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

Abstract

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-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 Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-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 Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-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 Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

“…Previously described petite mutants of T. brucei have γ-subunit mutations associated with mitochondrial ATP synthase uncoupling and kinetoplast-independent growth (6,10,12). We now describe 30 non-mitochondrial proteins and 3 multisubunit complexes that appear to have the same effect when depleted.…”

“…Dyskinetoplastic T. evansi and T. equiperdum are essentially "petite" mutants of T. brucei (10) with a γ-subunit mutation that uncouples the ATP synthase (12). This uncoupling renders the F o component obsolete and allows a soluble F 1 component to sustain electrogenic exchange and ΔΨ mito , likely in concert with an ADP 3− /ATP 4− carrier (6).…”

“…ATP synthase γ-subunit mutations support dyskinetoplasty by compensating for loss of the kinetoplast-encoded F o -A6 subunit (6); the mutations uncouple the F 1 and F o sectors, allowing the F 1 ATPase to operate independently (12). Oligomycin inhibits the coupled ATPase by binding the F o (the o indicates oligomycin sensitivity) sector (30).…”

“…Indeed, these dyskinetoplastic trypanosomes have been referred to as petite mutants of T. brucei (10), sharing similarities with petite mutants of yeast (11). Thus, dyskinetoplastic trypanosomes use an alternative, F o -independent mode of ΔΨ mito generation involving the uncoupled F 1 sector of ATP synthase and an ADP 3-/ATP 4-carrier (6,12).…”

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

“…Even though the α subunit is cleaved, the complex still hydrolyzes and synthesizes ATP, which is critical for the survival of Trypanosoma brucei, the sleeping sickness parasite (10,11,(14)(15)(16)(17)(18)(19). How this cleavage affects the structure and molecular mechanism of the enzyme is unknown.…”