FIG 2.

Epitope-tagged and TurboID-tagged GRA64 proteins are exposed to the host cell cytoplasm during intracellular infection. (A) IFA panels of parasites expressing GRA64 tagged at the N terminus with a 1xHA tag in tachyzoite vacuoles (2 days postinfection [dpi], pH 7) or induced bradyzoite vacuoles (3 dpi, pH 8). The digitonin concentration used for permeabilization in these experiments (0.001%) selectively permeabilizes the host cell membrane, but not the PVM. In contrast, 0.2% Triton X-100 fully permeabilizes the host cell and the PVM, as demonstrated by positive staining of MAG1, a parasite protein localizing to the lumen of parasitophorous vacuoles and to the cyst wall in differentiating parasite vacuoles. Scale bars = 5 μm. (B) IFA panels of parasites expressing GRA64 tagged at the N terminus with a 3×HA tag and the proximity-based biotinylating enzyme TurboID tag or untagged PruQ parasites in induced bradyzoite vacuoles (7 dpi, pH 8), supplemented with 150 μM biotin during the last 3 days of culture. Infected cells were permeabilized with digitonin (0.001%) to selectively permeabilizes the host cell membrane, but not the cyst membrane. Vacuoles were stained with αbiotin and αHA to confirm activity of the TurboID tag and expression of GRA64 via the 3×HA tag at the host cytoplasm interface, respectively. A lack of MAG1 labeling was used as an indicator for selective permeabilization. Scale bars = 5 μm.