Yeast Msp1 and human ATAD1 are required to limit the level of mitochondrially mislocalized TA proteins Pex15 and PEX26, respectively Representative images of GFP‐Pex15 localization. Yeast Msp1 and human ATAD1 are required to limit the level of mitochondrially mislocalized TA proteins Pex15 and PEX26, respectively Representative images of GFP‐Pex15 localization. The indicated yeast strains expressing GFP‐Pex15 from the GAL1 promoter and plasmid‐borne Mito‐RFP were grown overnight in SD medium, switched to galactose induction medium for 6 h, and visualized by fluorescence microscopy. The get3Δ msp1Δ images (1,000 ms) were exposed 5 times longer than the WT, msp1Δ, and get3Δ images (200 ms). The WT and get3Δ strain expressing GFP‐Pex15 from the GAL1 promoter and the plasmid‐borne Mito‐RFP were transformed with either EV or a Msp1 overexpressing construct (Msp1(2μ)) and subjected to the same procedure as described in (A). Pex15 co‐immunoprecipitates with the ‘trap mutant’ Msp1E193Q. Msp1 was immunoprecipitated by anti‐HA antibodies from digitonin‐solubilized mitochondria that were extracted from the indicated strains overexpressing GFP‐Pex15 from the GAL1 promoter and empty vector (−), wild‐type Msp1 (WT) or Msp1E193Q (E193Q). Pex15 expression was induced in galactose medium for 6 h. 4% of the crude lysates, and final eluates were immunoblotted with anti‐HA and GFP. Om45 is a mitochondrial outer membrane protein and is used as a loading control. Five‐fold dilutions of the indicated strains expressing Pex15 from the native PEX15 promoter or GAL1 promoter (PGAL1::GFP–PEX15) were grown overnight in SD medium, spotted on SD or synthetic galactose (SGal) plates, and cultured at 30°C. The get3Δ strain co‐expressing GFP‐Pex15 from the GAL1 promoter and wild‐type Msp1 or Msp1E193Q was pulsed in galactose medium for 5 h to induce GFP‐Pex15 accumulation and chased in glucose medium to shut off transcription. Whole‐cell lysates were prepared from cells harvested every 45 min and analyzed by immunoblot. Por1 is the loading control. HepG2 cells stably expressing GFP‐PEX26 were treated with scrambled siRNA (scr) or siRNAs (#1–3) targeting human ATAD1 for 6 days, stained with Mitotracker Red, and visualized by fluorescence microscopy with equivalent exposure times. Whole‐cell lysates of cells from (F) were analyzed by immunoblot using the indicated antibodies. QCR2 is a mitochondrial protein and is used as a loading control. Source data are available online for this figure. Yu‐Chan Chen et al. EMBO J. 2014;33:1548-1564 © as stated in the article, figure or figure legend