Characterization of Isolated Acidocalcisomes from Toxoplasma gondii Tachyzoites Reveals a Novel Pool of Hydrolyzable Polyphosphate*

Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and magnesium, as found before for acidocalcisomes. Staining with 4′,6-diamidino-2-phenylindole (DAPI) indicated that poly- phosphate (polyP) was preferentially localized in this fraction together with pyrophosphate (PPi). Using an enzyme-based method, millimolar levels (in terms of Pi residues) of polyP chains of less than 50 residues long and micromolar levels in polyP chains of about 700–800 residues long were found to be preferentially localized in this fraction. The fraction also contained the pyrophosphatase and polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using antibodies against proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii polyP levels rapidly decreased upon exposure of the parasites to a calcium ionophore (ionomycin), to an inhibitor of the V-H+-ATPase (bafilomycin A1), or to the alkalinizing agent NH4Cl. These changes were in parallel to an increase in intracellular Ca2+concentration, suggesting a close association between polyP hydrolysis and Ca2+ release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes, showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of polyP metabolism in response to cellular stress.

Acidocalcisomes are organelles characterized by their acidic nature, high electron density, and a matrix consisting of pyrophosphate (PP i ), 1 polyphosphate (polyP), calcium, magnesium, and other elements (1,2). First described in trypanosomatids (3,4), they are now known to be present in several other microorganisms and are similar to the organelles historically described as "volutin granules" or "polyP bodies" (1,2).
The functions of the acidocalcisome in cell growth and survival are poorly understood. Based on its chemical composition, rich in PP i and polyP (a linear polymer of hundreds of orthophosphate residues linked by high energy phosphoanhydride bonds), one possible function could be as an energy and phosphate reservoir. PolyP has been shown to have a function in the cellular response to nutrient limitation during the stationary phase of growth and in the chelation of metals (17)(18)(19). A critical role for the acidocalcisomal polyP in the adaptation processes of Trypanosoma cruzi to environmental changes has been demonstrated (20). In several protozoan parasites it has been shown that the acidocalcisome is the main calcium storage compartment (1,2). Because Ca 2ϩ signaling is involved in many processes, such as invasion of host cells by different parasites (2,21), it is possible, although it has not yet been demonstrated, that this compartment could also have a role as a source of releasable Ca 2ϩ .
Many aspects of acidocalcisomes, such as their transport and enzymatic activities, need further study to understand the physiological importance of these organelles in protozoan parasites. A crucial requirement for these studies is the isolation of the organelles free from interference of other organelles or enzymatic activities. In the present work, we report a modification of the cell fractionation method used for T. cruzi epimastigotes (22) for the purification of acidocalcisomes from Toxoplasma gondii tachyzoites. We also report for the first time the determination of short and long chain polyP in these parasites.
Our results indicate that the concentration of polyP changes drastically under alkaline stress or when the cells are incubated with calcium ionophores. Ca 2ϩ release from acidocalcisomes is associated with the hydrolysis of polyP.

EXPERIMENTAL PROCEDURES
Culture Methods-Tachyzoites of T. gondii RH strain were cultivated and purified by the method of Moreno and Zhong (5) in bovine turbinate cells (ATCC CRL 1390). Host cells were cultivated in tissue culture flasks using Dulbecco's minimum essential medium supplemented with 10% horse serum. Cells were infected with tachyzoites at a final hostto-parasite ratio of 1:5. Parasites were harvested 2-3 days after infection and purified as described previously (23).
Chemicals and Reagents-Horse serum, DNase, RNase, Dulbecco's minimum essential medium (D5523), Dulbecco's phosphate buffer saline (PBS), Hanks' solution, nocodazole, sodium orthovanadate, sodium pyrophosphate, silicon carbide (400 mesh), protease inhibitors mixture, and goat anti-mouse antibodies labeled with horseradish peroxidase were purchased from Sigma. Bafilomycin A 1 was from Kamiya Biomedicals, Thousand Oaks, CA. Iodixanol (Optiprep, Nycomed) was from Invitrogen. 2-Amino-6-mercapto-7-methylpurine ribonucleoside and purine nucleoside phosphorylase (these two components were from the EnzChek phosphate assay kit) and the tetraacetoxymethyl esters of fura 2 (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2Јamino-5Ј-methylphenoxy)-ethane-N,N,NЈ,NЈ-tetraacetic acid) and BCECF (2Ј,7Ј-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein), fura 2-AM and BCECF-AM, respectively, were from Molecular Probes, Eugene, OR. Isolation of Acidocalcisomes-Tachyzoites (ϳ2.4 ϫ 10 10 cells) were centrifuged at 500 ϫ g for 10 min, and the cell pellet was washed twice with Dulbecco's PBS and once in lysis buffer (125 mM sucrose, 50 mM KCl, 4 mM MgCl 2 , 0.5 mM EDTA, 20 mM K-Hepes, 5 mM dithiothreitol, protease inhibitors mixture (0.2% v/v), 12 g/ml DNase, 12 g/ml RNase, and 8 g/ml nocodazole, pH 7.2). The cell pellet was mixed with 1.8ϫ wet weight silicon carbide and lysed by grinding with a pestle and mortar for not more than 60 s (lysis was checked by microscopy every 15 s). The mixture of silicon carbide and lysed cells was resuspended in ϳ100 ml of lysis buffer, and the suspension was left for 3 min on ice to allow most of the silicon carbide to settle. The liquid phase was carefully transferred to another tube and centrifuged at 36 ϫ g for 5 min. The supernatant was collected and left on ice, whereas the pellet was resuspended in ϳ50 ml of lysis buffer and centrifuged again under the same conditions. Combined supernatants were centrifuged at 144 ϫ g for 10 min to remove debris and unbroken cells. The supernatant obtained from this last centrifugation was centrifuged at 15,000 ϫ g for 10 min. The pellet was resuspended in 2.7 ml of lysis buffer, homogenized with the aid of a 22-gauge needle about 8 -10 times until no clumps were observed, and mixed with 1.3 ml of 60% iodixanol. This mixture (20% iodixanol) was included as the middle layer of a discontinuous gradient, with the other (4-ml) steps containing 10, 15, 25, and 30% iodixanol (diluted in lysis buffer). The gradient was centrifuged at 50,000 ϫ g using a Beckman SW 28 rotor for 60 min. Thirteen fractions were collected corresponding to bands and interfaces (Fig. 1). The acidocalcisomal fraction (F13) pelleted on the bottom of the tube and was resuspended in lysis buffer. All these procedures were done at 4°C.
Enzyme Assays-ATPases, pyrophosphatase, and exopolyphosphatase activities were assayed by measuring phosphate release using the EnzCheck phosphate assay kit as described before (11)(12)(13) with the microtiter plate modification (11). Acid phosphatase (lysosome marker, Ref. 24) was assayed in microtiter plates by measuring phosphate release from p-nitrophenylphosphate. Fractions (5 l) were added to a 50-l reaction mixture containing 0.1 M sodium acetate, pH 5.5, and 10 mM p-nitrophenylphosphate and incubated for 30 min at 28°C. Reactions were stopped by the addition of 150 mM sodium hydroxide (100 l), and released p-nitrophenol was detected at 405 nm. Protein was determined using the Bio-Rad Coomassie Blue method. All assays were recorded in a PowerWave 340i plate reader (Bio-Tek Instruments). Polyphosphatase activity was determined measuring P i release in the presence and absence of polyP. Samples were incubated for 5 min at 30°C with 60 mM Tris-HCl, pH 7.5, 6.0 mM MgCl 2 , and 520 M purified polyP 500 -700 in a final volume of 75 l. One unit corresponded to the release of 1 pmol of P i per min. Release of P i was monitored by the method of Lanzetta et al. (25). PolyP 500 -700 was isolated from agarose gels with Maddrell salt solution as described Clark and Wood (26). The construction of normalized density distribution histograms was carried out as described before (15,16).
Fluorescence Microscopy-Cells (ϳ5 ϫ 10 7 ) obtained as described above were washed twice with Dulbecco's PBS. The pellet was resuspended in 2 ml of the same buffer, and 45 l of this suspension was incubated at room temperature with 10 g of 4Ј,6-diamidino-2-phenylindole (DAPI)/ml. After 10 min, the samples were mounted on a slide and observed with an Olympus Fluoview FV300 laser-scanning confocal microscope using optical sections of 0.1 m and an argon laser for detection of polyP (20). Acidocalcisomal fractions (0.5 mg of protein/ml) were incubated with DAPI as described above, and samples were observed with an epifluorescence microscope using an Olympus WIG filter (excitation 380 -385 nm; emission Ͼ580 nm) for polyP detection.
Purification of Recombinant Exopolyphosphatase (rPPX1) from Saccharomyces cerevisiae-Recombinant exopolyphosphatase (rPPX1) was obtained and purified as described before (20) from E. coli strain CA38 pTrcPPX1. This strain of bacteria is insertionally inactivated for endogenous polyphosphate kinase and exopolyphosphatase (27) and contains a plasmid with the His-tagged rPPX1 gene from S. cerevisiae (28).
Electron Microscopy and Elemental Microanalysis-For observation of acidocalcisomal fraction 13, the sample was washed twice in 250 mM sucrose and diluted before being applied to Formvar-coated copper or nickel grids. The fraction was allowed to adsorb for 5-10 min at room temperature, blotted dry, and observed directly by electron microscopy (11)(12)(13). X-ray microanalysis was performed at the Electron Microscopy Center, Southern Illinois University, as described before (11)(12)(13).
Extraction and Analysis of Long and Short Chain polyP and PP i -Long chain polyP extraction was performed as described by Ault-Riché et al. (34). For short chain polyP, extractions were done as described by Ruiz et al. (20). PolyP levels were determined by the amount of P i released upon treatment with an excess of purified recombinant exopolyphosphatase (rPPX1) from S. cerevisiae as previously described (20). PP i levels were determined by the amount of P i released upon treatment with inorganic pyrophosphatase (Sigma, final activity, 10 units/ml) as previously described (35).
Cell Volume Determination-The intracellular concentration of polyP was calculated taking into account the cell volume of tachyzoites measured by the [ 14 C]inulin exclusion method as previously described by Damper and Patton (36), with some modifications. Cells were washed twice in Hanks' balanced salt solution (HBSS) supplemented with 0.1% (w/v) glucose and 0.05% (w/v) albumin (HBSSA) and then resuspended in HBSSA containing 0.2 mg/ml [ 14 C]inulin at final concentrations of 1 ϫ 10 9 and 2 ϫ 10 9 cells/ml. Three 50-l aliquots were taken from each suspension and transferred to scintillation vials. The suspensions were then centrifuged in Eppendorf tubes at 14,000 ϫ g for 2 min, and three 50-l aliquots of the supernatant were transferred to scintillation vials. 5 ml of scintillation mixture was added to each vial, and the vials were counted. All cell manipulations were performed on ice and/or with chilled solutions. Cell volume was determined by inulin exclusion. The difference in radioactivity/ml between the cell suspension and the supernatant was used to calculate the cell volume (differ-ence divided by radioactivity of supernatant ϭ fraction of volume occupied by cells). Results were expressed as an average of the values obtained in two different suspensions from three independent experiments.
Cell Treatments-Spectrofluorometric determinations of tachyzoites loaded with fura 2-AM or BCECF-AM were performed as described previously (5). For the alkaline or ionophore treatments, tachyzoites (2.5 ϫ 10 8 ) were washed once with Dulbecco's PBS and resuspended in 0.55 ml of 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO 4 , 5.5 mM glucose, 1 mM EGTA, and 50 mM Hepes pH 7.4 (buffer A). At the times indicated, 40 mM NH 4 Cl, 1 M ionomycin, or 1 M bafilomycin were added. Aliquots of 50 l were withdrawn at the times indicated and mixed with 500 l of guanidine isothiocyanate lysis buffer (20) for long chain polyP determination or with 300 l of ice-cold 0.5 M HClO 4 for short chain polyP determination as described above.

Isolation and Characterization of Acidocalcisomes from T.
gondii Tachyzoites-The method used to isolate acidocalcisomes from various protozoa has been improved in stages (11)(12)(13)22) but has not proved useful for isolation of these organelles from apicomplexan parasites. A new procedure for the isolation of acidocalcisomes from T. gondii is depicted in Fig. 1. This method used generally lower concentrations of iodixanol in the gradient steps than those previously used for the purification of acidocalcisomes from T. cruzi epimastigotes (22) but with the T. gondii sample added in the middle of the gradient in a 20% iodixanol layer rather than applied to the top of the gradient without added iodixanol. This strategy allowed a better separation of the acidocalcisomes from the ghosts present in the intermediate fractions and a better recovery of the pellet fraction at the base of the gradient (fraction 13). When directly applied to Formvar-coated grids and observed by electron microscopy (Fig. 2B), fraction 13 was seen to contain electrondense structures, as expected for acidocalcisomes (1,2). The same electron-dense organelles were observed in whole cells when they were applied to EM grids in the same manner ( Fig.  2A). X-ray microanalysis of the granules present in fraction 13 (Fig. 3) showed that they have the same chemical composition as acidocalcisomes found in other protozoa (1, 2) and electrondense vacuoles in whole T. gondii (9), indicating that they are acidocalcisomes.
The distribution of different enzymatic activities and organelle markers was compared along with that of the established marker for acidocalcisomes, H ϩ -PPase (Figs. 4 and 5). The aminomethylenediphosphonate-sensitive pyrophosphatase activity peaks in three different regions along the gradient (Fig. 4). Observation of these fractions by electron microscopy showed that the only fraction containing the electron-dense vacuoles, as seen in Fig. 2B, was fraction 13. Fractions 1 and 5 contained many cell ghosts produced in the lysis procedure (data not shown).
A V-H ϩ -ATPase sensitive to bafilomycin has been described to be present in acidocalcisomes of T. gondii (5)(6). However, the V-H ϩ -ATPase activity seems to have a broader distribution if compared with the H ϩ -PPase (Fig. 4). Acid phosphatase activity, a marker for another acidic organelle, the lysosome, did not peak in fraction 13 (Fig. 4).
The acidocalcisomal fraction also lacks markers for other organelles present in T. gondii, such as rhoptries (30), micronemes (29), and dense granules (31), as depicted in the Western blots shown in Fig. 5. Likewise, antibodies against the surface antigen of T. gondii tachyzoites (SAG1) (32) show that the plasma membrane is distributed mainly in the middle of the gradient (Fig. 5). Antibodies against the H ϩ -PPase (Fig. 5) confirm the distribution of this protein, obtained by enzymatic measurements (shown in Fig. 4). The strong reaction of this antibody in fractions 4 -12 is because of the presence of plasma membrane fragments (as marked by SAG1) and the high amount of protein in these fractions (Fig 4). The H ϩ -PPase has been previously shown to be located on the cell surface of T. gondii as well as in acidocalcisomes (7). Importantly though, fraction 13, which contains low amounts of protein, showed a clear reaction with antibodies against the H ϩ -PPase and no reaction with antibodies against all other markers, thus indicating that this fraction contained only acidocalcisomes.
PP i and PolyP Levels in T. gondii Tachyzoites-T. gondii tachyzoites contain high levels of long and short chain polyP, as determined by measuring degradation of polyP with recombinant yeast exopolyphosphatase (rPPX1). Cellular concentrations of short chain polyP (less than 50 phosphate residues) were in the mM range (in terms of P i residues, 24.0 Ϯ 0.5 mM), whereas values for long chain polyP (700 -800 phosphate residues) were in the micromolar range (43.0 Ϯ 0.5 M), taking into account a calculated cell volume of 16.5 Ϯ 3 l/10 9 tachyzoites. Tachyzoites also contained very high levels of PP i (7.95 Ϯ 0.16 mM). Controls of uninfected host cells presented undetectable levels of polyP or PP i (data not shown).
Localization of PolyP and Polyphosphatase Activity in T. gondii-The localization of polyP in T. cruzi (20) and other unicellular eukaryotes (15, 16) has been investigated using DAPI. DAPI is a useful tool in the fluorometric analysis of DNA but can also be used to study polyP (20,37). T. gondii tachyzoites incubated in solutions of DAPI (10 g/ml) were mounted on slides and examined using confocal microscopy. Examination of tachyzoites showed small spherical bodies (Fig.  6A). The distribution of fluorescent vacuoles was identical to that of the electron dense granules identified as acidocalcisomes in T. gondii (9). To investigate the localization of T. gondii polyP, we analyzed iodixanol density gradient fractions. The fractions were extracted, and PP i and short and long chain polyPs were determined. The acidocalcisome fraction (fraction 13) contained significant amounts (30 and 60%, respectively) of the total short and long chain polyPs recovered (Fig. 7), which correlated well with the distribution of PP i and the acidocalcisomal marker, proton-translocating PPase activity (Fig. 4). Because polyP yields were between 30 and 60% for short chain and long chain polyP, respectively, in fraction 13, whereas the yield of protein was 1.5%, this represents a 20 -40-fold purification, respectively. These results suggest a preferential acidocalcisomal location of these compounds. To further confirm the acidocalcisomal localization of polyPs, acidocalcisomal fractions were incubated with DAPI, mounted on slides, and examined by epifluorescence microscopy. Fig. 6B shows the strong staining of the isolated acidocalcisomes with DAPI, corroborating the presence of a high content of polyP in these organelles. An exopolyphosphatase activity, previously described in acidocalcisomes of trypanosomatids (20), was also present in the acidocalcisomal fraction of T. gondii (Fig. 7). This activity was distributed along the gradient with the same pattern as the H ϩ -PPase (Fig. 4).
Changes in PolyP Levels Induced by Processes That Mobilize Ca 2ϩ -Previous work has demonstrated that alkaline stress results in an increase in the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) of T. gondii (5). Therefore, we investigated whether there was a correlation between Ca 2ϩ release from the acidic compartment containing most polyP (acidocalcisome) and polyP hydrolysis in T. gondii. The addition of bafilomycin A 1 , a specific inhibitor of the vacuolar type H ϩ ATPase, or the alkalinizing agent NH 4 Cl to tachyzoites resulted in significant decrease in long (Fig. 8A) and short chain (Fig. 8B) polyP. Ionomycin, a Ca 2ϩ ionophore that induces cell acidification and Ca 2ϩ release from intracellular compartments (5) (EGTA was present in the extracellular medium to avoid Ca 2ϩ entry), also induced degradation of long (Fig. 8A) and short chain (Fig. 8B) polyP. However, cytosolic acidification by the addition of propionic acid (5) did not increase [Ca 2ϩ ] i . Because acidocalcisomes are the main cellular store of polyP and the total amounts released (ϳ70% of long chain and ϳ50% of short chain polyP) correspond to the percent polyP present in them (Fig. 7), these organelles are certainly involved in these effects. Taken together, these results suggest that processes that lead to alkalinization of the acidocalcisomes (NH 4 Cl addition or treatment with bafilomycin A 1 ) and/or result in [Ca 2ϩ ] i increase (5) also result in polyP hydrolysis.
Simultaneous measurements of changes in pH i , [Ca 2ϩ ] i , and short and long chain polyP in tachyzoites are shown in Fig. 9. The addition of bafilomycin A 1 (in the absence of extracellular Ca 2ϩ ) caused a decrease in pH i and a rise in [Ca 2ϩ ] i (Fig. 9A). The addition of bafilomycin A 1 was accompanied by immediate hydrolysis of long and short chain polyP. Subsequent addition of ionomycin resulted in a further acidification and Ca 2ϩ release accompanied by further hydrolysis of long and short chain polyP (Fig. 9, B and C).

DISCUSSION
Acidocalcisomes are organelles characterized by their acidic nature and high calcium and phosphorus content (1, 2). They possess several transport systems involved in the maintenance of the acidic environment and the accumulation or release of inorganic ions. Although acidocalcisomes isolated from several organisms have some common characteristics, there are some differences between species and also between different stages within the same species. The isolation of these organelles by subcellular fractionation and gradient centrifugation has been a key step to their final characterization (11-13, 15, 16, 22).
We have modified the previously described gradient (22) for the isolation of acidocalcisomes by expanding the lower concentrations of iodixanol and eliminating the highest concentration. These changes allowed the separation of acidocalcisomes from other T. gondii organelles. A similar distribution of the H ϩ -PPase, with three peaks along the gradient, was found during the isolation of acidocalcisomes from Dictyostelium discoideum (16). In this organism, the activity at the top of the gradient (fraction 1) was associated with markers for the contractile vacuole (16). Similar structures, however, have not been described in apicomplexan parasites. Electron microscopy of fraction 1 showed that it was composed of cell ghosts and other unidentified structures (data not shown). Localization of the H ϩ -PPase in other compartments has been described in different organisms, in plasma membrane vesicles and the Golgi apparatus of the trypanosomatid T. cruzi (38), and in plants, the H ϩ -PPase has been shown to be present in the plasma membrane as well as the membrane of the vacuole (the tonoplast) (39).
Other activities known to be associated with acidocalcisomes showed the same distribution pattern as the H ϩ -PPase. Exopolyphosphatase is an enzyme involved in the degradation of polyP and has been shown to be present in different compartments of various cell types (17)(18)(19). Part of this enzymatic activity was found in the acidocalcisomal fraction, as occurs in T. cruzi epimastigotes (20). A bafilomycin A 1 -sensitive-V-H ϩ -ATPase activity has been identified in acidocalcisomes and plasma membrane of trypanosomatids and apicomplexan parasites (3)(4)(5)(6) as well as in other unicellular eukaryotes (15,16). Although it has a broader distribution along the gradient, this is similar to that detected for the H ϩ -PPase and exopolyphosphatase activities, showing three peaks of activity. This enzyme could become inactive during acidocalcisome purification, as occurs with the T. cruzi V-H ϩ -ATPase (22). This is the first report of the presence of long chain polyP in an apicomplexan parasite. In addition, our results indicate the presence of high levels of PP i and short chain polyP in T. gondii tachyzoites. We also demonstrated that PP i and polyP are located preferentially in the acidocalcisomes using two different approaches, by visualization of polyP using DAPI and by the biochemical identification of PP i and polyP in isolated acidocalcisomes. A possible function of the polyPs in the acidocalcisome was proposed based on results obtained with T. cruzi (20) in which there is a close association between polyP hydrolysis and intracellular Ca 2ϩ increase, suggesting that upon polyP hydrolysis Ca 2ϩ bound to polyP is released from acidocalcisomes.
The possible roles of polyP and PP i in microorganisms have been reviewed (17,19). Some of their possible functions are to serve as energy stores and/or as chelators of metal ions. PP i could be used in place of ATP as an energy donor in several reactions in T. gondii, such as the PP i -dependent phosphofructokinase (40) and the acidocalcisomal H ϩ -PPase that can drive proton uptake through cleavage of cytosolic PP i (7). Because PP i is a charged and polar molecule, the utilization of acidocalcisomal PP i for these activities implies the presence of a spe- cialized channel or transporter in the acidocalcisomal membrane. A transmembrane transporter that shuttles PP i between intracellular and extracellular compartments has been identified recently in several mammalian tissues (41). A similar channel in the acidocalcisomal membrane would explain PP i release to the cytosol to serve as substrate for the H ϩ -PPase and PP i -dependent phosphofructokinase. A role for polyP as an energy source, however, has been disputed on the basis of its low metabolic turnover as compared with that of ATP (42), and a more important regulatory role has been suggested (43). Long chain polyP, even at relatively low levels, has been shown to be essential for adaptation to various stresses and for survival of bacteria in stationary phase (43). Similar studies have been reported in eukaryotic cells such as yeast (44). We have reported (6) that influx of ammonia into tachyzoites induces a rapid alkalinization of the cells followed by recovery of the cytoplasmic pH. This recovery (6) occurs in parallel with hydrolysis of polyP (Fig. 8, A and B). Because H ϩ generation from polyP hydrolysis can neutralize up to 2.5 pH units of change in S. cerevisiae (44), a role for polyP hydrolysis in pH recovery from an alkaline load has been suggested (20).
Ca 2ϩ release from acidocalcisomes by combination of an inhibitor of the V-H ϩ -ATPase (bafilomycin A 1 ) and a calcium ionophore (ionomycin) was associated with short and long chain polyP hydrolysis (Fig. 9). The addition of bafilomycin A 1 leads to acidification of the cytosol and alkalinization of the acidocalcisomes by inhibition of the V-H ϩ -ATPase. This would favor Ca 2ϩ release through a Ca 2ϩ /H ϩ exchanger, the presence of which has been demonstrated in acidocalcisomes from other Closed diamonds are after the addition of bafilomycin A 1 and ionomycin. Results depicted in panels B and C are from a representative experiment with data points given as means Ϯ S.E. microorganisms (10). Alkalinization of the acidocalcisomes may result in activation of the polyP-hydrolyzing activities in the organelles, as it has been shown that other acidocalcisomes contain polyphosphatases with an alkaline pH optimum (20). It has been hypothesized that one of the roles of acidocalcisomes is calcium storage for use in intracellular signaling, particularly in invasive parasite stages (1,2). Enzymes cleaving short and long chain polyPs to P i in acidocalcisomes may, therefore, indirectly regulate the intracellular Ca 2ϩ concentration.
In conclusion, the isolation of acidocalcisomes provides definitive evidence that they are distinct from other previously recognized organelles present in T. gondii and will allow their further biochemical characterization. Our results indicate that the amount of polyP in acidocalcisomes rapidly decreases under alkaline stress. PolyP hydrolysis is accompanied by an increase in [Ca 2ϩ ] i of tachyzoites. These effects suggest an important role for acidocalcisomes in the adaptation of T. gondii to environmental changes.