Knowledge-based systems analysis and design

Acidocalcisomes of <i>Trypanosoma brucei</i> and the acidocalcisome-like vacuoles of <i>Saccharomyces cerevisiae</i> are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in <i>T. brucei</i> and yeast, respectively), but the roles of these transporters in growth and orthophosphate (P_i) transport are unclear. We found here that <i>Tbpho91</i>^-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased P_i content. Heterologous expression of <i>TbPHO91</i> in <i>Xenopus</i> oocytes followed by two-electrode voltage clamp recordings disclosed that <i>myo</i>-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and P_i conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na⁺/P_i-loaded giant vacuoles prepared from WT or from <i>TbPHO91</i>-expressing <i>pho91</i>Δ strains but not from the <i>pho91</i>Δ yeast strains or from the <i>pho91</i>Δ strains expressing <i>PHO91</i> or <i>TbPHO91</i> with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar P_i and Na⁺ efflux and that <i>myo</i>-inositol polyphosphates stimulate the Na⁺/P_i symporter activities through their SPX domains.