TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi.

Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a parasitic protozoan that invades mammalian cells and develops intracellularly as amastigotes. Invasion of cells by T. cruzi is dependent upon an elevation in the concentration of cytosolic free calcium in the invading trypomastigote (1,2). Unlike mammalian cells, T. cruzi possesses most of its intracellular Ca 2ϩ in an acidic compartment named the acidocalcisome (3)(4)(5)(6)(7)(8). The molecular and biochemical characterization of this organelle has provided evidence that it has an orthovanadate-sensitive plasma membrane-type Ca 2ϩ ATPase for Ca 2ϩ uptake (3,(5)(6)(7). Evidence for non-mitochondrial and endoplasmic reticulumlocated Ca 2ϩ uptake has been more elusive and based on the presence of a low capacity and high affinity orthovanadatesensitive Ca 2ϩ uptake in permeabilized cells and the ability of these cells to buffer [Ca 2ϩ ] in the range 0.05-0.1 M (9), features in common with the sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPases (SERCA) 1 of animal cells (10). In addition, like mammalian cells (11), calcium is needed for the correct folding and assembly of proteins in the endoplasmic reticulum of T. cruzi, which depends on chaperones such as the Ca 2ϩ -binding protein, calreticulin (12).
Biochemical distinction of the different Ca 2ϩ pumps present in T. cruzi has been hampered by the lack of distinguishing features such as specific inhibitor sensitivity. Orthovanadate inhibits all types of Ca 2ϩ ATPases (10), whereas thapsigargin, a specific inhibitor of animal SERCA-type Ca 2ϩ -ATPases (13), is ineffective in inhibiting Ca 2ϩ uptake in permeabilized T. cruzi (14,15). Thus, a molecular approach to studying individual pumps is necessary. Expression of genes identified as encoding Ca 2ϩ -ATPases and localization of the corresponding proteins is necessary, as the plasma membrane-type Ca 2ϩ ATPase/SERCA paradigm does not necessarily apply in nonanimal cells; a SERCA-type gene in tomato is expressed in different parts of the cell (16). Also, a Tca1 gene has been identified in T. cruzi (6), which encodes a protein with homology to mammalian plasma membrane Ca 2ϩ -ATPases but with characteristics that place it in a novel category of Ca 2ϩ -AT-Pases along with the vacuolar Ca 2ϩ -ATPases described in Saccharomyces cerevisiae (17), Dictyostelium discoideum (18), Entamoeba histolytica (19), and Toxoplasma gondii (20). The gene is expressed at a high level in the amastigote stage and is localized to acidocalcisomes and the plasma membrane of the parasite (6).
Here we demonstrate that a gene from T. cruzi (TcSCA) complemented yeast mutants defective in Ca 2ϩ pumps by restoring their growth in EGTA. The protein encoded by the TcSCA gene localizes to the endoplasmic reticulum of different stages of the parasite and, in contrast to the acidocalcisomal Ca 2ϩ -ATPase (6), it is expressed at similar levels in the different developmental stages of the parasite. Culture Methods-T. cruzi amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L 6 E 9 myoblasts by a modification of the method of Schmatz and Murray (21), as we have described before (14,15). The contamination of trypomastigotes with amastigotes and intermediate forms or of amastigotes with trypomastigotes or intermediate forms was always less than 5% unless otherwise stated. T. cruzi epimastigotes (Y strain) were grown at 28°C in liver infusion tryptose medium (22) supplemented with 10% newborn calf serum. S. cerevisiae strain K616 (MATa pmr1::HIS3 pmc1::TRP1 cnb1::LEU2, ura3) (23) was kindly provided by Kyle W. Cunningham, Department of Biology, The Johns Hopkins University, Baltimore, MD and was maintained in YPD agar plates (1% Difco yeast extract, 2% Bacto Peptone, 2% dextrose, and 2% agar).
Chemicals-Restriction enzymes and protease inhibitor mixture (P-8340) were purchased from Sigma. Yeast media were bought from Bio 101 (Vista, CA). Trizol reagent, reverse transcriptase, and Taq polymerase were from Life Technologies, Inc. The pGEM-T Easy vector, Riboprobe in vitro transcription system and Prime-a-Gene labeling system were from Promega (Madison, WI). The PfuTurbo DNA polymerase, the ZAP-Express phage and pBluescript KS(Ϫ) vectors were from Stratagene (La Jolla, CA). [␥-32 P]ATP, [␣-32 P]dCTP, and [␣-32 P]UTP were from Amersham Pharmacia Biotech. The pYES2 vector was from Invitrogen (Carlsbad, CA). Zeta-Probe GT nylon membranes and the protein assay were from Bio-Rad. The primers were purchased from Genosys Biotechnologies Inc. (Woodlands, TX). Plasmid pXG-GFPϩ2Ј was a gift from Stephen Beverley, Washington University (St. Louis, MO). T. cruzi expression vector pTEX was a gift from David Engman, Northwestern University (Chicago, IL). Antibodies against T. brucei BiP and T. cruzi calreticulin were kindly provided by James D. Bangs and Armando Parodi, respectively. The antibody against the acidocalcisomal Ca 2ϩ -ATPase was described before (6). The Alexafluor 564-conjugated goat anti-rabbit antibody was from Molecular Probes (Eugene, OR).
PCR Cloning and Screening of the Genomic Library-All basic recombinant DNA techniques followed standard procedures described previously (24) unless otherwise noted. To amplify the TcSCA gene, the polymerase chain reaction (PCR) was performed with 30 cycles of 94°C for 1 min, 40°C for 1 min, and 72°C for 1 min using 1.25 units of Taq DNA polymerase with 50 ng of T. cruzi genomic DNA, 1 M each oligonucleotide primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl 2 , and 0.2 mM each deoxynucleoside trisphosphate in a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, MA). The PCR products were separated in an agarose gel, purified, and cloned into pGEM-T-easy. The sequences of the primers used for the PCR were: F4, 5Ј-GC(T/C/A/G)GG(T/C/A/G)AT(T/C/A)(C/A)G(T/C/A/ G)GT(T/C/A/G)-3Ј, that corresponded to AGIRV in the loop region (amino acids 612-615 in TcSCA), and R2, 5Ј-(A/G)TC(T/C/A/G)GT(T/C/A/ G)AC(T/C/A/G)A(A/G)(A/G)TT(T/C/A/G)AC(A/G/C)(T/C)A-3Ј, which corresponded to WVNLVTD in the M6 region (amino acids 788 -794 in TcSCA). To make a T. cruzi subgenomic library, the genomic DNA was completely digested by BglII, and DNA fragments of 5-10 kilobases were purified from the gel and ligated into ZAP-Express vector. The resulting library was screened by plaque hybridization with the PCR clone as a probe in a manner described previously (24). DNA sequencing (25) was performed automatically with the Dye Terminator Cycle sequencing kit and a 373A DNA automatic sequencer (PerkinElmer Life Sciences) at the Biotechnology Center, University of Illinois at Urbana-Champaign. DNA and deduced amino acid sequences were analyzed with the Wisconsin Sequence Analysis Package (version 8.0, GCG, Madison, WI). Hydropathy plot analysis was performed by using the Kyte and Doolittle method (26). The cDNA was synthesized by using reverse transcriptase, total RNA, and oligo(dT) as a primer and amplified by PCR using primers corresponding to the T. cruzi splice leader sequence (5Ј-ATAGAACAGTTTCTGTAC-3Ј) and a TcSCA sequence (5Ј-CGAATTCAGCTCCCGTGTAA-3Ј). PCR conditions were the same as described above except that the annealing temperature was 60°C.
Construction of the Expression Plasmids-To obtain the vector for TcSCA expression in the yeast S. cerevisiae, a BamHI/EcoRI fragment that contained 261 bp of the 5Ј-end-untranslated region and 1951 bp of the 5Ј-end of the TcSCA open reading frame (ORF) was ligated into S. cerevisiae expression vector pYES2. The BamHI/SalI fragment at the 5Ј-end of the TcSCA ORF was replaced with the BamHI/SalI-digested PCR fragment amplified by the primer pair (5Ј-CCCGGATCCAGGAT-GGCGCTTCTTTCACTCCC-3Ј and 5Ј-CGTGGGAAGTTCATTAGTAC-3Ј) to give the yeast Kozak consensus sequence (ANNATGG) (27) and a BamHI site to the 5Ј-end of the ORF. The 1079 bp of the 3Ј-end of the TcSCA ORF were added to the plasmid at the EcoRI/HindIII site by the EcoRI/HindIII-digested PCR fragment amplified by the primer pair (5Ј-AGAGGCCATCTGCAGGAAAC-3Ј and 5Ј-CCGCCTCTAGAAGCTT-TTATTTTATTCGTCA-3Ј). The resulting plasmid was named TcSCA/ pYES2 and used to transform yeast cells. To obtain the vector for green fluorescence protein (GFP)-tagged TcSCA-encoding protein (TcSCA), a BamHI/HindIII fragment that contained the entire TcSCA ORF was ligated into the BamHI/HindIII site of pBluescript KS(-). Site-directed mutagenesis was carried out to introduce a unique NheI site in the ORF to insert GFP into the loop region of TcSCA. For this purpose, a pair of primers (5Ј-GATGGAGATCTTTACGCTAGCCCTGGACGGTAATCC-3Ј and 5Ј-GGATTACCGTCCAGGGCTAGCGTAAAGATCTCCATC-3Ј) that correspond to a part of the loop region (amino acids 371-381) containing a NheI site and complementary to each other were used to amplify the whole plasmid with the mutation by using PfuTurbo DNA polymerase. The DNA product was then digested by DpnI to eliminate the methylated, non-mutated parental DNA template and used to transform Escherichia coli. The GFP fragment with NheI sites was amplified by PCR with a primer pair (5Ј-GGGGGGCTAGCCATGGT-GAGCAAGGGCGAGGA-3Ј and 5Ј-GGGGGGCTAGCATCTTGTACA-GCTCGTCCATGCCGTG-3Ј), and the GFP-containing plasmid pXG-GFPϩ2Ј (28) was digested by NheI and ligated into the NheI site of the TcSCA ORF in pBluescript KS(Ϫ). The BamHI/HindIII fragment containing the entire TcSCA ORF with the GFP insertion was ligated into the BamHI/HindIII site of T. cruzi expression vector pTEX (29). The resulting vector was named TcSCA-GFP/pTEX and was used to transform T. cruzi epimastigotes.
Nucleic Acids Blotting-For Southern blotting, total DNA from epimastigotes (10 g/lane) was digested with BamHI, BglII, HindIII, SalI, SphI, and EcoRI, separated on 1.0% agarose with TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA (pH 8.0)) buffer, and transferred to Zeta-Probe GT nylon membrane. The blot was probed with [␣-32 P]dCTPlabeled TcSCA DNA. DNA was isolated by standard procedures (24). For the Northern blot analysis total RNA was isolated from trypomastigotes, epimastigotes, and amastigotes of T. cruzi with Trizol reagent according to the manufacturer's instructions. RNA was electrophoresed in 1.0% agarose gels with 2.2 M formaldehyde, 20 mM Mops (pH 7.0), 8 mM sodium acetate, and 1 mM EDTA and transferred to Zeta-Probe GT nylon membranes. DNA probes were prepared using random hexanucleotide primers and a Klenow fragment of DNA polymerase I (Prime-a-Gene labeling system) and [␣-32 P]dCTP. RNA probes were prepared from linearized double-stranded DNA templates with either T3 or T7 promoter sequences upstream of the probe sequence using T3 or T7 RNA polymerase (Riboprobe in vitro transcription system) and [␣-32 P]dUTP. The TcP0 (T. cruzi ribosomal protein 1; Ref. 30) fragment used as a control in the Northern blots was obtained by amplifying T. cruzi genomic DNA by PCR, with primers corresponding to nucleotides 3-54 and 918 -936 of the sequence of the TcP0 gene (30). Densitometric analyses of Northern blots were done with an ISI-1000 digital imaging system (Alpha Inotech Corp.) and standardized using the intensity of TcP0 transcripts and assuming a similar level of expression of this gene in all stages (31). Similar results were obtained when the densitometric values were compared by taking into account the amount of RNA added to each lane in three different experiments.
Production of Polyclonal Antibody against TcSCA-A DNA fragment encoding the 518-amino acid, COOH-terminal domain of TcSCA protein was generated by polymerase chain reaction using T. cruzi genomic DNA as a template. A 5Ј primer (primer 1) containing an NheI site (5Ј-GCTAGCGTGGCGATTGCGCTTGCCGT-3Ј) and a 3Ј primer (primer 2) encoding an HindIII site (5Ј-AAGCTTCAAGGCCTCCGGTAGAC-CAC-3Ј) were used. The product was subcloned into the NheI and HindIII sites of the pET-28a(ϩ) expression vector, resulting in a construct that encoded the protein fused next 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 within other sequences are denoted by dashes. The predicted transmembrane domains are indicated with lines above the alignment (M1-M10), and the position of the putative cAMP-dependent protein kinase phosphorylation sites are indicated by double lines above the alignment. Asterisks above the alignment indicate the residues previously identified in SERCA pumps as the high affinity Ca 2ϩ -binding sites. The phosphorylation and ATP binding domains are also indicated above the alignment. been obtained. The recombinant plasmid was transfected into the BL21(DE3) strain of E. coli, and cells were grown in LB medium. Gene expression was induced by adding isopropyl-1-D-galactopyranoside at a final concentration of 1 mM when the cell density reached an A 600 of 0.6. The cells were harvested after a 4-h incubation at 37°C, sonicated (4 ϫ 30 s with 30-s intervals at 4°C) in 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCL (pH 7.9), and centrifuged at 21,000 ϫ g at 4°C for 20 min for separation into pellet and supernatant fractions. The pellet, which contained inclusion bodies, was used to extract TcSCA for antibody production following the instructions outlined for inclusion body purification in the His⅐Bind® kit (Novagen, WI). The protein was renatured by dialysis and concentrated to one-tenth volume.
Rabbits received 250 g of fusion protein injected subcutaneously with Freund's complete adjuvant (Difco). Subsequent injections were performed at 3-week intervals using 250 g of fusion protein in incomplete Freund's adjuvant (Difco). Serum was collected before the initial injection (preimmune serum) (via the ear nick method) and 1 week after every immunization. Once the desired specific antibody titer had been achieved, the rabbit received a final booster injection and was terminated by exsanguination 1 week later. The antiserum was aliquoted and stored at Ϫ70°C.
Immunoblot Methods-Aliquots of sonicated lysates of different stages of T. cruzi containing 10 g of total protein were mixed with an equal amount of nonreducing 2ϫ SDS buffer (125 mM Tris-HCl (pH 6.6), 20% glycerol (v/v), 6.0% SDS (w/v), and 0.4% (w/v) bromphenol blue) and boiled for 5 min before application of SDS-polyacrylamide gels. Proteins were separated using 7.5% Ready Gels (Bio-Rad) and blotted onto nitrocellulose (NitroPure, MSI, Westborough, MA) using a Bio-Rad transblot apparatus by standard techniques. Subsequent processing steps were done in Dulbecco's phosphate-buffered saline containing 0.1% Tween 20. Blots were blocked overnight at 4°C with 5% nonfat dry milk, washed three times, and incubated with primary antibody (1: 5,000) for 1 h at room temperature. Blots were then washed three times, incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000), washed three times, and processed for chemiluminescence detection following the instructions of the manufacturer (Amersham Pharmacia Biotech). Photographic exposures of 5 s to 1 min were made.
Fluorescence Microscopy-After washing three times with phosphate-buffered saline, parasites were fixed with 4% formaldehyde in phosphate-buffered saline for 1 h at room temperature and allowed to adhere to poly-L-lysine-coated glass slides (Sigma) for 10 min. After permeabilization with 0.3% Triton X-100 for 3 min and blocking with 3% bovine serum albumin in phosphate-buffered saline for 1 h, the parasites were incubated with a 1:100 dilution of the antibody (anti-TcSCA) against the 55.3-kDa expressed protein followed by 1:100 of a fluorescein isothiocyanate-coupled goat anti-rabbit immunoglobulin G (IgG) secondary antibody, both at room temperature. Control preparations were incubated with preimmune serum (1:50) or without the primary antibody. Immunofluorescence images were obtained with an Olympus BX-60 fluorescence microscope digital image system (6,32). For dual labeling with GFP-tagged TcSCA, the same methods were used, except that the primary antibodies against calreticulin, BiP, or T. cruzi Tca1 Ca 2ϩ -ATPase was used at a 1:150 dilution, and an Alexafluor 564-conjugated goat anti-rabbit IgG secondary antibody was used at 1:300.
Cell Transformation and Growth-Yeast strain K616 was transformed with TcSCA/pYES2 or the vector alone by the lithium acetate method (33). Transformants were selected by plating them on synthetic complete medium minus uracil (SC-URA) (23). Cells obtained from a single colony were grown overnight in SC-URA liquid medium with 2% galactose at 30°C. The resulting cell suspension was used to inoculate the same medium to an initial A 600 of 0.01. Either CaCl 2 or EGTA was added to the medium at various concentrations to change free-Ca 2ϩ levels. Growth at 30°C was monitored by measuring A 600 after 24 or 48 h. For the assay of Mn 2ϩ sensitivity, yeast grown in SC-URA plus 2% glucose was inoculated into glucose or galactose medium at an initial A 600 of 0.4, and the A 600 was measured at daily intervals for 4 days. Epimastigotes of T. cruzi (Y strain) were transformed with TcSCA-GFP/ pTEX or vector alone by electroporation. Cells at the logarithmic phase of growth were washed once with Zimmerman postfusion medium (132 mM NaCl, 8 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 0.5 mM magnesium acetate, 90 M CaCl 2 ) and suspended in the same medium at a concentration of 1 ϫ 10 8 cell/ml. The cell suspension (0.5 ml) was mixed with the plasmid DNA (50 g), electroporated twice with a Bio-Rad gene pulser at 1.5 kV with a 0.4-cm path length, no resistance, and a 20-microfarad capacitance, and resuspended in 5 ml of liver infusion tryptose medium with 10% fetal calf serum. After an overnight incuba-tion at 28°C, G418 was added at 200 g/ml for selection of the stable transfectants.
Formation of Phosphoenzyme-32 P-labeled phosphoprotein formation was assayed according to Schatzmann and Burgin (34) with some modifications. The reaction mixture (150 l) contained 150 mM KCl, 1 mM EGTA, 0.02 mM MgCl 2 , 75 mM K-Hepes (pH 7.0), and 15 g of microsomal protein. Where indicated, 0.5 mM LaCl 3 was added to prevent dephosphorylation of the pump. To test cation dependence, CaCl 2 was added to a total concentration of 1.232 mM, resulting in a final free Ca 2ϩ concentration of 220 M. The reaction was started by adding [␥-32 P]ATP (2 Ci/reaction; 3,000 Ci/mmol) to a final concentration of 19 M and terminated after 30 s at 0°C by adding 0.2 ml of 50 mM NaH 2 PO 4 , 2 mM ATP, and 5% trichloroacetic acid followed by centrifugation. Where indicated, 0.8 mM hydroxylamine in 0.6 sodium acetate (pH 5.3) was added to the pellet and incubated for 15 min at room temperature. After two washes with trichloroacetic acid solution, the pellet was suspended in 20 l of sample buffer and subjected to acidic SDS/polyacrylamide gel electrophoresis (35) and autoradiography.

Cloning and Characterization of a SERCA-type Ca 2ϩ -
ATPase Gene-To clone the SERCA-type Ca 2ϩ -ATPase gene of T. cruzi, a region containing a conserved Ca 2ϩ -ATPase sequence was amplified from T. cruzi genomic DNA. To design degenerate oligonucleotide primers, amino acid sequences of SERCA-type Ca 2ϩ -ATPases of different species were retrieved from GenBank TM , and regions with the highest similarity were located. Two primers (F4 and R2) were selected according to these domains, and the PCR was carried out with T. cruzi genomic DNA as a template. One of the PCR products (ϳ650 bp) reacted strongly with a DNA probe from the Leishmania mexicana amazonensis putative SERCA-type Ca 2ϩ -ATPase (Lmaa1) (32) by Southern blotting, and it was cloned into pGEM-T-easy. The deduced amino acid sequence of this PCR clone was 77.6 and 84.2% identical to the sequences of the putative SERCA-type Ca 2ϩ -ATPases of L. m. amazonensis (Lmaa1) (32) and Trypanosoma brucei (TBA1) (36,37), respectively. A T. cruzi genomic library was constructed and screened using this PCR fragment as a probe. A genomic clone was found to contain the sequence of the 3Ј region (1278 bp) of the putative SERCA-type Ca 2ϩ -ATPase gene of T. cruzi. To obtain the sequence of the 5Ј region of the gene, cDNA was synthesized, and the gene was amplified by PCR using primers corresponding to the splice leader sequence and a specific sequence present in the genomic clone. PCR produced a DNA fragment that covered the 5Ј region (1957 bp) of the gene including a fragment of 205 bp overlapping with the sequence of the genomic clone. Together with the genomic clone sequence, the PCR product sequence gave the 3018-bp ORF of the gene, encoding 1006 amino acid residues, which was named TcSCA for T. cruzi SERCA-type Ca 2ϩ -ATPase (GenBank TM accession number AF093566).
Sequence Analysis of TcSCA-The TcSCA amino acid sequence is 73% identical to the T. brucei SERCA-type Ca 2ϩ -ATPase sequence (TBA1 (36,37)) and 61% identical to the L. m. amazonensis putative SERCA-type Ca 2ϩ -ATPase sequence (Lmaa1 (32)) ( Fig. 1). It has about 46 -48% identity to SERCAtype Ca 2ϩ -ATPase sequences of non-trypanosomatids and only 24.1-24.8% identity to plasma membrane-type Ca 2ϩ -ATPases sequences from different species. Analysis of the TcSCA amino acid sequence showed that this gene product contains all the conserved subdomains and invariant residues found in other P-type ATPases such as the phosphorylation and ATP binding domains (38,39). Hydropathy analysis of the deduced amino acid sequence revealed a profile very similar to those of other calcium pumps containing 10 transmembrane domains (M1-M10) (Fig. 1). This is in line with structures obtained from crystals of a mammalian SERCA-type Ca 2ϩ -ATPase at 8 Å of resolution (40). TcSCA also contains all the residues (Glu 315 , Glu 765 , Asn 790 , Thr 793 , Asp 794 , and Glu 895 , indicated by asterisks above the alignment in Fig. 1) that were previously identified as the high affinity Ca 2ϩ -binding sites in the center of the putative transmembrane domains M4, M5, M6, and M8 (41). As occurs with other SERCA-type pumps, TcSCA lacks the conserved amino acid sequence associated with calmodulin binding found near the COOH terminus of mammalian plasma membrane-type Ca 2ϩ ATPase isoforms (42). The amino acid sequence Lys-Asp-Asp-Lys-Pro-Val 402 , which was found to be critical for the functional association of the Ca 2ϩ -ATPase of cardiac sarcoplasmic reticulum with phospholamban (42), is absent in TcSCA. Interestingly the residues located in transmembrane segment 3 important for thapsigargin binding of SERCA Ca 2ϩ -ATPases (43) are different in TcSCA (10 of 20 residues in segment 3 are different as compared with SERCA pumps). In agreement with these results we were unable to detect any significant increase in [Ca 2ϩ ] i in fura 2-loaded cells in the presence of low concentrations of thapsigargin (0.1-4 M) (14,15). These results were also confirmed in experiments with the enzyme expressed in yeast (see below). Interestingly, TcSCA has three potential cAMP-dependent protein kinase phosphorylation sites, which are common to all three putative SERCA-type Ca 2ϩ -ATPases of kinetoplastid parasites but are  not present in those of other species (Fig. 1). A genomic Southern blot probed by TcSCA DNA showed a single hybridizing band in each lane except for EcoRI (results not shown), suggesting that TcSCA is a single-copy gene. The two DNA bands observed by EcoRI digestion are due to the presence of an internal EcoRI site in the TcSCA sequence (nucleotide 1957).

FIG. 4. Immunoblot analysis of TcSCA (inset in A) and immunofluorescence microscopy showing the localization of TcSCA in epimastigote (E) (panels A and E), trypomastigote (T) (panels B and F), and amastigote (A, panels C and G) forms of T. cruzi. Panels A-C show localization of
Expression of TcSCA in Different Stages of T. cruzi-Northern blot analysis showed the presence of a single TcSCA tran-script of approximately 5 kilobases in each of the three life cycle stages of T. cruzi (Fig. 2, upper panel). Analysis of the 5-kilobase band by densitometry indicated that the TcSCA gene is expressed at similar levels in all stages of T. cruzi. This is in contrast with lmaa1, the gene for the putative L. m. amazonensis SERCA-type Ca 2ϩ pump that is developmentally regulated and more abundantly expressed in intracellular amastigotes (32).
Localization of TcSCA-To determine the localization of TcSCA, the sequence of the GFP of Aequorea victoria was fused to the TcSCA ORF and ligated into the T. cruzi expression vector pTEX (28,29). The loop region in TcSCA was chosen for the site of insertion of GFP to avoid any possible interference with a potential targeting signal sequence. T. cruzi epimastigotes were transfected with this TcSCA-GFP plasmid, and the stable transfectants were observed by fluorescence microscopy. Transgenic parasites expressing the TsSCA-GFP construct exhibited GFP florescence as a ring surrounding the nucleus and in a network extending from its periphery (Fig. 3, A-C), suggesting an endoplasmic reticulum (ER) localization. To further confirm these results, transgenic cells were also stained with antibodies against BiP and calreticulin, two ER chaperones involved in the control of protein folding (44). Both BiP (Fig. 3D) and calreticulin (Fig.  3E) co-localized with TcSCA (Figs. 3, A and B), thus confirming the ER localization of TcSCA. In contrast, incubation of transgenic cells with antibodies against the acidocalcisomal Ca 2ϩ -ATPase of T. cruzi (6) resulted in a punctate staining (Fig. 3F), clearly different from the perinuclear and reticular localization of TcSCA (Fig. 3C).
We also investigated the localization of TcSCA in other developmental stages of T. cruzi. Total lysates from different stages of T. cruzi were subjected to Western blotting analysis with antibodies against a 518-amino acid, COOH-terminal domain of TcSCA. These antibodies detected a single band of ϳ110 kDa (Fig. 4, inset), close to the predicted molecular mass of TcSCA, in epimastigote (E), trypomastigote (T), and amastigote (A) lysates. No band was detected when using preimmune serum (data not shown). In indirect immunofluorescence assays using the anti-TcSCA antiserum, TcSCA was detected (Fig. 4) in epimastigotes (A), trypomastigotes (B), and amastigotes (C) as a ring surrounding the nucleus and in a network extending from its periphery, similar to the GFP protein labeling (Fig. 3). No detectable signal was observed when the preimmune serum was used (Fig. 4D).
Functional Complementation by TcSCA of the Ca 2ϩ -ATPase-deficient S. cerevisiae Strain K616 -To test the function of TcSCA, 3018 bp of the TcSCA ORF were subcloned into a yeast expression vector, pYES2, under the control of a galactose-inducible promoter, and the resulting construct or the vector alone was used to complement yeast mutant K616. The yeast triple mutant K616 is defective in both the Golgi (Pmr 1) and the vacuolar (Pmc 1) Ca 2ϩ pumps and also lacks calcineurin (CNB 1) function. This mutant provides an extremely valuable expression system for determining the nature of individual Ca 2ϩ pumps from other eukaryotes (23,45). The triple mutant transformed with vector alone grew poorly on a medium containing low Ca 2ϩ (2-10 mM EGTA) (Fig. 5). This effect was noticeable even at low concentrations of EGTA during the initial 24 h of growth (Fig. 5A) and decreased after 48 h (Fig. 5B). However, the triple mutant transformed with TcSCA became tolerant of EGTA, supporting the idea that TcSCA encodes a functional divalent cation pump. The likely transport of Ca 2ϩ was supported by results on phosphoenzyme formation (below). Transport of Mn 2ϩ was indicated by complementation of the Mn 2ϩ sensitivity of the triple mutant (Fig. 6). The mutant with or without TcSCA grew equally well in glucose medium, where the gene is not induced (results not shown). In galactose medium, though, there was little growth of the vector control strain in the presence of 1-3 mM MnCl 2 , whereas the TcSCA-transformed (and induced) strain showed significant growth (Fig. 6).  (Fig. 7A, lane 7) but was absent in yeast transformed with vector alone (Fig. 7A, lane 3). Phosphorylation was dependent on the presence of Ca 2ϩ . La 3ϩ enhanced the steady-state level of the phosphoprotein severalfold (Fig. 7A,  lane 8). The denatured phosphoprotein was sensitive to hydroxylamine (Fig. 7A, lane 9), indicating the hydrolysis of an acyl phosphate bond (48) probably formed by Asp 357 (Fig. 1). Together, these results provide compelling evidence that Tc-SCA is a P-type Ca 2ϩ -dependent ATPase.

Effect of Inhibitors on the Formation of a Ca 2ϩ -dependent
Cyclopiazonic acid, an inhibitor of animal SERCA pumps, inhibited the phosphorylation of TcSCA (Fig. 7B, lanes 6 and  7), whereas thapsigargin had no effect at low concentrations (Fig. 7B, lanes 2 and 3). The phosphorylation of TcSCA was also inhibited by erythrosin B (Fig. 7B, lanes 4 and 5), a haloge-nated derivative of fluorescein that binds to nucleotide-binding sites with high affinity and specificity (49). DISCUSSION Using yeast as a heterologous expression system (23), we provide the first direct evidence that a cloned SERCA-type T. cruzi gene encodes a functional Ca 2ϩ -ATPase. Expression of the T. cruzi TcSCA gene restored the growth in a medium containing submicromolar levels of Ca 2ϩ of a yeast mutant (K616) defective in Ca 2ϩ pumps (Fig. 4). Several lines of evidence indicate that TcSCA encodes an ER Ca 2ϩ pump; 1) its amino acid sequence shares 46 -48% identity with SERCA pumps and less identity (24.1-24.8%) with plasma membrane-type Ca 2ϩ -ATPases; 2) TcSCA contains ER retention motifs (50), KKXX-stop at the COOH terminus and RILL in the first transmembrane domain (Fig. 1); 3) TcSCA is mainly localized to the nuclear membrane and to a reticular structure in different stages of T. cruzi (Figs. 3 and 4); 4) TcSCA co-localizes with calreticulin and BiP, two well known endoplasmic reticulum chaperones (Fig. 3).
As occurs with plant SERCA-type Ca 2ϩ ATPases (48), TcSCA appears to be insensitive to thapsigargin (14,15). Residues located in the third transmembrane segment (M 3 ) and in the stalk segment (S 3 ) have been postulated to be important for thapsigargin binding and are conserved in all SERCA Ca 2ϩ -ATPases (43,51). Since the T. brucei SERCA pump TBA1 was reportedly sensitive to thapsigargin (37), whereas L. m. amazonensis putative SERCA pump LMAA1 was not (32), it was proposed that two amino acid substitutions present in LMAA1 could account for their different sensitivity: a Gly 271 in LMAA1 that replaces Lys 261 in TBA1 Other residues that are different in TcSCA as compared with TBA1 are conserved substitutions and possibly could not account for the differences in sensitivity to thapsigargin, except for a Thr 266 that replaces an Ile in TBA1 and LMAA1. There is only one difference in the S 3 segment; a Met 257 replaces a Val in TBA1 and LMAA1. Our conclusion is that these differences in transmembrane segment M 3 and S 3 could account for the differences in sensitivity to thapsigargin of these pumps.
In mammalian cells, SERCAs are important in refilling ER calcium stores used in signaling. Ca 2ϩ is released from the ER by inositol 1,4,5-trisphosphate (IP 3 ), cyclic ADP-ribose, or nicotinic acid adenine dinucleotide phosphate acting upon IP 3 or ryanodine receptors (52,53). Whether the same applies to T. cruzi is uncertain. The isolated ER has not been studied in trypanosomatids, but the most likely alternative store of calcium for signaling, the acidocalcisome (8), is not sensitive to calcium-releasing metabolites after isolation from T. cruzi (7). 2 Calcium signals certainly appear to be generated in trypanosomatids, by L. m. amazonensis during macrophage invasion (32) and by T. cruzi during the invasion of myoblasts (1). In both cases, chelation of intracellular Ca 2ϩ inhibited invasion.
Another important function of Ca 2ϩ -ATPases located in the ER and the Golgi of different cells is to supply intralumenal cations (not just Ca 2ϩ but also Mn 2ϩ ) required for the correct processing of proteins through the secretory pathway in eukaryotic cells. There is obviously some overlap in function between the Golgi and ER Ca 2ϩ -ATPases as the yeast pmr1 mutant can be complemented by ER Ca 2ϩ -ATPases from animals (54,55) and plants (23). The best-studied case of a Ca 2ϩ -ATPase also transporting Mn 2ϩ is the yeast Golgi PMR1 protein (56), but some Ca 2ϩ -ATPases located in the ER also probably transport Mn 2ϩ , including those of mammals (57), and plants (23). In the latter instance, the plant Ca 2ϩ -ATPase was found to alleviate Mn 2ϩ toxicity in the pmr1 mutant, similar to the results we report here. Within the ER and Golgi of yeast, Ca 2ϩ and Mn 2ϩ are involved in processes of protein folding, degradation of mis-folded proteins, sorting to the vacuole, and glycosylation (56). Similar results were found in earlier work with mammalian cells (57)(58)(59), although in some of these studies it was assumed that the observed effects were due to Ca 2ϩ without regard to Mn 2ϩ . In PC 12 cells, expression of a SERCA isoform is enhanced upon treatment of the cells with agents that interfere with protein folding or inhibit glycosylation or disrupt the Golgi body (60). Evidence from SERCA gene-knockout studies in Drosophila also showed a vital role for the pump in the processing and trafficking of several plasma membrane or cell junction transmembrane proteins (61). Therefore, in T. cruzi, the TcSCA transporter may be important in the maintenance of lumenal Ca 2ϩ and/or Mn 2ϩ required for proper trafficking and modification (glycosylation) of new proteins during differentiation and, particularly (from the point of view of infectivity), cell surface proteins involved in interactions with host cells.