Title: Knock-downs of Iron-Sulfur Cluster Assembly Proteins IscS and IscU Down-regulate the Active Mitochondrion of Procyclic Trypanosoma brucei
Abstract: Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle. Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle. Trypanosoma brucei is one of the most important protozoan pathogens, responsible for human sleeping sickness and nagana in livestock. Moreover, because the genome of T. brucei has recently been completely sequenced (1Berriman M. Ghedin E. Hertz-Fowler C. Blandin G. Renauld H. Bartholomeu D.C. Lennard N.J. Caler E. Hamlin N.E. Haas B. Bohme W. Hannick L. Aslett M.A. Shallom J. Marcello L. Hou L.H. Wickstead B. Alsmark U.C.M. Arrowsmith C. Atkin R.J. Barron A.J. Bringaud F. Brooks K. Carrington M. Cherevach I. Chillingworth T.J. Churcher C. Clark L.N. Corton C.H. Cronin A. Davies R.M. Doggett J. Djikeng A. Feldblyum T. Field M.C. Fraser A. Goodhead I. Hance Z. Harper D. Harris B.R. Hauser H. Hostetter J. Ivens A. Jagels K. Johnson D. Johnson J. Jones K. Kerhornou A.X. Koo H. Larke N. Landfear S. Larkin C. Leech V. Line A. Lord A. MacLeod A. Mooney P.J. Moule S. Martin D.M.A. Morgan G.W. Mungall K. Norbertczak H. Ormond D. Pai G. Peacock C.S. Peterson J. Quail M.A. Rabbinowitsch E. Rajandream M.A. Reitter C. Salzberg S.L. Sanders M. Schobel S. Sharp S. Simmonds M. Simpson A.J. Talton L. Turner C.M.R. Tait A. Tivey A.R. Van Aken S. Walker D. Wanless D. Wang S.L. White B. White O. Whitehead S. Woodward J. Wortman J. Adams M.D. Embley T.M. Gull K. Ullu E. Barry J.D. Fairlamb A.H. Opperdoes F. Barret B.G. Donelson J.E. Hall N. Fraser C.M. Melville S.E. El-Sayed N.M. Science. 2005; 309: 416-422Crossref PubMed Scopus (1351) Google Scholar), and the cells are amenable to approaches of involving reverse genetics (2Motyka S.A. Englund P.T. Curr. Opin. Microbiol. 2004; 7: 362-368Crossref PubMed Scopus (29) Google Scholar), T. brucei has become a new model organism, which is evolutionarily highly divergent from classical models such as Saccharomyces cerevisiae. While yeast and other fungi are more related to metazoa including humans (eukaryotic group Opisthokonta), trypanosomatids belong to the distant eukaryotic group called Excavata (3Simpson A.G. Roger A.J. Curr. Biol. 2004; 14: R693-R696Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 4Cavalier-Smith T. Proc. Biol. Sci. 2004; 271: 1251-1262Crossref PubMed Scopus (252) Google Scholar). This group is formed exclusively of unicellular eukaryotes, many of them with highly modified mitochondria (5Lukeš J. Hashimi H. Ziková A. Curr. Genet. 2005; 48: 277-299Crossref PubMed Scopus (174) Google Scholar). The mitochondrion of T. brucei is of particular interest, because it undergoes dramatic metabolic and structural changes during the cell cycle between the blood of the mammalian host and the digestive tract of the tsetse fly. An excess of glucose in the mammalian host permits the bloodstream stage to employ glycolysis for energy generation, a significant part of which is localized to specialized peroxisomes called glycosomes (6Michels P.A. Moyersoen J. Krazy H. Galland N. Herman M. Hannaert V. Mol. Membr. Biol. 2005; 22: 133-145Crossref PubMed Scopus (57) Google Scholar), producing pyruvate as a major end product. Consequently, the mitochondrion lacks the cytochrome-dependent electron transport chain and the activities of the citric acid cycle enzymes and is thus impaired in its ability to produce ATP by oxidative phosphorylation (7Clayton C.E. Michels P. Parasitol. Today. 1996; 12: 465-471Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Through its functions as a sink for reducing equivalents from glycolysis, the mitochondrion still plays an indispensable role in the metabolism of blood stages (8Chaudhuri M. Ajayi W. Hill G.C. Mol. Biochem. Parasitol. 1998; 95: 53-68Crossref PubMed Scopus (105) Google Scholar). In the insect vector, trypanosomes encounter a nutrient-poor environment, where they can only survive through an overall switch in their energy metabolism, primarily by activating the mitochondrion. In the active mitochondria of procyclics, pyruvate is degraded with the benefit of additional ATP synthesis, mainly to acetate and succinate (9van Hellemond J.J. Opperdoes F.R. Tielens A.G.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (117) Google Scholar). However, most of the excreted succinate is produced in glycosomes (10Besteiro S. Barrett M.P. Riviere L. Bringaud F. Trends Parasitol. 2005; 21: 185-191Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This transformation is accompanied by the de novo synthesis of iron-sulfur (FeS) 2The abbreviations used are: FeS, iron-sulfur; RNAi, RNA interference; HPLC, high pressure liquid chromatography; MRP2, mitochondrial RNA-binding protein 2. clusters and maturation of a number of FeS proteins including electron-transporting subunits of the mitochondrial respiratory chain (complexes I, II, and III) and aconitase, an enzyme with dual localization in the mitochondrion and cytosol (9van Hellemond J.J. Opperdoes F.R. Tielens A.G.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (117) Google Scholar). The biogenesis of FeS clusters is a recently discovered process essential for both prokaryotic and eukaryotic cells (11Lill R. Mühlenhoff U. Trends Biochem. Sci. 2005; 30: 133-141Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 12Johnson D. Dean D. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2004; 74: 247-281Crossref Scopus (1126) Google Scholar). In eukaryotes, the de novo formation of FeS clusters was first discovered in mitochondria (13Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) and later in other organelles of endosymbiotic origin including hydrogenosomes, mitosomes, and plastids (14Sutak R. Dolezal P. Fiumera H.L. Hrdy I. Dancis A. Delgadillo-Correa M. Johnson P.J. Müller M. Tachezy J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10368-10373Crossref PubMed Scopus (105) Google Scholar, 15Tovar J. Léon-Avila G. Sánchez L.B. Sutak R. Tachezy J. van der Giezen M. Hernández M. Müller M. Lucocq J.M. Nature. 2003; 426: 172-176Crossref PubMed Scopus (427) Google Scholar, 16LaGier M.J. Tachezy J. Stejskal F. Kutisová K. Keithly J.S. Microbiology (UK). 2003; 149: 3519-3530Crossref PubMed Scopus (65) Google Scholar, 17Katinka M.D. Duprat S. Cornillot E. Metenier G. Thomarat F. Prensier G. Barbe V. Peyretaillade E. Brottier P. Wincker P. Delbac F. El Alaoui H. Peyret P. Saurin W. Gouy M. Weissenbach J. Vivares C.P. Nature. 2001; 414: 450-453Crossref PubMed Scopus (848) Google Scholar, 18Balk J. Lobreaux S. Trends Plant Sci. 2005; 10: 324-331Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The machinery responsible for mitochondrial FeS cluster assembly consists of at least 10 different proteins, the key components being pyridoxal 5-phosphate-dependent cysteine desulfurase IscS, which generates sulfur (S0) from cysteine, and the metallochaperone IscU, which provides the molecular scaffold for the formation of a transient FeS cluster (11Lill R. Mühlenhoff U. Trends Biochem. Sci. 2005; 30: 133-141Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). The transient FeS cluster is transferred from IscU to apoproteins during FeS protein maturation (19Kispal G. Sipos K. Lange H. Fekete Z. Bedekovics T. Janaky T. Bassler J. Aguilar Netz D.J. Balk J. Rotte C. Lill R. EMBO J. 2005; 24: 589-598Crossref PubMed Scopus (204) Google Scholar). In S. cerevisiae, mitochondria were shown to play an essential role not only in the biosynthesis of mitochondrial FeS proteins but also in the maturation of FeS proteins localized in the cytosol and nucleus. For example, this essential requirement for the FeS cluster assembly machinery was demonstrated for Rli1, a cytosolic FeS protein that is indispensable to ribosomal functionality (19Kispal G. Sipos K. Lange H. Fekete Z. Bedekovics T. Janaky T. Bassler J. Aguilar Netz D.J. Balk J. Rotte C. Lill R. EMBO J. 2005; 24: 589-598Crossref PubMed Scopus (204) Google Scholar, 20Yarunin A. Panse V.G. Petfalski E. Dez C. Tollervey D. Hurt E.C. EMBO J. 2005; 24: 580-588Crossref PubMed Scopus (141) Google Scholar). A different model was proposed for the biogenesis of FeS clusters in human cells. In addition to mitochondria, several components of the mitochondrial FeS cluster assembly machinery were detected in the cytosol and nucleus (21Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 22Tong W.H. Jameson G.N.L. Huynh B.H. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9762-9767Crossref PubMed Scopus (186) Google Scholar, 23Tong W.H. Rouault T. EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (170) Google Scholar), and the ability of their cytosolic forms to promote de novo FeS cluster formation was demonstrated (24Li K. Tong W.H. Hughes R.M. Rouault T.A. J. Biol. Chem. 2006; 281: 12344-12351Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Because no information is available on FeS cluster assembly in trypanosomes, we investigated the function of mitochondria of T. brucei procyclics in this process. Initially, we identified the main components of its FeS cluster assembly machinery and constructed cell lines in which the expression of the IscS and IscU genes was down-regulated by means of RNA interference (RNAi). Analyses of the resulting phenotypes provide the first insight into FeS cluster biogenesis in the mitochondria of parasitic protists and support the hypothesis that the mitochondrion plays a fundamental and evolutionary conserved role in cellular FeS cluster assembly throughout the eukaryotes. Construction of Vectors, Transfection, Cloning, RNAi Induction, and Growth—A 424-bp fragment of the TbiscS2 gene (supplemental material) was amplified by PCR from the T. brucei 427 genomic DNA using oligonucleotides IscS-F1 (5′-CACCATATGGTAGAGATGAAGCGTGATT) and IscS-R1 (5′-CACAAGCTTTTTCCTTCCATCAGCAAGT) (added NdeI and HindIII restriction sites are underlined), was cloned into pCR2.1 TOPO® (Invitrogen) and subcloned in pZJM (25Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Similarly, a large portion of the TbiscU gene (nucleotides 44–528; see supplemental material) was amplified with the primers IscU-F1 (5′-CACCTCGAGCAGCCTCACTTCGGTCACT) and IscU-R1 (5′-TGCACGGATCCCCAACAGCCTCGGACTTAG) (added XhoI and BamHI restriction sites are underlined) and cloned into p2T7-177 (26Wickstead B. Ersfeld K. Gull K. Mol. Biochem. Parasitol. 2002; 125: 211-216Crossref PubMed Scopus (209) Google Scholar). The procyclic T. brucei strain 29-13, transgenic for T7 RNA polymerase and the tetracycline repressor, was grown in SDM-79 medium in the presence of hygromycin and G418 (27Wirtz E. Clayton C. Science. 1995; 268: 1179-1183Crossref PubMed Scopus (210) Google Scholar). Transfection, selection and cloning were performed as described elsewhere (28Vondrušková E. van den B.J. Ziková A. Ernst N.L. Stuart K. Benne R. Lukeš J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Synthesis of double-stranded RNA was induced by the addition of 2 μg/ml tetracycline. Growth curves of parental cells and clonal cell lines were obtained using the Z2 Cell Counter (Beckman Coulter Inc., Fullerton, CA) over a period of 8 days after the induction of RNAi. Following confirmation of successful RNAi (see below), one clone for each TbiscS2 and TbiscU was used for further experiments. Reverse Transcription-PCR Analysis and Northern Blotting—DNA enriched for TbiscS2 mRNA was synthesized by reverse transcription of poly(A)+ RNA with IscS-R2 (5′-CGAATTAAATCTGCCACTTGCGTC) and used as a template for amplification of the 5′ end region of this mRNA with IscS-R3 (5′-GGCAATATTGTTGGACTCCGTTGC) and the upstream spliced leader-specific primer SLTb1 (5′-AACTAACGCTATTATTAGAACAGT). Three sequenced clones were identical. Total RNA was isolated from 5 × 107 exponentially growing noninduced and RNAi-induced cells by extraction with Tri Reagent (Sigma-Aldrich). TbiscS2 or TbiscU gene probes were labeled by random priming with [α-32P]dATP (MP Biomedicals, Irvine, CA). Hybridization was carried out using standard procedure (28Vondrušková E. van den B.J. Ziková A. Ernst N.L. Stuart K. Benne R. Lukeš J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The radioactive signal was detected using storage phosphorimaging. Preparation of Antibodies and Immunoblot Analysis—The coding region of TbiscS2 and TbiscU was subcloned into a pQE30 vector (Qiagen) incorporating an N-terminal His6 tag. Soluble protein was obtained from induced bacterial cells under denaturating conditions using affinity chromatography as described in the manufacturer's instructions. Polyclonal antibodies against recombinant TbiscS were prepared by immunizing rabbits (28Vondrušková E. van den B.J. Ziková A. Ernst N.L. Stuart K. Benne R. Lukeš J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 29Foldynová-Trantírková S. Paris Z. Sturm N.R. Campbell D.A. Lukeš J. Int. J. Parasitol. 2005; 35: 359-366Crossref PubMed Scopus (18) Google Scholar). The polyclonal rabbit antibodies raised against recombinant TbiscU were commercially prepared (Seva-Imuno, Prague, Czech Republic). Rabbit γ-globulins were purified from sera by affinity chromatography in a Prosep®, a high capacity column (Bioprocessing Ltd., Consett, UK). Total cell lysates and subcellular fractions of noninduced and induced trypanosomes were separated on SDS-PAGE gels, blotted, and probed with polyclonal antibodies against TbisS2, TbiscU, aconitase (Ref. 40Panigrahi A.K. Schnaufer A. Carmean N. Igo Jr., R.P. Gygi S.P. Ernst N.L. Palazzo S.S. Weston D.S. Aebersold R. Salavati R. Stuart K.D. Mol. Cell Biol. 2001; 21: 6833-6840Crossref PubMed Scopus (98) Google Scholar; provided by M. Boshart), Hsp60 (provided by P. A. M. Michels), mitochondrial RNA-binding protein 2 (MRP2) (28Vondrušková E. van den B.J. Ziková A. Ernst N.L. Stuart K. Benne R. Lukeš J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and the La protein (29Foldynová-Trantírková S. Paris Z. Sturm N.R. Campbell D.A. Lukeš J. Int. J. Parasitol. 2005; 35: 359-366Crossref PubMed Scopus (18) Google Scholar). Secondary anti-rabbit or anti-mouse antibodies coupled to alkaline phosphatase (MP Biomedicals) were visualized with 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich). Digitonin Fractionation—Digitonin fractionation of procyclics was performed as described elsewhere (30Moyersoen J. Choe J. Kumar A. Voncken F.G. Hol W.G. Michels P.A. Eur. J. Biochem. 2003; 270: 2059-2067Crossref PubMed Scopus (43) Google Scholar) with the following modifications. The cells were washed twice and resuspended in ice-cold SHE buffer (25 mm Hepes, pH 7.4, 250 mm sucrose, 1 mm EDTA) at ∼5 × 109 cells/ml. Aliquots containing 1 mg of protein were resuspended in 350 μl of Hanks' balanced salt solution buffer (Invitrogen) and incubated for 4 min at 25 °C with increasing concentrations of digitonin (Merck) dissolved in dimethylformamide and centrifuged at 14,000 × g for 2 min. Whole cell lysates were prepared by incubating the same aliquots in 350 μl of Hanks' balanced salt solution buffer containing 0.1% Triton X-100 for 5 min on ice and centrifuged as above. The resulting supernatants were immediately assayed for the presence of the cytosolic (pyruvate kinase) and mitochondrial (threonine dehydrogenase) marker enzymes. To obtain cytosolic and mitochondria-rich fractions, the concentration of digitonin was used that released the maximum pyruvate kinase activity and no threonine dehydrogenase into the supernatant. This supernatant was considered to represent the cytosolic fraction. Pelleted intact mitochondria were washed once and then resuspended in 350 μl of Hanks' balanced salt solution buffer and incubated with 0.1% Triton X-100 for 5 min on ice. After centrifugation, the supernatant containing mitochondrial matrix proteins was collected (the mitochondrial fraction). Enzyme Assays and Determination of Metabolic End Products—The activities of pyruvate kinase and threonine dehydrogenase were monitored spectrophotometrically at 340 nm as a rate of NADH oxidation or NAD reduction, respectively (31Callens M. Opperdoes F.R. Mol. Biochem. Parasitol. 1992; 50: 235-243Crossref PubMed Scopus (20) Google Scholar, 32Heise N. Opperdoes F.R. Mol. Biochem. Parasitol. 1999; 99: 21-32Crossref PubMed Scopus (65) Google Scholar). The activity of aconitase was measured as the production of cis-aconitate, monitored at 240 nm (33Saas J. Ziegelbauer K. von H.A. Fast B. Boshart M. J. Biol. Chem. 2000; 275: 2745-2755Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Ubiquinol-cytochrome-c reductase activity (complex III) was measured in QCR buffer (40 mm sodium phosphate buffer, pH 7.4, 0.5 mm EDTA, 20 mm sodium malonate, 50 μm horse heart cytochrome c (Sigma-Aldrich), 0.005% dodecylmaltoside) as the rate of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol oxidation monitored at 550 nm (prior to use, decylubiquinone (Sigma-Aldrich) was reduced as described elsewhere (34Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar)). KCN was added to a final concentration of 200 μm as an inhibitor of interfering oxidase activity. To determine the metabolic end products, ∼8 × 107 cells were washed once with phosphate-buffered saline, resuspended in 200 μl of incubation buffer (phosphate-buffered saline buffer supplemented with 11 mm glucose and 24 mm NaHCO3, pH 7.3), and incubated for 2 h at 27 °C. After centrifugation for 10 min at 1,400 × g, the supernatant was analyzed by HPLC in a PL Hi-Plex H column as described elsewhere (35Vanacova S. Rasoloson D. Razga J. Hrdý I. Kulda J. Tachezy J. Microbiology (UK). 2001; 147: 53-62Crossref PubMed Scopus (53) Google Scholar). ATP Production in Isolated Mitochondria—Intact mitochondria were prepared from cell aliquots containing 1 mg of protein by digitonin fractionation as described above. The mitochondrial pellet was resuspended in 750 μl of the buffer for in organello ATP production assay (20 mm Tris-HCl, pH 7.4, 15 mm KH2PO4, 0.6 m sorbitol, 10 mm MgSO4, 2.5 mg/ml bovine serum albumin), and 75-μl aliquots were used for each measurement. ATP production was induced by the addition of substrates as published elsewhere (36Bochud-Allemann N. Schneider A. J. Biol. Chem. 2002; 277: 32849-32854Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The concentration of ATP was determined by a luminometer using the ATP Bioluminiscence assay kit CLS II (Roche Applied Science). Measurement of Mitochondrial Membrane Potential—Tetramethylrodamine ethyl ester (Molecular Probes, Eugene, OR) uptake was used as a measure of the mitochondrial membrane potential (37Horváth A. Horáková E. Dunajciková P. Verner Z. Pravdová E. Šlapetová I. Cuninková L. Lukeš J. Mol. Microbiol. 2005; 58: 116-130Crossref PubMed Scopus (67) Google Scholar). EPR Analysis of FeS Clusters—EPR spectra were recorded on a Bruker Elexsys E580 spectrometer operating in X-band continuous-wave mode, with an Oxford Instruments ESR9000 liquid helium flow cryostat. The measurement conditions were: temperature, 12 K; microwave power, 20 milliwatts; frequency, 9.38 GHz; and modulation amplitude, 1 millitesla. Genes Coding for TbiscS and TbiscU—The T. brucei genome project data base (www.sanger.ac.uk/) was searched for homologs of IscS and IscU using S. cerevisiae Nfs1p and Isu1p as queries. A BLAST search for IscS identified two homologs in the T. brucei genome, here named TbiscS1 and TbiscS2, with calculated molecular weights of 48,991 and 48,150, respectively. Alignment of their deduced amino acid sequences together with Nfs1p revealed significant differences between the two trypanosome genes (supplemental material). Similar to Nfs1p, TbiscS2 possesses conserved residues corresponding to the active site loop that are responsible for the targeted delivery of sulfur to IscU (38Lauhon C.T. Skovran E. Urbina H.D. Downs D.M. Vickery L.E. J. Biol. Chem. 2004; 279: 19551-19558Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), as well as the conserved C terminus essential for specific interaction with IscU (39Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In contrast, TbiscS1 lacks these regions. Phylogenetic analysis of the two T. brucei proteins indicated that TbiscS2 is closely related to other mitochondrial IscS homologs, whereas TbiscS1 clusters together with putative selenocysteine lyases (data not shown). Thus, TbiscS2 was selected for further studies. To prove that TbiscS2 is transcribed, the spliced leader RNA and primers from the 5′ end of the gene were used for reverse transcription-PCR. In three clones with identical sequences, the splice acceptor site was mapped to 25 bp upstream of the initial methionine, resulting in a very short 5′-untranslated region (data not shown). A BLAST search for Isu1p identified a single IscU homolog (TbiscU) in the T. brucei genome data base with a calculated molecular weight of 19,435. Alignment of the deduced TbiscU amino acid sequence with yeast Isu1p revealed that all three conserved cysteine residues required for the assembly of the transient FeS cluster are present in TbiscU (supplemental material). TbiscS2 and TbiscU Are Localized in the Mitochondrion—PsortII analysis (psort.nibb.ac.jp/) of TbiscS2 and TbiscU predicted N-terminal leader sequences for targeting the proteins into mitochondria as well as putative cleavage sites with the characteristic arginine at the –2 position that is recognized by mitochondrial processing peptidase (supplemental material). To verify the predicted cellular localization of these proteins in procyclic T. brucei, specific polyclonal antibodies were raised against recombinant TbiscS2 and TbiscU and used for immunoblot analysis. In subcellular fractions obtained by differential permeabilization of the cell membranes by increasing concentrations of digitonin (30Moyersoen J. Choe J. Kumar A. Voncken F.G. Hol W.G. Michels P.A. Eur. J. Biochem. 2003; 270: 2059-2067Crossref PubMed Scopus (43) Google Scholar), both TbiscS2 and TbiscU were detected in the mitochondrial fractions, whereas no signals were observed in the cytosol (Fig. 1). Antibodies against the MRP2 (28Vondrušková E. van den B.J. Ziková A. Ernst N.L. Stuart K. Benne R. Lukeš J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and the cytosolic La protein (29Foldynová-Trantírková S. Paris Z. Sturm N.R. Campbell D.A. Lukeš J. Int. J. Parasitol. 2005; 35: 359-366Crossref PubMed Scopus (18) Google Scholar) were used as controls in cell fractionation experiments. The low cellular abundance of TbiscS2 is probably responsible for difficulty in identifying the protein in the whole cell lysate (40Panigrahi A.K. Schnaufer A. Carmean N. Igo Jr., R.P. Gygi S.P. Ernst N.L. Palazzo S.S. Weston D.S. Aebersold R. Salavati R. Stuart K.D. Mol. Cell Biol. 2001; 21: 6833-6840Crossref PubMed Scopus (98) Google Scholar). Inhibition of TbiscS2 and TbiscU Gene Expression by RNAi—A 424-bp-long fragment of TbiscS2 and a 484-bp-long fragment of TbiscU (supplemental material) were cloned into the pZJM (25Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar) and p2T7-177 (26Wickstead B. Ersfeld K. Gull K. Mol. Biochem. Parasitol. 2002; 125: 211-216Crossref PubMed Scopus (209) Google Scholar) RNAi vectors, respectively. Transfection of both constructs into the procyclics resulted in stable integration into the trypanosome genome as confirmed by Southern hybridization of the digested total DNA (data not shown). Phleomycin-resistant transfectants were cloned by limiting dilution, and RNAi was induced by the addition of tetracycline into the SDM-79 medium. Total RNA was isolated from the noninduced and induced clonal cell lines at different time points and analyzed on Northern blots (Fig. 2). The analysis showed that in the TbiscS2 and TbiscU knock-downs, the corresponding mRNA was almost completely eliminated after 2–3 days of induction, followed by growth inhibition of both mutants. Although the growth of the parental strain 29-13 and noninduced RNAi cells were almost identical, the growth of TbiscS2 knock-down cells gradually slowed down, and the growth of the TbiscU-induced cells was completely inhibited (Fig. 3). The observed phenotypes indicate that both components of the mitochondrial FeS cluster assembly machinery are essential for the viability of the procyclics. Based on the growth curves, days 4 and 7 after RNAi induction were selected for all subsequent biochemical experiments. At these time points, the levels of TbiscS2 and TbiscU mRNA and corresponding proteins in the mitochondria of their respective knock-downs became undetectable by Northern and immunoblot analyses (Fig. 3).FIGURE 3Effect of TbiscS2 (A) and TbiscU (B) RNAi on cell growth. The cell density of the parental 29-13 cells (triangles), noninduced cells (diamonds), and cells induced with 2 μg/ml of tetracycline (squares) are indicated. The y axis is a log scale and represents the product of the measured cell densities and total dilution. The arrows indicate the time points chosen for biochemical experiments. The insets show a Northern blot of the total RNA (top panels) and immunoblots of mitochondrial fractions (middle and bottom panels) before (–) and after (+) RNAi induction by tetracycline. The mitochondrial protein MRP2 was used as a protein loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mitochondrial FeS Cluster Assembly Machinery Is Required for FeS Proteins in Mitochondria and Cytosol—Aconitase was chosen as a marker FeS protein to test whether TbiscS2 and TbiscU are required for FeS cluster assembly in the trypanosome organelle and whether the mitochondrial FeS cluster assembly machinery is also involved in the maturation of cytosolic FeS proteins. In procyclic trypanosomes, aconitase has dual localization, with 70 and 30% of its total active form being distributed in the mitochondrion and cytosol, respectively (33Saas J. Ziegelbauer K. von H.A. Fast B. Boshart M. J. Biol. Chem. 2000; 275: 2745-2755Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). After