Dissecting the subunit composition, structure and evolution of the the highly diverged mitochondrial F-type ATP synthase complex from Toxoplasma gondii

Abstract

The mitochondrial F-type ATP synthase (F-ATPase), a multisubunit nanomotor, is critical for maintaining cellular ATP level. In Toxoplasma gondii and other apicomplexan parasites many subunits required for proper assembly and functioning of this enzymes are appears to be missing in these parasites. Despite missing crucial subunits, the functionality of F-ATPase is established in apicomplexan parasites, indicating that novel proteins are probably associated with the complex to make it functional. Here, we characterize the subunit composition of mitochondrial F-ATPase enzymes in apicomplexan parasite T. gondii. We have partially purified and enriched the F-ATPase enzymes complex from detergent solubilized mitochondrial lysate using three different approaches - Blue Native PAGE, immunoprecipitation and by combination of ion exchange and gel filtration chromatography. Using mass spectrometry based proteomic analysis on partially purified ATP synthase complex, we have identified 20 novel subunit components of T. gondii F-ATPase. These studies also confirmed that the enzyme complex is present in monomeric (~600 kDa) and dimeric (>1 MDa) forms, as previously reported in other eukaryotes. Despite the extreme sequence diversification, conserved structure analysis helped in identification of missing key FO subunits a, b and d, which are necessary for assembly and function of the complex. Notably, orthologs of these novel subunits of T. gondii F-ATPase are present in all apicomplexan parasites, except in Cryptosporidium species lacking a true mitochondrion. Interestingly, these proteins are also conserved in other alveolate species, such as the chromerids and dinoflagellates, suggesting an ancient origin for these proteins. Further the structural and functional characterization of these highly divergent novel components of F-ATPase will facilitate the fundamental understanding of energy metabolism and development of novel antiparasitic agents against apicomplexan parasites.