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.

Institute of Parasitology Biology Centre CAS České Budějovice Czech Republic

Institute of Parasitology Biology Centre CAS České Budějovice Czech Republic; Faculty of Science University of South Bohemia České Budějovice Czech Republic

Zobrazit více v PubMed

Jamonneau V, Ilboudo H, Kabore J, Kaba D, Koffi M, et al. (2012) Untreated human infections by Trypanosoma brucei gambiense are not 100% fatal. PLoS Negl Trop Dis 6: e1691 10.1371/journal.pntd.0001691 PubMed DOI PMC

Stuart K, Brun R, Croft S, Fairlamb A, Gurtler RE, et al. (2008) Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest 118: 1301–1310. 10.1172/JCI33945 PubMed DOI PMC

Steverding D (2008) The history of African trypanosomiasis. Parasit Vectors 1: 3 10.1186/1756-3305-1-3 PubMed DOI PMC

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

Besteiro S, Barrett MP, Riviere L, Bringaud F (2005) Energy generation in insect stages of Trypanosoma brucei: metabolism in flux. Trends Parasitol 21: 185–191. PubMed

Hannaert V, Bringaud F, Opperdoes FR, Michels PA (2003) Evolution of energy metabolism and its compartmentation in Kinetoplastida. Kinetoplastid Biol Dis 2: 11 PubMed PMC

Brown SV, Hosking P, Li J, Williams N (2006) ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei. Eukaryot Cell 5: 45–53. PubMed PMC

Guler JL, Kriegova E, Smith TK, Lukes J, Englund PT (2008) Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei. Mol Microbiol 67: 1125–1142. 10.1111/j.1365-2958.2008.06112.x PubMed DOI PMC

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

Huang G, Vercesi AE, Docampo R (2013) Essential regulation of cell bioenergetics in Trypanosoma brucei by the mitochondrial calcium uniporter. Nat Commun 4: 2865 10.1038/ncomms3865 PubMed DOI PMC

Vercesi AE, Docampo R, Moreno SN (1992) Energization-dependent Ca2+ accumulation in Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria. Mol Biochem Parasitol 56: 251–257. PubMed

Kovarova J, Horakova E, Changmai P, Vancova M, Lukes J (2014) Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei. Eukaryot Cell 13: 353–362. 10.1128/EC.00235-13 PubMed DOI PMC

Mazet M, Morand P, Biran M, Bouyssou G, Courtois P, et al. (2013) Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability. PLoS Negl Trop Dis 7: e2587 10.1371/journal.pntd.0002587 PubMed DOI PMC

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

Buchet K, Godinot C (1998) Functional F1-ATPase essential in maintaining growth and membrane potential of human mitochondrial DNA-depleted rho degrees cells. J Biol Chem 273: 22983–22989. PubMed

Dean S, Gould MK, Dewar CE, Schnaufer AC (2013) Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A 110: 14741–14746. 10.1073/pnas.1305404110 PubMed DOI PMC

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

Stuart KD (1971) 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 49: 189–195. PubMed PMC

Lai DH, Hashimi H, Lun ZR, Ayala FJ, Lukes J (2008) 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 105: 1999–2004. 10.1073/pnas.0711799105 PubMed DOI PMC

Walker JE (2013) The ATP synthase: the understood, the uncertain and the unknown. Biochem Soc Trans 41: 1–16. 10.1042/BST20110773 PubMed DOI

Devenish RJ, Prescott M, Rodgers AJ (2008) The structure and function of mitochondrial F1F0-ATP synthases. Int Rev Cell Mol Biol 267: 1–58. 10.1016/S1937-6448(08)00601-1 PubMed DOI

Walker JE, Dickson VK (2006) The peripheral stalk of the mitochondrial ATP synthase. Biochim Biophys Acta 1757: 286–296. PubMed

Dickson VK, Silvester JA, Fearnley IM, Leslie AG, Walker JE (2006) On the structure of the stator of the mitochondrial ATP synthase. EMBO J 25: 2911–2918. PubMed PMC

Vaidya AB, Mather MW (2009) Mitochondrial evolution and functions in malaria parasites. Annu Rev Microbiol 63: 249–267. 10.1146/annurev.micro.091208.073424 PubMed DOI

Lapaille M, Escobar-Ramirez A, Degand H, Baurain D, Rodriguez-Salinas E, et al. (2010) Atypical subunit composition of the chlorophycean mitochondrial F1FO-ATP synthase and role of Asa7 protein in stability and oligomycin resistance of the enzyme. Mol Biol Evol 27: 1630–1644. 10.1093/molbev/msq049 PubMed DOI

Zikova A, Schnaufer A, Dalley RA, Panigrahi AK, Stuart KD (2009) The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog 5: e1000436 10.1371/journal.ppat.1000436 PubMed DOI PMC

Mather MW, Henry KW, Vaidya AB (2007) Mitochondrial drug targets in apicomplexan parasites. Curr Drug Targets 8: 49–60. PubMed

Balabaskaran Nina P, Dudkina NV, Kane LA, van Eyk JE, Boekema EJ, et al. (2010) Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila. PLoS Biol 8: e1000418 10.1371/journal.pbio.1000418 PubMed DOI PMC

Nishi A, Scherbaum OH (1962) Oxidative phosphorylation in synchronized cultures of Tetrahymena pyriformis. Biochim Biophys Acta 65: 419–424. PubMed

Uyemura SA, Luo S, Vieira M, Moreno SN, Docampo R (2004) Oxidative phosphorylation and rotenone-insensitive malate- and NADH-quinone oxidoreductases in Plasmodium yoelii yoelii mitochondria in situ. J Biol Chem 279: 385–393. PubMed

Vazquez-Acevedo M, Cardol P, Cano-Estrada A, Lapaille M, Remacle C, et al. (2006) The mitochondrial ATP synthase of chlorophycean algae contains eight subunits of unknown origin involved in the formation of an atypical stator-stalk and in the dimerization of the complex. J Bioenerg Biomembr 38: 271–282. PubMed

Hashimi H, Benkovicova V, Cermakova P, Lai DH, Horvath A, et al. (2010) The assembly of F(1)F(O)-ATP synthase is disrupted upon interference of RNA editing in Trypanosoma brucei. Int J Parasitol 40: 45–54. 10.1016/j.ijpara.2009.07.005 PubMed DOI

Chi TB, Brown BS, Williams N (1998) Subunit 9 of the mitochondrial ATP synthase of Trypanosoma brucei is nuclearly encoded and developmentally regulated. Mol Biochem Parasitol 92: 29–38. PubMed

Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241: 779–786. PubMed

Biegert A, Mayer C, Remmert M, Soding J, Lupas AN (2006) The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res 34: W335–339. PubMed PMC

Norais N, Prome D, Velours J (1991) ATP synthase of yeast mitochondria. Characterization of subunit d and sequence analysis of the structural gene ATP7. J Biol Chem 266: 16541–16549. PubMed

Walker JE, Runswick MJ, Poulter L (1987) ATP synthase from bovine mitochondria. The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs. J Mol Biol 197: 89–100. PubMed

Nugent T, Jones DT (2009) Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 10: 159 10.1186/1471-2105-10-159 PubMed DOI PMC

Vertommen D, Van Roy J, Szikora JP, Rider MH, Michels PA, et al. (2008) Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol 158: 189–201. 10.1016/j.molbiopara.2007.12.008 PubMed DOI

Ko YH, Delannoy M, Hullihen J, Chiu W, Pedersen PL (2003) Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP. J Biol Chem 278: 12305–12309. PubMed

Meyer B, Wittig I, Trifilieff E, Karas M, Schagger H (2007) Identification of two proteins associated with mammalian ATP synthase. Mol Cell Proteomics 6: 1690–1699. PubMed

Chen Y, Hung CH, Burderer T, Lee GS (2003) Development of RNA interference revertants in Trypanosoma brucei cell lines generated with a double stranded RNA expression construct driven by two opposing promoters. Mol Biochem Parasitol 126: 275–279. PubMed

Bowler MW, Montgomery MG, Leslie AG, Walker JE (2006) How azide inhibits ATP hydrolysis by the F-ATPases. Proc Natl Acad Sci U S A 103: 8646–8649. PubMed PMC

Appleby RD, Porteous WK, Hughes G, James AM, Shannon D, et al. (1999) Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA. Eur J Biochem 262: 108–116. PubMed

Garcia JJ, Ogilvie I, Robinson BH, Capaldi RA (2000) Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation—Comparison with the enzyme in Rho(0) cells completely lacking mtDNA. Journal of Biological Chemistry 275: 11075–11081. PubMed

Wittig I, Meyer B, Heide H, Steger M, Bleier L, et al. (2010) Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L. Biochim Biophys Acta 1797: 1004–1011. 10.1016/j.bbabio.2010.02.021 PubMed DOI

Stock D, Leslie AG, Walker JE (1999) Molecular architecture of the rotary motor in ATP synthase. Science 286: 1700–1705. PubMed

Strauss M, Hofhaus G, Schroder RR, Kuhlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27: 1154–1160. 10.1038/emboj.2008.35 PubMed DOI PMC

Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H (1998) Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J 17: 7170–7178. PubMed PMC

Burger G, Lang BF, Braun HP, Marx S (2003) The enigmatic mitochondrial ORF ymf39 codes for ATP synthase chain b. Nucleic Acids Res 31: 2353–2360. PubMed PMC

Heazlewood JL, Whelan J, Millar AH (2003) The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase. FEBS Lett 540: 201–205. PubMed

Miranda-Astudillo H, Cano-Estrada A, Vazquez-Acevedo M, Colina-Tenorio L, Downie-Velasco A, et al. (2014) 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 1837: 1–13. 10.1016/j.bbabio.2013.08.001 PubMed DOI

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

Chevallet M, Lescuyer P, Diemer H, van Dorsselaer A, Leize-Wagner E, et al. (2006) Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view. Electrophoresis 27: 1574–1583. PubMed PMC

Orian JM, Hadikusumo RG, Marzuki S, Linnane AW (1984) Biogenesis of mitochondria: defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated. J Bioenerg Biomembr 16: 561–581. PubMed

Paul MF, Velours J, Arselin de Chateaubodeau G, Aigle M, Guerin B (1989) The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex. Eur J Biochem 185: 163–171. PubMed

Rak M, Gokova S, Tzagoloff A (2011) Modular assembly of yeast mitochondrial ATP synthase. EMBO J 30: 920–930. 10.1038/emboj.2010.364 PubMed DOI PMC

Chen C, Ko Y, Delannoy M, Ludtke SJ, Chiu W, et al. (2004) Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP. J Biol Chem 279: 31761–31768. PubMed

Alibu VP, Storm L, Haile S, Clayton C, Horn D (2005) A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei. Mol Biochem Parasitol 139: 75–82. PubMed

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

Surve S, Heestand M, Panicucci B, Schnaufer A, Parsons M (2012) Enigmatic presence of mitochondrial complex I in Trypanosoma brucei bloodstream forms. Eukaryot Cell 11: 183–193. 10.1128/EC.05282-11 PubMed DOI PMC

Borst P, Fase-Fowler F, Gibson WC (1987) Kinetoplast DNA of Trypanosoma evansi. Mol Biochem Parasitol 23: 31–38. PubMed

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

Oeffinger M, Wei KE, Rogers R, DeGrasse JA, Chait BT, et al. (2007) Comprehensive analysis of diverse ribonucleoprotein complexes. Nat Methods 4: 951–956. PubMed

Panigrahi AK, Ogata Y, Zikova A, Anupama A, Dalley RA, et al. (2009) A comprehensive analysis of Trypanosoma brucei mitochondrial proteome. Proteomics 9: 434–450. 10.1002/pmic.200800477 PubMed DOI PMC

Panigrahi AK, Zikova A, Dalley RA, Acestor N, Ogata Y, et al. (2008) Mitochondrial complexes in Trypanosoma brucei: a novel complex and a unique oxidoreductase complex. Mol Cell Proteomics 7: 534–545. PubMed

Singha UK, Peprah E, Williams S, Walker R, Saha L, et al. (2008) Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei. Mol Biochem Parasitol 159: 30–43. 10.1016/j.molbiopara.2008.01.003 PubMed DOI PMC

Vondruskova E, van den Burg J, Zikova A, Ernst NL, Stuart K, et al. (2005) RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei. J Biol Chem 280: 2429–2438. PubMed

Hannaert V, Albert MA, Rigden DJ, da Silva Giotto MT, Thiemann O, et al. (2003) Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase. Eur J Biochem 270: 3205–3213. PubMed

Law RH, Manon S, Devenish RJ, Nagley P (1995) ATP synthase from Saccharomyces cerevisiae. Methods Enzymol 260: 133–163. PubMed

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

Acestor N, Zikova A, Dalley RA, Anupama A, Panigrahi AP, et al. (2011) Trypanosoma brucei Mitochondrial Respiratome: Composition and organization in procyclic form. Molecular Cell Proteomics resubmitted. PubMed PMC

Acestor N, Panigrahi AK, Ogata Y, Anupama A, Stuart KD (2009) Protein composition of Trypanosoma brucei mitochondrial membranes. Proteomics 9: 5497–5508. 10.1002/pmic.200900354 PubMed DOI PMC

Mayhew TM, Lucocq JM (2008) Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying). Histochem Cell Biol 129: 367–378. 10.1007/s00418-007-0375-6 PubMed DOI

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

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

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia

Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei

Isolation of plastids and mitochondria from Chromera velia

Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1

ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

The ADP/ATP carrier and its relationship to oxidative phosphorylation in ancestral protist trypanosoma brucei