Mitochondrial isolations and BN-PAGE were performed as previously described 62 (link),63 (link). Frozen cell pellets were thawed on ice in hypotonic buffer (83 mM sucrose, 10 mM MOPS, 1X cOmplete EDTA-free protease inhibitor cocktail (Roche, 4693159001)) and subsequently homogenized using a dounce homogenizer. After this, osmolarity was normalized using hypertonic sucrose buffer and the resulting solution was centrifuged at 1000xg for 5 minutes. The supernatant was then centrifuged for a second time to remove cell debris. Next, the supernatant containing mitochondria was centrifuged at 9,000xg for 10 minutes. The mitochondrial pellet was resuspended and washed twice in resuspension buffer (320 mM sucrose, 1 mM EDTA, 10 mM Tris pH 7.4, 1X protease inhibitor cocktail (Roche, 4693159001)). Protein content of each mitochondrial prep was quantified and mitochondria were divided into 100–200 μg aliquots that were flash frozen. Before running BN-PAGE, mitochondria were solubilized in NativePAGE 4x Sample Buffer (ThermoFisher Scientific, BN2003) at a 6 g/g digitonin/protein ratio for 20 minutes on ice. The insoluble portion was removed by centrifugation for 15 minutes at max speed. The soluble supernatant was then mixed with Coomassie G-250 (ThermoFisher Scientific, BN2004) prior to BN-PAGE.