Fractionation of mitochondrial membrane

Mitochondria were isolated, and submitochondrial fractionation was performed as previously described (37). Briefly, the pellet of crude mitochondria was prepared as described above. Following washing with SHE buffer [250 mM sucrose, 10 mM Hepes, and 1 mM EGTA (pH 7.4)] and centrifugation at 16,000g for 10 min, the mitochondrial pellet was resuspended in swelling buffer [10 mM KH₂PO₄ (pH 7.4)] at a concentration of 200 μg of mitochondrial protein per milliliter and incubated on ice for 20 min. Then, an equal volume of shrinking buffer [10 mM KH₂PO₄ (pH 7.4), 32% (wt/vol) sucrose, 30% (wt/vol) glycerol, and 10 mM MgCl₂] was added and centrifuged at 10,000g for 10 min. The supernatant containing the OMM and the mitochondrial intermembrane space fractions were transferred to a fresh tube. The mitoplast pellet was washed with 1:1 mixture of swelling and shrinking buffer and centrifuged at 10,000g for 10 min at 4°C. After washing, the mitoplasts were resuspended in swelling buffer and incubated on ice for 20 min. OMM/IMS (intermembrane space)- and IMM (inner mitochondrial membrane)–containing fractions were centrifuged at 14,000g for 1 hour. The resulting pellet contains OMMs and IMMs. The supernatant contains the IMS proteins. The supernatant was concentrated with centrifugal concentrators (Millipore). Fractions were resuspended in RIPA buffer and analyzed by SDS-PAGE and Western blot with corresponding antibodies, including mitochondrial membrane and intermembrane markers.