Mmessage sp6 transcription kit

In vitro mRNA transcription and protein translation was performed using mMessage SP6 transcription kit (Ambion) and rabbit reticulocyte lysate system (Promega) respectively. In vitro translation was performed in the presence of ³⁵S-methionine. Mitochondrial in vitro protein import assays were performed as described (Ryan et al., 2001 (link)). Radiolabeled proteins were incubated with isolated mitochondria resuspended at 1 mg/ml in import buffer (20 mM HEPES-KOH (pH 7.4), 250 mM sucrose, 5 mM magnesium acetate and 80 mM potassium acetate) supplemented with 10 mM sodium succinate, 1 mM 1,4-dithiothreitol and 5 mM ATP. Protein import was performed at 37°C for the desired amount of time in the presence or absence of membrane potential. To dissipate the membrane potential, a final concentration of 10 μM of Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich) was added to each import reaction. Import reaction was stopped at 4°C followed by incubation with 50 μg/mL of PK and subsequent treatment with 1 mM PMSF. Mitochondria were re-isolated and TCA precipitated for SDS-PAGE analysis or resuspended into digitonin-containing buffer for BN-PAGE analysis. Autoradiography was performed to visualise the radioactive signal using a Typhoon Phosphor Imager (GE Healthcare). Radioactive images were processed using Image J software.

Open reading frames (ORF) encoding mitochondrial precursor proteins were cloned into pGEM4Z (Promega). The desired ORFs were amplified by PCR using vector specific primers (M13_FORWARD and M13_REVERSE) and applied to in vitro transcription reactions using the mMessage SP6 transcription kit (Ambion). mRNA was isolated by LiCl precipitation according to the manufacturer’s instructions and applied to in vitro translation reactions using rabbit reticulocyte lysate (Promega) in the presence of [³⁵S]-methionine/cysteine (Perkin-Elmer). Isolated mitochondria were incubated with translation products in import buffer (20 mM HEPES-KOH pH 7.4, 250 mM sucrose, 80 mM KOAc, 5 mM MgOAc, 10 mM sodium succinate, 1 mM DTT and 5 mM ATP) at 37°C for various times as indicated in the figure legends. Samples subjected to protease treatment were incubated on ice for 10 min with 50 μg/ml proteinase K (Sigma-Aldrich), followed by the addition of 1 mM PMSF and incubation for 10 min on ice. For analysis of protein complexes by blue native electrophoresis and antibody-shift mitochondria were solubilised in 1% digitonin (at 1 mg/mL) and incubated with the desired antibody for 30 min on ice, prior to clarification and subsequent analysis by BN-PAGE. Following in vitro import samples were separated by SDS-PAGE and BN-PAGE and radiolabelled proteins were detected by digital autoradiography.