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A plasma membrane-type Ca2+-ATPase co-localizes with a vacuolar H+-pyrophosphatase to acidocalcisomes of Toxoplasma gondii

2001; Springer Nature; Volume: 20; Issue: 1 Linguagem: Inglês

10.1093/emboj/20.1.55

1460-2075

Shuhong Luo, Maurício Vieira, Jessica Graves, Zhong Li, Silvia N.J. Moreno,

Endoplasmic Reticulum Stress and Disease

Article15 January 2001free access A plasma membrane-type Ca2+-ATPase co-localizes with a vacuolar H+-pyrophosphatase to acidocalcisomes of Toxoplasma gondii Shuhong Luo Shuhong Luo Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Mauricio Vieira Mauricio Vieira Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Jessica Graves Jessica Graves Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Li Zhong Li Zhong Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Silvia N.J. Moreno Corresponding Author Silvia N.J. Moreno Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Shuhong Luo Shuhong Luo Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Mauricio Vieira Mauricio Vieira Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Jessica Graves Jessica Graves Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Li Zhong Li Zhong Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Silvia N.J. Moreno Corresponding Author Silvia N.J. Moreno Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA Search for more papers by this author Author Information Shuhong Luo1, Mauricio Vieira1, Jessica Graves1, Li Zhong1 and Silvia N.J. Moreno 1 1Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:55-64https://doi.org/10.1093/emboj/20.1.55 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ca2+-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca2+ concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium. Here, we report the cloning and sequencing of a gene encoding a plasma membrane-type Ca2+-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32–36% identity to vacuolar Ca2+-ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum. Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C-terminus. A hydropathy profile of the protein suggests 10 transmembrane domains. TgA1 suppresses the Ca2+ hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co-localizes with the vacuolar H+-pyrophosphatase to intracellular vacuoles identified morphologically and by X-ray microanalysis as the acidocalcisomes. This vacuolar-type Ca2+-ATPase could play an important role in Ca2+ homeostasis in T.gondii. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite of humans and animals that has emerged as a major opportunistic pathogen in people with AIDS. Infection with T.gondii is usually asymptomatic and results in the formation of dormant encysted bradyzoites that remain in the brain and other tissues for life. Only the developing fetus and the immunosuppressed patient are at substantial risk of severe disease. The tachyzoite is the rapidly growing asexual form that is also seen in acutely infected animals. Ca2+ signaling has been shown to play a key role in the process of mammalian cell invasion by T.gondii. An increase in the cytosolic Ca2+ concentration ([Ca2+]i) of tachyzoites occurs upon invasion (Vieira and Moreno, 2000), and pretreatment of tachyzoites with an intracellular Ca2+ chelator (BAPTA/AM) to prevent the increase in [Ca2+]i results in an inhibition of cellular invasion (Vieira and Moreno, 2000). Recent studies (Carruthers and Sibley, 1999; Carruthers et al., 1999) have also demonstrated that agents able to increase [Ca2+]i of tachyzoites were able to stimulate microneme secretion, suggesting a role for Ca2+ in this process that is essential for penetration of T.gondii into host cells (Carruthers and Sibley, 1997, 1999; Carruthers et al., 1999). Unlike mammalian cells, T.gondii possesses a great proportion of its intracellular Ca2+ in an acidic compartment named the acidocalcisome (Moreno and Zhong, 1996). Acidocalcisomes were first described in trypanosomatids (Vercesi et al., 1994; Docampo et al., 1995; Lu et al., 1997) and have recently been postulated to be similar to the organelles described historically as volutin granules or polyphosphate bodies in different microorganisms (Docampo and Moreno, 1999). Acidocalcisomes in trypanosomatids are characterized by their high electron density, high content of polyphosphates, calcium, magnesium, sodium and zinc (Scott et al., 1997; Urbina et al., 1999), and a number of pumps and exchangers in their limiting membrane, among them a Ca2+-ATPase, a vacuolar H+-ATPase, a Na+/H+ exhanger and a vacuolar H+-pyrophosphatase (V-H+-PPase) (Scott et al., 1995; Vercesi and Docampo, 1996; Lu et al., 1998; Scott et al., 1998; Rodrigues et al., 1999a,b). Morphologically similar structures have been described in T.gondii and termed black granules, and have been postulated to be the acidocalcisomes (Bouchot et al., 1999). In addition, the presence of a V-H+-ATPase and a V-H+-PPase located in intracellular vacuoles has been described in T.gondii, but the vacuoles were not identified at the electron microscope level (Moreno et al., 1998; Rodrigues et al., 2000). This study reports the identification in T.gondii of the TgA1 gene, which encodes a protein with homology to mammalian plasma membrane Ca2+-ATPases (PMCA) and with characteristics that place it in the novel category of Ca2+-ATPases formed by the acidocalcisomal Ca2+-ATPase of Trypanosoma cruzi (Lu et al., 1998) and the vacuolar Ca2+-ATPases described in Saccharomyces cerevisiae (Cunningham and Fink, 1994), Dictyostelium discoideum (Moniakis et al., 1995) and Entamoeba histolytica (Ghosh et al., 2000). Indirect immunofluorescence and immunoelectron microscopy analysis suggest that the product of this gene (TgA1) is associated not only with the plasma membrane but also with the acidocalcisomes, where it co-localizes with the V-H+-PPase. The gene is expressed at high levels in both tachyzoites and bradyzoites and is able to complement functionally the PMC1 gene, encoding the vacuolar Ca2+-ATPase of S.cerevisiae. Results Cloning and characterization of a Ca2+-ATPase gene Since T.gondii acidocalcisomes (Moreno and Zhong, 1996) are physiologically similar to the same organelles present in T.cruzi (Docampo et al., 1995), we looked for the presence of a gene encoding a Ca2+-ATPase with homology to the Ca2+-ATPase present in acidocalcisomes of T.cruzi (Lu et al., 1998). Degenerate oligonucleotides corresponding to two conserved domains in Ca2+-ATPases, a phosphorylation site and a site involved in ATP binding (Allen and Green, 1976; Pick and Bassilian, 1981), were used to amplify, by PCR, specific sequences from T.gondii genomic DNA. The PCR products were cloned and sequenced. Analysis of the deduced partial amino acid sequence of these clones revealed that a 1.4 kb PCR clone had the best score of sequence identity (50.2%) and similarity (56.6%) with the acidocalcisomal Ca2+-ATPase described in T.cruzi (Lu et al., 1998). To obtain the complete gene, this PCR clone (TgA1.4) was used as a probe to screen T.gondii genomic and cDNA libraries. Screening of 3 × 105 plaques from the genomic library yielded 51 positive clones. Mapping and sequencing of five clones revealed an 8.5 kb fragment containing a complete open reading frame (ORF), designated TgA1, with 4215 nucleotides. Interestingly, this ORF was interrupted by introns near the C-terminal region. The DNA sequence of the ∼1.4 kb PCR product was identical to the corresponding region of the gene obtained from the lambda EMBL3 genomic library. Screening of 5 × 105 plaques from the cDNA library with TgA1.4 yielded 12 positive cDNA clones, λc1 to λc12. Restriction enzyme digestion analysis indicated similar patterns but clone size varied from ∼0.8 to ∼5.4 kb. Four cDNA clones (λc1, λc4, λc7 and λc10) were selected for sequence analysis. Sequence data indicated that there were identical overlapping areas in all four clones. Comparison of the genomic sequence with the cDNA sequence revealed that the ORF of TgA1 contains three exons interrupted by two introns. The exons ranged in size from 320 to 3853 bp. The introns ranged between 330 and 479 bp with the 5′-G/GTRAGY and 3′-(Y)nAG/G conserved splice-site signals typical of eukaryotic nuclear introns (Breathnach and Chambon, 1981; Nagel and Boothroyd, 1988). According to the consensus translational initiation sequence ‘gNCAAaATGg’ for T.gondii genes (Seeber, 1997), which is similar to the Kozak sequence (GCCA/GCCATGG) of higher eukaryotes (Kozak, 1989), and the initiation codon ATG predicted, the ORF codes for a protein of 1405 amino acids with a predicted molecular mass of 152 827 Da (Figure 1). The first nucleotide of the longest clone, λc7 (5424 bp), was located 109 nt upstream of the start codon (base 2998). The exact transcription start site was not mapped. A computer-aided database search of nucleotides 1–2900 did not reveal eukaryotic promoter consensus sequences. However, eight sequences with nucleotides cgtct/gct/a (cis-acting elements) were identified (Mercier et al., 1996) within nucleotides 1–2500. Figure 1.Alignment of different Ca2+-ATPases. CLUSTAL W alignment (Thompson et al., 1994) of Ca2+-ATPases from T.gondii (DDBJ/EMBL/GenBank accession No. AF151371), T.cruzi (U70620) and S.cerevisiae (U03060). Similar residues are shaded. Amino acid residues not present within other sequences are denoted by dashes. The ATP-binding and autophosphorylation domains employed in the design of degenerate oligonucleotides for PCR are underlined, the predicted transmembrane domains in the T.gondii sequence are indicated by dashed lines above the alignment. The potential N-glycosylation sites are indicated with a dash above the alignment. Download figure Download PowerPoint Examination of the sequence of 1097 bases flanking TgA1 at the 3′-end of the ORF revealed some interesting features. The consensus poly(A) signal found in plants and higher eukaryotes, AATAAA, was not present. However, a related motif, ATAAA, was found twice, at 92 and 981 bases from the stop codon (bases 8225 and 9114), and a motif AATAAG was found 253 bases downstream of the TGA codon (base 8487). Three (TA)8 strings located at 486, 707 and 789 bases downstream of the stop codon (bases 8620, 8840 and 8922) were identified. In order to confirm the transcription of the TgA1 gene and determine the sequence of the 5′-end of the transcript, RT–PCR was performed as described in Materials and methods. Sequence analysis of the RT–PCR products (bands of 540 and 428 bp) indicated that they were derived from the TgA1 gene and that the predicted initiation codon of the ORF was preceded by 223 bp of 5′-untranslated sequence. Southern blotting was performed with TgA1.4 as a probe to confirm the presence of this gene in the T.gondii genome (Figure 2A). Genomic DNA from T.gondii was digested with several restriction enzymes (selected to demonstrate genome copy number) and hybridized at high stringency with the TgA1.4 probe. The different restriction enzymes used produced single bands, which varied in size (Figure 2A, lanes 1–5), suggesting that TgA1 is present as a single copy gene in the haploid Toxoplasma genome. No band was detected when bovine turbinate (BT) cells were used as control (Figure 2A, lane 6). Figure 2.Southern and northern analysis of TgA1. (A) Southern blot analysis of the TgA1 gene in genomic DNA from T.gondii. Total genomic DNA (10 μg/lane) was digested with various restriction enzymes and analyzed as described in Materials and methods. Size markers are indicated. Trypanosoma gondii DNA digested with the following restriction enzymes: lane 1, EcoRI; 2, BamHI; 3, HindIII; 4, PstI; 5, BglII. Control BT cell's DNA (10 μg/lane) was digested with EcoRI and BamHI (lane 6). The blots were hybridized with the 32P-labeled 1.4 kb PCR product (TgA1.4) and washed at high stringency. (B) Expression of TgA1 mRNA in tachyzoite (lanes 1 and 2) and bradyzoite (lane 3) forms of T.gondii. Upper panel, poly(A)+ RNA (3.7 μg/lane) was electrophoresed, blotted, and probed at high stringency with a 32P-labeled probe corresponding to the entire TgA1 ORF. Size markers are indicated on the right. Approximately equal 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 TUB1 gene from T.gondii as control. Exposure times were 3 days (upper panel) and 4 h (lower panel) at −80°C. Lane 1, tachyzoites, RH strain; lane 2, tachyzoites, ME49 strain; lane 3, bradyzoites, ME49 strain. Download figure Download PowerPoint Structure of the coding region and genomic organization of TgA1 Analysis of the TgA1 amino acid sequence (Figure 1) 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 (Allen and Green, 1976; Pick and Bassilian, 1981). Hydropathy analysis of the deduced amino acid sequence (Figure 1) revealed a profile very similar to those of other calcium pumps containing 10 transmembrane domains (dashed lines above TgA1 sequence in Figure 1). As occurs with the vacuolar Ca2+-ATPases described for T.cruzi (Lu et al., 1998), S.cerevisiae (Cunningham and Fink, 1994), D.discoideum (Moniakis et al., 1995) and E.histolytica (Ghosh et al., 2000), a TFASTA search of protein databases showed that TgA1 was closely related to the PMCA, with 37% identity (56% similarity) to the PMCA from human erythrocytes (Strehler et al., 1990). It also had 32–36% identity and 53–55% similarity over its entire length with the sequences of the vacuolar Ca2+-ATPases of T.cruzi (Lu et al., 1998), S.cerevisiae (Cunningham and Fink, 1994) and D.discoideum (Moniakis et al., 1995), respectively, and had 25–29% identity to sarcoplasmic (endoplasmic) reticulum-type Ca2+-ATPases (SERCA) and 22–26% identity to Na+,K+-ATPases from different species (Shull et al., 1986; Sverdlov et al., 1987). An alignment of the vacuolar Ca2+-ATPases with the T.cruzi, S.cerevisiae and D.discoideum sequences is shown in Figure 1 using CLUSTAL W (Thompson et al., 1994). TgA1 contains two potential N-glycosylation sites, Asn-Ser-Thr and Asn-Phe-Thr, indicated with a dash above the alignment in Figure 1. TgA1 lacks the conserved amino acid sequence associated with calmodulin binding that is found near the C-terminus of all mammalian PMCA isoforms (Strehler, 1991), as is also the case for the vacuolar Ca2+-ATPases from other eukaryotic organisms, such as S.cerevisiae Pmc1p (Cunningham and Fink, 1994) and D.discoideum PAT1 (Moniakis et al., 1995). Like PAT1 of D.discoideum (Moniakis et al., 1995) and tca1 of T.cruzi (Lu et al., 1998), TgA1 also has a long extension of ∼100 amino acids after transmembrane domain 10, which is absent in Pmc1p. Expression of TgA1 in tachyzoite and bradyzoite forms of T.gondii Northern blot analysis showed a single ∼5.2 kb transcript in tachyzoite and bradyzoite forms of T.gondii (Figure 2B, upper panel). Analysis of the ∼5.2 kb band by densitometry indicated that the TgA1 transcript is expressed at similar levels in tachyzoites and bradyzoites. Bands obtained after hybridization with a PCR product for the TUB1 gene, which is expressed at similar levels in tachyzoite and bradyzoite forms of T.gondii (Nagel and Boothroyd, 1988) (Figure 2B, lower panel), were used as reference controls. To detect the TgA1 gene product, antibodies were raised against a protein of 255 amino acids within the large cytosolic loop, fused to a His6 tag, and purified as described in Materials and methods. This region was chosen because it is the least conserved region of all known Ca2+-ATPases. Total cell lysates prepared from tachyzoite and bradyzoite forms of T.gondii were subjected to western blot analysis with the affinity-purified antibodies. These antibodies detected a single band of ∼135 kDa, close to the predicted molecular mass of TgA1 (Figure 3, lanes 4–6). A 33 kDa His6-TgA1 fusion protein was recognized by anti-TgA1 (Figure 3, lane 7). No background staining was observed when pre-immune serum was used as a control (Figure 3, lanes 1–3). Figure 3.Western blot analysis of TgA1. Total cell lysates containing 20 μg of protein from tachyzoites (RH strain, lanes 1 and 4; ME49 strain, lanes 2 and 5) and bradyzoites (ME49 strain, lanes 3 and 6) and 3 μg of purified recombinant His6-TgA1 fusion protein (lane 7) were subjected to SDS–PAGE on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with antibodies prepared as described in Materials and methods (lanes 4–7) or with pre-immune serum (lanes 1–3). Download figure Download PowerPoint Functional complementation of the PMC1 gene of S.cerevisiae with TgA1 Saccharomyces cerevisiae K665 with deletion of the genes encoding the high-affinity Ca2+-ATPase and low-affinity Ca2+/H+ antiporter (PMC1 and VCX1) is intolerant of high Ca2+ in the growth medium (Cunningham and Fink, 1994). Since the T.gondii TgA1 gene encodes a vacuolar-type Ca2+-ATPase with homology to PMC1, we investigated whether complementation of the vcx1 pmc1 yeast mutants with the TgA1 gene could suppress their Ca2+ hypersensitivity. Figure 4A and B shows that transformation of the vcx1 pmc1 K665 strain with pYES2-TgA1 restored growth on high Ca2+ almost completely, thus suggesting the function of TgA1 as a vacuolar Ca2+-ATPase in these mutants. K665 was transformed with a control vector (K665pYES2) or a vector containing the entire ORF of T.gondii TgA1 (K665pYES2-TgA1). Strain K661 has the PMC1 gene (Cunningham and Fink, 1994) and thus served as positive control (Figure 4A and B). Figure 4.Suppression of the Ca2+ hypersensitivity of the S.cerevisiae vcx1 pmc1 mutant by T.gondii TgA1. Saccharomyces cerevisiae vcx1 pmc1 strain K665 was transformed with a control vector (K665pYES2) or a vector containing the entire ORF of T.gondii TgA1 (K665pYES2-TgA1). Strain K661 has the PMC1 gene and thus served as the positive control. The cultures were streaked on YPD (1% Difco extract, 2% Bacto-Peptone, 2% dextrose pH 5.5) plates containing 200 mM CaCl2 (A), or were inoculated into YPD pH 5.5 with 0, 50, 100, 200 and 400 mM CaCl2, and growth was estimated by measuring the optical density at 600 nm (B), to identify Ca2+-tolerant transformants. Download figure Download PowerPoint Localization of T.gondii Ca2+-ATPase We investigated the localization of the Ca2+-ATPase in T.gondii by immunocytochemistry with the antibodies described above. The reaction of these antibodies in the tachyzoite and bradyzoite forms of T.gondii as revealed with fluorescein-labeled secondary antibodies was of variable intensity. We observed strong labeling in intracellular vacuoles and a weak labeling of the cell surface (Figure 5A and F). Live cells were not labeled with these antibodies (data not shown). No fluorescence was observed in control parasites incubated only in the presence of the secondary fluorescein-labeled goat anti-rabbit IgG (data not shown) or in the presence of the pre-immune serum (Figure 5C). A monoclonal antibody against BAG-5, a bradyzoite-specific antigen (Weiss et al., 1995, 1996), was used as bradyzoite marker (Figures 5H and 6C and D). No labeling was seen in tachyzoites when incubated with the monoclonal antibody against BAG-5 (Figure 5D). Similarly, the localization of TgA1 was observed in both tachyzoite (Figure 6E) and bradyzoite (Figure 6B and D) forms of T.gondii by using confocal laser scanning microscopy analysis, consistent with the results of the northern and western blot analysis. Figure 5.Immunofluorescence microscopy showing the localization of TgA1 in tachyzoites (A and B) and bradyzoites (E and F) of T.gondii. (C) Tachyzoites incubated with pre-immune serum (1:100). (D, G and H) Tachyzoites (D) and bradyzoites (G and H) incubated with monoclonal antibody against BAG-5 (1:1000). (B), (E) and (G) show the same cells as in (A), (F) and (H), respectively, by bright-field microscopy. Bar, 10 μm. Download figure Download PowerPoint Figure 6.Confocal laser scanning microscopy analysis showing the localization of the Ca2+-ATPase (green in B, D and E) and BAG-5 (red in C, D and F) in bradyzoites (A–D) and tachyzoites (E and F) of T.gondii ME49 strain. (D) An overlay of (B) and (C) with no evidence of co-localization. Bar, 10 μm. Download figure Download PowerPoint In order to analyze in more detail the structures labeled with the antibodies, immunoelectron microscopy was performed on thin sections of parasites embedded in the hydrophilic resin Unicryl. The results obtained confirmed that gold particles were seen in cytoplasmic vacuoles that appeared empty (Figure 7A–C, arrows, 10 nm gold particles), a characteristic feature of acidocalcisomes (Docampo and Moreno, 1999), and on the cell surface (Figure 7A, arrows), consistent with the results of immunofluorescence. Very few gold particles were observed in cytoplasmic structures. Figure 7.Immunocytochemical localization of Ca2+-ATPase (A–C), GRA2 (D) and V-H+-PPase (A–D) in tachyzoites of T.gondii, and western blot analysis of the V-H+-PPase (E). Note that 20 (A and D) and 15 (B and C) nm particles were used to localize the V-H+-PPase (arrowheads) and 10 nm particles were used for the Ca2+-ATPase (arrows, B and C), or GRA2 (D). V, vacuole; DG, dense granule. Bars, 500 (A), 100 (B), 120 (C) and 220 nm (D). (E) Total cell lysates containing 20 μg of protein from T.cruzi epimastigotes (Ep) and tachyzoites (RH strain, lane T) were subjected to SDS–PAGE on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with the antibody prepared as described in Materials and methods. Download figure Download PowerPoint Co-localization studies with the antibody against the Ca2+-ATPase (Figure 7A–C, 10 nm gold particles, arrows), and a polyclonal antibody that recognizes a peptide of 28 amino acids in the C-terminal region of the V-H+-PPase of T.cruzi [Figure 7A–C, arrowheads, 20 (A) or 15 nm (B and C) gold particles], confirmed the presence of both enzymes in the acidocalcisomes. Figure 7E, upper panel, shows that a single protein band with a molecular mass identical to that of the T.cruzi epimastigotes (Ep) V-H+-PPase was detected in tachyzoite lysates (T). No reaction was detected when pre-immune serum was used (lower panel). Co-localization studies with the antibody against the V-H+-PPase and an antibody against the dense granule marker GRA2 (Labruyere et al., 1999) were also performed to rule out that the organelles identified as acidocalcisomes were the dense granules. Figure 7D shows labeling of dense granules (DG) with antibodies against GRA2 (10 nm gold particles) and labeling of acidocalcisomes (V) and the plasma membrane with antibodies against the V-H+-PPase (arrowheads, 15 nm gold particles). Presence of electron-dense vacuoles in T.gondii Acidocalcisomes have been identified in T.cruzi (Scott et al., 1997), Leishmania donovani (Rodrigues et al., 1999a) and Trypanosoma brucei (Rodrigues et al., 1999b) as the electron-dense vacuoles present in unstained parasites. We have found that similar electron-dense vacuoles are present in unstained, unfixed preparations of T.gondii tachyzoites (Figure 8). The contrast of a given structure in these images arises solely from its mass density since these preparations were not stained. Approximately 10–12 spherical electron-dense vacuoles of varying sizes were observed in each tachyzoite by transmission electron microscopy (Figure 8A–C). They were distributed at random over the tachyzoite's cytoplasm or occasionally arranged in rows and towards the periphery of the cells (Figure 8B, arrowheads). Groups of them were observed in some cells (Figure 8C, arrowheads). These electron-dense vacuoles do not correspond to the dense granules detected in fixed, stained preparations. X-ray microanalysis of the electron-dense organelles (Figure 9A) yielded spectra characteristic of acidocalcisomes (Docampo and Moreno, 1999). The spectrum shown is the one that yielded the most counts in 100 s (out of 14 spectra obtained), but all the other spectra taken from dense organelles were qualitatively similar: counts for phosphorus were ∼2.5-fold greater than counts for calcium, which were ∼3.5-fold greater than counts for magnesium. Sodium was also detected in comparable amounts to magnesium. Zinc was detectable as a trace in only a few spectra. These peaks were not present in a spectrum taken from the background (Figure 9B). Sulfur was not detected in the electron-dense vacuoles, indicating that they do not contain much cysteine or methionine or proteins containing these amino acids. This is also in contrast to dense granules that are known to contain several proteins (Carey et al., 2000). Figure 8.Transmission electron microscopy of whole tachyzoites (A–C). Whole, unfixed and unstained cells were suspended in 0.25 M sucrose. Drops were applied to Formvar-coated grids, cells were allowed to adhere for 10 min, and then carefully blotted dry and observed directly with the Hitachi-600 electron microscope. Note the numerous electron-dense vacuoles of different sizes. Bar, 1 μm. Download figure Download PowerPoint Figure 9.X-ray microanalysis of the electron-dense organelles. (A and B) The X-ray spectra recorded from an electron-dense vacuole (A) or the background (B) in whole tachyzoites. Download figure Download PowerPoint Discussion In this work, we have demonstrated that TgA1, a gene encoding a functional Ca2+-ATPase, is present in the T.gondii genome. Comparison of the sequence of TgA1 from T.gondii with other P-type ATPases indicates that this ATPase gene is closely related to the family of PMCA pumps. The expression of TgA1 in a yeast mutant deficient in vacuolar Ca2+ accumulation (K665) provides genetic evidence that TgA1 encodes a vacuolar Ca2+ pump. This calcium pump was shown to be localized to acidocalcisomes and the plasma membrane of T.gondii, as indicated by immunofluorescence (Figures 5 and 6) and immunoelectron microscopy (Figure 7). This pump apparently lacks the calmodulin-binding domain present in other PMCA pumps (Strehler, 1991). This characteristic places this enzyme in a novel category together with the acidocalcisomal Ca2+-ATPase described in T.cruzi (Lu et al., 1998), and the vacuolar Ca2+-ATPases of S.cerevisiae (Cunningham and Fink, 1994), D.discoideum (Moniakis et al., 1995) and E.histolytica (Ghosh et al., 2000). As in the case of these other Ca2+-ATPases, we cannot rule out the possibility that calmodulin regulates TgA1 activity by interacting with as yet unidentified sequences on the enzyme. We also report, for the first time, the localization of the V-H+-PPase to the acidocalcisomes of T.gondii. A previous study (Rodrigues et al., 2000) reported a vacuolar and plasma membrane localization of this enzyme but the vacuoles were not identified at the electron microscope level. The co-localization of a proton pump and a calcium pump to the same vacuoles is consistent with their characteristic acidity and their high calcium content. As occurs with acidocalcisomes in trypanosomatids (Docampo and Moreno, 1999) and volutin or metachromatic granules in a number of microorganisms (Meyer, 1904; Wilkinson and Duguid, 1960; Harold, 1966; Jacobson et al., 1982; Kornberg, 1995), these intracellular vacuoles appear empty with conventional electron microscopy but have a high electron density when cells are observed directly without fixation and dehydration. This has been attributed to the presence of polyphosphates bound to different cations (Wilkinson and Duguid, 1960). In this regard, we previously reported (Rodrigues et al., 2000) that T.gondii possesses a high concentration of short chain polyphosphates.