Fig 1. Characteristics of the transport of P_i in Toxoplasma.

A. Kinetics of P_i uptake. Extracellular parasites were incubated in a P_i-depleted reaction medium at pH 7.4 containing 100 μM ³²P_i at the indicated times, before washing by filtration and radioactivity counting. Open circles: Na⁺-independent uptake in the presence of 140 mM choline chloride. Filled circles: Na⁺-dependent uptake in the presence of 140 mM NaCl. Data are means ± SEM of 4 independent assays. *, p<0.05 (unpaired Student’s t-test at matched time of reaction). B. Ion co-transport activities. ³²Pi uptake was assayed on parasites incubated in a P_i-depleted medium containing radioactive P_i as indicated in A, in the presence of NaCl, KCl, NH₄Cl or LiCl compared to choline chloride (control conditions). Data are means ± SEM (n = 3 independent assays). *, p<0.05 (unpaired Student’s t-test). C. Na⁺ dependence for P_i uptake. ³²P_i uptake was assayed on parasites incubated in a P_i-depleted medium containing radioactive P_i as indicated in A, with increasing NaCl concentrations. Medium osmolarity was maintained at 300 mOsM with choline chloride supplementation. Data are means ± SEM (n = 6 independent assays). D. Saturation curve of P_i transport. ³²Pi uptake by the parasites was monitored in medium with 140 mM NaCl and various concentrations of P_i at pH 7.4, and traced with 100 μM ³²P_i (25 μCi/ml assay) for 30 min. Data are means ± SEM (n = 6 independent assays). E. Competition assay for P_i transport. ³²P_i uptake by the parasite was monitored in medium with 140 mM NaCl and excess non-radioactive P_i at pH 7.4, and traced with ³²P_i as described in D. Data are means ± SEM (n = 3 independent assays).*, p<0.05 (unpaired Student’s t-test). F. Selectivity for P_i influx. Extracellular parasites were incubated in a P_i-depleted reaction medium at pH 7.4 containing 100 μM P_i, traced with 25 μCi of ³²P_i/ml for 30 min, with added of 0.3, 3 mM SO₄^2- or no addition (−). Data are means ± SEM (n = 4 independent assays). G. P_i uptake upon inhibition of Na⁺-H⁺-ATPase. Extracellular parasites were treated 10 min with 5 nM, 50 nM, 500 nM cipargamin or without drug (DMS0 control) prior to P_i uptake, washed and incubated 10 min in the presence of 100 μM P_i and traced with 250 μCi of ³²P_i at pH 7.4, in medium containing 140 mM NaCl. Data are means ± SEM (n = 3 independent assays). *, p = 0.0013; **, p = 0.0008; ***, p<0.0001 comparing with DMSO condition (unpaired Student’s t-test). H. P_i uptake upon condition of described in the presence of 100 μM P_i and traced with 250 μCi of ³²P_i at pH 7.4, in a medium containing 140 mM NaCl, plus 10 μM FCCP, 100 nM bafilomycin A₁, without drug (control) or vehicle (DMSO) for 10 min. Data are means ± SEM (n = 6 independent assays). *, p<0.05 comparing with DMSO (unpaired Student’s t-test). I. ³²P_i uptake was measured on parasites in a P_i-depleted medium containing 140 mM NaCl and traced with 100 μM ³²P_i (25 μCi/ml assay) for 30 min in different pH ranges. pH in the incubation medium was adjusted by adding concentrated HCl (5.5, 6.0 and 6.5) or KOH (7.0, 7.5, 8.0 and 8.5). The ratio H₂PO₄⁻/HPO₄²⁻ varied from 50:1 at pH 5.5 to 1:20 at pH 8.5. Data are means ± SEM (n = 4 independent assays). *, p<0.05 (One-way ANOVA using the Tukey’s test, comparing each value with that obtained at pH 5.5). J. Panel a: pH-dependence of the influx of ³²P_i into extracellular parasites resuspended in medium at pH 6.4, 7.4 or 8.4. Panel b: kinetic constants at pH 6.4, 7.4 or 8.4. The V_max and K_m values were extrapolated from the 3 curves in panel a. To determine the H₂PO₄^- and HPO₄^2- concentration dependences, the respective concentration in the medium of these ionic forms was calculated at the P_i concentrations shown in panel a, for each pH value: 6.4, 7.4 and 8.4. Asterisks, values obtained at the three different pH values differing significantly from one another (p<0.0001; One-way ANOVA using the Tukey’s test).