causing human and animal diseases, which mainly affect developing countries. The single mitochondrion of trypanosomes is essential across its entire life cycle. Most organellar proteins are imported by hetero-oligomeric protein complexes in the two mitochondrial membranes. Interestingly, the composition of the two import machineries is remarkably different from their corresponding counterparts in other organisms. In contrast, chaperones termed small Tims that escort hydrophobic proteins across the aqueous intermembrane space are conserved in almost all eukaryotes including trypanosomes. Here we show that a fraction of them interact tightly with the inner membrane translocase. Another fraction is present as soluble 70 kDa complexes, which likely consists of hexamers of small Tims without a defined subunit composition. In other eukaryotes these hexamers are usually composed of two alternating small Tims. Moreover, while some small Tims are involved in import of a core subunit of the inner membrane protein translocase, we found one small Tim that directly mediates the assembly of the translocase complex. Knowing which components of the trypanosomal protein import systems are conserved and which ones are not is essential to evaluate whether mitochondrial protein import might be a suitable drug target.