Abstract
Previous studies in Trypanosoma brucei have shown that intracellular pH homeostasis is affected by inhibitors of H+-ATPases, suggesting a major role for these pumps in this process (Vander-Heyden, N., Wong, J., and Docampo, R., (2000) Biochem. J. 346, 53-62). Here, we report the cloning and sequencing of three genes (TbHA1, TbHA2, and TbHA3) present in the genome of T. brucei that encode proteins with homology to fungal and plant P-type proton-pumping ATPases. Northern and Western blot analyses revealed that these genes are up-regulated in procyclic trypomastigotes. TbHA1, TbHA2, and TbHA3 complemented a Saccharomyces cerevisiae strain deficient in P-type H+-ATPase activity, providing genetic evidence for their function. Indirect immunofluorescence analysis showed that TbHA proteins are localized mainly in the plasma membrane of procyclic forms and in the plasma membrane and flagellum of bloodstream forms. T. brucei H+-ATPase genes were functionally characterized using double-stranded RNA interference methodology. The induction of double-stranded RNA (RNA interference) caused growth inhibition, which was more accentuated in procyclic forms and when expression of all TbHA proteins was decreased. Knockdown of TbHA1 and TbHA3, but not of TbHA2, resulted in cells with a lower steady-state pH(i) and a slower rate of pH(i) recovery from acidification. No evidence was found of an intracellular P-type H+-ATPase activity. These results establish that T. brucei H+-ATPases are plasma membrane enzymes essential for parasite viability.
Highlights
Mental pH is acidic [1], whereas the bloodstream form propagates in the host’s blood, where the extracellular pH is consistently 7.4
On the basis of studies performed with inhibitors of proton pumps, it has been suggested that, a plasma membrane Hϩ-ATPase is a major regulator of pHi [2] and membrane potential (⌬⌿) [4] in procyclic forms, it plays a minor role in bloodstream forms, where pyruvate efflux is more important for pHi regulation, and an ouabain-sensitive sodium pump is more relevant for ⌬⌿ regulation
Amino acid sequences of P-type Hϩ-ATPases from T. cruzi [9] were used to search all available sequence data bases using TBLASTN. This search yielded three putative open reading frames designated TbHA1, TbHA2, and TbHA3, which have a high identity to T. cruzi P-type Hϩ-ATPases (76%)
Summary
Mental (extracellular) pH is acidic [1], whereas the bloodstream form propagates in the host’s blood, where the extracellular pH is consistently 7.4. Gene products, total cell lysates prepared from procyclic and bloodstream stages of T. brucei were subjected to Western blot analysis with anti-TcHAf antibody [9].
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