Acetyl:Succinate CoA-transferase in Procyclic Trypanosoma brucei
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m407513200
ISSN1083-351X
AutoresLoïc Rivière, Susanne W.H. van Weelden, Patricia Glass, Patricia Vegh, Virginie Coustou, Marc Biran, Jaap J. van Hellemond, Frédéric Bringaud, Aloysius G. M. Tielens, Michael Boshart,
Tópico(s)Lysosomal Storage Disorders Research
ResumoAcetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of 13C-enriched and 14C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei. Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of 13C-enriched and 14C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei. Acetyl:succinate CoA-transferase (ASCT) 1The abbreviations used are: ASCT, acetyl:succinate CoA-transferase; BSD, blasticidin; BLE, phleomycin; PBS, phosphate-buffered saline; RNAi, RNA interference; wt, wild type. is an enzyme that transfers the CoA moiety of acetyl-CoA to succinate, yielding acetate and succinyl-CoA. ASCT was earlier characterized as an acetate-producing activity in Trypanosomatidae (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). Trypanosomatidae are the earliest branching eukaryotes that retained mitochondria. They belong to the order Kinetoplastida, characterized by the kinetoplast, a highly concatenated form of the genome of the single mitochondrion. These unicellular eukaryotes contain unique organelles, such as glycosomes, which harbor part of the glycolytic pathway (2Opperdoes F.R. J. Bioenerg. Biomembr. 1994; 26: 145-146Crossref PubMed Scopus (11) Google Scholar, 3Hannaert V. Bringaud F. Opperdoes F.R. Michels P.A. Kinetoplastid. Biol. Dis. 2003; 2: 11Crossref PubMed Scopus (139) Google Scholar). The energy metabolism of Trypanosomatidae is characterized by excretion of mainly partially oxidized end products like pyruvate, succinate, and acetate. All of the Trypanosomatidae investigated produce acetate to a certain extent during their life cycle (4Cazzulo J.J. FASEB J. 1992; 6: 3153-3161Crossref PubMed Scopus (175) Google Scholar, 5Blum J.J. Parasitol. Today. 1994; 9: 118-122Abstract Full Text PDF Scopus (57) Google Scholar, 6Opperdoes F.R. Marr J.M. London A. Biochemistry and Molecular Biology of Parasites. Academic Press, Inc., London, UK1995: 19-31Crossref Google Scholar, 7Van Hellemond J.J. Van Der Meer P. Tielens A.G.M. Parasitology. 1997; 114: 351-360Crossref Scopus (36) Google Scholar), but the amount of acetate produced differs widely depending on the species and the stage of development. Trypanosoma brucei, one of the causative agents of African trypanosomiasis, alternates during its life cycle between the bloodstream of its mammalian host and the blood-feeding tsetse insect vector Glossina spp. In the mammalian bloodstream long slender-form parasites proliferate. At the peak of parasitemia, nonproliferative short stumpy cells develop that are prepared to differentiate to procyclic insect stage parasites. The long slender stage depends entirely on glycolysis for energy generation and excretes pyruvate as the main end product of carbohydrate metabolism (8Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar, 9Opperdoes F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 10Clayton C. Michels P.A. Parasitol. Today. 1996; 12: 465-471Abstract Full Text PDF PubMed Scopus (106) Google Scholar). The procyclic stage, on the other hand, can also use amino acids for its energy generation, and its glucose metabolism is completely reorganized, with succinate and acetate as main end products. Recently it was shown that inside the glycosome succinate is produced by a soluble fumarate reductase (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In the procyclic stage the mitochondrion is directly involved in the degradation of substrates, in contrast to the situation in the long slender bloodstream stage. Pyruvate enters the mitochondrion and is converted by pyruvate dehydrogenase into acetyl-CoA. This acetyl-CoA is not degraded to carbon dioxide via the Krebs cycle but is converted into acetate by ASCT (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar, 12Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The ASCT reaction concomitantly produces succinyl-CoA, which is recycled to succinate by succinyl-CoA synthetase, an enzyme that also forms part of the Krebs cycle and produces ATP. ASCT activity is also known to occur in the anaerobically functioning mitochondria of metazoa that produce acetate, such as parasitic helminths like Fasciola hepatica and Ascarissuum (13Saz H.J. deBruyn B. de Mata Z. J. Parasitol. 1996; 82: 694-696Crossref PubMed Scopus (15) Google Scholar, 14McLaughlin G.L. Saz H.J. deBruyn B.S. Comp. Biochem. Physiol. B. 1986; 83: 523-527Crossref PubMed Scopus (12) Google Scholar). Furthermore, ASCT is also a key enzyme in the metabolism of a wide spectrum of anaerobic protists, including ciliates such as Nyctotherus ovalis, chytridiomycete fungi such as Neocallimastix, and parabasalids such as Trichomonas vaginalis (15Steinbuchel A. Muller M. Mol. Biochem. Parasitol. 1986; 20: 57-65Crossref PubMed Scopus (89) Google Scholar, 16Marvin-Sikkema F.D. Pedro Gomes T.M. Grivet J.P. Gottschal J.C. Prins R.A. Arch. Microbiol. 1993; 160: 388-396Crossref PubMed Scopus (67) Google Scholar, 17van Hoek A.H. Akhmanova A.S. Huynen M.A. Hackstein J.H. Mol. Biol. Evol. 2000; 17: 202-206Crossref PubMed Scopus (42) Google Scholar). In these protists ASCT is located inside their hydrogenosomes, anaerobic energy-generating organelles. Hydrogenosomes are H2-producing, membrane-enclosed organelles related to mitochondria, and they evolved independently in the various protists (18Embley T.M. Finlay B.J. Dyal P.L. Hirt R.P. Wilkinson M. Williams A.G. Proc. R. Soc. Lond. B Biol. Sci. 1995; 262: 87-93Crossref PubMed Scopus (134) Google Scholar, 19Cavalier-Smith T. Chao E.E. J. Mol. Evol. 1996; 43: 551-562Crossref PubMed Scopus (103) Google Scholar). Sequence comparison of several organelle-specific heat shock proteins or chaperonins and the presence of targeting signals at the N terminus of hydrogenosomal enzymes that resemble mitochondrial import signals showed that hydrogenosomes and mitochondria are evolutionary related (20Bui E.T. Bradley P.J. Johnson P.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9651-9656Crossref PubMed Scopus (231) Google Scholar, 21Germot A. Philippe H. Le Guyader H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14614-14617Crossref PubMed Scopus (162) Google Scholar, 22Horner D.S. Hirt R.P. Kilvington S. Lloyd D. Embley T.M. Proc. R. Soc. Lond. B Biol. Sci. 1996; 263: 1053-1059Crossref PubMed Scopus (133) Google Scholar, 23Roger A.J. Clark C.G. Doolittle W.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14618-14622Crossref PubMed Scopus (137) Google Scholar). They probably originated from the same prokaryotic endosymbiont of the α group of proteobacteria, and it is suggested that hydrogenosomes have evolved by adaptation to anaerobic conditions (24Martin W. Muller M. Nature. 1998; 392: 37-41Crossref PubMed Scopus (905) Google Scholar). The exact evolutionary relation between mitochondria and the various types of hydrogenosomes is, however, still debated. Although the main function in energy generation of both mitochondria and hydrogenosomes is the oxidation of pyruvate and acetyl-CoA, the two types of organelles do not share many similarities. The ASCT/succinyl-CoA synthetase cycle is the only metabolic pathway common to mitochondria and hydrogenosomes (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). For that reason, phylogenetic analysis of ASCTs from different sources could provide a valuable tool to unravel the evolutionary history of the respective energy-generating organelles, the various types of hydrogenosomes, and (an)aerobically functioning mitochondria. Unfortunately, the sequence of not a single ASCT gene is known yet. Here we have identified the gene encoding ASCT of T. brucei. Several reverse genetic strategies and subcellular localization studies were used to establish the link between ASCT gene sequence and mitochondrial ASCT enzyme activity. Furthermore, two independent methods of metabolic analysis using 13C-enriched and 14C-labeled glucose were applied to wild type and transgenic mutant clones of T. brucei. The combined biochemical and genetic approach resulted in the gene identification of T. brucei ASCT and also demonstrated that this enzyme is indeed involved in acetate production. However, ASCT is not an essential gene as ASCT is not the only enzyme producing acetate in T. brucei. Plasmid Constructs—The sheared DNA clone 7D10 from The Institute for Genomic Research (GenBank™ accessions AQ652285 and AQ652287) was obtained from Dr. Najib M. A. El-Sayed. The complete sequence of that part of chromosome 11 is now available from the TrypDB genome data base (Tb11.02.0290); it is identical with results of sequencing of clone 7D10. A MluI (blunted)/MscI fragment of the insert of 7D10 was excised and replaced in the reading frame orientation by a blasticidin S resistance BSD (25Kimura M. Takatsuki A. Yamaguchi I. Biochim. Biophys. Acta. 1994; 1219: 653-659Crossref PubMed Scopus (89) Google Scholar) cassette (SmaI/StuI fragment from pHD887 (courtesy of C. Clayton, Heidelberg) containing the PmlI fragment nucleotides 2189–2687 from pcDNA6/V5-His C (Invitrogen) or by a phleomycin resistance BLE cassette (BamH1/NheI, both blunted, from pLew20 (26Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1126) Google Scholar)) to generate the targeting vectors pBER2 and pBER3, respectively. Before transfection, the targeting vectors were linearized with BsrGI plus BsmI. To produce double-stranded RNA using head-to-head promoters, the N-terminal fragment of the T. brucei ASCT (nucleotides position 35 to position 665) was PCR-amplified from 7D10 and inserted into p2T7 (27LaCount D.J. Bruse S. Hill K.L. Donelson J.E. Mol. Biochem. Parasitol. 2000; 111: 67-76Crossref PubMed Scopus (157) Google Scholar) to give p2T7.ASCT. Plasmid pLew-ASCT-SAS contains a "sense/antisense" cassette targeting a 572-bp fragment of the T. brucei ASCT (nt position 85–657) in the pLew100 expression vector (kindly provided by E. Wirtz and G. Cross) (26Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1126) Google Scholar) and was constructed as previously reported for repression of a flagellar protein (FTZC) gene expression (28Bringaud F. Robinson D.R. Barradeau S. Biteau N. Baltz D. Baltz T. Mol. Biochem. Parasitol. 2000; 111: 283-297Crossref PubMed Scopus (47) Google Scholar). For overexpression in T. brucei, the complete ASCT-coding region was assembled from a N-terminal PCR product amplified from T. brucei 927/4 genomic DNA with the primers 5′-ggcagaattcCAGCTCCACATCACCTTCCAGG (EcoRI site) and 5′actgaagcttATGCTCCGCCGAACAAATTTT (HindIII site) and from a C-terminal fragment excised from plasmid 7D10 digested with EcoRI and BamHI. The two fragments were inserted into a HindIII/BamHI-opened pTSArib (29Xong H.V. Vanhamme L. Chamekh M. Chimfwembe C.E. Van Den Abbeele J. Pays A. Van Meirvenne N. Hamers R. De Baetselier P. Pays E. Cell. 1998; 95: 839-846Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Southern blot analysis was done as described before (12Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) with an ASCT-specific probe (nucleotides 1–314 of the coding region) generated by PCR. Trypanosomes, Cell Culture, and Transfections—The procyclic T. brucei cells were cultured at 27 °C in SDM79 medium containing 10% (v/v) heat-inactivated fetal calf serum, 100 units·ml–1 penicillin, 100 units·ml–1 streptomycin, 1 mm glycerol, and 3.5 mg·ml–1 hemin (12Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 30Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar) at cell densities between 2 × 106 ml–1 and 1 × 107 ml–1. The EATRO1125 procyclic line used in this work was originally isolated from a tsetse experimentally infected with AnTat1.1E bloodstream trypanosomes (31Delauw M.F. Pays E. Steinert M. Aerts D. Van Meirvenne N. Le Ray D. EMBO J. 1985; 4: 989-993Crossref PubMed Scopus (42) Google Scholar). The AnTat1.1 procyclic line was obtained from the same bloodstream AnTat1.1E clone by differentiation in culture (32Vassella E. Boshart M. Mol. Biochem. Parasitol. 1996; 82: 91-105Crossref PubMed Scopus (53) Google Scholar). The EATRO1125-T7T and the stock 427 derived 29-13 clone 6 procyclic cells expressing the T7 RNA polymerase gene and the tetracycline repressor under control of a T7 RNA polymerase promoter are described in Bringaud et al. (28Bringaud F. Robinson D.R. Barradeau S. Biteau N. Baltz D. Baltz T. Mol. Biochem. Parasitol. 2000; 111: 283-297Crossref PubMed Scopus (47) Google Scholar) and in Wirtz et al. (26Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1126) Google Scholar), respectively. Trypanosome transfection and selection of drug-resistant clones were performed as previously reported (33McCulloch R. Vassella E. Burton P. Boshart M. Barry J.D. Methods Mol. Biol. 2004; 262: 53-86PubMed Google Scholar) with the following modifications; for washing and electroporation, cytomix was used at 4 °C, and 1 × 107 cells were electroporated. After electroporation, procyclic cells were directly cloned in 30–50% conditioned SDM79 supplemented with 5–10 μg·ml–1 phleomycin (Cayla), 10 μg·ml–1 hygromycin S (Calbiochem), or 15 μg·ml–1 hygromycin B (Roche Applied Science). For generation of clonal lines, the cells were diluted (1:100 and 1:10) in 30% conditioned SDM79 after 24 h and selected in microtiter plates. Production of Anti-ASCT Antibodies—A recombinant fragment corresponding to the first 414 amino acids of ASCT followed by a C-terminal histidine tag was expressed in the Escherichia coli BL21 using the pET16b expression vector (Novagen). After 2 h of induction at 37 °C with 1 mm isopropyl-β-d-thiogalactopyranoside, the cells were harvested by centrifugation, and proteins were purified by nickel chelate affinity chromatography according to the manufacturer's instructions (Novagen). Antisera were raised in rabbits by 3 injections at 15-day intervals of 200 μg of recombinant purified protein, electro-eluted after separation on SDS-PAGE, and emulsified with complete (first injection) or incomplete Freund's adjuvant. The antiserum was affinity-purified on polyvinylidene difluoride membrane-bound recombinant protein (amino acids 12–493 fused to an N-terminal His tag in vector pQE32 (Qiagen)). Elution with 0.2 m glycine, 1 mm EGTA, pH 2.2, for 10 min was followed by immediate neutralization, stabilization in 200 μg·ml–1 bovine serum albumin, and dialysis against PBS containing 0.02% NaNO3. One specific 53-kDa band was detected on the Western blot. Western Blot and Immunofluorescence Analyses—For the Western blot analysis total protein lysates of procyclic form T. brucei (1.75 × 105 cells) were separated by SDS-PAGE (10%) and blotted on Immobilon-P filters (Millipore) (34Harlow E. Lane D. Antibodies: A Laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Harbor, NY1988Google Scholar). The membranes were blocked with PBS, 5% milk powder for 1 h at room temperature. Primary antibodies and secondary antibodies were diluted in PBS, 0.1% Tween 20 with 1% milk powder: rabbit anti T. brucei phosphoglycerate kinase C 1:5,000; rabbit anti-T. brucei ASCT 1:500; mouse anti-T. brucei heat shock protein 60 (hsp60) 1:10,000; IRD800 goat anti-mouse IgG (Biomol) 1:5,000; Alexa 680 goat anti-rabbit IgG (Molecular Probes) 1:5,000. Antibody incubations were for 1 h at room temperature. For detection, the membrane was dried and scanned with the Odyssey™ dual wavelength infrared fluorescence scanner (Li-Cor Biosciences, Lincoln, NE). For immunofluorescence, log phase cells were fixed with formaldehyde as previously described (35Bringaud F. Baltz D. Baltz T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7963-7968Crossref PubMed Scopus (109) Google Scholar). Slides were incubated with 1:100 diluted affinity-purified rabbit anti-ASCT, and the undiluted H95 monoclonal mouse anti-hsp60 (36Bringaud F. Peyruchaud S. Baltz D. Giroud C. Simpson L. Baltz T. Mol. Biochem. Parasitol. 1995; 74: 119-123Crossref PubMed Scopus (35) Google Scholar) was followed by fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody diluted 1:100 (Bio-Rad) and ALEXA Fluor 568-conjugated goat anti-mouse secondary antibody diluted 1:100 (Molecular Probes). Cells were observed with a Zeiss UV microscope, and images were captured by a MicroMax-1300Y/HS camera (Princeton Instruments) and MetaView software (Universal Imaging Corp.) and assembled in Adobe Photoshop. Digitonin Fractionations—Digitonin fractionations were done as described in Saas et al. (37Saas J. Ziegelbauer K. von Haeseler A. Fast B. Boshart M. J. Biol. Chem. 2000; 275: 2745-2755Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Proteinase K digestion was adapted from Sveshnikova et al. (38Sveshnikova N. Grimm R. Soll J. Schleiff E. Biol. Chem. 2000; 381: 687-693Crossref PubMed Scopus (61) Google Scholar). The pellet and concentrated supernatant were suspended in 500 μl of trypanosome homogenization buffer without leupeptin (37Saas J. Ziegelbauer K. von Haeseler A. Fast B. Boshart M. J. Biol. Chem. 2000; 275: 2745-2755Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and 8 μl of proteinase K (10 mg·ml–1) were added on ice. After 3 min at 37 °C the reaction was stopped by adding 6 μl of phenylmethylsulfonyl fluoride (100 mm diluted in isopropanol) on ice. For thermolysin digestion, the pellet and concentrated supernatant were suspended in 90 and 40 μl, respectively, of 0.1 m Hepes-HCl, pH 7.2, 2 mm CaCl2, 0.2 μg·ml–1 leupeptin, 0.6 m saccharose, 1 mm dithiothreitol. The pellet and supernatant were incubated with 10 and 5 μl, respectively, of thermolysin (1 mg·ml–1) for 30 min at 45 °C. The digestion was stopped by adding EDTA on ice. ASCT Activity Assay—Homogenates of procyclic T. brucei were prepared from 5 × 107 cells in 20 mm HEPES buffer, pH 7.4, containing 1% Triton X-100 using a Teflon glass homogenizer. Subsequently, cell debris was removed by centrifugation for 5 min at 500 × g, 4 °C. The activity of ASCT was determined by a radioactive assay as described previously (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). In brief, the assay mix contained 50 mm succinate, pH 7.4, 1 mm [1-14C]acetyl-CoA (0.2 Mbq, Amersham Biosciences), 50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, and 0.05% (v/v) Triton X-100. The assay was started by the addition of the homogenate (50–100 μg protein), incubated at 20 °C for 10 min, and terminated by the addition of ice-cold trichloroacetic acid (10% w/v, final concentration). Reaction products were then separated by anion-exchange chromatography and quantified by liquid scintillation counting. Immunoprecipitation—Procyclic cells (1 × 108 cells) were harvested by centrifugation at 500 × g for 5 min. The cells were washed twice and resuspended in 20 mm Tris-HCl buffer, pH 7.8, containing 1% Triton X-100. Antiserum was added to the cell lysate (20% v/v) and incubated on ice for 2 h. Subsequently, protein A-conjugated acrylic beads (Sigma, 150 μm) in 20 mm Tris-HCl buffer, pH 7.8, were added to the lysate and rotated for 2 h at 4 °C. The antigen-bead preparation was pelleted by centrifugation (500 × g for 10 s). The supernatant was collected, and the pelleted beads were washed once with 20 mm Tris-HCl buffer, pH 7.8, and then resuspended in the same buffer. Both the supernatant and the resuspended beads were assayed for ASCT activity. Cell lysates treated with protein-A acrylic beads only were used as the control. Nuclear Magnetic Resonance (NMR) Experiments—4 × 109T. brucei procyclic cells grown in the SDM79 medium (up to 1 × 107 cells·ml–1) were incubated in 10 ml of incubation buffer (PBS buffer supplemented with 24 mm NaHCO3, pH 7.3) containing 110 μmole d-[1-13C]glucose (11 mm) for 90–180 min at 27 °C as described before (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 39Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The cell viability and motility, checked every 30 min, were not affected during the incubation time. d-Glucose concentration in the medium was determined at the beginning and the end of the incubation with the d-glucose Trinder kit (Sigma). The supernatant was lyophilized and re-dissolved in 500 μl of D2O, and 15 μl of pure dioxane was added as an external reference. 13C NMR spectra were collected as described before (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 39Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The relaxation delay was 8 s for a nearly complete longitudinal relaxation. The specific 13C enrichment of lactate (carbon C3), acetate (carbon C2), and succinate (carbons C2 and C3) was determined from 1H-observed/13C-edited NMR (1H/13C NMR) spectra acquired under 13C decoupling (40Freeman R. Mareci T.H. Morris G.A. J. Magn. Reson. 1981; 42: 341-345Google Scholar, 41Rothman D.L. Behar K.L. Hetherington H.P. den Hollander J.A. Bendall M.R. Petroff O.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1633-1637Crossref PubMed Scopus (204) Google Scholar) as described before (39Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The reproducibility and accuracy of the method were assessed using pure solutions of succinate, acetate, lactate, malate, pyruvate, or β-hydroxybutyrate; the relative errors on the 13C enrichment determinations were <5%. 2M. Biran and P. Canioni, unpublished data. The amount of 13C-enriched molecules (including d-glucose) was calculated on the basis of the dioxane peak. For each experiment, the amount of remaining d-glucose calculated from the NMR spectra corresponded ±5–25% to the concentration measured with the d-glucose Trinder kit. Metabolic Incubations—Proliferating T. brucei cells (starting at 5 × 106 ml–1) were incubated for 18–24 h at 27 °C in sealed 25-ml Erlenmeyer flasks containing 5 ml of SDM79 incubation medium. Before sealing, the flasks were flushed for 1 min with a gas phase of air and CO2 (95/5%). The incubations were performed after the addition of 5 μCi of d-[6-14C]glucose. Incubations were terminated by the addition of 40 μl of 6 m HCL to lower the pH to 3.5. Preceding acidification, 0.1 ml 1 m NaHCO3 was added through the rubber stopper, and the flasks were placed on ice. Immediately after acidification, the incubation flasks were flushed with nitrogen for 90 min at 0 °C. In this way all carbon dioxide was removed, whereas acetate remained in the incubation medium. The carbon dioxide was trapped in a series of four scintillation vials, each filled with 1 ml of 0.3 m NaOH and 15 ml of Tritisol scintillation fluid modified according to the method described by Pande (42Pande S.V. Anal. Biochem. 1976; 74: 25-34Crossref PubMed Scopus (91) Google Scholar, 43Horemans A.M. Tielens A.G. van den Bergh S.G. Parasitology. 1991; 102: 259-265Crossref PubMed Scopus (20) Google Scholar). After removal of carbon dioxide, the acidified supernatant was separated from the cells by centrifugation (4 °C, 10 min at 500 × g) and neutralized by the addition of 40 μlof6 m NaOH. Analysis of the labeled end products occurred by anion-exchange chromatography on a Dowex 1X8, 100–200 mesh column (Serva), 60 × 1.1 cm in chloride form (44Tielens A.G. van der Meer P. van den Bergh S.G. Mol. Biochem. Parasitol. 1981; 3: 205-214Crossref PubMed Scopus (38) Google Scholar). The column was eluted successively with 200 ml of 5 mm HCl, 130 ml of 0.2 m NaCl, and 130 ml of 0.5 m NaCl. The fractions were collected and counted with 2 ml of Lumac LCS in a scintillation counter. All values were corrected for blank incubations. Labeled end products were identified by their RF values. Glucose concentrations were determined enzymatically using a standard procedure (45Bergmeyer H.U. Bernt E. Schmidt F. Stork H. Bergmeyer H.U. Methoden der Enzymatischen Analyse. Verlag Chemie, Weinheim, Germany1970: 1163-1165Google Scholar). Protein was determined with a Lowry method using defatted and dialyzed bovine serum albumin (Roche Applied Science) as the standard (46Bensadoun A. Weinstein D. Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2740) Google Scholar). Identification of an ASCT Candidate Gene—The transfer of the coenzyme A (CoA) group is catalyzed by a large family of prokaryotic and eukaryotic CoA transferases (47Heider J. FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (110) Google Scholar) that share conserved protein domains (CoA_trans in protein families data base, accession PF01144) and have a broad range of substrate specificities. Acetoacetate, a preferred substrate of mammalian succinyl-CoA:3-ketoacid-CoA transferase, is structurally comparable with acetate, the substrate of ASCT. Therefore, we first searched for succinyl-CoA:3-ketoacid-CoA transferase-related gene sequences in the available T. brucei genome databases using BLAST. Only a single high scoring segment pair was identified, and the target shares up to 55% amino acid identity over its full length with succinyl-CoA:3-ketoacid-CoA transferase from several vertebrate species. A single copy (see Fig. 3 and data not shown) of this gene maps to chromosome 11 of the T. brucei genome reference strain TREU 927/4 (see TrypDB, release 2.1, systematic name Tb1102.0290). The gene encodes a 53-kDa protein (493 amino acids) with two CoA transferase domains in tandem, the hallmark of eukaryotic CoA transferases (48Bateman K.S. Brownie E.R. Wolodko W.T. Fraser M.E. Biochemistry. 2002; 41: 14455-14462Crossref PubMed Scopus (23) Google Scholar). All nine amino acids found in immediate proximity of the catalytic center in the crystal structure of porcine succinyl-CoA:3-ketoacid-CoA transferase (48Bateman K.S. Brownie E.R. Wolodko W.T. Fraser M.E. Biochemistry. 2002; 41: 14455-14462Crossref P
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