A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi

Abstract

The proton pumping pyrophosphatase (H(+) -PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii , and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H(+) -PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K(+) ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H(+) -PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H(+) -PPase. Taken together, these results are consistent with the presence of a functional H(+) -PPase in the plasma membrane of these parasites.