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Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles.
Potapenko E
,
Cordeiro CD
,
Huang G
,
Docampo R
.
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Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes.IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.
FIG 1. Effect of PPi on Pi-elicited currents in oocytes expressing TbPho91 and Pi and PPi uptake by the same oocytes. (A) Representative currents recorded after the addition of 10 mM Na+/Pi or 10 mM Na+/PPi to oocytes expressing TbPho91. (B) Quantification of results from several experiments as described for panel A. (C) 32P incorporation of Na+/32Pi, K+/32Pi, Na+/32PPi, or NMDG/32PPi into oocytes expressing TbPho91. (D) Representative currents after sequential addition of 10 mM Na+/Pi or 10 mM Na+/PPi to oocytes expressing TbPho91. Values in panels B and C are means ± SEM; n = 6 (B) and n = 3 (C). *, P < 0.05 (Student's t test); ns, not significant.
FIG 2. Currents elicited by PPi and polyPs in oocytes expressing TbPho91 and Pho91p. (A) Representative currents recorded after the addition of 10 mM Na+/PPi, Na+/polyP3, Na+/polyP100, and Na+/polyP700 to oocytes expressing TbPho91. The right panel shows the quantification of currents elicited from four experiments. (B) Representative currents recorded after the addition of 10 mM Na+/PPi, Na+/polyP3, Na+/polyP100, and Na+/polyP700 to oocytes expressing Pho91p. The right panel shows the quantification of currents elicited from four experiments. (C to E) Currents recorded in response to the addition of 10 mM Na+/polyP3 at different pH levels (C and D) and quantification of the results of three experiments (E). (F to H) Currents recorded in response to the addition of 10 mM Na+/polyP3 at different Ca2+ concentrations (F) and quantification of the current intensity (G) or current duration (H) of several experiments. Values in panels E, G, and H are means ± SEM; n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant (Student's t test). Concentrations of Na+/polyP100 and Na+/polyP700 are expressed in phosphate units.
FIG 3. Effect of PPi on Pi-elicited currents in oocytes expressing TbPho91. (A) Representative currents recorded when the addition of 10 mM Na+/Pi was done in the absence or presence of the indicated concentrations of PPi. (B) Quantification of the results of several experiments as described for panel A. C, control. (C) Representative currents recorded when the addition of 10 mM Na+/Pi was done in the absence or presence of the indicated concentrations of polyP3. (D) Quantification of the results of several experiments as described for panel C. C, control. Values in panels B and D are means ± SEM; n = 4. *, P < 0.05; **, P < 0.01 (Student's t test).
FIG 4. PPi induces activation of Na+/Pi currents in Pho91- and TbPho91-expressing yeast vacuoles. (A) Activation of Na+/Pi outward currents in vacuoles from wild-type yeast after the addition of 10 mM PPi. (B) pho91Δ vacuoles do not produce currents after application of PPi. (C) Complementation of pho91Δ with TbPHO91 restores vacuole response to PPi. Data are representative of two to four independent experiments and are quantified in panel D. We used at least four successful current recordings for each experiment. About 80% of the vacuoles showed clear responses. WT, wild type; KO, knockout.
FIG 5. Schematic representation of PPi signaling function. Large amounts of Pi are needed for biosynthetic pathways, which generate PPi as a by-product. PPi stimulates the vacuolar Pho91 Na+/Pi symporters through their SPX domains, increasing the release of Pi needed for ATP biosynthesis.
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