Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii
Fig 3
Identifying the dimer and monomer forms of T. gondii F-type ATP synthase by BNP analysis.
(A) Mitochondria lysates were prepared from tachyzoites stage transgenic parasites expressing TgATPβ-YFP-HA protein were separated by BNP. Lane M, native molecular weight markers; lane A, Coomassie blue staining of BNP gel; lane B, western blotting of BNP gel using α-HA antibodies. The dimer and monomer forms are indicated by arrows. The boxed regions in lane A correspond to the excised gel pieces, which were processed for LC-MS/MS analysis. (B) In-gel ATPase activity assays following BNP separation confirms that the dimer and monomer forms of F-type ATP synthase are functionally intact. BNP, blue native PAGE; HA, hemagglutinin; LC-MS/MS, liquid chromatography–tandem mass spectrometry; TgATP-β, T. gondii ATP synthase β subunit; TgATP-OSCP, T. gondii ATP synthase OSCP subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.