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J. Biol. Chem., Vol. 277, Issue 46, 44497-44506, November 15, 2002

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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^*

Shuhong Luo, David A. Scott, and Roberto Docampo

From the Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

Received for publication, March 7, 2002, and in revised form, August 30, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, whereas TcHA2 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 the T. cruzi life cycle.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). A sequence analysis of conserved core sequences of all P-type ATPases has grouped them in five subfamilies designated types I-V (2). 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 (2). Because the L. donovani sequences (LHA1A and LHA1B) are obviously distinct from the plant and yeast H⁺-ATPase sequences, some authors (3) have raised the question of whether they are indeed H⁺-ATPases, as was inferred from sequence homology (4-7). 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 (2). Among P-type H⁺-ATPases, this has been done until now only with plant and fungal transporters (8-12). 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 (13, 14). 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 (15), although PMA2 is expressed at very low levels and is not essential for growth (16).

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 (17). Chagas' disease remains a problem because of limited therapeutic choices and adverse reactions to the two drugs available, nifurtimox and benznidazole (17, 18). 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 of T. 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Culture Methods-- T. cruzi amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L₆E₉ myoblasts as described before (19). T. cruzi epimastigotes (Y strain) were grown at 28 °C in liver infusion tryptose medium (20) supplemented with 10% heat-inactivated newborn calf serum.

PCR Amplification-- Genomic DNA isolation and genomic DNA library construction were done as described (21, 22). Oligonucleotide primers were designed to recognize the ATP phosphorylation site and the ATP binding site of cationic ATPase genes (23, 24), 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.

Library Screening-- 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 [-³²P]dCTP-labeled probes according to standard procedures. Positive plaques identified in the screen were serially plaqued to homogeneity.

Southern and Northern Hybridization-- Southern and Northern hybridization were done by standard procedures (21). 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 mM sodium acetate, 5 mM EDTA, 100 mM MOPS,¹ pH 8.0. Northern hybridization was done by standard procedures (21) using probes a (218 bp), b (89 bp), c (780 bp), d (1,035 bp), and e (1,542 bp) (see Fig. 1). The TcP0 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 (25). 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 (25). 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.

Reverse Transcription (RT)-PCR-- 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.

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).

Expression of Complete and Truncated TcHA1 and TcHA2 in Yeast-- Plasmids pMP625, derived from YEp351 (26) and containing the promoter and terminator of PMA1, and pRS890 (8), 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 5-1 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 and SpeI 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 (TcHA148/pMP625), pRD204 (TcHA240/pMP625), and pRD205 (TcHA2N-50/pMP625) with the right orientation for expression. All PCR amplifications were carried out using Pfu 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.

Yeast Strains and Culture Conditions-- 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 (26). 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 mM adenine, 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. (27). 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 mM succinic acid adjusted to pH 5.5 (or other pH values in pH growth experiments) with Tris. Solid media contained 2% agar (Difco).

Yeast Lysis and Plasma Membrane Preparation-- Yeast strain RS1002, RD2011, or RD2055 grown in 300 ml of SDAH to an A₆₀₀ 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 mM Hepes, 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 mM MgCl₂, 1 mM EGTA, 5 mM dithiothreitol, 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 (28). This fraction was recovered from the 43/53% interface, diluted 5× in water, and centrifuged at 80,000 × g for 20 min. Pellets were resuspended in glycerol buffer and stored at 80 °C before use.

Reconstitution of Functional H⁺-ATPases-- 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. (10). 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.

Preparation and Purification of Antibodies-- 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 and BamHI. The fragment was then subcloned into the BamHI 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.

SDS Electrophoresis and Preparation of Western Blots-- Samples of yeast fractions (10 µg of protein) were mixed with 10 µl of 125 mM Tris-HCl, pH 7, 10% w/v -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).

Proton Transport Assays-- 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 (29, 30), with the replacement of pyrophosphate by 1 mM ATP. In addition, the assay buffer contained 5 mM sodium azide and 100 nM bafilomycin A₁ to suppress mitochondrial and vacuolar H⁺-ATPase activities, respectively (8, 31), and 50 mM potassium nitrate to provide a membrane-permeant anion (28).

ATPase Assays-- The mixture for assaying ATP hydrolysis activity in plasma membrane preparations was similar to that of Villalba et al. (8): 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. (32). 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 K_m 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. K_m 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).

Immunofluorescence Microscopy-- Fixation and immunofluorescence microscopy of yeast cells were performed as described by Pringle et al. (33). 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 (34).

Chemicals-- 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-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 GEM11, host strain LE392, the Packagene System, EMBL3 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). [-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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequencing of P-type H⁺-ATPase Genes from T. cruzi-- Degenerate oligonucleotides corresponding to two conserved domains of P-type ATPases, a phosphorylation site and a site involved in ATP binding (23, 24), 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 clone (TcHAf) had the best scores of sequence identity (83%) and similarity (90%) with the putative H⁺-ATPase genes LDHA1A and LDHA1B from L. donovani (4-7).

Southern blotting was performed with TcHAf as a probe to confirm the presence of this gene in the T. cruzi genome (data not shown). Most restriction enzymes used produced multiple hybridization bands. This suggested that TcHA was present as a multiple copy gene. Restriction enzymes HindIII, KpnI, and SacI gave bands of similar sizes, which is characteristic of the presence of tandem repeated genes.

To obtain complete gene(s), TcHAf was used as a probe to screen a genomic library of T. cruzi in bacteriophage vector GEM11 (22). 35 positive plaques were identified in the screen, and one (phage 5-1) was selected for further characterization. DNA from 5-1 was analyzed by restriction endonuclease digestion and hybridization with radiolabeled TcHAf. Digestion with SacI produced ~2.9-, ~3.8-, and ~5.5-kb fragments. The restriction enzyme cleavage pattern of 5-1 was identical to the pattern predicted by Southern analysis of T. cruzi genomic DNA (data not shown). The fragments of ~3.8 and ~5.5 kb strongly hybridized with TcHAf. We purified these fragments and subcloned them into the pBluescript II KS() vector for sequence analysis and mapping. This revealed two similar complete open reading frames, designated TcHA1 and TcHA2, and two small partial open reading frames at either end (Fig. 1). One of these partial sequences was identical to the C-terminal 21 nucleotides of TcHA2, and the other was identical to the N-terminal 48 nucleotides of TcHA1 (Fig. 1), suggesting that TcHA is present as a tandem repeat of more than two copies. Genes repeated in tandem with a high degree of sequence homology are frequent in trypanosomatids (7) including T. cruzi (35, 36). There are two linked repeated regions, designated repI and repII, in noncoding regions of the sequence (Fig. 1). The nucleotide sequence from the 5-1 clone was deposited in GenBank^TM with accession number AF254412.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Physical map of the H+-ATPase gene locus. Map of the DNA sequence of the ATPase gene locus from 5-1. Top line, SacI digestion pattern, with arrowheads indicating 21-bp fragments cut out by SacI. The RT-PCR products are shown as lines of 600 and 500 bp below the fragments. Lower drawing, solid boxes represent predicted protein coding sequences. Bent arrows show spliced leader acceptor sites or poly(A) sites. The shaded and open boxes show repeated noncoding regions I (repI) and II (repII), respectively. The lines marked a-e, above the drawing, indicate the DNA probes prepared to analyze gene expression (Fig. 3). The two transcripts of 3.86 and 5.35 kb identified by 5'-RACE and 3'-RACE mapping are also indicated.

To confirm the transcription of the TcHA genes and the sequence of the 5'-end of the transcripts, RT-PCR was performed as described under "Experimental Procedures," using the spliced leader sequence as a primer (Tc-5'-SL) and a specific primer for both isoforms (RTP3'2). Sequence analysis of the RT-PCR products (bands of 600 and 500 bp, respectively, Fig. 1), indicated that they were derived from the TcHA genes and that the predicted translation initiation sites of TcHA1 and TcHA2 were preceded by 312 and 40 bp of 5'-untranslated sequence, respectively. The remainder of the sequence of the PCR products was identical to the 5'-ends of the coding sequences of the respective genes (Fig. 1).

Structure of the Coding Region of the TcHA Genes-- TcHA1 and TcHA2 have open reading frames of 2,625 and 2,751 bp, predicted to encode proteins of 875 and 917 amino acids, with molecular masses of 96.3 and 101.2 kDa, respectively. These sizes are consistent with those reported for other proton pumps in plants and fungi (12) and with the size of a polypeptide present in plasma membrane preparations of T. cruzi epimastigotes that forms an acylphosphate intermediate (37). A BLASTP search of protein data bases showed that TcHA1 and TcHA2 were closely related to the putative P-type H⁺-ATPases from other trypanosomatids. TcHA1 and TcHA2 have 80 and 85% identity and 87 and 91% similarity to the putative H⁺-ATPase from L. donovani and to the sequence of a putative H⁺-ATPase from T. brucei (AF145721 and chromosome 10: TRYP10.0.001893, from the T. brucei genome data base, www.sanger.ac.uk/Projects/T_brucei/), respectively. The next highest BLAST matches were identified as putative H⁺-ATPases from plants, fungi, various algae, the slime mold Dictyostelium, and the apicomplexan parasite Toxoplasma (29-35% identity; 46-53% similarity). Hydropathy analysis revealed a profile very similar to those of other P-type ATPases, with 10 transmembrane domains, as marked in Fig. 2. The sequence contains two motifs common to all P-type ATPases, which were the basis of the original PCR primers (underlined in Fig. 2). The first of these is DKTGT[LIVM][TIS] (Prosite motif PS00154; www.expasy.org/prosite), which starts with the aspartate (D) that is phosphorylated during substrate transport. The second of these is GDG-ND (2), the hinge sequence linking the large cytosolic domain to the C-terminal, membrane-associated domain of P-type ATPases (13). The TcHA sequences also contain all of the amino acid residues and short peptides that are common to type IIIA P-type ATPases but are not preserved in other subgroups of P-type ATPases (boxed residues in Fig. 2; Ref. 2).

View larger version (103K): [in this window] [in a new window]   Fig. 2.   ClustalW alignment of putative H+-ATPase amino acid sequences from T. cruzi (TcHA1 and TcHA2), T. brucei (TbHA1, chromosome 10: TRYP10.0.001893, www.sanger.ac.uk/Projects/T_brucei/), L. donovani (LDH1A, AF109296), and S. cerevisiae (PMA1, Z72530). Identical residues are shaded. Amino acid residues absent from other sequences are denoted by dashes. The amino acid sequences corresponding to the conserved catalytic autophosphorylation and ATP binding domains employed for the design of degenerate oligodeoxyribonucleotides for PCR are underlined. Transmembrane domains (I-X) and potential N-glycosylation sites are indicated by dashed lines and asterisks above the alignment, respectively. Boxes are motifs specific to type IIIA P-type ATPases. Arrowheads show conserved residues that have been studied by site-directed mutagenesis (38, 39).

The T. cruzi proteins also conserve a number of amino acids known, from site-directed mutagenesis studies, to have a role in other H⁺-ATPases. Mutation of the S. cerevisiae Gly¹⁵⁸ residue (residues Gly⁷⁸ and Gly¹²⁸ in TcHA1 and 2, respectively; first arrowhead in Fig. 2) confers a hygromycin resistance phenotype (38). Mutation of Asp⁷³⁰ in S. cerevisiae (T. cruzi Asp⁶⁵⁹ and Asp⁷⁰⁹; second arrowhead in Fig. 2) abolishes ATPase activity and proton transport (39). The combined presence of these features suggests a close relationship of the T. cruzi enzymes to the fungal and plant group of proton-pumping ATPases.

There are two potential glycosylation sites (Asn-Aln-Thr, Asn-Tyr-Thr) present in TcHA1 (amino acids 74-76 and 358-360) and TcHA2 (asterisks above the alignment in Fig. 2). The 8-amino acid extension at the C terminus of TcHA1 creates a third potential glycosylation site (Asn-Glu-Ser). The significance of this is unknown. There is no evidence at this time as to whether any of these sites are glycosylated in the T. cruzi ATPases.

3'-RACE mapping using primers specific for sequences in TcHA1 (nucleotides 5490-5507) and TcHA2 (nucleotides 10407-10423), 1062 nucleotides downstream from the stop codon, amplified fragments of 1035 and 1542 nucleotides, respectively, which mapped the TcHA1 and TcHA2 polyadenylation sites to GAA trinucleotides 6432-6434 and 11902-11904, as well as the presumptive polyadenylation site for the upstream gene at nucleotides 2443-2445 (Fig. 1, lower drawing). Sequence analysis of the 3'-RACE products indicated that they were derived from the TcHA genes, and the polyadenylation sites (nucleotides 6433 and 11903) of TcHA1 and TcHA2 were preceded by 920 and 2558 nucleotides of 3'-untranslated sequences, respectively (Fig. 1, lower drawing).

Expression of T. cruzi P-type ATPase Genes-- To analyze the expression of the T. cruzi H⁺-ATPase genes, DNA probes were prepared from five regions (see Fig. 1). Probes a and b covered the upstream regions or part of the coding regions of each of the two proteins; probe c (TcHAf) covered the center, almost identical, part of the coding region of both proteins; and probes d and e covered part of the coding region or the downstream region of each of the two proteins. The probes were hybridized to Northern blots of T. cruzi mRNA. Probe c, which would hybridize to transcripts from either ATPase gene, revealed the existence of two main transcripts of 3.86 and 5.35 kb (Fig. 3, panel 1). Probes that would hybridize to transcripts from TcHA1 (probes a, panel 2, and d, panel 4) detected mainly the 3.86-kb band, whereas probes that would hybridize to transcripts from TcHA2 (probes b, panel 3, and e, panel 5) detected mainly the 5.35-kb band. These messages were present in all T. cruzi stages but at different levels. The 3.86-kb transcript (TcHA1) was more abundant in epimastigotes than in trypomastigotes and amastigotes, whereas the 5.35-kb transcript (TcHA2) was more abundant in trypomastigotes. Bands obtained after hybridization with a PCR product of the TcP0 gene, which is expressed at similar levels in the three stages of T. cruzi (25), were used as a reference control (Fig. 3, panel 6).

View larger version (68K): [in this window] [in a new window]   Fig. 3.   Expression of TcHA mRNA in different stages of T. cruzi. Poly(A) RNA (3 µg/lane) isolated from amastigotes (A), epimastigotes (E), or trypomastigotes (T) was electrophoresed, blotted, and probed at high stringency with different 32P-labeled probes (a-e, see Fig. 1) prepared as described under "Experimental Procedures." Equal amounts of mRNA were observed under UV light in each lane. The membrane was probed, then stripped and reprobed with 32P-labeled probes c, a, b, d, e and TcP0 in panels 1, 2, 3, 4, 5, and 6, respectively. Exposure time was 72 h except for panel 6 (5 h).

Complementation of Yeast Mutants by TcHA1 and TcHA2-- To investigate whether TcHA1 and TcHA2 encode functional proton pumps we expressed them in mutant yeast (8). The yeast H⁺-ATPase gene PMA1 is essential and rate-limiting for growth (40); therefore, a mutant strain, RS-72, which has PMA1 under the control of a galactose-dependent promoter, cannot grow on glucose medium (41). This strain may then be transformed with a yeast multicopy vector carrying a heterologous H⁺-ATPase gene under the control of a constitutive PMA1 promoter. On galactose medium the transformed yeast strain expresses both PMA1 and the heterologous H⁺-ATPase, whereas on glucose medium growth is dependent on the heterologous H⁺-ATPase alone. When this method was used to express genes encoding Arabidopsis H⁺-ATPases, it was found that removal of a region encoding a C-terminal autoinhibitory domain of the protein was necessary to complement the yeast H⁺-ATPase genetically. The autoinhibitory domain (in Arabidopsis AHA2, ⁸⁶¹AFTMKKDYGKEEREAQWALAQRTLHGLQ⁸⁸⁸) may be modulated by proteolytic removal, lysolecithin binding, fusicoccin binding, or changes in phosphorylation (1, 42). TcHA1 and TcHA2 are 32-35% identical to the plant H⁺-ATPase AHA2, and although they lack an extended C-terminal region they have some of the amino acids of the highly conserved autoinhibitory domain in their C-terminal regions (Fig. 4C). Therefore, four expression vectors were constructed to express the complete and C-terminal truncated forms of TcHA1 and TcHA2 in the yeast strain RS-72. We investigated the ability of these strains to grow on glucose medium and galactose medium at the optimal pH, 5.5. The results are shown in Fig. 4. The positive control (the strain RS1002 with the yeast PMA1 under its own promoter) grew well at pH 5.5 on both media (Fig. 4A, lane 7). The negative control (the strain MP625 with the yeast PMA1 under the GAL1 promoter) grew well in galactose medium only (Fig. 4A, lane 6). The strains RD2033 and RD2044 with TcHA2 and TcHA240 (truncated form) did not support yeast growth on glucose medium (Fig. 4A, lanes 3 and 4), but strains RD2011 and RD2022 with the expression of the complete and truncated (48) forms of TcHA1 complemented the yeast H⁺-ATPase (Fig. 4A, lanes 1 and 2). Therefore, TcHA1 apparently lacks an autoinhibitory domain in the C-terminal region because the complete form of TcHA1 supported yeast growth as well as the truncated form in glucose medium. In liquid medium, the growth properties of strains RD2011 and RD2022, and RD2033 and RD2044 were comparable with RS1002 and MP625, respectively, when 10³ cells/5 ml were inoculated, although growth supported by TcHA1 (full-length or truncated) was somewhat lower than in yeast expressing the homologous yeast H⁺-ATPase (Fig. 4B). Similar results were obtained when 10⁴ or 10⁵ cells/5 ml were inoculated (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Expression of TcHA in yeast. A, drop test for the growth of yeast strains on galactose (SGAH) or glucose (SDAH) medium. In SGAH, all strains (except MP625 and RS1002) could potentially express both yeast PMA1 and transfected TcHA genes or truncated genes. In SDAH, only the TcHA constructs could be expressed (except in RS1002). Lane 1, strain RD2011 (expressing TcHA1); lane 2, strain RD2022 (expressing TcHA148); lane 3, strain RD2033 (expressing TcHA2); lane 4, strain RD2044 (expressing TcHA240); lane 5, strain RD2055 (expressing TcHA2N-50); lane 6, control strain MP625 (expressing yeast PMA1 only on galactose medium); lane 7, control strain RS1002 (expressing yeast PMA1 on both media). Cells were grown to saturation on galactose medium, and about 103 cells in 5 µl were spotted on agar plates containing medium as indicated. Growth was recorded after 4 days at 30 °C. B, growth of yeast strains in liquid media. Cells were grown to saturation in SGAH, and about 103 cells in 5 µl were inoculated into 5 ml of SGAH or SDAH. Growth was estimated by measuring the optical density at 660 nm after 4 days at 30 °C. The data shown are the means ± S.D. of five independent experiments. C, ClustalW alignment of the C-terminal region of putative H+-ATPases from T. cruzi (TcHA1 and TcHA2, GenBankTM accession number AF254412), Arabidopsis thaliana (AHA1, P20649; AHA2, P19456; and AHA3, P204310). Identical residues are shaded. Amino acid residues not present within other sequences are denoted in dashes. The autoinhibitory domain is underlined. The dashed line above the alignment shows the transmembrane domain X. The box shows the YTV motif.

We speculated that the failure of TcHA2 (full-length or C-terminal truncated) to complement the H⁺-ATPase mutant yeast was because of the presence of a 50-amino acid extension at the N terminus which is absent in TcHA1 (Fig. 2). This is the only major difference between TcHA1 and TcHA2, especially in the C-terminal truncated forms. To investigate the possibility that this N-terminal fragment could be affecting the functional expression of TcHA2 in RS-72 yeast or the targeting of the protein to the yeast plasma membrane, an N-terminal, 50-amino acid, deletion mutant of TcHA2 was generated by PCR and transfected into yeast to produce strain RD2055. Yeast growth was fully supported by this modified TcHA2. The growth properties of RD2055 were very similar to those of RD2011, expressing full-length TcHA1, at the optimal pH (5.5) (Fig. 4, lane 5).

To observe the growth properties of RD2011 (expressing the full-length TcHA1) and RD2055 (expressing the N-terminal truncated TcHA2) at different pH levels, they were grown in SDAH medium at pH 3.0, 4.0, 5.0, 6.0, and 7.0. The results indicated that there was no significant difference in the growth properties among the strains at pH 4.0, 5.0, and 6.0 (Fig. 5). At a lower (3.0) or higher pH (7.0), however, RD2055 grew better than RD2011, suggesting TcHA2 may be better able to sustain growth at extreme pH values.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Growth at different pH levels of yeast cells expressing the full-length TcHA1 and N-terminal deletion form of TcHA2. The yeast strains RS1002, MP625, RD2011, and RD2055 (for definitions, see Fig. 4 legend) were grown to saturation in SGAH medium at pH 5.5. A, yeast (103 in 5 µl) spotted onto solid SDAH at pH 3.0, 4.0, 5.0, 6.0, and 7.0. Growth was recorded after 4 days at 30 °C. B, same inoculum into 5 ml of liquid SDAH at pH 3.0, 4.0, 5.0, 6.0, and 7.0. Growth was estimated by measuring the optical density at 660 nm after 4 days at 30 °C. The data shown are the means ± S.D. of three independent experiments.

Localization of TcHA1 in Yeast Plasma Membrane-- The localization of TcHA1 and the N-terminal truncated TcHA2 in the yeast plasma membrane was confirmed by their immunological detection in formaldehyde-fixed yeast spheroplasts permeabilized with methanol and acetone (Fig. 6). A strong positive reaction was detected with polyclonal antibody against the T. cruzi H⁺-ATPase in cells grown in both glucose (Fig. 6, right panel) and galactose (Fig. 6, left panel) medium. In contrast, only weak reactions were observed in the control strain RS1002, probably reflecting some cross-reaction with the yeast H⁺-ATPase. A predominantly intracellular reaction was detected with polyclonal antibody against T. cruzi H⁺-ATPase in yeast transformed with TcHA2 or TcHA240 and grown in galactose. Expression of these forms was confirmed by Western blotting analysis of yeast homogenates (data not shown), suggesting that these proteins remain trapped at an early stage of the secretory pathway or are trafficked to an intracellular compartment.

View larger version (58K): [in this window] [in a new window]   Fig. 6.   Immunofluorescence staining of yeast cells. Yeast were grown in galactose (SGAH, left panel) or glucose (SDAH, right panel) medium. Lower panels show bright field images of the same cells. Yeast strains are as in Fig. 4. Bar, 5 µm.

Western blot analysis of yeast extracts demonstrated that T. cruzi H⁺-ATPases TcHA1 (in strain RD2011) and N-terminal truncated TcHA2 (in strain RD2055) were expressed (Fig. 7). Two bands of size 108 and 100 kDa were observed in total homogenates of T. cruzi epimastigotes (Tc in Fig. 7), 6 and 4 kDa greater than the expected molecular mass of the two H⁺-ATPase isoforms, respectively. The molecular masses of the bands from RD2011 and RD2055 were 104 (arrowhead) and 96 (arrow) kDa, respectively, compared with expected values of 96 and 95 kDa. All experimental values were close to the expected values, although part of the observed differences may be caused by alternative post-translational modifications of the proteins in the different cells. In yeast expressing its own H⁺-ATPase (RS1002) there was no reaction except for a weak cross-reacting band of ~70 kDa which was present in all samples.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   Expression of TcHA1 and TcHA2 in yeast. Samples (10 µg of protein/lane) of total lysate (Lys), plasma membrane fraction from 43/53% sucrose gradient interfaces (Plm), and 20,000 × g pellet (Pel) from RS1002, RD2011, and RD2055 and total cell homogenates from T. cruzi epimastigotes (Tc) were subjected to SDS-PAGE on 10% gels and transferred to nitrocellulose membranes. Membranes were probed with affinity-purified anti-TcHAf antibody prepared as described under "Experimental Procedures." Migration positions of prestained molecular mass standards are shown to the right of the blots.

Analysis of T. cruzi H⁺-ATPase Activity in Yeast-- H⁺-ATPase activity in transformed yeast was analyzed both by proton transport assay and assay of ATPase activity. Proton transport activity was detected in preparations of plasma membrane reconstituted into proteoliposomes (Fig. 8; a traces indicate uninhibited activity in preparations from yeast RS1002 (A), RD2011 (B), and RD2055 (C)). Reduction in absorbance of acridine orange using the wavelength pair 493-530 nm indicates acidification of vesicles in the preparation. This acidification was reversed by the addition of 10 mM NH₄Cl, which, being a weak base, accumulates in the vesicles and neutralizes the acidity. The activity was inhibited by the H⁺-ATPase inhibitors N,N'-dicyclohexylcarbodiimide (50 µM), diethylstilbestrol (5 µM), and o-vanadate (10 µM) (43) (Fig. 8, traces b-d, respectively). Inhibition by these compounds was measured in proton transport assays using reconstituted H⁺-ATPase from three preparations of each type of yeast. These data are summarized in Table I. In all cases, using the above concentrations of these inhibitors, the degree of inhibition was in the order vanadate > diethylstilbestrol > N,N'-dicyclohexylcarbodiimide. The N-terminal truncated TcHA2 (in strain RD2055) was less inhibited by N,N'-dicyclohexylcarbodiimide than the other H⁺-ATPases, however (p < 0.01 compared with the yeast activity (strain RS1002) by t test).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   ATP-driven proton transport into proteoliposomes prepared from plasma membrane vesicles purified from H+-ATPase-deficient yeast transfected with yeast or T. cruzi H+-ATPase genes; effects of inhibitors. Each assay contained proteoliposomes prepared from 32 µg of plasma membrane protein. A decrease in absorbance indicates acidification of membrane vesicles. A, yeast strain RS1002 (yeast H+-ATPase PMA1); B, yeast strain RD2011 (T. cruzi H+-ATPase TcHA1); C, yeast strain RD2055 (T. cruzi H+-ATPase N-terminal truncated TcHA2). In each panel, trace a is the control; trace b assays include 50 µM N,N'-dicyclohexylcarbodiimide; trace c, 5 µM diethylstilbestrol; and trace d, 10 µM o-vanadate. In the case of diethylstilbestrol and N,N'-dicyclohexylcarbodiimide, the inhibitor was added to the assay mixture 5 min before the addition of ATP. 1 mM ATP and 10 mM NH4Cl were added at the points indicated by arrows. The figure is representative of the activity obtained in different experiments, except that in A is unusually high.

To quantify the H⁺-ATPase activity better and to try to distinguish the T. cruzi and yeast activities further, ATPase (phosphate release) assays were done on the plasma membrane fractions. It was found that the ATPase activity in these fractions was sensitive to vanadate to different extents, with the T. cruzi TcHA1 activity significantly less sensitive than the yeast PMA1 enzyme over the range of 1-20 µM (IC₅₀ 10 µM versus 2 µM), whereas the truncated TcHA2 form was almost identical in its sensitivity (Fig. 9). Note that in this figure, the maximal inhibition obtained was ~87%, indicating that there was some minor ATPase or phosphatase activity in the preparation which was not sensitive to vanadate or the other ATPase/phosphatase inhibitors (azide, nitrate, and molybdate) included in the assay mixture. The K_m and V_max for ATP for the H⁺-ATPases were determined in assays over the range of [ATP] 0.05-10 mM. Illustrative results are shown in Fig. 10, and the results summarized from assays of three preparations of each type of transformed yeast are shown in Table II. Kinetic data for all three H⁺-ATPases were similar.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Differential sensitivity to vanadate of ATPase activity in plasma membrane vesicles from transformed yeast. Vanadate sensitivity of ATPase activity in plasma membrane fractions from H+-ATPase-deficient yeast transfected with TcHA1 (diamonds) or N-terminal truncated TcHA2 (triangles), or yeast PMA1 (squares) is shown. TcHA1 activity was significantly less sensitive to vanadate than the yeast activity at 1, 5, 10, and 20 µM (p < 0.01, 0.002, 0.02, and 0.05, respectively, by t test). Data points represent the average percent inhibition of ATPase activity from three to five separate preparations; error bars are S.D. ATPase specific activity (in µmol/min/mg of protein) was in the range 0.23-0.61 for the TcHA1-transfected yeast, 0.10-0.34 for the truncated TcHA2-transfected yeast, and 0.12-0.47 for the PMA1-transfected yeast (strain RS1002).

View larger version (11K): [in this window] [in a new window]   Fig. 10.   Plots of ATPase activity versus total ATP concentration for plasma membrane preparations from yeast transformed with different H+-ATPases. Data points are shown for H+-ATPases: yeast expressing T. cruzi TcHA1 (filled diamonds) or N-terminal truncated T. cruzi TcHA2 (open squares), or yeast PMA1 (open triangles). Lines fitted to the data by least squares methodology using the Michaelis-Menten equation are continuous (TcHA1), long dashes (TcHA2), or short dashes (PMA1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our laboratory has previously reported a major role for a plasma membrane H⁺-ATPase in the regulation of intracellular pH in different stages of T. cruzi (19, 44). In this work, we have demonstrated that a pair of genes linked in tandem encoding proteins with homology to P-type H⁺-ATPases are present in the T. cruzi genome (TcHA1 and TcHA2). TcHA1 and an N-terminal truncated version of TcHA2 could complement a yeast strain deficient in H⁺-ATPase, providing evidence that they encode functional proton pumps. Use of antibodies to a region of the P-type H⁺-ATPase common to the two proteins revealed the plasma membrane localization of both proteins in the transformed yeast (Figs. 6 and 7). Although very similar to TcHA1, the full-length TcHA2 was not able to complement yeast deficient in H⁺-ATPase, and the protein product appeared to be located intracellularly. This may represent mistargeting, or trapping at an early stage of the secretory pathway in yeast, of a protein that is expressed on the cell surface in T. cruzi. Such "trapping" occurs with the Arabidopsis AHA2 H⁺-ATPase isoform when it is expressed in yeast (8). The N-terminal extension of TcHA2 may prevent interaction with yeast proteins required for packaging of plasma membrane H⁺-ATPase into COPII vesicles for export from the endoplasmic reticulum (12), or association with lipid rafts for transport from the Golgi to the plasma membrane (46). An alternative, more interesting explanation, is that the TcHA2 isoform has targeting information in the N-terminal section, which directs it to an intracellular location in the T. cruzi cell (and also yeast). Previously, we found evidence for intracellular P-type H⁺-ATPase activity in T. cruzi (47), and P-type H⁺-ATPase activity has been found associated with the endoplasmic reticulum of certain plant cells (48). Work is in progress to identify the subcellular localization of TcHA1 and TcHA2 in different stages of T. cruzi.

Phylogenetic analysis (39) of the family of P-type H⁺-ATPases aligns TcHA1 and TcHA2 with a cluster that includes the Leishmania and T. brucei putative proton pumps. Like the L. donovani putative proton pumps, the T. cruzi ATPases lack the C-terminal peptide present in yeast PMA1 which constitutes the nonessential inhibitory domain involved in the regulation of the enzyme by glucose metabolism (38). Likewise, the C-terminal regulatory domain present in plant H⁺-ATPases is missing. However, TcHA1 (but not TcHA2 or the Leishmania proteins) possesses a C-terminal sequence (Pro-Thr-Val) that is similar to the C-terminal motif (Tyr-Thr-Val), which has been found in several plant H⁺-ATPases (Fig. 4C), and is a 14-3-3 protein binding sequence (1, 49). Phosphorylation of the penultimate residue (Thr) in this motif allows the enzyme to form a stable complex with 14-3-3 regulatory proteins, resulting in activation of the enzyme (49). Studies on 14-3-3 proteins in trypanosomatids have not been described, but partial sequences for 14-3-3-like proteins in T. brucei and T. cruzi are present in the GenBank^TM data base.

While this paper was in preparation, a report describing the cloning of a T. cruzi (Sylvio/X10/7 strain) P-type ATPase gene (TCH3, AF000161) appeared (50). TcHA1 and TcHA2 have 94% identity (95% similarity) to the protein encoded by TCH3. TCH3 has an open reading frame of 2778 bp encoding a protein of 925 amino acids. The main differences among TcHA1, TcHA2, and TCH3 are located in the N-terminal 50 and C-terminal 18 amino acids. Only 24 amino acid differences were observed in the central regions. One amino acid change was located in hydrophobic domain I, but this change did not alter the hydrophobicity of the domain. Similarly, the single amino acid changes in domains III, IV, V, and VIII were also conservative. No studies were reported (50) concerning the function of this protein. This work raises the question, though, as to how many H⁺-ATPase isoforms there are in T. cruzi. Our data suggest that, at least in the Y strain that we used, there are only two forms, which may each be encoded by multiple genes. Although the genomic sequence (Fig. 1) implies that there are at least four genes, the upstream and downstream gene fragments corresponded exactly to TcHA2 and TcHA1, respectively. The RT-PCR experiments yielded only two bands, which derived from TcHA2 and TcHA1, as did extensive Northern blot analysis (Fig. 3). Western blotting of yeast transformed with TcHA1 or truncated TcHA2 (Fig. 7) gave bands with apparent molecular masses slightly different from those obtained from a homogenate of T. cruzi, but this may be caused by alternative post-translational modifications in the yeast and trypanosomes. The Silvio/X10 and Y strains of T. cruzi are from separate phylogenetic branches of the T. cruzi lineage. There are considerable differences in equivalent DNA sequences between the lineages (51), and therefore TCH3 from the Silvio/X10 strain may be a homolog of either TcHA1 or TcHA2.

The expression of TcHA2 and TcHA1, to similar extents (Fig. 3) but in different life cycle stages, implies that both isoforms have significant roles in the growth and survival of T. cruzi. TcHA2 is the H⁺-ATPase expressed predominantly in the trypomastigote stage, which is exposed to drastic changes in environmental pH such as those present in the parasitophorous vacuole or the intestine of the insect vector, and (N-terminal truncated) TcHA2 allowed yeast to grow at more extreme pH than did TcHA1 (Fig. 5). It would be interesting if this reflected an ability in native TcHA2 in T. cruzi to protect against extremes of pH, but this interpretation is tentative, given the truncated nature of the expressed protein and possible alternative post-translational processing (e.g. glycosylation and phosphorylation) of the proteins in yeast, as was noted for plant H⁺-ATPases expressed in yeast (1). Assay of the ATPase activity of the enzymes at different pH values indicated a broad pH optimum between pH 6 and 7 for both T. cruzi H⁺-ATPases, as well as the yeast activity (results not shown).

In conclusion, our work provides strong evidence that T. cruzi possess functional P-type H⁺-ATPases. This is the first report showing conclusive evidence of a plasma membrane P-type H⁺-ATPase in an organism different from plants and fungi. The absence of electrogenic P-type H⁺-ATPases from mammalian cells (2, 13) and their presence in fungi has led to the proposal that these pumps are promising targets for antifungal therapy (45). The present work implies a similar situation in trypanosomatids. Analysis of the role of these proteins in parasite survival and multiplication will determine their suitability as possible drug targets.

	ACKNOWLEDGEMENTS

We thank Drs. Michael Palmgren and R. Serrano for the gifts of yeast plasmids and strain RS-72; Wen Yan, Hong-gang Lu, and Li Zhong for initial help in this project; and Linda Brown for technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AI-23259 (to R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF254412.

To whom correspondence should be addressed: Laboratory of Molecular Parasitology, Dept. of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Ave., Urbana, IL 61802. Tel.: 1-217-333-3845; Fax: 1-217-244-7421; E-mail: rodoc@uiuc.edu.

Published, JBC Papers in Press, September 6, 2002, DOI 10.1074/jbc.M202267200

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES