J Vis Exp. 2026 Aug 21.
Mitochondria are central hubs in bioenergetic metabolism and are the primary source of ATP. The inner mitochondrial membrane houses the oxidative phosphorylation system, which includes electron transport chain complexes (CI, CII, CIII2, and CIV) and the ATP synthase (CV). In mammals, CI, CIII2, and CIV form higher-order structures called supercomplexes (SCs) such as SC I+III2+IV, SC I+III2, and SC III2+IV. Although the physiological factors favoring SC formation remain unclear, it has been proposed that SC formation may enhance electron-transfer rates between complexes, reduce reactive oxygen species production, or prevent nonspecific protein aggregation within the densely packed mitochondrial inner membrane. Structural and functional studies of respiratory SCs have relied heavily on detergent-extracted complexes. While these studies have improved our understanding of the electron transport chain, the lack of a sealed membrane bilayer limits their ability to probe the functional benefits of supercomplex assembly. Recent advances, however, have shown that membrane proteins can be structurally characterized in reconstituted, native-like membrane environments, offering a more physiological context for these investigations. Here, we present a simple, quick, and reproducible protocol for reconstituting respiratory SCs into liposomes. This method allows for testing the effects of varying lipid compositions, protein concentration, and membrane potential on the function of respiratory SCs, providing a valuable tool for future mechanistic studies.