Figure 1. TgTRPPL-2 localizes to the endoplasmic reticulum and periphery of T. gondii.

(A) Schematic representation of the InterPro Domain annotation of TgTRPPL-2 in the GT1 strain. Red line indicates coverage of the sequence by mass spectrometry. (B) Predicted topology for TgTRPPL-2 in GT1 strain. Model was generated with the Protter application (Omasits et al., 2014). The PKD domain is shown in orange. The domain used to generate antibodies is highlighted in green. Predicted Transmembrane Domains (TMDs) are highlighted in purple. The red arrowhead indicates the predicted cleavage site for TgTRPPL2. (C) Schematic representation of C-terminal tagging of TgTRPPL-2 in TatiΔKu80 parasites and western blots of TgTRPPL-2-smHA membranes using αHA (1:1000) showing a major band at approximately 150 kDa (red arrowhead). (D) Immunofluorescence analysis (IFA) of extracellular tachyzoites using αHA antibody and co-localization with αSAG1 (1:1000) and αSERCA (1:1000) showing partial co-localization with both markers. (E) IFAs of extracellular and intracellular (F) tachyzoites respectively with αTgTRPPL-2 (1:100) showing labeling of the protein at the periphery, co-localized with αSAG1 (1:1000) and with αTgSERCA (1:1000). (G) Super-resolution of extracellular IFAs using αTgTRPPL-2 (1:100) co-localized with αSAG1 (1:1000) but not with αGAP45 (1:1000). Scale bars in D-G represent 5 µm.

Figure 1—figure supplement 1. Validation of C-terminal tagging of TgTRPPL-2-smHA.

(A) Amplification of ~2 kB in the TgTRPPL-2-smHA cell line validating the correct integration of TgTRPPL-2-smHA. (B) Western blot of three biological replicates of immunoprecipitated TgTRPPL-2-smHA using αHA antibody (1:1000) shows a band of ~150 kDa (highlighted by red boxes and arrow). No band is present in the control cell line. (C) Immunofluorescence analysis (IFA) of intracellular tachyzoites with αHA (1:100), αSAG1 (1:1000), and αGAP45 (1:1000) . (D) IFAs of intracellular and extracellular tachyzoites with αHA (1:100) show that TgTRPPL-2 did not co-localize with either αGAP45 (1:1000) or αGra1 (1:500). (E) IFAs of extracellular tachyzoites preincubated with α-toxin showing co-localization of TgTRPPL-2 with αSag1 (1:1000) but not with αGap45. (F) Quantification of Pearson’s coefficient of the co-localization of TgTRPPL2 with SAG1 or GAP45.