Two classes of plant-like vacuolar-type H+-pyrophosphatases in malaria parasites

In plants, cytosolic inorganic pyrophosphate (PP,) is hydrolyzed by energy- conserving vacuolar-type H+-pyrophosphatases (V-PPases) that harness the fr ee energy of PP, hydrolysis to establish transmembrane H+ gradients. Here w e describe the identification and cloning of two genes, PfVP1 and PfVP2, fr om the malaria parasite Plasmodium falciparum. Inferred to encode type I (K +-dependent) and type II (K+-independent) V-PPases, respectively, PfVP1 and PfVP2 appeared more sequence divergent from each other than from their typ e I and type II counterparts in plants. The steady state levels of PfVP1 mR NA were high in comparison to PfVP2 mRNA throughout the erythrocytic phases of infection. Western analyses of trophozoite membranes using generic V-PP ;ise antibodies (PAB(HK) and PAB(TK)) demonstrated appreciable amounts of a Mr 67000 polypeptide whose associated aminomethylenediphosphonate- (AMDP) inhibitable PPase activity was markedly stimulated by K+. Immunofluorescenc e microscopy of infected erythrocytes revealed PfVP antigen associated with both the parasite plasma membrane and punctate intracellular inclusions. T ransient transfection of a pfVP1-GFP fusion further supported the localizat ion of PfVP1 to the parasite plasma membrane. Based on these findings and t he growth-retarding effects of AMDP, P. falciparum is concluded to possess both type I and type II V-PPases of which thr former has the greatest poten tial for contributing to the establishment of H+ gradients across the paras ite plasma membrane under conditions of energy limitation. (C) 2001 Elsevie r Science B.V. All rights reserved.