Coa3 is a mitochondrial inner membrane protein facing the IMS. (A) Isolated wild-type (WT) and Shy1^ProtA mitochondria were solubilized and subjected to IgG chromatography. After tobacco etch virus (TEV) protease cleavage, eluates were analyzed by SDS-PAGE, Western blotting, or peptide LC/tandem MS. The asterisk marks ADP/ATP carrier, an impurity of purification. Cytochrome bc₁ complex components identified are not depicted (Mick et al., 2007). (B) Immunofluorescence analysis using MitoTracker red and Coa3-specific antibodies. Bars, 5 µm. (C) Alignment of Coa3 homologues (ClustalW 2.0.11). Black boxes indicate identical residues in at least five species; gray boxes indicate similar amino acids. Black underlining indicates the predicted transmembrane segment. Sc, S. cerevisiae; Nc, Neurospora crassa; Yl, Yarrowia lipolytica; Pp, Pichia pastoris; Ag, Ashbya gossypii; and Kl, Kluyveromyces lactis. (D and E) Membrane association and submitochondrial localization of Coa3 as described in Materials and methods. T indicates the total; S and P indicate the supernatant and pellet, respectively, after ultracentrifugation. TX-100, Triton X-100; PK, proteinase K.