Preparation of mitochondria

For each experiment, three to five 15-cm plates each of MEFs stably expressing either mitochondria-targeted TagRFP or CFP were grown to ∼90% confluency. Cells were harvested by cell scraping, pelleted, and washed in mitochondrial isolation buffer (MIB) (0.2 M sucrose, 10 mM Tris-MOPS, pH 7.4, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid). The cell pellet was resuspended in one cell pellet volume of cold MIB, and cells were homogenized by ∼15 strokes on ice with a Kontes Potter-Elvehjem tissue grinder set at 400 rpm. The homogenate was centrifuged (500 × g, 5 min, 4°C) to remove nuclei and unbroken cells, and homogenization of the pellet fraction was repeated followed by centrifugation at 500 × g, 5 min, 4°C. The supernatant fractions were combined and centrifuged again at 500 × g, 5 min, 4°C to remove remaining debris. The supernatant was transferred to a clean microfuge tube and centrifuged (7400 × g, 10 min, 4°C) to pellet a crude mitochondrial fraction. The crude mitochondrial pellet was resuspended in a small volume of MIB. Protein concentration of fractions was determined by Bradford assay (Bio-Rad Laboratories).