In organello translation assays were carried out in isolated heart mitochondria as described before (18, 19, 23, 41). Heart or skeletal muscle mitochondria (500 μg) were incubated in 750 μl of translation buffer [100 mM mannitol, 10 mM succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KPi, 25 mM Hepes (pH 7.4), 5 mM ATP, 20 μM GTP, 6 mM creatine phosphate, creatine kinase (60 μg/ml), and all amino acids (60 μg/ml) except methionine and cysteine]. Mitochondria were supplemented with 150 μCi of [35S]methionine/cysteine (PerkinElmer) for 1 hour at 37°C. After labeling, mitochondria were washed in translation buffer and resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentration was measured, and 50 μg of mitochondrial protein was resolved by SDS-PAGE and visualized by autoradiography.

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