Fig. 4

AtErv1 exhibits a dominant-negative activity on yeast Mia40. a Mitochondria isolated from a Δerv1 strain expressing AtErv1 were treated with 1 mM cleavable cross-linker dithiobis succinimidyl propionate for 15 min at 25 °C and lysed with 1% SDS. The extract was used for immunoprecipitation with Mia40-specific antibodies or with pre-immune serum (PIS). Hemagglutinin-tagged AtErv1 protein was visualized by western blotting. Arrows indicate immunoprecipitated AtErv1. Total samples contain 10% of the material used per immunoprecipitation reaction. b A multicopy plasmid carrying the yeast MIA40 gene was transformed into wild-type or Δerv1 cells. Whole cell extracts were prepared and analyzed by western blotting. Note that the levels of Atp23 and Tim10 were largely restored upon overexpression of Mia40 despite the absence of yeast Erv1 in these mutants (blue arrows). c An extra copy of yeast Mia40 (ScMia40) partially rescues the growth defect of the AtErv1 mutant on non-fermentative medium. d Mitochondria were isolated from wild-type cells lacking or carrying an expression plasmid for AtErv1. The Mia40 substrates Cmc1 and Tim9, as well as the matrix protein Oxa1, were incubated with these mitochondria at 25 °C for the times indicated. Non-imported material was removed by protease treatment. The amounts of imported radiolabeled proteins were quantified. Mean values and standard deviations of at least three replicates are shown