Extended Data Fig. 6: Generation and characterization of ApiCox10-KO line.
From: Structure, assembly and inhibition of the Toxoplasma gondii respiratory chain supercomplex
(a) Schematic of the strategy used to C-terminally HA-epitope tag the Cox2a protein. The expected size of integration PCRs are shown. (b) PCR to test integration of HA-epitope tag and CAT selection cassette into the endogenous locus, as outlined in (A). (c) Immunoblot analysis of whole cell lysate extracted from Cox2a-HA and parental parasites. Samples were separated by SDS-PAGE, blotted, and detected using anti-HA and anti-TOM40 as a loading control. (d) Schematic of the strategy used to replace the coding sequence of ApiCox10 with a DHFR selection cassette. The expected size of integration PCRs are shown. (e) PCR to test integration of the DHFR selection cassette into the endogenous locus, in Cox2a-HA, QCR2-HA and mNEON::Δku80 parental background, as outlined in (D). (f) Immunoblot analysis of ApiCox10-KO and complementation line (cApiCox10-Ty) parasites, detected using anti-Ty and anti-TOM40. (g) Immunofluorescence assay of ApiCox10-KO and cApiCox10-Ty parasites, labeled with anti-Ty and anti-TOM40. Scale bar is 5 μM. (h) Volcano plot of proteomic data from Cox2a-HA vs ApiCox10/Cox2a-HA immunoprecipitations, showing the -log10 P values and the log2 fold changes of proteins detected by mass spectrometry in 3 or more replicates, from 4 independent experiments. P values were calculated using a two-tailed t-test. Horizontal dotted gray line denotes P= 0.05 and P = 0.01. Vertical dotted line denotes 5x enrichment. Complex III subunits are labeled in red and complex IV subunits are labeled in the blue. (i) Native-PAGE and immunoblot analysis of Cox2a-HA, ApiCox10-KO/ Cox2a-HA and the complemented cApiCox10-Ty lines. Total lysate was treated with digitonin and separated by BN-PAGE, followed by immunoblot analysis. Positions of complexes are indicated. (j) Transmission electron microscopy (TEM) of parasites to visualize mitochondrial cristae. Scale bar is 200 nm. (k,l) Quantification of number of cristae cross sections per mitochondrial surface area (K) and mitochondrial area (L) in parental and ApiCox10-KO parasites from TEM. Data points from 100 mitochondrial profiles, with mean shown ( ± s.d. in K). P value from a two-tailed unpaired t-test. (m) MitoSOX labeling to detect mitochondrial ROS at steady state or upon treatment with ferric ammonium chloride (FAC) in ApiCox10 knockout and parental parasites in mNEON background via flow cytometry analysis. Left: red fluorescence of T. gondii, in the absence or presence of ferric ammonium chloride (FAC), stained with MitoSOX. Population to the right of the dotted gray line are MitoSOX positive. Right: Quantification of population that is positive for MitoSOX signal. Graphs show mean ± s.d., from 4 independent experiments One-way ANOVA followed by Turkey’s multiple pairwise comparisons was performed, and P value from relevant pairs displayed. ns, no significant difference; ** P < 0.01 (parental vs parental + FAC P = 0.0037; KO vs KO + FAC P = 0.0015). (n) Surface electrostatics of the III2-IV (calculated with the Adaptive Poisson-Boltzmann Solver, APBS4) reveals a negative lumenal patch. Cytochrome-c binding sites were inferred from overlays of PDBs 5iy5 and 3cx5. (o) Quantification of number of parasites per vacuole for parental and ApiCox10-KO parasites. Error bars are mean ±s.d. from 4 independent experiments, for which over 250 vacuoles were counted for each replicate. The P value determined by multiple two-tailed t-tests with a Holm-Sidak correction applied. (p) Mixed culture growth competition assay of ApiCox10-KO or parental mNEON fluorescent parasites with tdTomato parasites. Relative abundance (compared to passage 0) of ApiCox10-KO parasites across 6 passages. Points are mean ± s.d., from 4 independent experiments, P value determined from a one-way ANOVA, corrected for multiple comparisons (Dunnett) (ns, no significant difference; P3 P = 0.0242; P4 P = 0.0022; P5 P = 0.0004; P6 P < 0.0001).
