Isolation and Fractionation of Mitochondria

Mitochondria were prepared from 10⁷ cells grown overnight in 15 cm² dishes and isolated as described before (22 (link)). Rat liver mitochondria were prepared by homogenization in 250 mM sucrose, 5 mM Tris–HCl pH 7.4 and 1 mM EGTA (STE) followed by differential centrifugation (23 ) and the protein concentration determined by the biuret assay using BSA as a standard (24 (link)). Rat liver mitochondria were washed and suspended in STE containing 0.1% (w/v) fat-free BSA (STE/BSA) instead of STE for the final spin. The final pellet was suspended in a minimal amount of STE/BSA and the protein concentration was measured by the biuret assay. The mitochondria were diluted with STE/BSA to a final concentration of 100 mg ml^–1 and stirred on ice. One millilitre of digitonin (12.5 mg ml^–1) was added dropwise over 2 min and the solution was kept on ice for a further 13 min. Three volumes of STE/BSA were added and the mixture was homogenized four times with a tight homogenizer. The mitoplasts were collected by centrifugation for 10 min at 10 000g and the concentration was measured using the biuret assay. The supernatant was kept and centrifuged at 144 000g for 20 min at 5°C to separate the intermembrane fraction (supernatant) from the outer membrane (pellet). The mitoplasts were suspended at 30 mg ml^–1 in STE containing 0.16 mg lubrol mg^–1 mitoplast protein and incubated on ice for 20 min. This solution was diluted with three volumes of STE and centrifuged at 144 000g for 50 min at 5°C to separate the matrix (supernatant) from the inner membrane (pellet). Both membrane fractions were suspended in 1% lauryl maltoside in STE. The protein concentration of each fraction was determined using the BCA assay (25 (link)) and immunoblotting was carried out to confirm the fractionation.