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Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit- g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit- g together with subunit- e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit- g/e module as a structural component determining ATP synthase oligomeric assemblies. Mitochondrial ATP synthase assemble into oligomers. Here, authors resolve the structure of trypanosomal ATP synthase, showing that its dimerization is essential for function and evolutionary conserved.

The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

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.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the F o F 1 -ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the F o F 1 -ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨ m . This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨ m . In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans.

Mitochondria have undergone profound diversification in separate lineages that have radiated since the last common ancestor of eukaryotes some eons ago. Most eukaryotes are unicellular protists, including etiological agents of infectious diseases, like Trypanosoma brucei . Mitochondrial cristae are polymorphic invaginations of the inner membrane that are the fabric of cellular respiration. Both the mitochondrial contact site and cristae organization system (MICOS) and the F 1 F O -ATP synthase are vital for sculpting cristae by opposing membrane-bending forces. While MICOS promotes negative curvature at crista junctions, dimeric F 1 F O -ATP synthase is crucial for positive curvature at crista rims. Crosstalk between these two complexes has been observed in baker’s yeast, the model organism of the Opisthokonta supergroup. Here, we report that this property is conserved in Trypanosoma brucei , a member of the Discoba clade that separated from the Opisthokonta ∼2 billion years ago. Specifically, one of the paralogs of the core MICOS subunit Mic10 interacts with dimeric F 1 F O -ATP synthase, whereas the other core Mic60 subunit has a counteractive effect on F 1 F O -ATP synthase oligomerization. This is evocative of the nature of MICOS–F 1 F O -ATP synthase crosstalk in yeast, which is remarkable given the diversification that these two complexes have undergone during almost 2 eons of independent evolution. Furthermore, we identified a highly diverged, putative homolog of subunit e, which is essential for the stability of F 1 F O -ATP synthase dimers in yeast. Just like subunit e, it is preferentially associated with dimers and interacts with Mic10, and its silencing results in severe defects to cristae and the disintegration of F 1 F O -ATP synthase dimers. Our findings indicate that crosstalk between MICOS and dimeric F 1 F O -ATP synthase is a fundamental property impacting crista shape throughout eukaryotes. IMPORTANCE Mitochondria have undergone profound diversification in separate lineages that have radiated since the last common ancestor of eukaryotes some eons ago. Most eukaryotes are unicellular protists, including etiological agents of infectious diseases, like Trypanosoma brucei . Thus, the study of a broad range of protists can reveal fundamental features shared by all eukaryotes and lineage-specific innovations. Here, we report that two different protein complexes, MICOS and F 1 F O -ATP synthase, known to affect mitochondrial architecture, undergo crosstalk in T. brucei , just as in baker’s yeast. This is remarkable considering that these complexes have otherwise undergone many changes during their almost 2 billion years of independent evolution. Thus, this crosstalk is a fundamental property needed to maintain proper mitochondrial structure even if the constituent players considerably diverged.

Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes) that catalyze RNA editing but the relative roles of each protein are not known. The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect) and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the F o F 1 -ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the F o F 1 -ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨ m . This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨ m . In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.