Characterization of a Vacuolar Pyrophosphatase in Trypanosoma brucei and Its Localization to Acidocalcisomes

Abstract

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH 4 Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H ؉ -pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 M) and unaffected by bafilomycin A 1 (40 nM), concanamycin A (5 nM), sodium ovanadate (500 M), oligomycin (1 M), N-ethylmaleimide (100 M), and KNO 3 . AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H ؉ -ATPase, H ؉ -PPase, and (ADP-dependent) H ؉ /Na ؉ antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H ؉ -PPase (both stages) and H ؉ /Na ؉ exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

“…The linear epitope-tagging cassette was transfected into PCF and BSF trypanosomes, where it integrated into the original TbVtc4 locus via homologous recombination. TbVtc4 co-localized with the vacuolar proton pyrophosphatase (TbVP1), an acidocalcisomal marker (31,40), in both PCF (Fig. 1B) and BSF trypanosomes (Fig.…”

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…Kinetic studies have shown that acidocalcisomes maintain a low internal pH due to the presence of a V-H + -ATPase [22][23][24] and a V-H + -PPase [8,11,13,15,[25][26][27] which pump H + into the lumen of the organelle. They also posses a Ca 2+ -H + translocating ATPase, a Ca 2+ -H + exchanger and a Na + -H + exchanger which permit a complex regulation of these ions by the cell It was proposed that acidocalsisomes are involved in mechanisms of Ca 2+ signaling, osmoregulation, pH homeostasis and energy storage [16,28].…”

Cell fractionation of parasitic protozoa: a review

“…Immunochemical approaches have detected some enzymes that could be responsible for these transport activities: a vacuolar H + ATPase ), a Ca 2+ -H + translocating ATPase and a vacuolar-type H + -pyrophosphatase. This last enzyme was considered similar to plant pyrophosphatases, as its activity was stimulated by K + and inhibited by Na + , Luo et al 1999, Rodrigues et al 1999a, 1999b, 2000.…”

“…Docampo and Moreno have recently (1999) suggested some possibilities, such as a storage role in calcium homeostasis and in energy metabolism, in view of the large accumulation of inorganic pyrophosphate (Urbina et al 1999) Other possibilities include pH control and osmoregulation, and the presence of Na + -H + exchange activity (Rodrigues et al 1999b).…”

“…This led to great loss of acidocalcisomes when Percoll was washed out. Similar methods were adopted to isolate acidocalcisomes from L. donovani (Rodrigues et al 1999a) and T. brucei (Rodrigues et al 1999b). Recently, a new protocol was established by Scott and Docampo (2000) using iodixinol, a density gradient solute that does not precipitate, allowing acidocalcisome isolation in good yield (Fig.…”