brucei is the causative agent of sleeping sickness or African trypanosomiasis. These organelles were first described in the protozoan parasites Trypanosoma brucei and Trypanosoma cruzi, but have since been identified in an evolutionary diverse array of organisms from bacteria to humans. Acidocalcisomes also contain large amounts of PP i (pyrophosphate) and bivalent cations such as Ca 2+, Mg 2+ and Zn 2+. High levels of poly P accumulate in acidic organelles known as acidocalcisomes. Poly P (polyphosphate) is a linear chain of P i (inorganic phosphate) moieties linked by high-energy phosphoanhydride bonds widely distributed from bacteria to mammals. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis. We propose that the PP i-driven H + pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H + and Ca 2+ content, and decreased synthesis of poly P. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H + uptake and Ca 2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H +-PPase (vacuolar H +-pyrophosphatase), a marker for acidocalcisomes. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. We propose that the PP(i)-driven H+ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H+ and Ca2+ content, and decreased synthesis of poly P. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H+ uptake and Ca2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H+-PPase (vacuolar H+-pyrophosphatase), a marker for acidocalcisomes.
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