De novo mitochondrial translation was assessed by incubation (1 hour, 37°C, on rotating wheel) of 1.5 mg of freshly isolated mitochondria in 1 ml of 35S-translation buffer [100 mM mannitol, 10 mM Na-succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KH2PO4, 25 mM Hepes (pH 7.4), 5 mM ATP, 200 μM GTP, 6 mM creatine phosphate, creatine kinase (60 μg/ml), cysteine (60 μg/ml), tyrosine (60 μg/ml), amino acids (60 μg/ml) (Ala, Arg, Asp, Asn, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, and Val), 35S-methionine (7 μl/ml)]. Subsequently, mitochondria were pelleted (12,000g, 2 min) and resuspended in 1 ml of nonradioactive translation buffer containing methionine instead of 35S-methionine. Half of the sample (“pulse fraction”) was pelleted again, resuspended in 100 μl of SDS–polyacrylamide gel electrophoresis (PAGE) loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue], and lysed (30 min, room temperature) before transfer at −20°C. For the “cold chase” allowing to estimate the protein turnover, the remaining 500 μl of resuspended mitochondria was incubated for 3 hours at 37°C on a rotating wheel. Subsequently, the “chase fraction” was pelleted, resuspended in 100 μl of SDS-PAGE loading buffer, and lysed as the “pulse sample” before.

Separation of mitochondrial proteins was achieved by SDS-PAGE. Ten microliters per sample was loaded on a 15-cm-long, 15% polyacrylamide gel and run in a “SE600X Chroma Deluxe Dual Cooled Vertical Protein Electrophoresis Unit” (Hoefer) overnight at 80 V continuously. After fixing (50% methanol and 10% acetic acid) for 30 min, staining in Coomassie solution, and destaining (20% methanol and 10% acetic acid) of the polyacrylamide gel, the latter one was placed on Whatman paper (GE Healthcare) and dried (2 hours, 80°C) in a gel dryer. For detection of radioactive signals of de novo synthetized proteins, Amersham Hyperfilm MP (GE Healthcare) was exposed to the dried polyacrylamide gel.

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