PRESENCE OF A PLANT-LIKE PROTON-PUMPING PYROPHOSPHATASE IN ACIDOCALCISOMES OF TRYPANOSOMA-CRUZI

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper-d emonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyro phosphate promoted organellar acidification in permeabilized amastigot es, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lac ked markers for mitochondria, lysosomes, glycosomes, cytosol, and plas ma membrane. Antiserum raised against specific sequences of the plant V-H+-RPase cross-reacted with a T. cruzi protein, which was also detec table in the dense Percoll fraction. The organelles in this fraction a ppeared by electron microscopy to consist mainly of acidocalcisomes (a cidic calcium storage organelles). This identification was confirmed b y x-ray microanalysis. Immunofluorescence and immunoelectron microscop y indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyropho sphate was able to drive calcium uptake in permeabilized T, cruzi, Thi s uptake depended upon a proton gradient and was reversed by a specifi c V-H+-PPase inhibitor. Our results imply that the phylogenetic distri bution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.