TgTEP and TgAP4 interact in clathrin-mediated transport to the PLVAC. (A) Colocalization experiments demonstrate that Halo-tagged AP-4ε (in yellow) colocalizes with mCherry-tagged TgTEP (in magenta). AP-4ε localization at the Golgi disappears in absence of TgTEP. All scale bars are 5 μm. (B) Western blot analysis of reciprocal co-IP assays confirms the physical interaction between TgTEP–GFP and AP4ε–HA. Beads alone, anti-GFP, and anti-HA–conjugated pull-downs are shown. Full blots are provided in Source Data. (C and D) Mass spectrometry of co-IP elutes reveals a significant enrichment of AP4 complex subunits and clathrin in both TgTEP and AP4ε pull-downs, displayed as volcano plots. Notably, CRT, a PLVAC transporter, is also enriched, supporting a role for this complex in PLVAC-directed trafficking. (E) CPL is found in small cytoplasmic vesicles in intracellular parasites and typically shows a diffuse localization. Depletion of TgTEP (in magenta) resulted in an accumulation of CPL (in yellow). This accumulation was seen as early as 48 h after induction, and fragmentation occurred at 72 h after induction of TgTEP knockout. Scale bars are 5 μm. (F) Colocalization of CPL (in yellow) with SortLR-Halo (in magenta) upon deletion of TgTEP demonstrates that CPL accumulation occurs in the trans-Golgi prior to its fragmentation. Scale bars are 5 μm. Source data are available for this figure: SourceData F5.
Figure 5.

TgTEP and TgAP4 interact in clathrin-mediated transport to the PLVAC. (A) Colocalization experiments demonstrate that Halo-tagged AP-4ε (in yellow) colocalizes with mCherry-tagged TgTEP (in magenta). AP-4ε localization at the Golgi disappears in absence of TgTEP. All scale bars are 5 μm. (B) Western blot analysis of reciprocal co-IP assays confirms the physical interaction between TgTEP–GFP and AP4ε–HA. Beads alone, anti-GFP, and anti-HA–conjugated pull-downs are shown. Full blots are provided in Source Data. (C and D) Mass spectrometry of co-IP elutes reveals a significant enrichment of AP4 complex subunits and clathrin in both TgTEP and AP4ε pull-downs, displayed as volcano plots. Notably, CRT, a PLVAC transporter, is also enriched, supporting a role for this complex in PLVAC-directed trafficking. (E) CPL is found in small cytoplasmic vesicles in intracellular parasites and typically shows a diffuse localization. Depletion of TgTEP (in magenta) resulted in an accumulation of CPL (in yellow). This accumulation was seen as early as 48 h after induction, and fragmentation occurred at 72 h after induction of TgTEP knockout. Scale bars are 5 μm. (F) Colocalization of CPL (in yellow) with SortLR-Halo (in magenta) upon deletion of TgTEP demonstrates that CPL accumulation occurs in the trans-Golgi prior to its fragmentation. Scale bars are 5 μm. Source data are available for this figure: SourceData F5.

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