MitOS is a conserved mitochondrial inner membrane–associated complex. (A) Schematic representation of predicted structural features of MitOS components. As described in Materials and methods, the amino acid sequences of MitOS components were subject to bioinformatic analyses to identify conserved features. Fcj1 contains a conserved Mitofilin domain, Mos1 contains a conserved eukaryotic DUF, and Aim37 and Mos2 are both similar to apolipoproteins, predicted to form extended amphipathic α helices (Lamant et al., 2006). Aim13 contains a conserved fungal-specific DUF and a CHCHD-like motif in its C-terminal region, which is marked by two conserved cysteine residues (Cavallaro, 2010). Significantly, a human CHCHD3 protein was reported to be in a complex with Mitofilin isolated from human heart mitochondria (Xie et al., 2007). (B) Protease protection analysis of mitochondria isolated from strains expressing FLAG-tagged versions of MitOS components. Intact mitochondria (lanes 1 and 4), mitoplasts (lane 3), or solubilized mitochondria (lane 2) before treatment with (+) or without (−) trypsin were analyzed by SDS-PAGE and immunoblotting with the indicated antisera. (C) Separation of soluble and membrane proteins by alkaline extraction. Mitochondria were treated with 0.1 M NaCO₃ and centrifuged into pellet (P) and soluble (S) fractions, which were analyzed by SDS-PAGE and immunoblotting with the indicated anti-sera. T, total.