Dataset Information

A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

ABSTRACT: Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) 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 Ca(2+)-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-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 Ca(2+) hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca(2+) 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 Ca(2+)-ATPase could play an important role in Ca(2+) homeostasis in T.gondii.

SUBMITTER: Luo S

PROVIDER: S-EPMC140201 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

Luo S S Vieira M M Graves J J Zhong L L Moreno S N SN

The EMBO journal 20010101 1-2

Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) 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 Ca(2+)-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-ATPases of Trypanosoma ...[more]

PMID: 11226155

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Project description:Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H(+)-PPase and V-H(+)-ATPase in these organelles.

Dataset Information

A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

Publications

A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

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