Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex
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.