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

Abstract

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma

“…The metabolic changes encountered by ISC- deficient parasites do not cause their rapid demise, as they are able to initiate conversion to the bradyzoite stage, which has been suggested to rely essentially on glycolysis for energy production [93]. Our analysis of the viability of other mitochondrial mutants confirmed results obtained by others showing that the organelle, and in particular the ETC, seem important for parasite fitness [61,62,75]. Yet, perhaps because of their metabolic flexibility, we have shown that mutant parasites seem to retain the ability to survive as tachyzoites, or initiate a switch to bradyzoites.…”

“…Several (19%) are predicted to be apicoplast-resident proteins, including radical SAM enzymes lipoate synthase (LipA) [57] and MiaB, a tRNA modification enzyme [58], as well as the IspG and IspH oxidoreductases of the non-mevalonate isoprenoid pathway [59]. Finally, for the most part (43%), candidate Fe-S proteins were predicted to be mitochondrial, with noticeably several important proteins of the respiratory chain (SDHB, the Fe-S subunit of the succinate dehydrogenase complex, Rieske protein and TgApiCox13) [60–62], but also enzymes involved in other metabolic pathways such as heme or molybdopterin synthesis. CRISPR/Cas9 fitness scores [38] confirmed many of these putative Fe-S proteins likely support essential functions for parasite growth.…”

“…This ETC comprises four complexes in Apicomplexa (which typically lack complex I), in which several Fe-S proteins have important function. As mentioned earlier, they include the Fe-S subunit of the succinate dehydrogenase complex (SDHB, part of complex II), the Rieske protein (part of complex III) and TgApiCox13 (part of complex IV) [60–62]. Not only these three Fe-S cluster proteins were found to be less expressed upon TgISU1 depletion, but about 60% and 80% of complexes III and IV components (including recently characterized parasite-specific subunits [61, 62]), respectively, were also significantly less abundant (Table S4, Figure 8A).…”

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

“…This is based on a stoichiometry of 10 ATPc subunits for the c-ring, canonical stoichiometry for orthologues to known components and single copies of novel components, predicting a monomer mass of~1 MDa (Table 1). This, along with the recent elucidation of the ATP synthase structure in T. gondii 61 and similar observations in a recent T. gondii complexome profile 62 , suggests that the~1 MDa complex observed in other studies probably represent the monomer rather than the dimer or is based on flawed mass calibration 7,59,63 (Supplementary Fig. 9).…”

“…As detection of CII components was limited to ABS3D and GCT3D (Supplementary Table 1), sample size is small compared to other complexes discussed in this study. In addition, as complexome profiling provides no direct evidence of interaction and only one comparable study in T. gondii 62 has suggested a similar composition, further studies will be needed to verify our findings.…”