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.