TcSCA Complements Yeast Mutants Defective in Ca 2+ Pumps and Encode a Ca 2+ -ATPase That Localizes to the Endoplasmic Reticulum of Trypanosoma cruzi

TcSCA Complements Yeast Mutants Defective in Ca2� Pumps andEncodes a Ca2�-ATPase That Localizes to the EndoplasmicReticulum of Trypanosoma cruzi*

Received for publication, May 3, 2001, and in revised form, May 24, 2001Published, JBC Papers in Press, May 29, 2001, DOI 10.1074/jbc.M104000200

Tetsuya Furuya‡, Michael Okura§, Felix A. Ruiz, David A. Scott, and Roberto Docampo¶

From the Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University ofIllinois at Urbana-Champaign, Urbana, Illinois 61802

Intracellular Ca2� in Trypanosoma cruzi is mainly lo-cated in an acidic compartment named the acidocalcisome,which among other pumps and exchangers possesses aplasma membrane-type Ca2�-ATPase. Evidence for an en-doplasmic reticulum-located Ca2� uptake has been moreelusive and based on indirect results. Here we report thecloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca2�-ATPase from T. cruzi.The protein (TcSCA) predicted from the nucleotide se-quence of the gene has 1006 amino acids and a molecularmass of 109.7 kDa. Several sequence motifs found in sarco-plasmic-endoplasmic reticulum-type Ca2�-ATPases werepresent in TcSCA. Expression of TcSCA in yeast mutantsdeficient in the Golgi and vacuolar Ca2� pumps (pmr1 pmc1cnb 1) restored growth on EGTA. Membranes were isolatedfrom the pmr1 pmc1 cnb1 mutant transformed with TcSCA,and it was found that the TcSCA polypeptide formed aCa2�-dependent and hydroxylamine-sensitive 32P-labeledphosphoprotein of 110 kDa in the presence of [�-32P]ATP.Cyclopiazonic acid, but not thapsigargin, blocked this phos-phoprotein formation. Transgenic parasites expressingconstructs of TcSCA with green fluorescent protein exhib-ited co-localization of TcSCA with the endoplasmic reticu-lum proteins BiP and calreticulin. An endoplasmic reticu-lum location was also found in amastigotes andtrypomastigotes using a polyclonal antibody against aCOOH-terminal region of the protein. The ability of TcSCAto restore growth of mutant pmr1 pmc1 cnb 1 on mediumcontaining Mn2� suggests that TcSCA may also regulateMn2� homeostasis by pumping Mn2� into the endoplasmicreticulum of T. cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas’ disease, isa parasitic protozoan that invades mammalian cells and devel-ops intracellularly as amastigotes. Invasion of cells by T. cruziis dependent upon an elevation in the concentration of cytosolicfree calcium in the invading trypomastigote (1, 2). Unlike mam-malian cells, T. cruzi possesses most of its intracellular Ca2� in

an acidic compartment named the acidocalcisome (3–8). Themolecular and biochemical characterization of this organellehas provided evidence that it has an orthovanadate-sensitiveplasma membrane-type Ca2� ATPase for Ca2� uptake (3, 5–7).Evidence for non-mitochondrial and endoplasmic reticulum-located Ca2� uptake has been more elusive and based on thepresence of a low capacity and high affinity orthovanadate-sensitive Ca2� uptake in permeabilized cells and the ability ofthese cells to buffer [Ca2�] in the range 0.05–0.1 �M (9), fea-tures in common with the sarcoplasmic/endoplasmic reticulumCa2�-ATPases (SERCA)1 of animal cells (10). In addition, likemammalian cells (11), calcium is needed for the correct foldingand assembly of proteins in the endoplasmic reticulum of T.cruzi, which depends on chaperones such as the Ca2�-bindingprotein, calreticulin (12).

Biochemical distinction of the different Ca2� pumps presentin T. cruzi has been hampered by the lack of distinguishingfeatures such as specific inhibitor sensitivity. Orthovanadateinhibits all types of Ca2� ATPases (10), whereas thapsigargin,a specific inhibitor of animal SERCA-type Ca2�-ATPases (13),is ineffective in inhibiting Ca2� uptake in permeabilized T.cruzi (14, 15). Thus, a molecular approach to studying individ-ual pumps is necessary. Expression of genes identified as en-coding Ca2�-ATPases and localization of the correspondingproteins is necessary, as the plasma membrane-type Ca2�

ATPase/SERCA paradigm does not necessarily apply in non-animal cells; a SERCA-type gene in tomato is expressed indifferent parts of the cell (16). Also, a Tca1 gene has beenidentified in T. cruzi (6), which encodes a protein with homol-ogy to mammalian plasma membrane Ca2�-ATPases but withcharacteristics that place it in a novel category of Ca2�-AT-Pases along with the vacuolar Ca2�-ATPases described in Sac-charomyces cerevisiae (17), Dictyostelium discoideum (18), En-tamoeba histolytica (19), and Toxoplasma gondii (20). The geneis expressed at a high level in the amastigote stage and islocalized to acidocalcisomes and the plasma membrane of theparasite (6).

Here we demonstrate that a gene from T. cruzi (TcSCA)complemented yeast mutants defective in Ca2� pumps by re-storing their growth in EGTA. The protein encoded by theTcSCA gene localizes to the endoplasmic reticulum of differentstages of the parasite and, in contrast to the acidocalcisomalCa2�-ATPase (6), it is expressed at similar levels in the differ-ent developmental stages of the parasite.

* This work was supported by National Institutes of Health GrantAI-23259 (to R. D.). The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

‡ Supported by a predoctoral fellowship of the American Heart Asso-ciation, Midwest affiliate. Present address: Seattle Biomedical Re-search Institute, 4 Nickerson St., Suite 200, Seattle, WA 98109-1651.

§ Supported by a United States Department of Agriculture traininggrant.

¶ To whom correspondence should be addressed: Laboratory of Mo-lecular Parasitology, Dept. of Pathobiology, College of Veterinary Med-icine, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61802.Tel.: 217-333-3845; Fax: 217-244-7421; E-mail: [email protected].

1 The abbreviations used are: SERCA, sarcoplasmic-endoplasmic re-ticulum-type Ca2�-ATPase; TcSCA, T. cruzi SERCA-type Ca2�-ATPase;PCR, polymerase chain reaction; bp, base pair(s); ORF, open readingframe; GFP, green fluorescent protein; Mops, 4-morpholinepropanesul-fonic acid; SC-URA, synthetic complete medium minus uracil; ER,endoplasmic reticulum.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 35, Issue of August 31, pp. 32437–32445, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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FIG. 1. Alignment of different SERCA-type Ca2�-ATPases. CLUSTALW alignment of Ca2�-ATPases from T. cruzi (GenBankTM accessionnumber AF093566), T. brucei (AAA30227; TBA1), L. m. amazonensis (U70620; Lmaa1), Homo sapiens SERCA 1 (1586563; SERCA1), andArabidopsis thaliana ECA3 (AJ132388; RCA3). Identical residues are in black, and similar residues are shaded. Amino acid residues not present

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EXPERIMENTAL PROCEDURES

Culture Methods—T. cruzi amastigotes and trypomastigotes (Ystrain) were obtained from the culture medium of L6E9 myoblasts by amodification of the method of Schmatz and Murray (21), as we havedescribed before (14, 15). The contamination of trypomastigotes withamastigotes and intermediate forms or of amastigotes with trypomas-tigotes or intermediate forms was always less than 5% unless otherwisestated. T. cruzi epimastigotes (Y strain) were grown at 28 °C in liverinfusion tryptose medium (22) supplemented with 10% newborn calfserum. S. cerevisiae strain K616 (MATa pmr1::HIS3 pmc1::TRP1cnb1::LEU2, ura3) (23) was kindly provided by Kyle W. Cunningham,Department of Biology, The Johns Hopkins University, Baltimore, MDand 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 Bio101 (Vista, CA). Trizol reagent, reverse transcriptase, and Taq polym-erase were from Life Technologies, Inc. The pGEM-T Easy vector,Riboprobe in vitro transcription system and Prime-a-Gene labelingsystem were from Promega (Madison, WI). The PfuTurbo DNA polym-erase, the �ZAP-Express phage and pBluescript KS(�) vectors werefrom Stratagene (La Jolla, CA). [�-32P]ATP, [�-32P]dCTP, and[�-32P]UTP were from Amersham Pharmacia Biotech. The pYES2 vec-tor was from Invitrogen (Carlsbad, CA). Zeta-Probe GT nylon mem-branes and the protein assay were from Bio-Rad. The primers werepurchased from Genosys Biotechnologies Inc. (Woodlands, TX). PlasmidpXG-GFP�2� was a gift from Stephen Beverley, Washington University(St. Louis, MO). T. cruzi expression vector pTEX was a gift from DavidEngman, 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 theacidocalcisomal Ca2�-ATPase was described before (6). The Alexafluor564-conjugated goat anti-rabbit antibody was from Molecular Probes(Eugene, OR).

PCR Cloning and Screening of the Genomic Library—All basic re-combinant DNA techniques followed standard procedures describedpreviously (24) unless otherwise noted. To amplify the TcSCA gene, thepolymerase chain reaction (PCR) was performed with 30 cycles of 94 °Cfor 1 min, 40 °C for 1 min, and 72 °C for 1 min using 1.25 units of TaqDNA polymerase with 50 ng of T. cruzi genomic DNA, 1 �M eacholigonucleotide primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM

MgCl2, and 0.2 mM each deoxynucleoside trisphosphate in a PTC-100programmable thermal controller (MJ Research, Inc., Watertown, MA).The PCR products were separated in an agarose gel, purified, andcloned into pGEM-T-easy. The sequences of the primers used for thePCR were: F4, 5�-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)-3�, that corresponded to AGIRV in the loop region (ami-no acids 612–615 in TcSCA), and R2, 5�-(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-3�, whichcorresponded to WVNLVTD in the M6 region (amino acids 788–794 inTcSCA). To make a T. cruzi subgenomic library, the genomic DNA wascompletely digested by BglII, and DNA fragments of 5–10 kilobaseswere purified from the gel and ligated into �ZAP-Express vector. Theresulting library was screened by plaque hybridization with the PCRclone as a probe in a manner described previously (24). DNA sequencing(25) was performed automatically with the Dye Terminator Cycle se-quencing kit and a 373A DNA automatic sequencer (PerkinElmer LifeSciences) at the Biotechnology Center, University of Illinois at Urbana-Champaign. DNA and deduced amino acid sequences were analyzedwith the Wisconsin Sequence Analysis Package (version 8.0, GCG,Madison, WI). Hydropathy plot analysis was performed by using theKyte and Doolittle method (26). The cDNA was synthesized by usingreverse transcriptase, total RNA, and oligo(dT) as a primer and ampli-fied by PCR using primers corresponding to the T. cruzi splice leadersequence (5�-ATAGAACAGTTTCTGTAC-3�) and a TcSCA sequence (5�-CGAATTCAGCTCCCGTGTAA-3�). PCR conditions were the same asdescribed above except that the annealing temperature was 60 °C.

Construction of the Expression Plasmids—To obtain the vector forTcSCA expression in the yeast S. cerevisiae, a BamHI/EcoRI fragmentthat contained 261 bp of the 5�-end-untranslated region and 1951 bp ofthe 5�-end of the TcSCA open reading frame (ORF) was ligated into S.

cerevisiae expression vector pYES2. The BamHI/SalI fragment at the5�-end of the TcSCA ORF was replaced with the BamHI/SalI-digestedPCR fragment amplified by the primer pair (5�-CCCGGATCCAGGAT-GGCGCTTCTTTCACTCCC-3� and 5�-CGTGGGAAGTTCATTAGTAC-3�) to give the yeast Kozak consensus sequence (ANNATGG) (27) and aBamHI site to the 5�-end of the ORF. The 1079 bp of the 3�-end of theTcSCA ORF were added to the plasmid at the EcoRI/HindIII site by theEcoRI/HindIII-digested PCR fragment amplified by the primer pair(5�-AGAGGCCATCTGCAGGAAAC-3� and 5�-CCGCCTCTAGAAGCTT-TTATTTTATTCGTCA-3�). The resulting plasmid was named TcSCA/pYES2 and used to transform yeast cells. To obtain the vector for greenfluorescence protein (GFP)-tagged TcSCA-encoding protein (TcSCA), aBamHI/HindIII fragment that contained the entire TcSCA ORF wasligated into the BamHI/HindIII site of pBluescript KS(-). Site-directedmutagenesis was carried out to introduce a unique NheI site in the ORFto insert GFP into the loop region of TcSCA. For this purpose, a pair ofprimers (5�-GATGGAGATCTTTACGCTAGCCCTGGACGGTAATCC-3�and 5�-GGATTACCGTCCAGGGCTAGCGTAAAGATCTCCATC-3�)that correspond to a part of the loop region (amino acids 371–381)containing a NheI site and complementary to each other were used toamplify the whole plasmid with the mutation by using PfuTurbo DNApolymerase. The DNA product was then digested by DpnI to eliminatethe methylated, non-mutated parental DNA template and used totransform Escherichia coli. The GFP fragment with NheI sites wasamplified by PCR with a primer pair (5�-GGGGGGCTAGCCATGGT-GAGCAAGGGCGAGGA-3� and 5�-GGGGGGCTAGCATCTTGTACA-GCTCGTCCATGCCGTG-3�), and the GFP-containing plasmid pXG-GFP�2� (28) was digested by NheI and ligated into the NheI site ofthe TcSCA ORF in pBluescript KS(�). The BamHI/HindIII fragmentcontaining the entire TcSCA ORF with the GFP insertion was ligatedinto the BamHI/HindIII site of T. cruzi expression vector pTEX (29).The resulting vector was named TcSCA-GFP/pTEX and was used totransform T. cruzi epimastigotes.

Nucleic Acids Blotting—For Southern blotting, total DNA from epi-mastigotes (10 �g/lane) was digested with BamHI, BglII, HindIII, SalI,SphI, and EcoRI, separated on 1.0% agarose with TAE (40 mM Tris, 20mM acetic acid, 1 mM EDTA (pH 8.0)) buffer, and transferred to Zeta-Probe GT nylon membrane. The blot was probed with [�-32P]dCTP-labeled TcSCA DNA. DNA was isolated by standard procedures (24).For the Northern blot analysis total RNA was isolated from trypomas-tigotes, epimastigotes, and amastigotes of T. cruzi with Trizol reagentaccording to the manufacturer’s instructions. RNA was electrophoresedin 1.0% agarose gels with 2.2 M formaldehyde, 20 mM Mops (pH 7.0), 8mM sodium acetate, and 1 mM EDTA and transferred to Zeta-Probe GTnylon membranes. DNA probes were prepared using random hex-anucleotide primers and a Klenow fragment of DNA polymerase I(Prime-a-Gene labeling system) and [�-32P]dCTP. RNA probes wereprepared from linearized double-stranded DNA templates with eitherT3 or T7 promoter sequences upstream of the probe sequence using T3or T7 RNA polymerase (Riboprobe in vitro transcription system) and[�-32P]dUTP. The TcP0 (T. cruzi ribosomal protein 1; Ref. 30) fragmentused as a control in the Northern blots was obtained by amplifying T.cruzi genomic DNA by PCR, with primers corresponding to nucleotides3–54 and 918–936 of the sequence of the TcP0 gene (30). Densitometricanalyses of Northern blots were done with an ISI-1000 digital imagingsystem (Alpha Inotech Corp.) and standardized using the intensity ofTcP0 transcripts and assuming a similar level of expression of this genein all stages (31). Similar results were obtained when the densitometricvalues were compared by taking into account the amount of RNA addedto each lane in three different experiments.

Production of Polyclonal Antibody against TcSCA—A DNA fragmentencoding the 518-amino acid, COOH-terminal domain of TcSCA proteinwas generated by polymerase chain reaction using T. cruzi genomicDNA as a template. A 5� primer (primer 1) containing an NheI site(5�-GCTAGCGTGGCGATTGCGCTTGCCGT-3�) and a 3� primer (prim-er 2) encoding an HindIII site (5�-AAGCTTCAAGGCCTCCGGTAGAC-CAC-3�) were used. The product was subcloned into the NheI andHindIII sites of the pET-28a(�) expression vector, resulting in a con-struct that encoded the protein fused next to a six-histidine tag thatallowed its purification on nickel-agarose columns. This plasmid waschecked 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), andthe position of the putative cAMP-dependent protein kinase phosphorylation sites are indicated by double lines above the alignment. Asterisksabove the alignment indicate the residues previously identified in SERCA pumps as the high affinity Ca2�-binding sites. The phosphorylation andATP binding domains are also indicated above the alignment.

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been obtained. The recombinant plasmid was transfected into theBL21(DE3) strain of E. coli, and cells were grown in LB medium. Geneexpression was induced by adding isopropyl-1-D-galactopyranoside at afinal concentration of 1 mM when the cell density reached an A600 of 0.6.The cells were harvested after a 4-h incubation at 37 °C, sonicated (4 �30 s with 30-s intervals at 4 °C) in 5 mM imidazole, 500 mM NaCl, and20 mM Tris-HCL (pH 7.9), and centrifuged at 21,000 � g at 4 °C for 20min for separation into pellet and supernatant fractions. The pellet,which contained inclusion bodies, was used to extract TcSCA for anti-body production following the instructions outlined for inclusion bodypurification in the His�Bind® kit (Novagen, WI). The protein was rena-tured by dialysis and concentrated to one-tenth volume.

Rabbits received 250 �g of fusion protein injected subcutaneouslywith Freund’s complete adjuvant (Difco). Subsequent injections wereperformed at 3-week intervals using 250 �g of fusion protein in incom-plete Freund’s adjuvant (Difco). Serum was collected before the initialinjection (preimmune serum) (via the ear nick method) and 1 week afterevery immunization. Once the desired specific antibody titer had beenachieved, the rabbit received a final booster injection and was termi-nated by exsanguination 1 week later. The antiserum was aliquotedand stored at �70 °C.

Immunoblot Methods—Aliquots of sonicated lysates of differentstages of T. cruzi containing 10 �g of total protein were mixed with anequal amount of nonreducing 2� 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 blottedonto nitrocellulose (NitroPure, MSI, Westborough, MA) using a Bio-Radtransblot apparatus by standard techniques. Subsequent processingsteps were done in Dulbecco’s phosphate-buffered saline containing0.1% Tween 20. Blots were blocked overnight at 4 °C with 5% nonfat drymilk, 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-rabbitIgG antibody (1:10,000), washed three times, and processed for chemi-luminescence detection following the instructions of the manufacturer(Amersham Pharmacia Biotech). Photographic exposures of 5 s to 1 minwere made.

Fluorescence Microscopy—After washing three times with phos-phate-buffered saline, parasites were fixed with 4% formaldehyde inphosphate-buffered saline for 1 h at room temperature and allowed toadhere to poly-L-lysine-coated glass slides (Sigma) for 10 min. Afterpermeabilization with 0.3% Triton X-100 for 3 min and blocking with3% bovine serum albumin in phosphate-buffered saline for 1 h, theparasites were incubated with a 1:100 dilution of the antibody (anti-TcSCA) against the 55.3-kDa expressed protein followed by 1:100 of afluorescein isothiocyanate-coupled goat anti-rabbit immunoglobulin G(IgG) secondary antibody, both at room temperature. Control prepara-tions were incubated with preimmune serum (1:50) or without theprimary antibody. Immunofluorescence images were obtained with anOlympus BX-60 fluorescence microscope digital image system (6, 32).For dual labeling with GFP-tagged TcSCA, the same methods wereused, except that the primary antibodies against calreticulin, BiP, or T.cruzi Tca1 Ca2�-ATPase was used at a 1:150 dilution, and an Alexafluor564-conjugated goat anti-rabbit IgG secondary antibody was used at1:300.

Cell Transformation and Growth—Yeast strain K616 was trans-formed with TcSCA/pYES2 or the vector alone by the lithium acetatemethod (33). Transformants were selected by plating them on syntheticcomplete medium minus uracil (SC-URA) (23). Cells obtained from asingle colony were grown overnight in SC-URA liquid medium with 2%galactose at 30 °C. The resulting cell suspension was used to inoculatethe same medium to an initial A600 of 0.01. Either CaCl2 or EGTA wasadded to the medium at various concentrations to change free-Ca2�

levels. Growth at 30 °C was monitored by measuring A600 after 24 or48 h. For the assay of Mn2� sensitivity, yeast grown in SC-URA plus 2%glucose was inoculated into glucose or galactose medium at an initialA600 of 0.4, and the A600 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 phaseof growth were washed once with Zimmerman postfusion medium (132mM NaCl, 8 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 0.5 mM magne-sium acetate, 90 �M CaCl2) and suspended in the same medium at aconcentration of 1 � 108 cell/ml. The cell suspension (0.5 ml) was mixedwith the plasmid DNA (50 �g), electroporated twice with a Bio-Radgene pulser at 1.5 kV with a 0.4-cm path length, no resistance, and a20-microfarad capacitance, and resuspended in 5 ml of liver infusiontryptose 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 stabletransfectants.

Isolation of Yeast Microsomes—Yeast microsomes were isolated by amodification of a previous procedure (23). Yeast (�8-ml packed volume)were added to 4 ml of YL buffer (10% sucrose, 25 mM K-Hepes, 2 mM

MgCl2, 1 mM EGTA, 10 mM benzamidine, 5 mM dithiothreitol, 1.5% (v/v)protease inhibitor mixture (pH 7.5), and 12 ml of 0.5-mm glass beads(Biospec, Bartlesville, OK) in the chamber of a Biospec Bead Beater,and vortexed for 3 min, which resulted in 80–90% cell lysis. The beadswere washed by gravity with YL buffer, and the supernatant fractionswere centrifuged at 3000 � g for 5 min. The supernatants from thiscentrifugation were layered on top of a step gradient of 25%/45% su-crose in 25 mM K-Hepes, 2 mM MgCl2, 10 mM benzamidine, 5 mM

dithiothreitol (pH 7.5) and centrifuged in a Beckman SW28 rotor at108,000 � gmax for 2 h. The 25%/45% interface was diluted 5-fold in 25mM Na-Hepes (pH 7.2), 1 mM dithiothreitol, 2 mM MgCl2 and centri-fuged at 105,000 � g for 50 min. The microsome pellet was resuspendedin 125 mM sucrose, 20 mM K-Hepes, 65 mM KCl, 2 mM MgCl2 (pH 7.2).Protein was assayed with the Bio-Rad protein assay.

Formation of Phosphoenzyme—32P-labeled phosphoprotein forma-tion was assayed according to Schatzmann and Burgin (34) with somemodifications. The reaction mixture (150 �l) contained 150 mM KCl, 1mM EGTA, 0.02 mM MgCl2, 75 mM K-Hepes (pH 7.0), and 15 �g ofmicrosomal protein. Where indicated, 0.5 mM LaCl3 was added to pre-vent dephosphorylation of the pump. To test cation dependence, CaCl2was added to a total concentration of 1.232 mM, resulting in a final freeCa2� concentration of 220 �M. The reaction was started by adding[�-32P]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

NaH2PO4, 2 mM ATP, and 5% trichloroacetic acid followed by centrifu-gation. 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 roomtemperature. After two washes with trichloroacetic acid solution, thepellet was suspended in 20 �l of sample buffer and subjected to acidicSDS/polyacrylamide gel electrophoresis (35) and autoradiography.

RESULTS

Cloning and Characterization of a SERCA-type Ca2�-ATPase Gene—To clone the SERCA-type Ca2�-ATPase gene ofT. cruzi, a region containing a conserved Ca2�-ATPase se-quence was amplified from T. cruzi genomic DNA. To designdegenerate oligonucleotide primers, amino acid sequences ofSERCA-type Ca2�-ATPases of different species were retrievedfrom GenBankTM, and regions with the highest similarity werelocated. Two primers (F4 and R2) were selected according tothese domains, and the PCR was carried out with T. cruzigenomic DNA as a template. One of the PCR products (�650bp) reacted strongly with a DNA probe from the Leishmaniamexicana amazonensis putative SERCA-type Ca2�-ATPase(Lmaa1) (32) by Southern blotting, and it was cloned intopGEM-T-easy. The deduced amino acid sequence of this PCRclone was 77.6 and 84.2% identical to the sequences of theputative SERCA-type Ca2�-ATPases of L. m. amazonensis

FIG. 2. Expression of TcSCA mRNA in amastigote (A), trypo-mastigote (T), and epimastigote (E) forms of T. cruzi. Upperpanel, total RNA (10 �g/lane) was electrophoresed, blotted, and probedat high stringency with a 32P-labeled probe corresponding to the entireTcSCA ORF. Size markers are indicated on the left. Approximatelyequal amounts of RNA were observed in the three lanes under UV light.Lower panel, the membranes were stripped and reprobed with a 32P-labeled PCR fragment of the TcP0 gene from T. cruzi as control. Expo-sure time was 2 h for the upper panel and 10 min for the lower panel. kb,kilobases.

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(Lmaa1) (32) and Trypanosoma brucei (TBA1) (36, 37), respec-tively. A T. cruzi genomic library was constructed and screenedusing this PCR fragment as a probe. A genomic clone was foundto contain the sequence of the 3� region (1278 bp) of the puta-tive SERCA-type Ca2�-ATPase gene of T. cruzi. To obtain thesequence of the 5� region of the gene, cDNA was synthesized,and the gene was amplified by PCR using primers correspond-ing to the splice leader sequence and a specific sequence pres-ent in the genomic clone. PCR produced a DNA fragment thatcovered the 5� region (1957 bp) of the gene including a fragmentof 205 bp overlapping with the sequence of the genomic clone.Together with the genomic clone sequence, the PCR productsequence gave the 3018-bp ORF of the gene, encoding 1006amino acid residues, which was named TcSCA for T. cruziSERCA-type Ca2�-ATPase (GenBankTM accession numberAF093566).

Sequence Analysis of TcSCA—The TcSCA amino acid se-quence is 73% identical to the T. brucei SERCA-type Ca2�-ATPase sequence (TBA1 (36, 37)) and 61% identical to the L. m.amazonensis putative SERCA-type Ca2�-ATPase sequence(Lmaa1 (32)) (Fig. 1). It has about 46–48% identity to SERCA-type Ca2�-ATPase sequences of non-trypanosomatids and only24.1–24.8% identity to plasma membrane-type Ca2�-ATPasessequences from different species. Analysis of the TcSCA aminoacid sequence showed that this gene product contains all theconserved subdomains and invariant residues found in otherP-type ATPases such as the phosphorylation and ATP bindingdomains (38, 39). Hydropathy analysis of the deduced amino

acid sequence revealed a profile very similar to those of othercalcium pumps containing 10 transmembrane domains (M1-M10) (Fig. 1). This is in line with structures obtained fromcrystals of a mammalian SERCA-type Ca2�-ATPase at 8 Å ofresolution (40). TcSCA also contains all the residues (Glu315,Glu765, Asn790, Thr793, Asp794, and Glu895, indicated by aster-isks above the alignment in Fig. 1) that were previously iden-tified as the high affinity Ca2�-binding sites in the center of theputative transmembrane domains M4, M5, M6, and M8 (41).As occurs with other SERCA-type pumps, TcSCA lacks theconserved amino acid sequence associated with calmodulinbinding found near the COOH terminus of mammalian plasmamembrane-type Ca2� ATPase isoforms (42). The amino acidsequence Lys-Asp-Asp-Lys-Pro-Val402, which was found to becritical for the functional association of the Ca2�-ATPase ofcardiac sarcoplasmic reticulum with phospholamban (42), isabsent in TcSCA. Interestingly the residues located in trans-membrane segment 3 important for thapsigargin binding ofSERCA Ca2�-ATPases (43) are different in TcSCA (10 of 20residues in segment 3 are different as compared with SERCApumps). In agreement with these results we were unable todetect any significant increase in [Ca2�]i in fura 2-loaded cellsin the presence of low concentrations of thapsigargin (0.1–4�M) (14, 15). These results were also confirmed in experimentswith the enzyme expressed in yeast (see below). Interestingly,TcSCA has three potential cAMP-dependent protein kinasephosphorylation sites, which are common to all three putativeSERCA-type Ca2�-ATPases of kinetoplastid parasites but are

FIG. 3. Fluorescence microscopyanalysis of the SERCA-type Ca2�-ATPase fused with GFP. The figureshows the co-localization of the Ca2�-ATPase (A, B, and C) with BiP (D) orcalreticulin (E) and a distinct localizationof the plasma membrane-type Ca2�-ATPase (F). Bar, 10 �m.

FIG. 4. Immunoblot analysis ofTcSCA (inset in A) and immunofluo-rescence microscopy showing the lo-calization 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 TcSCA inthe endoplasmic reticulum in all three lifecycle stages. Panel D shows the lack ofreaction when preimmune serum wasused with epimastigotes. Lower panelsshow the same cells as in the upper panelsby bright-field microscopy. Bar, 10 �m(A–H). Inset shows bands of the sameapparent molecular weight in parasitelysates (10 �g/well) of different stages ofT. cruzi.

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not present in those of other species (Fig. 1). A genomic South-ern blot probed by TcSCA DNA showed a single hybridizingband in each lane except for EcoRI (results not shown), sug-gesting that TcSCA is a single-copy gene. The two DNA bandsobserved by EcoRI digestion are due to the presence of aninternal EcoRI site in the TcSCA sequence (nucleotide 1957).

Expression of TcSCA in Different Stages of T. cruzi—North-ern blot analysis showed the presence of a single TcSCA tran-

script of approximately 5 kilobases in each of the three life cyclestages of T. cruzi (Fig. 2, upper panel). Analysis of the 5-kilo-base band by densitometry indicated that the TcSCA gene isexpressed at similar levels in all stages of T. cruzi. This is incontrast with lmaa1, the gene for the putative L. m. ama-zonensis SERCA-type Ca2� pump that is developmentallyregulated and more abundantly expressed in intracellularamastigotes (32).

Localization of TcSCA—To determine the localization ofTcSCA, the sequence of the GFP of Aequorea victoria wasfused to the TcSCA ORF and ligated into the T. cruzi expres-sion vector pTEX (28, 29). The loop region in TcSCA waschosen for the site of insertion of GFP to avoid any possibleinterference with a potential targeting signal sequence. T.cruzi epimastigotes were transfected with this TcSCA-GFPplasmid, and the stable transfectants were observed by fluo-rescence microscopy. Transgenic parasites expressing theTsSCA-GFP construct exhibited GFP florescence as a ringsurrounding the nucleus and in a network extending from itsperiphery (Fig. 3, A–C), suggesting an endoplasmic reticulum(ER) localization. To further confirm these results, trans-genic cells were also stained with antibodies against BiP andcalreticulin, two ER chaperones involved in the control ofprotein folding (44). Both BiP (Fig. 3D) and calreticulin (Fig.3E) co-localized with TcSCA (Figs. 3, A and B), thus confirm-ing the ER localization of TcSCA. In contrast, incubation oftransgenic cells with antibodies against the acidocalcisomal

FIG. 5. TcSCA restored the growthof yeast mutant K616 in Ca2�-de-pleted medium. S. cerevisiae pmr1 pmc1cnb1 strain K616 was transformed with acontrol vector (pYES) or a vector contain-ing the entire open reading frame of T.cruzi TcSCA (TcSCA/pYES). The cultureswere inoculated into SC-URA containinggalactose plus either 0, 2, 4, 6, 8, or 10 mM

EGTA or 10, 50 or 200 mM CaCl2, andincubated for 24 (A) or 48 (B) h. Growthwas monitored by measuring A600.

FIG. 6. TcSCA complements sensitivity of yeast K616 to Mn2�.S. cerevisiae K616 was transformed as in Fig. 5 and grown in SC-URAcontaining galactose. Bars represent the average increase in A600 after4 days of growth in 3 experiments; error bars are S.D. MnCl2 was addedat the indicated concentrations; Control, no added manganese.

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Ca2�-ATPase of T. cruzi (6) resulted in a punctate staining(Fig. 3F), clearly different from the perinuclear and reticularlocalization of TcSCA (Fig. 3C).

We also investigated the localization of TcSCA in other de-velopmental stages of T. cruzi. Total lysates from differentstages of T. cruzi were subjected to Western blotting analysiswith antibodies against a 518-amino acid, COOH-terminal do-main of TcSCA. These antibodies detected a single band of�110 kDa (Fig. 4, inset), close to the predicted molecular massof TcSCA, in epimastigote (E), trypomastigote (T), andamastigote (A) lysates. No band was detected when using pre-immune serum (data not shown). In indirect immunofluores-cence assays using the anti-TcSCA antiserum, TcSCA wasdetected (Fig. 4) in epimastigotes (A), trypomastigotes (B), andamastigotes (C) as a ring surrounding the nucleus and in anetwork extending from its periphery, similar to the GFP pro-tein labeling (Fig. 3). No detectable signal was observed whenthe preimmune serum was used (Fig. 4D).

Functional Complementation by TcSCA of the Ca2�-ATPase-deficient S. cerevisiae Strain K616—To test the func-tion of TcSCA, 3018 bp of the TcSCA ORF were subclonedinto a yeast expression vector, pYES2, under the control of agalactose-inducible promoter, and the resulting construct orthe 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) Ca2� pumps and also lackscalcineurin (CNB 1) function. This mutant provides an ex-tremely valuable expression system for determining the na-ture of individual Ca2� pumps from other eukaryotes (23, 45).The triple mutant transformed with vector alone grew poorlyon a medium containing low Ca2� (2–10 mM EGTA) (Fig. 5).This effect was noticeable even at low concentrations ofEGTA during the initial 24 h of growth (Fig. 5A) and de-creased after 48 h (Fig. 5B). However, the triple mutanttransformed with TcSCA became tolerant of EGTA, support-ing the idea that TcSCA encodes a functional divalent cationpump. The likely transport of Ca2� was supported by resultson phosphoenzyme formation (below). Transport of Mn2� wasindicated by complementation of the Mn2� sensitivity of thetriple mutant (Fig. 6). The mutant with or without TcSCAgrew equally well in glucose medium, where the gene is notinduced (results not shown). In galactose medium, though,there was little growth of the vector control strain in thepresence of 1–3 mM MnCl2, whereas the TcSCA-transformed(and induced) strain showed significant growth (Fig. 6).

Effect of Inhibitors on the Formation of a Ca2�-dependentPhosphoenzyme Intermediate—To test if TcSCA was able toform a phosphorylated intermediate like other P-type Ca2�-ATPases (46, 47), we isolated microsomes from TcSCA-trans-formed K616 and incubated them with [�-32P]ATP undervarious conditions. A major phosphoprotein of 110 kDa wasformed in membranes isolated from TcSCA-transformedK616 (Fig. 7A, lane 7) but was absent in yeast transformedwith vector alone (Fig. 7A, lane 3). Phosphorylation wasdependent on the presence of Ca2�. La3� enhanced thesteady-state level of the phosphoprotein severalfold (Fig. 7A,lane 8). The denatured phosphoprotein was sensitive to hy-droxylamine (Fig. 7A, lane 9), indicating the hydrolysis of anacyl phosphate bond (48) probably formed by Asp357 (Fig. 1).Together, these results provide compelling evidence that Tc-SCA is a P-type Ca2�-dependent ATPase.

Cyclopiazonic acid, an inhibitor of animal SERCA pumps,inhibited the phosphorylation of TcSCA (Fig. 7B, lanes 6 and7), whereas thapsigargin had no effect at low concentrations(Fig. 7B, lanes 2 and 3). The phosphorylation of TcSCA was alsoinhibited by erythrosin B (Fig. 7B, lanes 4 and 5), a haloge-

nated derivative of fluorescein that binds to nucleotide-bindingsites with high affinity and specificity (49).

DISCUSSION

Using yeast as a heterologous expression system (23), weprovide the first direct evidence that a cloned SERCA-type T.cruzi gene encodes a functional Ca2�-ATPase. Expression ofthe T. cruzi TcSCA gene restored the growth in a mediumcontaining submicromolar levels of Ca2� of a yeast mutant(K616) defective in Ca2� pumps (Fig. 4). Several lines ofevidence indicate that TcSCA encodes an ER Ca2� pump; 1)its amino acid sequence shares 46–48% identity with SERCApumps and less identity (24.1–24.8%) with plasma mem-brane-type Ca2�-ATPases; 2) TcSCA contains ER retentionmotifs (50), KKXX-stop at the COOH terminus and RILL inthe first transmembrane domain (Fig. 1); 3) TcSCA is mainlylocalized to the nuclear membrane and to a reticular struc-ture in different stages of T. cruzi (Figs. 3 and 4); 4) TcSCAco-localizes with calreticulin and BiP, two well known endo-plasmic reticulum chaperones (Fig. 3).

As occurs with plant SERCA-type Ca2� ATPases (48),TcSCA appears to be insensitive to thapsigargin (14, 15).Residues located in the third transmembrane segment (M3)and in the stalk segment (S3) have been postulated to beimportant for thapsigargin binding and are conserved in allSERCA Ca2�-ATPases (43, 51). Since the T. brucei SERCApump TBA1 was reportedly sensitive to thapsigargin (37),whereas L. m. amazonensis putative SERCA pump LMAA1was not (32), it was proposed that two amino acid substitu-tions present in LMAA1 could account for their differentsensitivity: a Gly271 in LMAA1 that replaces Lys261 in TBA1

FIG. 7. Formation of a Ca2�-dependent phosphoprotein is in-hibited by cyclopiazonic acid. A, Ca2�-dependent phosphoenzymeformation. Membranes were isolated from triple mutants, K616, trans-formed with either TcSCA (right) or with YES vector alone (left). Mem-branes were incubated with 19 �M [32P]ATP for 30 s with 1 mM EGTAalone (lanes 1 and 5), with added 0.5 mM LaCl3 (lanes 2, 4, 6, 8, and 9),or with added CaCl2 to give a final concentration of 220 �M Ca2� (lanes3, 4, 7, 8, and 9). The reaction was stopped with trichloroacetic acid, andthe proteins were analyzed by acidic SDS/polyacrylamide gel electro-phoresis and autoradiography. To test the sensitivity of phosphoenzymeformation to hydroxylamine (lane 9), the trichloroacetic acid pellet wasincubated with 10 �l of 0.8 mM hydroxylamine in 0.6 mM sodium acetate(pH 5.3) for 15 min before SDS/polyacrylamide gel electrophoresis. B,inhibition of phosphoenzyme formation by erythrosin B (EB) and cyclo-piazonic acid (CPA) but not by thapsigargin (TG). Membranes isolatedfrom triple mutant transformed with TcSCA were incubated withMe2SO (not shown) or inhibitors for 20 min before assay, Phosphoen-zyme formation was assayed with 220 �M free Ca2� and 20 �M ATP for30 s without La3�.

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and a Phe279 in LMAA1 that replaces Val269 in TBA1 (32).However, one of these amino acids is also present in T. cruziSERCA pump TcSCA; a Lys259 is in the same position inTcSCA as Lys261 in TBA1. In addition, instead of a Val269 inTBA1, there is an Ala269 in TcSCA, which is a conservedsubstitution. A comparison of the sequence of this transmem-brane segment M3 in TcSCA with those of other Ca2�-AT-Pases indicates that TcSCA shares 12 out of 20 residues withSERCA pumps and 15 out of 20 residues with T. brucei Ca2�-ATPase TBA1 (37). (See Scheme I, showing the alignment ofTcSCA with TBA1, and LMAA1; the colons and dots indicateidentical and similar amino acid residues, respectively).

Other residues that are different in TcSCA as compared withTBA1 are conserved substitutions and possibly could not ac-count for the differences in sensitivity to thapsigargin, exceptfor a Thr266 that replaces an Ile in TBA1 and LMAA1. There isonly one difference in the S3 segment; a Met257 replaces a Valin TBA1 and LMAA1. Our conclusion is that these differencesin transmembrane segment M3 and S3 could account for thedifferences in sensitivity to thapsigargin of these pumps.

In mammalian cells, SERCAs are important in refilling ERcalcium stores used in signaling. Ca2� is released from the ERby inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose, or nic-otinic acid adenine dinucleotide phosphate acting upon IP3 orryanodine receptors (52, 53). Whether the same applies to T.cruzi is uncertain. The isolated ER has not been studied intrypanosomatids, but the most likely alternative store of cal-cium for signaling, the acidocalcisome (8), is not sensitive tocalcium-releasing metabolites after isolation from T. cruzi (7).2

Calcium signals certainly appear to be generated in trypano-somatids, by L. m. amazonensis during macrophage invasion(32) and by T. cruzi during the invasion of myoblasts (1). Inboth cases, chelation of intracellular Ca2� inhibited invasion.

Another important function of Ca2�-ATPases located in theER and the Golgi of different cells is to supply intralumenalcations (not just Ca2� but also Mn2�) required for the correctprocessing of proteins through the secretory pathway in eu-karyotic cells. There is obviously some overlap in functionbetween the Golgi and ER Ca2�-ATPases as the yeast pmr1mutant can be complemented by ER Ca2�-ATPases from ani-mals (54, 55) and plants (23). The best-studied case of a Ca2�-ATPase also transporting Mn2� is the yeast Golgi PMR1 pro-tein (56), but some Ca2�-ATPases located in the ER alsoprobably transport Mn2�, including those of mammals (57),and plants (23). In the latter instance, the plant Ca2�-ATPasewas found to alleviate Mn2� toxicity in the pmr1 mutant,similar to the results we report here. Within the ER and Golgiof yeast, Ca2� and Mn2� are involved in processes of proteinfolding, degradation of mis-folded proteins, sorting to the vac-uole, and glycosylation (56). Similar results were found inearlier work with mammalian cells (57–59), although in some

of these studies it was assumed that the observed effects weredue to Ca2� without regard to Mn2�. In PC 12 cells, expressionof a SERCA isoform is enhanced upon treatment of the cellswith agents that interfere with protein folding or inhibit gly-cosylation or disrupt the Golgi body (60). Evidence fromSERCA gene-knockout studies in Drosophila also showed avital role for the pump in the processing and trafficking ofseveral plasma membrane or cell junction transmembrane pro-teins (61). Therefore, in T. cruzi, the TcSCA transporter may beimportant in the maintenance of lumenal Ca2� and/or Mn2�

required for proper trafficking and modification (glycosylation)of new proteins during differentiation and, particularly (fromthe point of view of infectivity), cell surface proteins involved ininteractions with host cells.

Acknowledgments—We thank Kyle Cunningham for the K616 yeaststrain, Steven Beverley for plasmid pXG-GFP�2�, David Engman forvector pTEX, James Bangs for antibody against BiP, Armando Parodifor antibody against calreticulin, and Linda Brown for technicalassistance.

REFERENCES

1. Moreno, S. N. J., Silva, J., Vercesi, A. E., and Docampo, R. (1994) J. Exp. Med.180, 1535–1540

2. Docampo, R., and Moreno, S. N. J. (1996) Parasitol. Today 12, 61–653. Docampo, R., Scott, D. A., Vercesi, A. E., and Moreno, S. N. J. (1995) Biochem.

J. 310, 1005–10124. Scott, D. A., Docampo, R., Dvorak, J. A., Shi, S., and Leapman, R. D. (1997)

J. Biol. Chem. 272, 28020–280295. Scott, D. A., de Souza, W., Benchimol, M., Zhong, L., Lu, H.-G., Moreno,

S. N. J., and Docampo, R. (1998) J. Biol. Chem. 273, 22151–221586. Lu, H.-G., Zhong, L., de Souza, W., Benchimol, M., Moreno, S. N. J., and

Docampo, R. (1998) Mol. Cell. Biol. 18, 2309–23237. Scott, D. A., and Docampo, R. (2000) J. Biol. Chem. 275, 24215–242218. Docampo, R., and Moreno, S. N. J. (1999) Parasitol. Today 15, 443–4489. Vercesi, A. E., Hoffmann, M. E., Bernardes, C. F., and Docampo, R. (1991) Cell

Calcium 12, 361–36910. Carafoli, E., and Brini, M. (2000) Curr. Opin. Chem. Biol. 4, 152–16111. Parodi, A. (2000) Annu. Rev. Biochem. 69, 69–9312. Labriola C., Cazzulo J. J., and Parodi, A. (1999) Mol. Biol. Cell 10, 1381–139413. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P.

(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466–247014. Moreno, S. N. J., Vercesi, A. E, Pignataro, O. P., and Docampo, R. (1992) Mol.

Biochem. Parasitol. 52, 251–26215. Docampo, R., Moreno, S. N. J., and Vercesi, A. E. (1993) Mol. Biochem.

Parasitol. 59, 305–31416. Ferrol, N., and Bennett, A. B. (1996) Plant Cell 8, 1159–116917. Cunningham, K. W., and Fink, G. R. (1994) J. Cell Biol. 124, 351–36318. Moniakis, J., Coukell, M. B., and Forer, A. (1995) J. Biol. Chem. 270,

28276–2828119. Ghosh, S. K., Rosenthal, B., Rogers, R., and Samuelson, J. (2000) Mol. Bio-

chem. Parasitol. 108, 125–13020. Luo, S., Vieira, M., Graves, J., Zhong, L., and Moreno, S. N. J. (2001) EMBO

J. 20, 55–6421. Schmatz, D. M., and Murray, P. K. (1982) Parasitology 85, 115–12522. Bone, G. J., and Steinert, M. (1956) Nature 178, 308–30923. Liang, F., Cunningham, K. W., Harper, J. F., and Sze, H. (1997) Proc. Natl.

Acad. Sci. U. S. A. 94, 8579–858424. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A

Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, NY

25. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci, U. S. A.74, 5463–5467

26. Kyte, J., and Doolittle, D. F. (1982) J. Mol. Biol. 157, 105–13227. Kozak, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8301–830528. Ha, D. S., Schwarz, J. K., Turco, S. J., and Beverley, S. M. (1996) Mol. Biochem.

Parasitol. 77, 57–6429. Kelly, J. M., Ward, H. M., Miles, M. A., and Kendall, G. (1992) Nucleic Acids

Res. 20, 3963–396930. Skeiky, Y. A. W., Benson, D. R., Parsons, M., Elkon, K. B., and Reed, S. G.

(1992) J. Exp. Med. 176, 201–21131. Furuya, T., Kashuba, C., Docampo, R., and Moreno, S. N. J. (2000) J. Biol.

Chem. 275, 6428–643832. Lu, H. G., Zhong, L., Chang, K. P., and Docampo, R. (1997) J. Biol. Chem. 272,

9464–947333. Chen, D. C., Yang, B. C., and Kuo, T. -T. (1992) Curr. Genet. 21, 83–8434. Schatzmann, H. J., and Burgin, H. (1978) Ann. N. Y. Acad. Sci. 307, 125–14735. Sarkadi, B., Enyedi, A., Foldes-Papp, Z., and Gardos, G. (1986) J. Biol. Chem.

261, 9552–955736. Revelard, P., and Pays, E. (1991) Mol. Biochem. Parasitol. 46, 241–25237. Nolan, D. P., Revelard, P., and Pays, E. (1994) J. Biol. Chem. 269,

26045–2605138. Allen, G., and Green, N. M. (1976) FEBS Lett. 63, 188–19239. Pick, U., and Bassilian, S. (1981) FEBS Lett. 123, 127–13040. Zhang, P., Toyoshima, C., Yonekura, K., Green, N. M., and Stokes, D. L. (1998)

Nature 392, 835–8392 D. A. Scott, unpublished information.

SCHEME 1

A SERCA-type Ca2�-ATPase from T. cruzi32444

by guest on June 27, 2016http://w

ww

.jbc.org/D

ownloaded from

41. Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. (1989) Nature 339,476–478

42. Toyofuku, T., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1994)J. Biol. Chem. 269, 22929–22932

43. Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem. 269,26598–26601

44. Hebert, D. N., Simons, J. F., Peterson, J. R., and Helenius, A. (1995) ColdSpring Harbor Symp. Quant. Biol. 60, 405–415

45. Liang, F., and Sze, H. (1998) Plant Physiol. 118, 817–82546. de Meis, L. (1988) Methods Enzymol. 157, 190–20647. Schatzmann, H. J. (1989) Annu. Rev. Physiol. 51, 473–48548. Mignaco, J. A., Barrabin, H., and Scofano, H. M. (1996) J. Biol. Chem. 271,

18423–1843049. Antebi, A., and Fink, G. R. (1992) Mol. Biol. Cell 3, 633–65450. Geisler, M., Axelsen, K. B., Harper, J. F., and Palmgren, M. G. (2000) Biochim.

Biophys. Acta 1465, 52–7851. Zhong, L., and Inesi, G. (1998) J. Biol. Chem. 273, 12994–12998

52. Berridge, M. J. (1997) J. Physiol. (Lond.) 499, 291–30653. Cancela, J. M., Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V., and

Petersen, O. H. (2000) EMBO J. 19, 2549–255754. Talla, E., de Mendonca, R. L., Degand, I., Goffeau, A., and Ghislain, M. (1998)

J. Biol. Chem. 273, 27831–2784055. Degand, I., Catty, P., Talla, E., Thines-Sempoux, D., de Kerchove d’Exaerde,

A., Goffeau, A., and Ghislain, M. (1999) Mol. Microbiol. 31, 545–55656. Durr, G., Strayle, J., Plemper, R., Elbs, S., Klee, S. K., Catty, P., Wolf, D. H.,

and Rudolph, H. K. (1998) Mol. Biol. Cell 9, 1149–116257. Kaufman, R. J., Swaroop, M., and Murtha-Riel, P. (1994) Biochemistry 33,

9813–981958. Lodish, H. F., and Kong, N. (1990) J. Biol. Chem. 265, 10893–1089959. Kuznetsov, G., Brostrom, M. A., and Brostrom, C. O. (1992) J. Biol. Chem. 267,

3932–393960. Caspersen, C., Pedersen, P. S., and Treiman, M. (2000) J. Biol. Chem. 275,

22363–2237261. Periz, G., and Fortini, M. E. (1999) EMBO J. 18, 5983–5993

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Tetsuya Furuya, Michael Okura, Felix A. Ruiz, David A. Scott and Roberto Docampo Trypanosoma cruzi-ATPase That Localizes to the Endoplasmic Reticulum of 2+ Pumps and Encodes a Ca2+ Complements Yeast Mutants Defective in CaTcSCA

doi: 10.1074/jbc.M104000200 originally published online May 29, 20012001, 276:32437-32445.J. Biol. Chem. 

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