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Originally published In Press as doi:10.1074/jbc.M208990200 on October 11, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48650-48656, December 13, 2002
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Characterization of Isolated Acidocalcisomes from Toxoplasma gondii Tachyzoites Reveals a Novel Pool of Hydrolyzable Polyphosphate*

Claudia O. RodriguesDagger , Felix A. RuizDagger , Peter Rohloff, David A. Scott, and Silvia N. J. Moreno§

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

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.

* This work was supported by National Institutes of Health Grant AI-43614 (to S. N. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ A Burroughs Wellcome Fund New Investigator in Molecular Parasitology. To whom correspondence should be addressed: Laboratory of Molecular Parasitology, Dept. of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Ave., Urbana, IL 61802. Tel.: 217-333-2746; Fax: 217-244-7421; E-mail: s-moreno@uiuc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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