TgTEP and TgAP4ε are essential for PLVAC trafficking and parasite survival. (A) Plaque assay of WT and loxP-AP4ε-HA parasites shows a drastic diminution of plaques in parasites induced with 50 nM of rapamycin. (B) Quantification of plaque area. One-way ANOVA with Tukey’s multiple comparison test was done. The P values are represented as follows: ns ≥ 0.05; * = 0.01; **** ≤0.0001. (C) Immunofluorescence imaging shows CPL (magenta), a PLVAC marker, accumulates in AP4ε-KO parasites (AP4ε shown in green) from 48 h after induction, mirroring the phenotype observed in TgTEP-KO. Scale bar: 5 μm. (D) Temporal distribution of phenotype appearances in TgTEP knockout parasites showing that disruption of CPL trafficking precedes Golgi fragmentation. Mitochondria collapse occurred significantly later. (E)TgTEP was tagged with TurboID at the C terminus to carry out proximity-based biotinylation and find interaction partners via the addition of 150 μM biotin. Immunofluorescence assays using fluorescently conjugated streptavidin show the localization of these biotinylated proteins (in magenta). Biotinylation for different lengths of time show different intensities and localizations of biotinylated proteins. Naturally occurring biotinylated proteins within the apicoplast are also labelled with the fluorescently conjugated streptavidin. The apicoplast was co-labelled with antibodies against G2-Trx (in yellow). All images are normalized. Scale bars are 5 μm. (F) All proteins identified at the 30-min time point were also identified at the 6-h time point. Proteins of high interest identified are listed and included those typically associated with the Golgi and postGolgi compartments (in blue), actin-binding proteins (in pink), proteins associated with parasite’s endocytic micropore (in green), and an uncharacterized AP-4 subunit (in orange). KO, knockout.
Figure 6.

TgTEP and TgAP4ε are essential for PLVAC trafficking and parasite survival. (A) Plaque assay of WT and loxP-AP4ε-HA parasites shows a drastic diminution of plaques in parasites induced with 50 nM of rapamycin. (B) Quantification of plaque area. One-way ANOVA with Tukey’s multiple comparison test was done. The P values are represented as follows: ns ≥ 0.05; * = 0.01; **** ≤0.0001. (C) Immunofluorescence imaging shows CPL (magenta), a PLVAC marker, accumulates in AP4ε-KO parasites (AP4ε shown in green) from 48 h after induction, mirroring the phenotype observed in TgTEP-KO. Scale bar: 5 μm. (D) Temporal distribution of phenotype appearances in TgTEP knockout parasites showing that disruption of CPL trafficking precedes Golgi fragmentation. Mitochondria collapse occurred significantly later. (E)TgTEP was tagged with TurboID at the C terminus to carry out proximity-based biotinylation and find interaction partners via the addition of 150 μM biotin. Immunofluorescence assays using fluorescently conjugated streptavidin show the localization of these biotinylated proteins (in magenta). Biotinylation for different lengths of time show different intensities and localizations of biotinylated proteins. Naturally occurring biotinylated proteins within the apicoplast are also labelled with the fluorescently conjugated streptavidin. The apicoplast was co-labelled with antibodies against G2-Trx (in yellow). All images are normalized. Scale bars are 5 μm. (F) All proteins identified at the 30-min time point were also identified at the 6-h time point. Proteins of high interest identified are listed and included those typically associated with the Golgi and postGolgi compartments (in blue), actin-binding proteins (in pink), proteins associated with parasite’s endocytic micropore (in green), and an uncharacterized AP-4 subunit (in orange). KO, knockout.

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