Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) Genes Complement Yeast Mutants Defective in H+ Pumps and Encode Plasma Membrane P-type H+-ATPases with Different Enzymatic Properties
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m202267200
ISSN1083-351X
AutoresShuhong Luo, David A. Scott, Roberto Docampo,
Tópico(s)Trypanosoma species research and implications
ResumoPrevious studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H+-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi:TcHA1 is expressed maximally in epimastigotes, whereasTcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H+-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H+-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1-and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in theT. cruzi life cycle. Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H+-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi:TcHA1 is expressed maximally in epimastigotes, whereasTcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H+-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H+-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1-and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in theT. cruzi life cycle. H+-ATPases within the P-type ATPase family are proton pumps driven by the hydrolysis of ATP. These pumps have been found almost exclusively in the plasma membrane of plants and fungi (1Palmgren M.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 817-845Crossref PubMed Scopus (661) Google Scholar). A sequence analysis of conserved core sequences of all P-type ATPases has grouped them in five subfamilies designated types I–V (2Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (757) Google Scholar). Type III covers H+-ATPases (type IIIA) and a small group of Mg2+-ATPases from bacteria (type IIIB). All fungal P-type H+-ATPases comprise one subcluster within type IIIA, the plant enzymes comprise a second subcluster, and sequences found in the trypanosomatid parasite Leishmania donovani make up a third subcluster (2Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (757) Google Scholar). Because the L. donovani sequences (LHA1A and LHA1B) are obviously distinct from the plant and yeast H+-ATPase sequences, some authors (3Wach A. Schlesser A. Goffeau A. J. Bioenerg. Biomembr. 1992; 24: 309-317PubMed Google Scholar) have raised the question of whether they are indeed H+-ATPases, as was inferred from sequence homology (4Meade J.C. Shaw J. Lemaster S. Gallagher G. Stringer J. Mol. Cell. Biol. 1987; 7: 3937-3946Crossref PubMed Scopus (64) Google Scholar, 5Meade J.C. Hudson K.M. Stringer S.L. Stringer J.R. Mol. Biochem. Parasitol. 1989; 33: 81-92Crossref PubMed Scopus (35) Google Scholar, 6Meade J.C. Coombs G.H. Mottram J.C. Steele P.E. Stringer J.R. Mol. Biochem. Parasitol. 1991; 45: 29-38Crossref PubMed Scopus (14) Google Scholar, 7Stiles J.K. Hicock P.I. Kong L. Meade J.C. Mol. Biochem. Parasitol. 1999; 103: 105-109Crossref PubMed Scopus (4) Google Scholar). Confirmation of the substrate specificities of cloned P-type ATPases requires, in addition to demonstration of amino acid identity to biochemically well characterized proteins, expression of the genes followed by biochemical characterization of the gene products (2Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (757) Google Scholar). Among P-type H+-ATPases, this has been done until now only with plant and fungal transporters (8Villalba J.M. Palmgren M.G. Berberián G.E. Ferguson C. Serrano R. J. Biol. Chem. 1992; 267: 12341-12349Abstract Full Text PDF PubMed Google Scholar, 9Palmgren M.G. Christensen G. J. Biol. Chem. 1994; 269: 3027-3033Abstract Full Text PDF PubMed Google Scholar, 10De Kerchove d'Exaerde A. Supply P. Dufour J.-P. Bogaerts P. Thines D. Goffeau A. Boutry M. J. Biol. Chem. 1995; 270: 23828-23837Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Luo H. Morsomme P. Boutry M. Plant Physiol. 1999; 119: 627-634Crossref PubMed Scopus (46) Google Scholar, 12Ferreira T. Mason A.B. Slayman C.W. J. Biol. Chem. 2001; 276: 29613-29616Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Plant H+-ATPases belong to multigene families, with individual members expressed in particular cell types. In some cases up to three H+-ATPase genes may be expressed in the same cell type at the same developmental stage, suggesting that isoforms with distinct catalytic or regulatory properties may coexist in the same cell (13Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar, 14Morsomme P. Boutry M. Biochim. Biophys. Acta. 2000; 1465: 1-16Crossref PubMed Scopus (320) Google Scholar). In unicellular organisms the presence of several genes encoding H+-ATPases is also frequent. The PMA2 gene product in yeast shows 89% identity to the PMA1 gene product (15Schlesser A. Ulaszewski S. Ghislain M. Goffeau A. J. Biol. Chem. 1988; 263: 19480-19487Abstract Full Text PDF PubMed Google Scholar), althoughPMA2 is expressed at very low levels and is not essential for growth (16Supply P. Wach A. Goffeau A. J. Biol. Chem. 1993; 268: 19753-19759Abstract Full Text PDF PubMed Google Scholar). Trypanosoma cruzi is the etiologic agent of Chagas' disease or American trypanosomiasis. T. cruzi has been recognized as a significant cause of morbidity and mortality in Mexico and Central and South America (17Anonymous 13th Program Report, UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, Progress 1995–1996. World Health Organization, Geneva1997: 112-123Google Scholar). Chagas' disease remains a problem because of limited therapeutic choices and adverse reactions to the two drugs available, nifurtimox and benznidazole (17Anonymous 13th Program Report, UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, Progress 1995–1996. World Health Organization, Geneva1997: 112-123Google Scholar, 18Kirchhoff L.V. New Engl. J. Med. 1993; 329: 639-644Crossref PubMed Scopus (339) Google Scholar). Therefore, it is important to identify enzymes and metabolic processes in T. cruzi which might be potential targets for drug development.T. cruzi has three main developmental stages: the epimastigote, which is found in the insect vector and can be grown in axenic culture; the amastigote or intracellular form, which lives in the cytosol of nucleated cells; and the trypomastigote, which is the terminal differentiation stage in the vector (metacyclic form) or is found in the bloodstream from mammalian hosts (bloodstream form). In the present study, we report the cloning and sequencing of a pair of genes linked in tandem from T. cruzi which encode proteins with homology to the L. donovani putative P-type H+-ATPase cluster. The T. cruzi genes are expressed differentially in the different developmental stages ofT. cruzi and can complement a yeast strain deficient in P-type H+-ATPase, providing genetic evidence for their function. The protein products of these genes localize to the yeast plasma membrane. Reconstitution of plasma membranes into proteoliposomes permits the detection of ATP-driven proton transport, and the two T. cruzi H+-ATPases show different biochemical properties. Together, these results provide the first evidence for the presence of a functional plasma membrane P-type H+-ATPase in organisms other than plants and fungi. T. cruzi amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L6E9 myoblasts as described before (19VanderHeyden N. Docampo R. Mol. Biochem. Parasitol. 2000; 105: 237-251Crossref PubMed Scopus (36) Google Scholar).T. cruzi epimastigotes (Y strain) were grown at 28 °C in liver infusion tryptose medium (20Bone G.J. Steinert M. Nature. 1956; 178: 308-309Crossref PubMed Scopus (133) Google Scholar) supplemented with 10% heat-inactivated newborn calf serum. Genomic DNA isolation and genomic DNA library construction were done as described (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar, 22Hill J.E. Scott D.A. Luo S.H. Docampo R. Biochem. J. 2000; 351: 281-288Crossref PubMed Scopus (50) Google Scholar). Oligonucleotide primers were designed to recognize the ATP phosphorylation site and the ATP binding site of cationic ATPase genes (23Allen G. Green N.M. FEBS Lett. 1976; 63: 188-192Crossref PubMed Scopus (69) Google Scholar, 24Pick U. Bassilian S. FEBS Lett. 1981; 123: 127-130Crossref PubMed Scopus (104) Google Scholar), i.e.5′-CGGGATCCGTNATNTGYWSNGAYAA-3′ and 5′-CGGAATTCGSRTCRTTNRYNCCR-3′ as the 5′-primer and 3′-primer, respectively. PCR was performed in a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, MA) at 94 °C for 1 min, 55–62 °C for 2 min, and 72 °C for 3 min/cycle (30 cycles) using Taq polymerase. PCR products were cloned into the pGEM-T vector according to the manufacturer's instructions. For library screening, 3.0 × 105 plaque-forming units (approximately three times the content of the library) were plated at a density of 2 × 104 plaque-forming units/90-mm plate on host strain LE392. Plaques were allowed to develop to ∼1.0 mm in diameter before being lifted onto nylon membranes. Membranes were probed with [α-32P]dCTP-labeled probes according to standard procedures. Positive plaques identified in the screen were serially plaqued to homogeneity. Southern and Northern hybridization were done by standard procedures (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Total RNA was isolated with Trizol reagent according to the manufacturer's recommendations. The polyadenylated RNA was obtained using the poly(A) tract mRNA isolation system. mRNA was electrophoresed in 1% agarose gels with 2.2 m formaldehyde, 40 mmsodium acetate, 5 mm EDTA, 100 mmMOPS, 1The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; RT, reverse transcription pH 8.0. Northern hybridization was done by standard procedures (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) using probesa (218 bp), b (89 bp), c (780 bp),d (1,035 bp), and e (1,542 bp) (see Fig. 1). TheTcP0 fragment used as a control in Northern blots was obtained by amplifying T. cruzi genomic DNA by PCR, with primers corresponding to nucleotides 3–54 and 918–936 in the sequence of the TcP0 gene (25Skeiky Y.A.W. Benson D.R. Parsons M. Elkon K.B. Reed S.G. J. Exp. Med. 1992; 176: 201-211Crossref PubMed Scopus (60) Google Scholar). Densitometric analyses of Northern blots was done using an ISI-1000 digital imaging system (Alpha Inotech Corp.). Comparison in levels of the H+-ATPase transcripts in the different stages was done taking as a reference the densitometric values obtained with the TcP0 transcripts and assuming a similar level of expression of this gene in all stages (25Skeiky Y.A.W. Benson D.R. Parsons M. Elkon K.B. Reed S.G. J. Exp. Med. 1992; 176: 201-211Crossref PubMed Scopus (60) Google Scholar). Similar results were obtained when the densitometric values were compared taking into account the amount of RNA added to each lane in three different experiments. First strand cDNA synthesis was primed with an oligonucleotide that annealed to 911 bp downstream from the putative start codon of the TcHA2 open reading frame (RTP3′1: 5′-GGAATGGACACCACAAGCAC-3′, 3627–3646, 7483–7502 bp) in a reaction containing 1 mm dNTPs, 2.5 mm MgCl2, 10 mm dithiothreitol, 1× SuperScript PCR buffer, 200 units SuperScript II reverse transcriptase, and total RNA (5 μg). Target sequences were amplified in a standard PCR using the first strand cDNA as template and primers Tc-5′-SL (5′-GCGGTCCATAGAACAGTTTCTGTAC-3′), which annealed to the 5′-spliced leader sequence of T. cruzi mRNA, and a downstream primer that annealed to a sequence just 461 bp upstream from the primer used for first strand cDNA synthesis (RTP3′2: 5′-TTCTTCAGCGCAGCCACAGC-3′, 3147–3166, 7003–7022 bp). The product of the amplification reaction was ligated into vector pCR2.1TOPO for sequence analysis. DNA sequence data were generated at the High Throughput Sequencing and Genotyping Unit of the Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign. Sequence analysis was done using the Biology Workbench 3.0 utility (workbench.sdsc.edu) and the Wisconsin Sequence Analysis Package (Version 8.0, Genetics Computer Group, Madison, WI). Hydropathy analysis was done with the Gene Jockey sequence processor (Biosoft, Cambridge, UK). Plasmids pMP625, derived from YEp351 (26Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google Scholar) and containing the promoter and terminator of PMA1, and pRS890 (8Villalba J.M. Palmgren M.G. Berberián G.E. Ferguson C. Serrano R. J. Biol. Chem. 1992; 267: 12341-12349Abstract Full Text PDF PubMed Google Scholar), containing the yeast PMA1 gene, were kindly provided by Dr. Palmgren (University of Copenhagen, Denmark). The full-length coding regions of TcHA1 and TcHA2 were amplified from λ5-1 clone DNA using the primers YA1P5, 5′-CTCGAGATGGTACCGCCGTCCAAGGG-3′ (2886–2905 bp), which includes an underlined XhoI site, and YA1P31, 5′-ACTAGTTTACACCGTGGGTTCCTTTG-3′ (5494–5513 bp), with an underlined SpeI site; YA2P51, 5′-CTCGAGATGGACCAGAAGAACGATAA-3′ (6592–6611 bp) and YA2P31, 5′-ACTAGTTTAATTGGCAGGCTCAGTGA-3′ (9326–9345 bp). The PCR products were subcloned into XhoI andSpeI sites of pMP625 to generate plasmid pRD201 (TcHA1/pMP625) and pRD203 (TcHA2/pMP625). To delete the C terminus of TcHA1 and TcHA2, PCRs were made utilizing primers YA1P5 and YA1P32, 5′-ACTAGTTTAAGCGTCCTGAATAAGCC-3′, which include an underlined SpeI site followed by an antisense stop codon and the antisense nucleotides 5350–5366 bp; YAP51 and YAP32, 5′-ACTAGTTTAAGCGTCCTGAATAAGCC-3′ (9206–9222 bp). The PCR products were truncated by either 144 bp (last 48 amino acids of TcHA1) or 120 bp (last 40 amino acids of TcHA2). To delete the N terminus of TcHA2, the PCR amplification was performed by using primers YA2P52, 5′-CTCGAGATGGTACCGCCGTCCAAGGG-3′ (6742–6760 bp) and YA2P31. The PCR product was truncated by 150 bp (first 50 amino acids of TcHA2). The shortened genes were subcloned into XhoI and SpeI sites of pMP625 to obtain pRD202 (TcHA1Δ48/pMP625), pRD204 (TcHA2Δ40/pMP625), and pRD205 (TcHA2N-Δ50/pMP625) with the right orientation for expression. All PCR amplifications were carried out usingPfu DNA polymerase, which exhibits the lowest error rate of any thermostable DNA polymerase. The PCRs were performed in a total reaction volume of 50 μl for 25 cycles of 96 °C for 1 min, 55–60 °C for 1 min, and 72 °C for 1.5 min using a thermal cycler. All constructs were sequenced to confirm their identity. Saccharomyces cerevisiae strain RS-72 (MATa, ade1–100 his4–519 leu2–3, 112; 10), carrying the yeast PMA1 gene under the control of the galactokinase gene (GAL1) promoter, was used for transformation with LEU2 plasmids (26Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google Scholar). Yeast were grown on synthetic medium (SGAHL) containing 2% (w/v) galactose, 0.7% (w/v) yeast nitrogen base without amino acids (Difco), 0.2 mmadenine, 0.4 mm histidine, and 1 mm leucine. Yeast were made competent for plasmid uptake by treatment with lithium acetate and polyethyleneglycol according to Gietz et al.(27Gietz D., St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Positive transformants were selected on SGAH medium (SGAHL without leucine) after 4 days of growth at 30 °C. The new strains (bearing the respective plasmids) were named MP625 (pMP625), RS1002 (pRS890), RD2011 (pRD201), RD2022 (pRD202), RD2033 (pRD203), RD2044 (pRD204), and RD2055 (pRD205). Transformants were maintained in SGAH or transferred to medium containing 2% (w/v) glucose in place of galactose (SDAH). The media were buffered with 50 mmsuccinic acid adjusted to pH 5.5 (or other pH values in pH growth experiments) with Tris. Solid media contained 2% agar (Difco). Yeast strain RS1002, RD2011, or RD2055 grown in 300 ml of SDAH to anA 600 of ∼5 was recovered by centrifugation (1,300 × g), washed once in water, and suspended in 1 ml of lysis buffer (250 mm sucrose, 25 mmHepes, 2 mm MgCl2, 1 mm EGTA, 10 mm benzamidine, 15 mm dithiothreitol, 1.5% protease inhibitor mixture, pH 7.5). An equal volume of glass beads (0.5-mm diameter) was added, and the mixture was vortexed for 3–5 min, until 80–90% of the yeast was lysed, as quantified by microscopy of yeast diluted in water. The glass beads were washed by gravity with 20% v/v glycerol, 25 mm Hepes, 2 mmMgCl2, 1 mm EGTA, 5 mmdithiothreitol, pH 7.5 (glycerol buffer). The supernatant (lysate) was centrifuged at 3,000 × g for 5 min to remove unbroken cells and debris, and the supernatant from this was centrifuged at 20,000 × g for 20 min. The pellet fraction was suspended in 4 ml of glycerol buffer and applied to a sucrose step gradient: 8 ml of 43% w/w sucrose over 4 ml of 53% w/w sucrose (both with 25 mm Hepes, 2 mm MgCl2, 1 mm EGTA, pH 7.5). The gradient was centrifuged for 6 h at 25,000 rpm (Beckman SW28 rotor) to prepare a plasma membrane fraction (28Serrano R. Methods Enzymol. 1988; 157: 533-544Crossref PubMed Scopus (233) Google Scholar). This fraction was recovered from the 43/53% interface, diluted 5× in water, and centrifuged at 80,000 × gfor 20 min. Pellets were resuspended in glycerol buffer and stored at −80 °C before use. The H+-ATPase proteins expressed in yeast plasma membrane were reconstituted into proteoliposomes by a modification of the method of de Kerchove d'Exaerde et al. (10De Kerchove d'Exaerde A. Supply P. Dufour J.-P. Bogaerts P. Thines D. Goffeau A. Boutry M. J. Biol. Chem. 1995; 270: 23828-23837Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Plasma membrane preparations were diluted to 0.5–2 mg of protein/ml in 10 mm MES, 50 mm K2SO4, 20% glycerol, pH 6.6 (MKG buffer). Liposomes were prepared by suspending 50 mg/ml soybean phospholipids in MKG buffer and sonicating until dispersed, adding 0.3 volume of 10% w/v sodium deoxycholate in MKG buffer, and diluting with a further 0.7 volume of MKG buffer. Liposomes (1.5 ml) were added to 1 ml of diluted plasma membrane and left on ice for 10 min, with shaking every 30 s before centrifugation for 1 h at 100,000 × g. Pellets were resuspended in MKG buffer and stored at −80 °C before use. The soybean phospholipids used for preparation of liposomes were checked for lack of ATPase activity using the assay described below. A 0.78-kb PCR fragment, named TcHAf (probe c) encoding a 260-amino acid non-transmembrane domain of the TcHA2 protein, was cloned into the pGEM-T vector and digested by EcoRI andBamHI. The fragment was then subcloned into theBamHI and EcoRI sites of the pET-28a(+) expression vector, resulting in a construct that encoded the protein fused to a six-histidine tag that allowed its purification on nickel-agarose columns. This plasmid was checked by DNA sequencing to ensure that the correct construct had been obtained. The recombinant plasmid was transfected into the DE3 strain of Escherichia coli, the fusion protein was induced, and the expressed protein of about 35 kDa, present in inclusion bodies, was solubilized and purified according to the manufacturer's instruction (Novagen). Rabbits were injected subcutaneously with 1 mg of fusion protein emulsified in Freund's complete adjuvant, followed 2 weeks later by subcutaneous injection of 1 mg of fusion protein in Freund's incomplete adjuvant. At 6, 10, and 14 weeks after the initial injection, rabbits were boosted with 1 mg of fusion protein in PBS containing a 10 mg/ml suspension of Al(OH)3. Serum was collected before the initial injection (preimmune serum) and 7–10 days after each boost. Affinity purification of anti-TcHAf antibody was performed using cyanogen bromide-activated matrices. Briefly, purified TcHAf fusion protein was coupled in 0.1 m NaHCO3 buffer containing 0.5 m NaCl, pH 8.5, and mixed with cyanogen-bromide activated resin for 2 h at room temperature. After being blocked with 0.2 m glycine, pH 8.0, for 2 h at room temperature and washed extensively with basic coupling buffer, pH 8.5, and with 0.1 m acetate buffer, pH 4, containing 0.5 m NaCl, the column was incubated with the anti-TcHAf serum for 1 h at room temperature to bind the specific antibody to the TcHAf protein. Then the column was washed with PBS three times, and the antibody was eluted with elution buffer (1 mm EDTA, 0.1 m glycine, pH 2.8) supplemented with azide to a final concentration of 0.05% and stored at 4 °C. Samples of yeast fractions (10 μg of protein) were mixed with 10 μl of 125 mm Tris-HCl, pH 7, 10% w/v β-mercaptoethanol, 20% w/v glycerol, 4.0% w/v SDS, and 4.0% w/v bromophenol blue as tracking dye and boiled for 5 min before application to SDS-polyacrylamide gels (10%). Electrophoresed proteins were transferred to nitrocellulose with a Bio-Rad transblot apparatus. After transfer, the nitrocellulose was blocked in 5% nonfat dry milk in 0.1% Tween 20-PBS overnight (Tween-PBS) at 4 °C. A 1:10,000 dilution of affinity-purified antiserum in Tween-PBS was then applied at room temperature for 60 min. The nitrocellulose was washed three times for 15 min each with Tween-PBS and incubated with secondary antibody (1:20,000) at room temperature for 60 min. Immunoblots were visualized on radiographic film using the ECL enhanced chemoluminescence detection kit and according to the instructions of the manufacturer (Amersham Biosciences). ATP-driven proton transport into proteoliposomes reconstituted from plasma membrane preparations was measured by following spectral changes in acridine orange absorbance using a method described previously (29Scott D.A. de Souza W. Benchimol M. Zhong L., Lu, H.-G. Moreno S.N.J. Docampo R. J. Biol. Chem. 1998; 273: 22151-22158Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 30Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), with the replacement of pyrophosphate by 1 mm ATP. In addition, the assay buffer contained 5 mm sodium azide and 100 nmbafilomycin A1 to suppress mitochondrial and vacuolar H+-ATPase activities, respectively (8Villalba J.M. Palmgren M.G. Berberián G.E. Ferguson C. Serrano R. J. Biol. Chem. 1992; 267: 12341-12349Abstract Full Text PDF PubMed Google Scholar, 31Dröse S. Altendorf K. J. Exp. Biol. 1997; 200: 1-8Crossref PubMed Google Scholar), and 50 mm potassium nitrate to provide a membrane-permeant anion (28Serrano R. Methods Enzymol. 1988; 157: 533-544Crossref PubMed Scopus (233) Google Scholar). The mixture for assaying ATP hydrolysis activity in plasma membrane preparations was similar to that of Villalba et al. (8Villalba J.M. Palmgren M.G. Berberián G.E. Ferguson C. Serrano R. J. Biol. Chem. 1992; 267: 12341-12349Abstract Full Text PDF PubMed Google Scholar): 50 mm MES, adjusted to pH 6.5 with Tris (or between pH 5.75 and pH 7.5 for pH optimum studies), 5 mm MgSO4, 50 mm KNO3, 5 mm sodium azide, 2 mm sodium molybdate, and 2 mm ATP. Assays were done at room temperature in microtiter plate wells in a volume of 50 μl containing 4 μg of plasma membrane protein. At intervals, 50 μl of 12% SDS was added to stop the reaction in individual wells. Color development to measure free phosphate was then done as per Chifflet et al. (32Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar). The plate was read at 800 nm on a Power Wave 340i microplate reader (Bio-tek Instruments, Winooski, VT) and calibrated using phosphate standards. For estimation of Km for ATP, ATP concentrations in the range 0.05–10 mm were used, and the assay mixture contained additionally 5 units/ml pyruvate kinase and 2 mm phosphoenolpyruvate as an ATP-regenerating system. MgSO4 concentrations in these assays were increased to 10 mm. Km values were calculated using the Solver function in MS Excel to calculate sum of least squares in fitting the Michaelis-Menten equation to the experimental data (for methodology, see orion1.paisley.ac.uk/kinetics/contents.html). Fixation and immunofluorescence microscopy of yeast cells were performed as described by Pringle et al. (33Pringle J.R. Adams A. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (601) Google Scholar). Permeabilization was accomplished by immersion in methanol at −20 °C for 6 min and then in acetone at −20 °C for 30 s. A 1:100 dilution of affinity-purified antibody against the 35-kDa expressed protein in PBS was applied at room temperature for 30 min, and a fluorescein isothiocyanate-coupled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:150) was then applied at room temperature for 30 min. Control preparations were incubated with preimmune serum. Slides were observed using an Olympus BX-60 microscope, and digital images were obtained using the system described previously (34Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar). Fetal and newborn calf serum, Dulbecco's PBS, EGTA, sodium o-vanadate, diethylstilbestrol,N,N′-dicyclohexylcarbodiimide, sodium deoxycholate, soybean phospholipids (type IIS phosphatidylcholine), proteinase K, RNase A, Tween 20, cyanogen bromide-activated matrices, RNase A, leupeptin,trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64), N α-p-tosyl-l-lysine chloromethyl ketone (TLCK), poly-l-lysine-treated slides, and protease inhibitor mixture (P-8340) were purchased from Sigma. Pepstatin came from Roche Molecular Biochemicals. Glass beads were from Biospec (Bartlesville, OK). Fluorescein-labeled antibodies were from Molecular Probes, Inc. (Eugene, OR). Trizol reagent, SuperScript PCR buffer, SuperScript II reverse transcriptase, the DNA ladder, pCR2.1TOPO cloning kit, and Taq polymerase were from Invitrogen (Carlsbad, CA). The bacteriophage vector λGEM11, host strain LE392, the Packagene System, λEMBL3 phage, restriction enzymes, the poly(A) tract mRNA isolation system, and pGEM-T vectors were from Promega (Madison, WI). Sequenase was from U. S. Biochemical Corporation. The pET-28a+ expression system, the His.Bind kit, and the E. coli DE3 strain were from Novagen (Madison, WI). [α-32P]dCTP (3000 Ci/mmol) was from Amersham Biosciences. Zeta-Probe GT nylon membranes, prestained molecular mass standards, and the protein assay were from Bio-Rad. Pfu polymerase was from Stratagene (La Jolla, CA). All other reagents were analytical grade. Degenerate oligonucleotides corresponding to two conserved domains of P-type ATPases, a phosphorylation site and a site involved in ATP binding (23Allen G. Green N.M. FEBS Lett. 1976; 63: 188-192Crossref PubMed Scopus (69) Google Scholar, 24Pick U. Bassilian S. FEBS Lett. 1981; 123: 127-130Crossref PubMed Scopus (104) Google Scholar), were used to amplify, by PCR, specific sequences from T. cruzi genomic DNA. The PCR products were cloned and sequenced. Analysis of the deduced partial amino acid sequences of these clones revealed that a 0.78-kb PCR clo
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