Trypanosoma brucei Plasma Membrane-Type Ca super(2+)-ATPase 1 (TbPMC1) and 2 (TbPMC2) Genes Encode Functional Ca super(2+)-ATPases Localized to the Acidocalcisomes and Plasma Membrane, and Essential for Ca super(2+) Homeostasis and Growth

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (14), p.14427-14439
• Main Authors: Luo, S, Rohloff, P, Cox, J, Uyemura, SA, Docampo, R
• Format: Article
• Language: English
• Subjects: Trypanosoma brucei
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309978200

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca super(2+) pumps (Ca super(2+)-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca super(2+)-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H super(+)-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca super(2+) hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca super(2+) accumulation. T. brucei Ca super(2+)-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca super(2+)-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca super(2+) and decreased mobilization of Ca super(2+) from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca super(2+)-ATPases are essential for parasite viability and validate them as targets for drug development.