Fig. 1: Four conserved AP2 domain-containing proteins in Alveolata (Myzozoa) are fitness-conferring and targeted to the mitochondrion of T. gondii.
From: Apicomplexan mitoribosome from highly fragmented rRNAs to a functional machine
a Summary of the four mtAP2s in T. gondii and their conservation in Alveolates. Fitness score based on the genome-wide CRISPR fitness screen30, mitochondrial targeting peptide predictions using MitoProt II, mitochondrial proteome based on the BirA* and APEX73, and localization information based on hyperLOPIT data27. Conservation in Alveolates is presented with a filled circle, absence with an empty one. b Graphic representation of mtAP2s and their predicted cleavage sites indicated by arrowheads. c Immunofluorescence assays (IFAs) of mtAP2s (anti-Ty or anti-HA, magenta) and the mitochondrial marker HSP70 (cyan). Three independent experiments. Scale bar, 5 μm. d Immunoblot of endogenous C-terminally 6×Ty tagged mtAP2-(1 to 3). Anti-Ty antibodies were used to detect three mtAP2 proteins and anti-actin antibodies were used as a loading control. Three independent experiments. e IFAs of mtAP2-(1 to 3)-Ty iKD parasites treated with ATc or vehicle for 48 h. Anti-Ty (magenta) and anti-GAP45 (cyan). Three independent experiments. Scale bar, 5 μm. f Immunoblots of mtAP2-(1 to 3)-Ty parasites treated with ATc or vehicle for 1-3 days. Anti-Ty (mtAP2s) and anti-actin or HSP70 antibodies (loading control). Three independent experiments. g Plaque formation by mtAP2-(1 to 4)-Ty iKD parasites growing on HFF monolayers for 7 days ± ATc. h Quantification of plaques, mean ± SD (n = 3 independent biological replicates). Each mtAP2-regulatable parasite line treated with ATc was individually analyzed for statistical significance using an unpaired two-tailed Student’s t-test (mtAP2s versus RH), ns: P > 0.05. i, Intracellular replication of parental line (RH) and mtAP2-(1 to 4)-Ty iKD lines after treatment with ATc or vehicle. Data represented as mean ± SD (n = 3 independent biological replicates). j, k Plaque formation (j) and intracellular replication (k) of RH, ΔmtAP2-2-(a and b) and ΔmtAP2-2::mtAP2-2-HA lines. Data (k) represented as mean ± SD (n = 2 independent biological replicates). l Survival curves of infected mice. Source data are provided as a Source Data file.
