Fig. 1.

Three-dimensional electrophoresis of yeast mitochondria to identify Cox26, a novel protein co-migrating with respiratory supercomplexes. III₂IV₂, III₂IV₁, respiratory supercomplexes containing dimeric complex III and two or one copy of complex IV, respectively. V_Dim. V_Mon, IV_Dim. IV_Mon. III_Dim, dimeric and monomeric complexes V, IV, and III. (A) Yeast mitochondria were solubilized by digitonin and the mitochondrial (super)complexes were separated by BNE. (B) A gel strip from 1-D BNE was used for 2-D resolution by modified BNE, i.e. DDM was added to the cathode buffer to release individual complexes from the supercomplexes. (C) A gel strip from 2-D BNE was used for 3-D SDS-PAGE. Subunits of dimeric complex III (III_Dim) and the detergent-labile subunits (assigned on the right side) were marked red. Subunits of dimeric and monomeric complex IV (IV_Dim and IV_Mon) and the detergent-labile subunits were marked green. The subunits were assigned according to previous Edman sequencing [29] and/or detection by specific antibodies. One single protein, Cox26, was identified as a novel protein component of supercomplexes. It was identified as the smallest protein in the group of detergent-labile subunits (assigned on the right side of Fig. 1C). (D) Sequence and structural features of Cox26 protein. The preprotein contains a presequence of eight amino acids (shaded grey). Basic and acidic residues are marked blue and red, respectively. A cysteine residue (C41 marked yellow) is located within a predicted transmembrane helix (underlined).