In vivo site-specific photo cross-linking of Tom22 IMS domain to Mgr1 and Mgr3. (A) Cartoon illustration of the in vivo site-specific photo–cross-linking method. (B and C) The MGR1-FLAG yme1^E541Q and MGR3-FLAG yme1^E541Q strains harboring plasmids for the expression of Tom22-HA with BPA incorporated at the indicated sites were irradiated with UV for 15 min. Whole-cell extracts were prepared and subjected to anti-FLAG IP as described in the Site-specific in vivo photo cross-linking and IP section of Materials and methods. Immunoprecipitates were analyzed by SDS-PAGE. About 30 µl out of 100 µl immunoprecipitates were loaded for the anti-HA blots. Red and green boxes highlight representative residue positions that are only clearly cross-linked to Mgr1-FLAG or Mgr3-FLAG, respectively. (D and E) The MGR1-FLAG strains (D) and MGR3-FLAG strains (E) in WT or yme1^E541Q background were transformed with plasmids expressing BPA-incorporated Tom22-HA and analyzed as in B and C. Red boxes highlight Tom22 residue positions showing similar level of cross-linking in WT and yme1^E541Q cells. Molecular masses are shown in kilodaltons.