Saponin-Mediated Isolation of Megacomplexes of the Mitochondrial Oxidative Phosphorylation System

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) supercomplexes determines electron flux and ATP synthesis. Although several techniques have been developed to isolate these supercomplexes, their native assembly states within the inner mitochondrial membrane have remained unclear. Here, we developed a separation technique based on saponins from Sapindus saponaria pericarps that enables the isolation of megacomplexes from mitochondria. Using size-exclusion chromatography (SEC) coupled with Western blot, we found that these megacomplexes may represent the predominant assembly state of the mitochondrial OXPHOS system. Furthermore, co-immunoprecipitation followed by Western blot assays revealed a megacomplex comprising respiratory chain complexes I-IV and ATP synthase. Notably, these supercomplexes remained intact under both fasting and a 24-h high-fat diet, underscoring their stability in vitro. Collectively, this study defines the primary architectural organization of OXPHOS system and establishes the saponin-based method as a mild alternative for extracting membrane protein supercomplexes.