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PMID: 9620872 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

The Biochemical journal ·Vol. 332 ( Pt 3) ·1998-06-15 ·Pages 695-702

Benchimol M, De Souza W, Vanderheyden N, Zhong L, Lu HG, Moreno SN, Docampo R

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.

MeSH Terms
Animals Anti-Bacterial Agents/pharmacology Cell Membrane/drug effects,enzymology Enzyme Inhibitors/pharmacology Fluorescent Antibody Technique, Indirect Hydrogen-Ion Concentration Intracellular Fluid/drug effects,enzymology Macrolides Microscopy, Confocal Microscopy, Immunoelectron Proton-Translocating ATPases/biosynthesis,physiology Protozoan Proteins/biosynthesis,physiology Trypanosoma cruzi/enzymology,growth & development,ultrastructure Vacuoles/drug effects,enzymology
Chemicals
Anti-Bacterial Agents Enzyme Inhibitors Macrolides Protozoan Proteins bafilomycin A1 Proton-Translocating ATPases
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Benchimol M
Universidade Santa Ursula, Rio de Janeiro, RJ 22231-010, Brazil.
De Souza W
Vanderheyden N
Zhong L
Lu H G
Moreno S N
Docampo R
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3320695
Published
1998-06-15
Pages
695-702
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1219530
Subset
IM
Grants
NIAID NIH HHS · AI-23259 · United States
FIC NIH HHS · TW-00476 · United States
External Links
PubMed source DOI: 10.1042/bj3320695 PMC Full Text View journal details
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