Trypanosoma brucei Plasma Membrane-Type Ca2+-ATPase 1 (TbPMC1) and 2 (TbPMC2) Genes Encode Functional Ca2+-ATPases Localized to the Acidocalcisomes and Plasma Membrane, and Essential for Ca2+ Homeostasis and Growth

Abstract

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca 2؉ pumps (Ca 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 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 ؉ -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 2؉ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca 2؉ accumulation. T. brucei Ca 2؉ -ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca 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 2؉ and decreased mobilization of Ca 2؉ from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca 2؉ -ATPases are essential for parasite viability and validate them as targets for drug development.

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…However this organelle accumulates also Ca and other divalent cations such as Zn and Fe which co-precipitates with large amounts of polyphosphates. For this reason, they have been suggested to participate in several cellular processes such as osmoregulation [37], storage of high-energy polyphosphate for mobilization in case of stress or great energy demands [47,48], in pHstasis [23] and the Ca 2+ homeostasis [25,49]. We have previously shown that the endoplasmic reticulum of Herpetomonas sp.…”

KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei

“…During this period, most cells had reduced motility, and formation of cell debris was observed, mostly at days 6-8, suggesting cell death (data not shown). Growth resumed after z9 d and was accompanied by the reappearance of KREPA6 protein based on Western analysis (data not shown), suggesting loss of KREPA6 repression as sometimes observed (for example, see O'Hearn et al 2003;Luo et al 2004). Quantitative real-time RT-PCR (qPCR) analysis indicated that the level of KREPA6 mRNA was reduced substantially (Fig.…”

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

“…Acidic stores include lysosomes (Lloyd-Evans et al, 2008;Churchill et al, 2002), secretory lysosomes (Holmsen and Weiss, 1979), acidocalcisomes (Luo et al, 2004) and vacuoles (Patel and Docampo, 2010). Moreover, recent studies have demonstrated that such pools are also mobilisable (Churchill et al, 2002;Lange et al, 2009).…”