ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ(m)) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F(o)F(1)-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ(m) that manifests as a decreased growth phenotype, indicating that the F(o)F(1)-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F(o)-moiety. These Dk cells generate the Δψ(m) by combining the hydrolytic activity of the matrix-facing F(1)-ATPase and the electrogenic exchange of ATP(4)- for ADP(3)- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F(o)F(1)-ATPase complexes were identified. In fact, the immunoprecipitation of a F(1)-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ(m) of Dk cells. Thus, even in the absence of subunit a, a portion of the F(o)F(1)-ATPase is assembled in Dk cells.