Fig 1.
mETC and F1Fo-ATP synthase in mammals and apicomplexans.
A schematic showing the main features of the mitochondrial electron transport chain (mETC) and ATP synthase in mammals (A) and in apicomplexans (B). The four complexes are depicted in orange (Complex I: NADH:ubiquinone oxidoreductase; Complex II: succinate dehydrogenase; Complex III: cytochrome bc1 complex; Complex IV: cytochrome c oxidase; and F1Fo-ATP synthase). Mobile electron carriers (ubiquinone/ubiquinol and the intermembrane space protein cytochrome c) are depicted in green. Main alternative entry points (the dehydrogenases DHODH: dihydrooratate dehydrogenase; G3PDH:glycerol 3-phosphate dehydrogenase; MQO:malate:quinone oxidoreductase and NDH2: type 2 NADH dehydrogenase (Toxoplasma possesses two copies)) are depicted in grey. The path of electron flow across the mETC complexes is depicted by blue arrows and the path of protons across the inner mitochondrial membrane (IMM) from the matrix to intermembrane space (IMS) and back is depicted with black arrows.
Fig 2.
Complexome profiling of Toxoplasma respiratory complexes.
(A) Percoll gradient used for density-gradient fractionation. Samples were taken from the top, 18%/23% and 23%/40% Percoll interfaces (marked with dashes lines) for analysis via immunoblot (shown in B). The fraction recovered from the 23%/40% Percoll interface was used for complexome profiling. (B) Immunoblot analysis of fractions from total cell lysate (total) and the density-gradient fractionation (top, 18/23 and 23/40) with antibodies against marker proteins for several cell components. Protein concentration from each fraction was quantified with a Bradford assay and 5 μg loaded per lane. Equal loading was confirmed by comassie staining (bottom panel). The following antibodies were used: Mys (mitochondrion), CPN60 (apicolplast), SAG1 (plasma membrane), IMC1 (inner membrane complex) and aldolase (cytosol). (C) Proportion of three mitochondrial proteome datasets: LOPIT mitochondrial membrane, LOPIT mitochondrial soluble [36] and mitochondrial matrix proteome [7] found in the complexome profile dataset. (D) Complexome profile heatmap of the Toxoplasma respiratory complexes subunits. Protein IDs or names are shown on the left of their respective profiles. Previously identified subunits are labelled in black, and putative novel subunits are labelled in blue. Heatmaps x-axis depict the 61 gel slices of the BN-PAGE gel from the top (left) to the bottom (right) of the gel. Molecular weight markers are based on the migrations of mammalian mitochondrial complexes of known size. Red indicates the highest relative abundance (1) and black the lowest (0). Asterisks mark subunits CytC1 of complex III and ATPTG1 of ATP synthase, which are both encoded by the same gene, TGGT1_246540, and therefore have the same profile (see S4 Fig). The full complexome profiling dataset is provided in S1 Table.
Fig 3.
Analysis of putative Toxoplasma complex II subunits.
(A) Immunoblot analysis of complex II subunit SDHB endogenously tagged with an HA epitope. Proteins from total lysate were separated by SDS-PAGE and detected using anti-HA antibodies. Parasites from the parental line (TATIΔku80) were analysed as negative control. (B) Total lysate from SDHB-HA separated by BN-PAGE and immunolabelled with anti-HA antibodies. (C) Immunofluorescence assay with parasites expressing the endogenously HA-tagged SDHB (cyan), showing co-localisation with the mitochondrial marker protein TOM40 [48] (magenta), along with merge and phase. Scale bar is 5 μm. (D) Table showing the previously predicted (known) and complexome identified putative novel (novel) complex II subunits and their homology distribution across key groups. Homology searches were performed using the HMMER tool [57]. Coloured circles refer to the e-value from the HMMER search: white indicates a hit with an e-value above 0.0001, black indicates no hits, and red indicates hits with an e-value below 0.0001, as indicated in the coloured scale. Full data are given in S7 Table. Hh: Hammondia hammondi; Bb: Babesia bovis; Pf: Plasmodium falciparum; Pb: Plasmodium berghei; Cp: Cryptosporidium parvun; Cyryptosporidium muris; Vb: Vitrella brassicaformis; Pm: Perkinsus marinus; Sm: Symbiodinium microadriaticum; Pt: Paramecium tetraurelia.
Table 1.
Details of putative complex II subunits.
Phenotype scores were taken from Sidik et al., 2016 [56]; Matrix proteome from Seidi et al., 2018 [7] (Y–present in proteome, N–absent); LOPIT from Barylyuk et al., 2020 [36] (mm–mitochondrial membrane, ms- mitochondrial soluble N/A–not identified in dataset). TMD: Transmembrane domain, predicted by TMHMM.
Table 2.
Details of nuclear encoded complex III subunits.
Phenotype scores were taken from Sidik et al., 2016 [56]; Matrix proteome from Seidi et al., 2018 [7]; (Y–present in proteome, N–absent); LOPIT from Barylyuk et al., 2020 [36] (mm–mitochondrial membrane, N/A–not identified in dataset). TMD: Transmembrane domain, predicted by TMHMM server. TGGT1_297160 was initially identified as a potential complex III subunit but was later excluded.
Fig 4.
Analysis of putative Toxoplasma complex III subunits.
(A) Table showing the previously predicted (known) and complexome identified putative novel (novel) complex III subunits and their homology distribution across key groups. Homology searches were performed using the HMMER tool [57]. Coloured circles refer to the e-value from the HMMER search: white indicates a hit with an e-value above 0.0001, black indicates no hits, and red indicates hits with an e-value below 0.0001, as indicated in the coloured scale. Full data are given in S7 Table. Hh: Hammondia hammondi; Pb: Plasmodium berghei; Cp: Cryptosporidium parvun; Cyryptosporidium muris; Vb: Vitrella brassicaformis; Pm: Perkinsus marinus; Sm: Symbiodinium microadriaticum; Pt: Paramecium tetraurelia; At: Arabidopsis thaliana; Sc: Saccharomyces cerevisiae; Hs: Homo sapiens. (B) Immunoblot analysis of putative complex III subunits endogenously tagged with an triple-HA epitope tag. Proteins from total lysate were separated by SDS-PAGE, blotted and detected using anti-HA antibodies. Parasites from the parental line (Δku80) were analysed as negative control. Antibodies against the mitochondrial protein Mys (TGME49_215430) [84] were used as a loading control. (C) Immunofluorescence assay of parasites with endogenously HA-tagged putative complex III subunits (cyan), showing co-localisation with the mitochondrial marker protein TOM40 (magenta), along with merge and phase. Scale bars are 5 μm. (D) Immunoblot analysis of complex III subunits CytC1-HA and QCR7-HA. Proteins from total lysate were separated by SDS-PAGE, blotted and detected using anti-HA antibodies. Antibodies against the mitochondrial protein TOM40 [48] were used as a loading control. (E) Total lysate from tagged complex III subunits separated by BN-PAGE, blotted and immunolabelled with anti-HA antibodies. (F) Total lysate from tagged complex III subunits, solubilised with different detergents, DDM or digitonin, separated by BN-PAGE, blotted and immunolabelled with anti-HA antibodies.
Fig 5.
Loss of the new complex III subunits affects parasite growth and mitochondrial membrane potential.
(A) Plaque assays of the parental line (parental) and four promoter replacement lines (rQCR8/9/11/12 / Rieske-HA). Parasites were grown for 9–10 days in the absence (-ATc) or presence of ATc (+ATc) before being fixed and stained with crystal violet. (B) Mitochondrial membrane potential detected using the JC-1 probe of parental (parental) and each of the four promoter replacement lines (rQCR8/9/11/12 / Rieske-HA) in the absence (blue) or presence (orange) of ATc, or valinomycin/atovaquone/antimycin a. Representative histograms of red fluorescence, which is dependent on mitochondrial membrane potential, recorded by flow cytometry are shown.
Fig 6.
Loss of each of the four putative subunits affects stability of complex III.
(A-D) Total lysate from parasites from promoter replacement lines in the Rieske-HA background (rQCR8/9/11/12 / Rieske-HA) grown in the absence (0) or presence of ATc for 1–3, were separated by BN-PAGE, blotted and immunolabelled with antibodies against HA. Samples were also separated by SDS-PAGE and immunolabelled with anti-HA antibodies. (E) Total lysate from the parental Rieske-HA line grown in the absence (0) or presence of ATc for 3 days were separated by BN-PAGE, blotted and immunolabelled with anti-HA antibodies. (F) Total lysate from promoter replacement lines in the Rieske-HA background (rQCR8/9/11/12 / Rieske-HA) grown in the absence (0) or presence of ATc for 3 days (3) were separated by BN-PAGE and immunolabelled with anti-TOM40. Samples were also separated by SDS-PAGE and immunolabelled with anti-HA and anti-SAG1 antibodies. (G) Total lysate from promoter replacement lines in the Rieske-HA background (rQCR8/9/11/12 / Rieske-HA) grown in the absence (0) or presence of ATc for 3 days (3) were separated by clear native-PAGE and complex IV oxidation activity was performed. A line where ribosomal protein mS35 is downregulated was used as positive control for loss of complex IV [34].