ESPript 3.0 output of multiple sequence alignment (MSA) is shown with secondary structure of the crystal structure of AP4-interacting ENTH domain.(A) Secondary structure element shown as squiggly lines represents the seven alpha helices of the crystal structure. Below the MSA is the bar representing solvent accessibility; blue, white, and teal colors represent accessible, buried, and intermediate residues of the protein. Residues highlighted in yellow demonstrate the alignment residue similarity; red highlight demonstrates identity. Purple arrows represent functional residues responsible for tepsin interaction with AP4. tENTH: Toxoplasma ENTH domain. (B) Tagging and floxing strategy for TgTEP (TGGT1_301410). (C) Genotyping of the WT (524 bp) parasite strain as well as parasite strains obtained wherein TGGT1_301410 is endogenously tagged with mCherry (1290 bp), SYFP2 (1299 bp), Halo (1529 bp), and TurboID (1557 bp). Primer design corresponds to the red arrows in panel B. (D) Orthogonal views of the merged image in Fig. 2 C show that despite TgTEP and SortLR come in very close proximity, they do not always colocalize. (E) Knockout of tagged TGGT1_301410 results in a band size of 514 bp. The floxed, endogenously tagged protein (-Rapa) could not be amplified due to its huge size. Primer design corresponds to black arrows in B. (F) The clone expressing TgTEP-mCherry was used to quantify loss of protein via IFA. 95% of parasite vacuoles lost the protein by 48 hpi. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was performed, with P values being represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. (G) Invasion assays were done, using parasites that were grown in the presence of 50 nM rapamycin or DMSO for a period of 48 h, after which these were manually released and allowed to invade fresh HFFs. All assays were done thrice, with a minimum of 100 parasites/vacuoles quantified per condition per replicate. All data are plotted as mean ± SD. Unpaired two-tailed Student’s t test was where P = 0.8193. hpi, h post-induction. Source data are available for this figure: SourceData FS1.
Figure S1.

ESPript 3.0 output of multiple sequence alignment (MSA) is shown with secondary structure of the crystal structure of AP4-interacting ENTH domain. (A) Secondary structure element shown as squiggly lines represents the seven alpha helices of the crystal structure. Below the MSA is the bar representing solvent accessibility; blue, white, and teal colors represent accessible, buried, and intermediate residues of the protein. Residues highlighted in yellow demonstrate the alignment residue similarity; red highlight demonstrates identity. Purple arrows represent functional residues responsible for tepsin interaction with AP4. tENTH: Toxoplasma ENTH domain. (B) Tagging and floxing strategy for TgTEP (TGGT1_301410). (C) Genotyping of the WT (524 bp) parasite strain as well as parasite strains obtained wherein TGGT1_301410 is endogenously tagged with mCherry (1290 bp), SYFP2 (1299 bp), Halo (1529 bp), and TurboID (1557 bp). Primer design corresponds to the red arrows in panel B. (D) Orthogonal views of the merged image in Fig. 2 C show that despite TgTEP and SortLR come in very close proximity, they do not always colocalize. (E) Knockout of tagged TGGT1_301410 results in a band size of 514 bp. The floxed, endogenously tagged protein (-Rapa) could not be amplified due to its huge size. Primer design corresponds to black arrows in B. (F) The clone expressing TgTEP-mCherry was used to quantify loss of protein via IFA. 95% of parasite vacuoles lost the protein by 48 hpi. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was performed, with P values being represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. (G) Invasion assays were done, using parasites that were grown in the presence of 50 nM rapamycin or DMSO for a period of 48 h, after which these were manually released and allowed to invade fresh HFFs. All assays were done thrice, with a minimum of 100 parasites/vacuoles quantified per condition per replicate. All data are plotted as mean ± SD. Unpaired two-tailed Student’s t test was where P = 0.8193. hpi, h post-induction. Source data are available for this figure: SourceData FS1.

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