Mitoplast production

Mitoplasts were formed at 4°C by standard procedures that yield outside-out, stable transport vesicles. Protease inhibitor (cOmplete Ultra, Roche) was present in all steps. HEK 293 cells from a 15-cm dish were pelleted, resuspended in 2-mL mitochondria resuspension buffer (MRB, 250 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.2-KOH), and lysed by passing through a 27.5 g needle 15 – 20 times. Nuclei and cell debris were removed by spinning the cell lysate at 1000 g for 10 min. The supernatant was spun down at 10,000 g for 10 min, resuspended in 2-mL MRB, and then spun down again to pellet crude mitochondria. To obtain mitoplasts, mitochondria were resuspended in 800-μL hypotonic shock buffer (5 mM sucrose, 5 mM HEPES, 1mM EGTA, pH 7.2-KOH), and subjected to osmotic shock for 10 min. Then 200 μL of high-salt storage buffer (750 mM KCl, 100 mM HEPES, 2.5 mM EGTA, pH 7.2-KOH) was added, and mitoplasts were subsequently sedimented by centrifugation at 20,000 g for 10 min. The supernatant, which contains proteins in the outer membrane and the intermembrane space, was collected if further analysis is required.