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Molecular and Cellular Biology, November 1999, p. 7712-7723, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Claudia O. Rodrigues, David A. Scott, and Roberto Docampo*

Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

Received 25 May 1999/Returned for modification 19 July 1999/Accepted 23 August 1999

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 NH4Cl. 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 µM) and unaffected by bafilomycin A1 (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 µM), oligomycin (1 µM), N-ethylmaleimide (100 µM), and KNO3. 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.

* Corresponding author. Mailing address: Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave., Urbana, IL 61802. Phone: (217) 333-3845. Fax: (217) 244-7421. E-mail: rodoc{at}uiuc.edu.

Molecular and Cellular Biology, November 1999, p. 7712-7723, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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