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4. After synthesis of mitochondrially encoded proteins in the presence of [ 35 S]methionine for 20 min at 30°C mitochondria were reisolated and washed twice with 1 ml of cold SHKCl buffer [0.6 M sorbitol 80 mM KCl 50 mM Hepes-KOH (pH 7.2)] to remove nonincorporated [ 35 S]methionine. Mitochondria were then carefully resuspended in translation buffer at a concentration of 1.5 mg/ml. For proteolysis samples of 30 or 100 μl (for gel filtration analysis) were further incubated at 37°C. At the time points indicated samples were immediately centrifuged at 4°C for 4 min at 13 200 g. Radioactivity recovered in the supernatant was determined after the addition of scintillation fluid (Ultima Gold Canberra Packard) (1 ml) and vigorous vortexing. Mitochondrial pellets were resuspended in SHKCl buffer (30 μl) and precipitated with TCA [12.5% (w/v)] for 15 min at −80°C. After centrifugation for 15 min at 36 000 g TCA-soluble material was recovered and radioactivity determined as above. TCA-insoluble material was resuspended in 30 μl of LiDS sample buffer and processed for counting as above. To monitor the integrity of mitochondrial membranes we routinely analyzed duplicate samples before TCA precipitation by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotting with antisera directed against the matrix protein Mge1 and the intermembrane space protein cytochrome b 2 .
5. Recovered supernatants (100 μl; ∼700 000 cpm) were subjected to gel filtration on a Sephadex Peptide column (7.5/300; Pharmacia) that was equilibrated with 40% (v/v) acetonitrile in 0.1% (v/v) TFA. Fractionation was carried out at room temperature at a flow rate of 0.3 ml/min. Fractions (0.3 ml) were collected and counted after the addition of scintillation fluid (1 ml). More than 95% of the radioactivity loaded was recovered from the column in each case. Rat gastrin (2126 daltons) substance P (1348 daltons) penta-glycine (303 daltons) methionine (149 daltons) and diglycine (132 daltons) were used for calibration.