Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi

Abstract

Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4+/-2.8 (S.D.) nmol of H+/min per 10(8) amastigotes and 6. 37+/-0.8 nmol of H+/min per 10(8) trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9+/-2.2 nmol of H+/min per 10(8) cells), but not by trypomastigotes (5.1+/-1.7 nmol of H+/min per 10(8) cells). The localization of the V-H+-ATPase of T. cruzi was investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…V-PPase may contribute to parasitic cytosolic pHstasis by pumping H + into the acidocalcisomes [17,23]. However this organelle accumulates also Ca and other divalent cations such as Zn and Fe which co-precipitates with large amounts of polyphosphates.…”

“…However this organelle accumulates also Ca and other divalent cations such as Zn and Fe which co-precipitates with large amounts of polyphosphates. For this reason, they have been suggested to participate in several cellular processes such as osmoregulation [37], storage of high-energy polyphosphate for mobilization in case of stress or great energy demands [47,48], in pHstasis [23] and the Ca 2+ homeostasis [25,49]. We have previously shown that the endoplasmic reticulum of Herpetomonas sp.…”

“…V-H + -PPases have been shown to localize in vacuoles of higher plants cells [34,41], and in acidocalcisomes [11,25,31] and plasma membrane of trypanosomatids [23]. We therefore investigated whether it is also present in 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].…”

The reservosome of Trypanosoma cruzi epimastigotes: an organelle of the endocytic pathway with a role on metacyclogenesis

“…Imunocytochemical quantification using DAMP as a probe showed that reservosomes have a luminal pH of about 6.0 (Soares et al 1992). However, it is still a matter of speculation how these organelles are acidified, as they were not labeled with antibodies against a vacuolar-type H + -ATPase, which however recognized other intracellular vacuoles, possibly the acidocalcisomes (Benchimol et al 1998). A 52-kDa protein sharing sequence homology with glutathione S-transferase (Tc52) has been also localized in reservosomes (Ouassi et al 1995).…”