In organello import

In vitro synthesis of [³⁵S]methionine-labeled precursor proteins was performed with the mMessage mMachine SP6 transcription kit (Ambion, Cat.# 1340) and the Flexi rabbit reticulocyte translation kit (Promega, Cat. # L4540), or with the TNT SP6 coupled reticulocyte transcription/translation kit (Promega, Cat. # L2080). The following plasmids were used as templates: pGEM4Z-AAC (Neurospora crassa), pGEM-F₁β (S. cerevisiae), pGEM4Z-b₂(220)-DHFR, pGEM4Z-Mpc1, pGEM4Z-Mpc2, and pGEM4Z-Mpc3. The radiolabeled precursors were imported into the isolated mitochondria at 25 °C in import buffer (10 mM MOPS-KOH pH 7.2, 3% [w/v] bovine serum albumin, 250 mM sucrose, 80 mM KCl, 5 mM MgCl₂, 2 mM KH₂PO₄, 5 mM methionine) with 2–4 mM NADH and an ATP-regenerating system including 2–4 mM ATP, 5–10 mM creatine phosphate, and 0.1–0.2 mg/ml creatine kinase. Import reactions into tim12-21 and the control wild-type mitochondria were performed at 30 °C. tim17-4 mitochondria and tim17-5 mitochondria and the corresponding wild-type mitochondria were heat-shocked for 10 min at 37 °C in import buffer prior to the addition of NADH, the ATP-regenerating system, and the radiolabeled precursor proteins (in reticulocyte lysate), followed by the import reaction at 25 °C. Reactions included a control sample where the membrane potential was dissipated with AVO mix (8 μM antimycin A, 1 μM valinomycin, 20 μM oligomycin) before the addition of precursor. The import reactions were stopped by the addition of AVO and transfer on ice. Non-imported precursor was removed by a 15-min incubation with 50 μg/ml proteinase K on ice, unless indicated otherwise. After the inactivation of proteinase K with 2 mM PMSF, the mitochondria were reisolated and washed in SEM buffer. To generate mitoplasts after the import reaction, the mitochondria were resuspended in hypotonic EM buffer (1 mM EDTA, 10 mM MOPS-KOH pH 7.2). The mitoplasts were treated with 50 μg/ml proteinase K and subsequently treated with PMSF and re-isolated as described above. Quantification of import/assembly efficiency was performed with Fiji ImageJ software. Replicates used for quantification were independent import and assembly assays of incubation of isolated yeast mitochondria (wild-type and mutant mitochondria) with radiolabeled precursor proteins, followed by independent gel separation and analysis. The individual data values from independent replicates are listed in Additional file 6: Table S1 and Additional file 7: Table S2.