Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.

• Klíčová slova
• constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
• MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• genomika MeSH
• molekulární evoluce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

• Klíčová slova
• Trypanosoma brucei, gene fragmentation, mitochondrial ATP synthase, proton path, proton translocation, subunit-a,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• Eukaryota metabolismus   MeSH
• mitochondriální protonové ATPasy * genetika   chemie   metabolismus   MeSH
• protonové ATPasy * metabolismus   MeSH
• protony MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mitochondriální protonové ATPasy * MeSH
• protonové ATPasy * MeSH
• protony MeSH

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.

• MeSH
• lipidy MeSH
• mitochondriální protonové ATPasy * metabolismus   MeSH
• podjednotky proteinů metabolismus   MeSH
• protony MeSH
• savci MeSH
• voda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidy MeSH
• mitochondriální protonové ATPasy * MeSH
• podjednotky proteinů MeSH
• protony MeSH
• voda MeSH

Mitochondrial F-type adenosine triphosphate (ATP) synthases are commonly introduced as highly conserved membrane-embedded rotary machines generating the majority of cellular ATP. This simplified view neglects recently revealed striking compositional diversity of the enzyme and the fact that in specific life stages of some parasites, the physiological role of the enzyme is to maintain the mitochondrial membrane potential at the expense of ATP rather than to produce ATP. In addition, mitochondrial ATP synthases contribute indirectly to the organelle's other functions because they belong to major determinants of submitochondrial morphology. Here, we review current knowledge about the trypanosomal ATP synthase composition and architecture in the context of recent advances in the structural characterization of counterpart enzymes from several eukaryotic supergroups. We also discuss the physiological function of mitochondrial ATP synthases in three trypanosomatid parasites, Trypanosoma cruzi, Trypanosoma brucei and Leishmania, with a focus on their disease-causing life cycle stages. We highlight the reversed proton-pumping role of the ATP synthase in the T. brucei bloodstream form, the enzyme's potential link to the regulation of parasite's glycolysis and its role in generating mitochondrial membrane potential in the absence of mitochondrial DNA.

• Klíčová slova
• ATP synthase, cryo-EM, mitochondria, mitochondrial membrane potential, oxidative phosphorylation,
• MeSH
• genetické inženýrství * MeSH
• Leishmania enzymologie   MeSH
• membránový potenciál mitochondrií MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• Trypanosoma cruzi enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mitochondriální protonové ATPasy MeSH
• protozoální proteiny MeSH

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 F1FO-ATP synthase are vital for sculpting cristae by opposing membrane-bending forces. While MICOS promotes negative curvature at crista junctions, dimeric F1FO-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 F1FO-ATP synthase, whereas the other core Mic60 subunit has a counteractive effect on F1FO-ATP synthase oligomerization. This is evocative of the nature of MICOS-F1FO-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 F1FO-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 F1FO-ATP synthase dimers. Our findings indicate that crosstalk between MICOS and dimeric F1FO-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 F1FO-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.

• Klíčová slova
• ATP synthase, MICOS, Trypanosoma, evolution, mitochondria,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• ATP synthase, ATPase, Trypanosoma brucei, alternative oxidase, bioenergetics, electron transport, mitochondria, mitochondrial membrane potential, oxidative phosphorylation, respiration,
• MeSH
• adenosintrifosfát genetika   metabolismus   MeSH
• buněčný cyklus * MeSH
• energetický metabolismus * MeSH
• membránový potenciál mitochondrií MeSH
• mitochondrie genetika   metabolismus   MeSH
• protonové ATPasy nedostatek   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• protonové ATPasy MeSH
• protozoální proteiny MeSH

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.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné dýchání účinky léků   MeSH
• buněčné linie MeSH
• elektrony MeSH
• glukosa farmakologie   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metabolické sítě a dráhy účinky léků   MeSH
• metabolomika * MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• oxidace-redukce MeSH
• oxidoreduktasy metabolismus   MeSH
• prolin metabolismus   MeSH
• proteom metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• signální transdukce MeSH
• transkriptom genetika   MeSH
• transport elektronů účinky léků   MeSH
• Trypanosoma brucei brucei účinky léků   genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• alternative oxidase MeSH Prohlížeč
• glukosa MeSH
• mitochondriální proteiny MeSH
• oxidoreduktasy MeSH
• prolin MeSH
• proteom MeSH
• protozoální proteiny MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH

Trypanosoma brucei is an extracellular parasite that alternates between an insect vector (procyclic form) and the bloodstream of a mammalian host (bloodstream form). While it was previously reported that mitochondrial release factor 1 (TbMrf1) is essential in cultured procyclic form cells, we demonstrate here that in vitro bloodstream form cells can tolerate the elimination of TbMrf1. Therefore, we explored if this discrepancy is due to the unique bioenergetics of the parasite since procyclic form cells rely on oxidative phosphorylation; whereas bloodstream form cells utilize glycolysis for ATP production and FoF1-ATPase to maintain the essential mitochondrial membrane potential. The observed disruption of intact bloodstream form FoF1-ATPases serves as a proxy to indicate that the translation of its mitochondrially encoded subunit A6 is impaired without TbMrf1. While these null mutants have a decreased mitochondrial membrane potential, they have adapted by increasing their dependence on the electrogenic contributions of the ADP/ATP carrier to maintain the mitochondrial membrane potential above the minimum threshold required for T. brucei viability in vitro. However, this inefficient compensatory mechanism results in avirulent mutants in mice. Finally, the depletion of the codon-independent release factor TbPth4 in the TbMrf1 knockouts further exacerbates the characterized mitchondrial phenotypes.

• MeSH
• fyziologická adaptace * MeSH
• membránový potenciál mitochondrií genetika   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie * genetika   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• oxidativní fosforylace MeSH
• protonové ATPasy genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje * MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• protonové ATPasy MeSH
• protozoální proteiny MeSH

The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F1-ATPases determined previously. The α3β3-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the β-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F1-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme.

• Klíčová slova
• ATP synthase, Trypanosoma brucei, catalytic domain, p18 subunit, structure,
• MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• molekulární modely MeSH
• podjednotky proteinů MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• Trypanosoma brucei brucei enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální protonové ATPasy MeSH
• podjednotky proteinů MeSH
• protozoální proteiny MeSH