Fig. 4.

Δvp1 tachyzoites have a TgCPL maturation defect in the presence of bafilomycin A₁. (A) Tachyzoites were pre-incubated for 15 min with 50 nM bafilomycin A₁ or DMSO as control. Metabolically pulse-labeled tachyzoites from Δku80, Δvp1 and Δvp1-cm clones were either kept on ice (0 min) or chased with medium containing unlabelled Met/Cys for the indicated times (10, 30 or 60 min). CPL proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Arrows indicate positions of the immature (pro-TgCPL) and mature (m-TgCPL) forms of CPL. Tachyzoites of the Δvp1 clone show impaired maturation of pro-TgCPL after blocking TgVATPase with bafilomycin A₁. (B, C) Quantification of TgCPL maturation in tachyzoites from Δku80, Δvp1 and Δvp1-cm clones for each chase time point in the absence (B, DMSO) or presence (C) of 50 nM bafilomycin A₁. Values are mean ± SD. Data shown are the combined results of three independent experiments. (D) Plaque numbers after pre-incubation of tachyzoites from Δku80, Δvp1 and Δvp1-cm clones in Ringer buffer contain 50 nM bafilomycin A₁. The effect of bafilomycin A₁ was more severe in tachyzoites from the Δvp1 clone. (E) Percentage of plaques as compared with controls without bafilomycin A₁ considered as 100%, after incubation of tachyzoites of the different clones in Ringer buffer. Tachyzoites from all clones produced ~20% less plaques after incubation with bafilomycin A₁ but tachyzoites from the Δvp1 clone produced ~55% less plaques than tachyzoites from Δku80 and Δvp1-cm clones. Data are the combined results of three independent experiments. Data were analyzed by GraphPad Prism 5. To compare two groups of data we used One-Way ANOVA test. *, p <0.05; **, p <0.01; ***, p <0.001.