Abstract
Polyphosphate (polyP) is an anionic polymer of orthophosphate groups linked by high energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until it was demonstrated that the protein named Vtc4p (vacuolar transporter chaperone 4) is a long chain polyP kinase that localizes to the yeast vacuole. Here, we report that TbVtc4 (Vtc4 ortholog of Trypanosoma brucei) encodes, in contrast, a short chain polyP kinase that localizes to acidocalcisomes. The subcellular localization of TbVtc4 was demonstrated by fluorescence and electron microscopy of cell lines expressing TbVtc4 in its endogenous locus fused to an epitope tag and by purified polyclonal antibodies against TbVtc4. Recombinant TbVtc4 was expressed in bacteria, and polyP kinase activity was assayed in vitro. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line. This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. The wild-type phenotype was recovered when an ectopic copy of the TbVtc4 gene was expressed in the presence of doxycycline. The mutant also exhibited a defect in volume recovery under osmotic stress conditions in vitro, underscoring the relevance of polyP in osmoregulation.
Highlights
Polyphosphate accumulates in an acidic calcium store named the acidocalcisome
The linear epitope-tagging cassette was transfected into procyclic form (PCF) and bloodstream form (BSF) trypanosomes, where it integrated into the original TbVtc4 locus via homologous recombination
TbVtc4 co-localized with the vacuolar proton pyrophosphatase (TbVP1), an acidocalcisomal marker [31, 40], in both PCF (Fig. 1B) and BSF trypanosomes (Fig. 1C), as detected by immunofluorescence (Fig. 1B) and immunoelectron microscopy (Fig. 1C), respectively
Summary
Polyphosphate (polyP) accumulates in an acidic calcium store named the acidocalcisome. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. Short and long chain polyP accumulate in acidocalcisomes, acidic calcium stores [17] where polyP is complexed with several cations [18, 19] These organelles were first described in Trypanosoma brucei [20] but later identified in a broad range of organisms from bacterial to human cells [18] and are involved in Ca2ϩ signaling, as inferred from the presence in them of an inositol 1,4,5-trisphosphate receptor [21]. Because Vtc proteins are absent in vertebrates, we propose this enzyme as a potential target for drug development and disease control
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