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The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments we show that trypanosomal Msp1 localizes to both glycosomes and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of a-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates suggesting they act in concert with Msp1. pATOM36 is a functional analogue of the yeast MIM complex and possibly of human MTCH2 suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all eukaryotes.

International audience; The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.

The mitochondrial F

The mitochondrial F1Fo ATP synthase of Trypanosoma brucei has been studied in detail. Whereas its F1 moiety is relatively highly conserved in structure and composition, the same is not the case for the Fo part and the peripheral stalk. A core subunit of the latter, the normally conserved subunit b, could not be identified in trypanosomes suggesting that it might be absent. Here we have identified a 17 kDa mitochondrial protein of the inner membrane that is essential for normal growth, efficient oxidative phosphorylation and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicate that the protein is associated with the F1Fo ATP synthase. Its ablation reduces the levels of Fo subunits, but not those of F1, and disturbs the cell cycle. HHpred analysis showed that the protein has structural similarities to subunit b of other species, indicating that the Fo part of the trypanosomal ATP synthase does contain a highly diverged subunit b. Thus, the Fo part of the trypanosomal ATPase synthase may be more widely conserved than initially thought.

Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all mitochondria.

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1–ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1–ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1–ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1–ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1–ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1–ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1–ATP synthase loss in insect versus mammalian forms of the parasite.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity., SCOPUS: ar.j, info:eu-repo/semantics/published

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F 1 F O –ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing F O -independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei , the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F 1 F O –ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites.