FIGURE 4.

APT1 homologue present in coccidian species only. A, indirect immunofluorescence microscopy of T. gondii tachyzoites. The upper panel shows endogenous epitope Ty-tagged TgASH1 in green (TgASH1-3Ty) and parasite pellicle shown with α-TgGAP45 in red. The lower panel shows endogenous TgASH1 detected by specific α-ASH1 antibodies in green and co-localized with cytoplasmic α-actin in red. B, subcellular fractionation of parasites indicates that TgASH1 is readily soluble in PBS, in high salt (1 m NaCl), and at high pH (0.1 m Na₂CO₃, pH 11.5). Complete solubilization occurs in the presence of 1% Triton X-100. C, Western blot analysis of an epitope-tagged second copy of TgASH1 (TgASH1-Ty) and epitope-tagged endogenous TgASH1 (knock-in TgASH1-3Ty) strains indicate a higher level of expression compared with endogenous TgASH1 (TgASH1e) in wild type strain (RH). The size shifts observed are due to the addition of one or three Ty tags. * indicates an unspecific band identified by the α-ASH1 Ab. D, subcellular fractionation of ku80-KO and TgASH1-Ty parasites in different salt and detergent conditions indicates that overexpression of TgASH1 does not impact the solubility of the palmitoylated protein TgISP1.