Fig. 2

AtErv1 fails to oxidize the catalytic cysteines in the CPC motif of Mia40. Western blot analysis of extracts of whole cells (a) or isolated mitochondria (b) of the indicated strains show a strong depletion of Mia40 substrates in strains that express AtErv1 in the absence of yeast Erv1. Panel a shows the shuffle strain before and after shuffling out the ScErv1-encoding URA3 plasmid. c To monitor the redox state of Mia40 in the different strains, proteins of the indicated strains were TCA-precipitated (in order to ‘freeze’ the redox state of thiol groups), denatured in SDS, treated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) and the alkylating compound methyl-polyethylene glycol₂₄-maleimide (mmPEG₂₄), and visualized by SDS-PAGE and western blotting. For this experiment a Mia40 variant was used that lacked the long membrane linker, which leads to much more reliable results in this kind of shift assay [18, 25]. TCEP reduced all thiols in Mia40 such that its six cysteines were alkylated, resulting in a shift of approximately 12 kDa (2 kDa per mmPEG₂₄). In wild-type cells, in the absence of TCEP, Mia40 was not shifted since all cysteines were oxidized (arrowhead). Additionally, in the Δerv1 mutant that was complemented by yeast Erv1, the cysteine residues of Mia40 remained largely inaccessible. In the AtErv1-expressing mutant, however, almost no oxidized Mia40 was detectable. The shift by 4 kDa corresponds to the alkylation of the two redox-active cysteines of Mia40, indicative of the reduced form of Mia40. It should be noted that the two structural disulfides that are critical for the formation of the substrate-binding domain of Mia40 were formed in this mutant. d Helical wheel representation of the Mia40 interaction region in Cox17, Tim9, yeast Erv1 (Sc), and AtErv1 [9, 10, 14, 17]. The hydrophobic (black) and hydrophilic (grey) faces of the helix are indicated as half circles. Note that the docking cysteines in Cox17 and Tim9, as well as cysteines of the shuttle disulfide in yeast Erv1 (yellow), are part of an amphipathic helix structure whereas cysteines of the shuttle disulfide of AtErv1 are not. e, f Levels of IMS proteins were analyzed by western blotting (e). Growth of the indicated mutants on non-fermentable medium (f). Gal galactose