title: Molecular characterization of Trypanosoma brucei P-type H+-ATPases.
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.
authors.affiliation: Center for Tropical and Emerging Global Diseases and the Department of Cellular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Shuhong
authors.initials: S
authors.lastname: Luo
authors.affiliation: Center for Tropical and Emerging Global Diseases and the Department of Cellular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Fang
authors.affiliation: Center for Tropical and Emerging Global Diseases and the Department of Cellular Biology, University of Georgia, Athens, Georgia 30602. Electronic address: rdocampo@cb.uga.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Docampo
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D017494:Proton Pumps / Q000235:genetics*; D006180:Proton-Translocating ATPases / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development
chemical_list: D008565:Membrane Proteins; D017494:Proton Pumps; D015800:Protozoan Proteins; C469751:HA1 protein, Trypanosoma cruzi; C469752:HA2 protein, Trypanosoma cruzi; D006180:Proton-Translocating ATPases