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.

Department of Medical Biochemistry Semmelweis University Budapest Hungary

Institute of Immunology and Infection Research University of Edinburgh United Kingdom

Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic; Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic; Institute of Immunology and Infection Research University of Edinburgh United Kingdom

Zobrazit více v PubMed

Walker J.E. The ATP synthase: The understood, the uncertain and the unknown. Biochem. Soc. Trans. 2013;41:1–16. PubMed

Kuhlbrandt W. Structure and mechanisms of F-type ATP synthases. Annu. Rev. Biochem. 2019;88:515–549. PubMed

Miranda-Astudillo H., Cano-Estrada A., Vazquez-Acevedo M., Colina-Tenorio L., Downie-Velasco A., Cardol P., Remacle C., Dominguez-Ramirez L., Gonzalez-Halphen D. Interactions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp. Biochim. Biophys. Acta. 2014;1837:1–13. PubMed

van Lis R., Mendoza-Hernandez G., Groth G., Atteia A. New insights into the unique structure of the F0F1-ATP synthase from the chlamydomonad algae Polytomella sp. and Chlamydomonas reinhardtii. Plant Physiol. 2007;144:1190–1199. PubMed PMC

Salunke R., Mourier T., Banerjee M., Pain A., Shanmugam D. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii. PLoS Biol. 2018;16 PubMed PMC

Muhleip A., McComas S.E., Amunts A. Structure of a mitochondrial ATP synthase with bound native cardiolipin. Elife. 2019;8 PubMed PMC

Balabaskaran Nina P., Dudkina N.V., Kane L.A., van Eyk J.E., Boekema E.J., Mather M.W., Vaidya A.B. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila. PLoS Biol. 2010;8 PubMed PMC

Zikova A., Schnaufer A., Dalley R.A., Panigrahi A.K., Stuart K.D. The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog. 2009;5 PubMed PMC

Gahura O., Subrtova K., Vachova H., Panicucci B., Fearnley I.M., Harbour M.E., Walker J.E., Zikova A. The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit. FEBS J. 2018;285:614–628. PubMed

Montgomery M.G., Gahura O., Leslie A.G.W., Zikova A., Walker J.E. ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites. Proc. Natl. Acad. Sci. U. S. A. 2018;115:2102–2107. PubMed PMC

Subrtova K., Panicucci B., Zikova A. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic Trypanosomes. PLoS Pathog. 2015;11 PubMed PMC

Bringaud F., Riviere L., Coustou V. Energy metabolism of trypanosomatids: Adaptation to available carbon sources. Mol. Biochem. Parasitol. 2006;149:1–9. PubMed

Hellemond J.J., Bakker B.M., Tielens A.G. Energy metabolism and its compartmentation in Trypanosoma brucei. Adv. Microb. Physiol. 2005;50:199–226. PubMed

Smith T.K., Bringaud F., Nolan D.P., Figueiredo L.M. Metabolic reprogramming during the Trypanosoma brucei life cycle. F1000Res. 2017;6 F1000 Faculty Rev-683. PubMed PMC

Nolan D.P., Voorheis H.P. The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase. Eur. J. Biochem. 1992;209:207–216. PubMed

Schnaufer A., Clark-Walker G.D., Steinberg A.G., Stuart K. The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J. 2005;24:4029–4040. PubMed PMC

Futai M., Kanazawa H. Structure and function of proton-translocating adenosine triphosphatase (F0F1): Biochemical and molecular biological approaches. Microbiol. Rev. 1983;47:285–312. PubMed PMC

Chinopoulos C., Adam-Vizi V. Mitochondria as ATP consumers in cellular pathology. Biochim. Biophys. Acta. 2010;1802:221–227. PubMed

Chinopoulos C. Mitochondrial consumption of cytosolic ATP: Not so fast. FEBS Lett. 2011;585:1255–1259. PubMed

Chen X.J., Clark-Walker G.D. The petite mutation in yeasts: 50 years on. Int. Rev. Cytol. 2000;194:197–238. PubMed

Appleby R.D., Porteous W.K., Hughes G., James A.M., Shannon D., Wei Y.H., Murphy M.P. Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA. Eur. J. Biochem. 1999;262:108–116. PubMed

Dean S., Gould M.K., Dewar C.E., Schnaufer A.C. Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc. Natl. Acad. Sci. U. S. A. 2013;110:14741–14746. PubMed PMC

Campanella M., Casswell E., Chong S., Farah Z., Wieckowski M.R., Abramov A.Y., Tinker A., Duchen M.R. Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1. Cell Metab. 2008;8:13–25. PubMed

Panicucci B., Gahura O., Zikova A. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite. PLoS Negl. Trop. Dis. 2017;11 PubMed PMC

Gahura O., Panicucci B., Vachova H., Walker J.E., Zikova A. Inhibition of F1 -ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1. FEBS J. 2018;285:4413–4423. PubMed

Vertommen D., Van Roy J., Szikora J.P., Rider M.H., Michels P.A., Opperdoes F.R. Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol. Biochem. Parasitol. 2008;158:189–201. PubMed

Frazier A.E., Taylor R.D., Mick D.U., Warscheid B., Stoepel N., Meyer H.E., Ryan M.T., Guiard B., Rehling P. Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery. J. Cell Biol. 2006;172:553–564. PubMed PMC

Chaudhuri M., Ott R.D., Hill G.C. Trypanosome alternative oxidase: From molecule to function. Trends Parasitol. 2006;22:484–491. PubMed

Brand M.D. The sites and topology of mitochondrial superoxide production. Exp. Gerontol. 2010;45:466–472. PubMed PMC

Maxwell D.P., Wang Y., McIntosh L. The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proc. Natl. Acad. Sci. U. S. A. 1999;96:8271–8276. PubMed PMC

Fernandez-Ayala D.J., Sanz A., Vartiainen S., Kemppainen K.K., Babusiak M., Mustalahti E., Costa R., Tuomela T., Zeviani M., Chung J., O'Dell K.M., Rustin P., Jacobs H.T. Expression of the Ciona intestinalis alternative oxidase (AOX) in Drosophila complements defects in mitochondrial oxidative phosphorylation. Cell Metab. 2009;9:449–460. PubMed

El-Khoury R., Dufour E., Rak M., Ramanantsoa N., Grandchamp N., Csaba Z., Duvillie B., Benit P., Gallego J., Gressens P., Sarkis C., Jacobs H.T., Rustin P. Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits mitochondrial ROS overproduction. PLoS Genet. 2013;9 PubMed PMC

Chinopoulos C. The “B space” of mitochondrial phosphorylation. J. Neurosci. Res. 2011;89:1897–1904. PubMed

Bakker B.M., Michels P.A., Opperdoes F.R., Westerhoff H.V. What controls glycolysis in bloodstream form Trypanosoma brucei? J. Biol. Chem. 1999;274:14551–14559. PubMed

Alberto Luevano-Martinez L., Girard R., Alencar M.B., Silber A.M. ATP regulates the activity of an alternative oxidase in Trypanosoma brucei. FEBS Lett. 2020 doi: 10.1002/1873-3468.13790. PubMed DOI

Nowikovsky K., Reipert S., Devenish R.J., Schweyen R.J. Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy. Cell Death Differ. 2007;14:1647–1656. PubMed

Bauerschmitt H., Mick D.U., Deckers M., Vollmer C., Funes S., Kehrein K., Ott M., Rehling P., Herrmann J.M. Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation. Mol. Biol. Cell. 2010;21:1937–1944. PubMed PMC

Perez E., Lapaille M., Degand H., Cilibrasi L., Villavicencio-Queijeiro A., Morsomme P., Gonzalez-Halphen D., Field M.C., Remacle C., Baurain D., Cardol P. The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae. Mitochondrion. 2014;19 Pt B:338–349. PubMed

Horvath A., Horakova E., Dunajcikova P., Verner Z., Pravdova E., Slapetova I., Cuninkova L., Lukes J. Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei. Mol. Microbiol. 2005;58:116–130. PubMed

Gnipova A., Panicucci B., Paris Z., Verner Z., Horvath A., Lukes J., Zikova A. Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits. Mol. Biochem. Parasitol. 2012;184:90–98. PubMed

Coustou V., Besteiro S., Biran M., Diolez P., Bouchaud V., Voisin P., Michels P.A., Canioni P., Baltz T., Bringaud F. ATP generation in the Trypanosoma brucei procyclic form: Cytosolic substrate level is essential, but not oxidative phosphorylation. J. Biol. Chem. 2003;278:49625–49635. PubMed

Lamour N., Riviere L., Coustou V., Coombs G.H., Barrett M.P., Bringaud F. Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose. J. Biol. Chem. 2005;280:11902–11910. PubMed

Krauth-Siegel R.L., Comini M.A. Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim. Biophys. Acta. 2008;1780:1236–1248. PubMed

Prochazkova M., Panicucci B., Zikova A. Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1. Sci. Rep. 2018;8:5135. PubMed PMC

Chowdhury S.R., Djordjevic J., Albensi B.C., Fernyhough P. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria. Biosci. Rep. 2015;36 PubMed PMC

Helfert S., Estevez A.M., Bakker B., Michels P., Clayton C. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei. Biochem. J. 2001;357:117–125. PubMed PMC

Lai D.H., Hashimi H., Lun Z.R., Ayala F.J., Lukes J. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc. Natl. Acad. Sci. U. S. A. 2008;105:1999–2004. PubMed PMC

Schnaufer A. Evolution of dyskinetoplastic trypanosomes: How, and how often? Trends Parasitol. 2010;26:557–558. PubMed PMC

Carnes J., Anupama A., Balmer O., Jackson A., Lewis M., Brown R., Cestari I., Desquesnes M., Gendrin C., Hertz-Fowler C., Imamura H., Ivens A., Koreny L., Lai D.H., MacLeod A. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. PLoS Negl. Trop. Dis. 2015;9 PubMed PMC

Brun R., Hecker H., Lun Z.R. Trypanosoma evansi and T. equiperdum: Distribution, biology, treatment and phylogenetic relationship (a review) Vet. Parasitol. 1998;79:95–107. PubMed

Motta M.C. Kinetoplast as a potential chemotherapeutic target of trypanosomatids. Curr. Pharm. Des. 2008;14:847–854. PubMed

Shapiro T.A., Englund P.T. Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proc. Natl. Acad. Sci. U. S. A. 1990;87:950–954. PubMed PMC

Giordani F., Morrison L.J., Rowan T.G., De Koning H.P., Barrett M.P. The animal trypanosomiases and their chemotherapy: A review. Parasitology. 2016;143:1862–1889. PubMed PMC

Eze A.A., Gould M.K., Munday J.C., Tagoe D.N., Stelmanis V., Schnaufer A., De Koning H.P. Reduced mitochondrial membrane potential is a late adaptation of trypanosoma brucei brucei to Isometamidium preceded by mutations in the gamma subunit of the F1Fo-ATPase. PLoS Negl. Trop. Dis. 2016;10 PubMed PMC

Gould M.K., Schnaufer A. Independence from kinetoplast DNA maintenance and expression is associated with multidrug resistance in Trypanosoma brucei in vitro. Antimicrob. Agents Chemother. 2014;58:2925–2928. PubMed PMC

Agbe A., Yielding K.L. Kinetoplasts play an important role in the drug responses of Trypanosoma brucei. J. Parasitol. 1995;81:968–973. PubMed

Young M.J., Copeland W.C. Human mitochondrial DNA replication machinery and disease. Curr. Opin. Genet. Dev. 2016;38:52–62. PubMed PMC

Ghozlane A., Bringaud F., Soueidan H., Dutour I., Jourdan F., Thebault P. Flux analysis of the Trypanosoma brucei glycolysis based on a multiobjective-criteria bioinformatic approach. Adv. Bioinformatics. 2012;2012:159423. PubMed PMC

Kiaira J.K., Njogu M.R. Oligomycin-sensitivity of hexose-sugar catabolism in the bloodstream form of Trypanosoma brucei brucei. Biotechnol. Appl. Biochem. 1994;20:347–356. PubMed

Miller P.G., Klein R.A. Effects of oligomycin on glucose utilization and calcium transport in African trypanosomes. J. Gen. Microbiol. 1980;116:391–396. PubMed

Heinrich R., Rapoport T.A. A linear steady-state treatment of enzymatic chains. General properties, control and effector strength. Eur. J. Biochem. 1974;42:89–95. PubMed

Bakker B.M., Walsh M.C., ter Kuile B.H., Mensonides F.I., Michels P.A., Opperdoes F.R., Westerhoff H.V. Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A. 1999;96:10098–10103. PubMed PMC

Schaaff I., Heinisch J., Zimmermann F.K. Overproduction of glycolytic enzymes in yeast. Yeast. 1989;5:285–290. PubMed

Ruyter G.J., Postma P.W., van Dam K. Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli. J. Bacteriol. 1991;173:6184–6191. PubMed PMC

Koebmann B.J., Westerhoff H.V., Snoep J.L., Nilsson D., Jensen P.R. The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 2002;184:3909–3916. PubMed PMC

Wickstead B., Ersfeld K., Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol. Biochem. Parasitol. 2002;125:211–216. PubMed

Poon S.K., Peacock L., Gibson W., Gull K., Kelly S. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor. Open Biol. 2012;2:110037. PubMed PMC

Engstler M., Boshart M. Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei. Genes Dev. 2004;18:2798–2811. PubMed PMC

Flaspohler J.A., Jensen B.C., Saveria T., Kifer C.T., Parsons M. A novel protein kinase localized to lipid droplets is required for droplet biogenesis in trypanosomes. Eukaryot. Cell. 2010;9:1702–1710. PubMed PMC

Wirtz E., Leal S., Ochatt C., Cross G.A. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 1999;99:89–101. PubMed

Stuart K.D. Evidence for the retention of kinetoplast DNA in an acriflavine-induced dyskinetoplastic strain of Trypanosoma brucei which replicates the altered central element of the kinetoplast. J. Cell Biol. 1971;49:189–195. PubMed PMC

Panigrahi A.K., Zikova A., Dalley R.A., Acestor N., Ogata Y., Anupama A., Myler P.J., Stuart K.D. Mitochondrial complexes in Trypanosoma brucei: A novel complex and a unique oxidoreductase complex. Mol. Cell. Proteomics. 2008;7:534–545. PubMed

Hannaert V., Albert M.A., Rigden D.J., da Silva Giotto M.T., Thiemann O., Garratt R.C., Van Roy J., Opperdoes F.R., Michels P.A. Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase. Eur. J. Biochem. 2003;270:3205–3213. PubMed

Wittig I., Karas M., Schagger H. High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol. Cell. Proteomics. 2007;6:1215–1225. PubMed

Acestor N., Zikova A., Dalley R.A., Anupama A., Panigrahi A.K., Stuart K.D. Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form. Mol. Cell. Proteomics. 2011;10 M110 006908. PubMed PMC

Acestor N., Panigrahi A.K., Ogata Y., Anupama A., Stuart K.D. Protein composition of Trypanosoma brucei mitochondrial membranes. Proteomics. 2009;9:5497–5508. PubMed PMC

Akerman K.E., Wikstrom M.K. Safranine as a probe of the mitochondrial membrane potential. FEBS Lett. 1976;68:191–197. PubMed

Chinopoulos C., Vajda S., Csanady L., Mandi M., Mathe K., Adam-Vizi V. A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT. Biophys. J. 2009;96:2490–2504. PubMed PMC

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation

An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases

Repurposing of MitoTam: Novel Anti-Cancer Drug Candidate Exhibits Potent Activity against Major Protozoan and Fungal Pathogens

Redesigned and reversed: architectural and functional oddities of the trypanosomal ATP synthase

Mitochondrial Contact Site and Cristae Organization System and F1FO-ATP Synthase Crosstalk Is a Fundamental Property of Mitochondrial Cristae