Isolation and Characterization of TgVP1, a Type I Vacuolar H+-translocating Pyrophosphatase fromToxoplasma gondii

Abstract

Here we report the isolation and characterization of a type I vacuolar-type H ؉ -pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire Nterminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmidborne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K ؉ -activated pyrophosphate (PP i ) hydrolysis and PP i -energized H ؉ translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 M had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I 50 for the inhibition of TgVP1 activity in vitro (0.9 M) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisomelike vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.Vacuolar-type H ϩ -translocating inorganic pyrophosphatases (V-PPases) 1 are primary electrogenic proton pumps that derive their energy from the hydrolysis of inorganic pyrophosphate (PP i ) (1). Long considered to be restricted to plants and certain photosynthetic bacteria, V-PPases have recently been identified in a wide range of organisms, including prokaryotic extremophiles and the kinetoplastid protists Trypanosoma and Leishmania, the causative agents of Chagas' disease and leishmaniasis, and the apicomplexan protists Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis (reviewed in Ref.2). The discovery of V-PPases in these parasitic protists has attracted much attention. The seemingly complete absence of V-PPases from their animal hosts has given rise to the exciting possibility that this enzyme might serve as an effective drug target for a nu...

The P-glycoprotein Inhibitor GF120918 Modulates Ca2+-Dependent Processes and Lipid Metabolism in Toxoplasma Gondii

“…Surprisingly, our localization data revealed that P-gp was found not only on the plasma membrane of the parasite, but also in organelles morphologically resembling acidocalcisomes and labeled with the acidocalcisome marker VP1, further supporting a P-gp involvement in Ca 2+ regulation. Similarly to the observations with the acidocalcisome marker type I vacuolar-type H + -pyrophosphatase [51], P-gp transiently re-localized in a collar-like structure at the apical end of extracellular parasites. Thus, we can propose a model where P-gp may participate in the Ca 2+ homeostasis via facilitating Ca 2+ uptake in acidocalcisomes by contributing to the establishment of the proton gradient in the organelles and, in addition, via promoting the function of TgA1.…”

A Toxoplasma gondii protein with homology to intracellular type Na+/H+ exchangers is important for osmoregulation and invasion

“…A vacuolar type H + -translocating pyrophosphatase (TgVP1) has been described in T. gondii and shown to localize to both the acidocalcisomes [36, 37] and the newly described PLV/VAC [14, 15]. Co-staining with antibodies against TgNHE3 and TgVP1 [14] indicated that TgNHE3 co-localizes with TgVP1 (Fig.…”

New Insights into Roles of Acidocalcisomes and Contractile Vacuole Complex in Osmoregulation in Protists

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