In vivo site-specific photo cross-linking of the Tom22 cytoplasmic domain to Mgr3. (A–C) The MGR3-FLAG strains in yme1^E541Q (A), WT (B), or yme1^E381Q (C) background were transformed with plasmids expressing Tom22-HA with BPA incorporated at the indicated sites and analyzed as in Fig. 4 C. Red boxes highlight a positive control, which is the cross-linking of Tom22 (143 BPA) with Mgr3 in yme1^E541Q background. White arrows highlight cross-linked bands. Molecular masses are shown in kilodaltons. (D) Cartoon illustration showing that the Yme1^E541Q mutant (protease dead but ATPase active) but not the Yme1^E381Q mutant (ATPase dead but protease active) can cross-link to Tom22 cytosolic domain after the dislocation of the entire protein into IMS. (E) Working model for the proteolysis of Tom22 and Om45 by the Yme1^-Mgr1-Mgr3 complex.