Fig. 5
Plant Erv1 can directly oxidize IMS proteins. a Radiolabeled A. thaliana CCMH was incubated for 2 min with isolated mitochondria from the AtErv1-expressing Δerv1. Mitochondria were treated with 400 μM cleavable cross-linker dithiobis succinimidyl propionate for 5 min at 25 °C and lysed with 1% SDS. The extract was used for immunoprecipitation with a combination of Erv1- and hemagglutinin-specific antibodies or with pre-immune serum (PIS). The crosslinker was cleaved with DTT when indicated. The radiolabeled protein was visualized by autoradiography. Total samples contain 10% of the material used per immunoprecipitation reaction. Arrow depicts radiolabeled CCMH pulled down with AtErv1. b Cytochrome c reduction by plant and yeast Erv1 with glutathione (GSH) as an electron donor. The reduction of cytochrome c (40 μM) was followed at 550 nm over 10 min after incubation with 5 mM GSH in the presence or absence of 8 μM AtErv1 or ScErv1. All of the reduced cytochrome c was obtained with 50 μM DTT, as shown in Fig. 1c. c In vitro-translated radioactive Cox19 was incubated in the absence or presence of 30 μM purified AtErv1 for the indicated times. Subsequently, samples were TCA-precipitated, treated with 15 mM mmPEG24 for 1 h at 25 °C, and then subjected to non-reducing SDS-PAGE and analyzed by autoradiography