Generation of knockout and stable cell lines

Immortalized WT and Yme1l^−/− MEFs and HeLa cells were described previously¹⁸. In brief, primary MEFs were isolated from Yme1l^loxP/loxP animals and immortalized using SV40 large T antigen-encoding plasmids. Upon transduction with Cre recombinase, individual clones were expanded and genotyped to identify WT (Yme1l^loxP/loxP) and Yme1l^−/− MEFs. YME1L^−/− HeLa cells and Slc25a33^−/− MEFs were generated using CRISPR–Cas9-mediated gene targeting. Guide RNA targeting YME1L (5′-GGAACCGACCATATTACAACAGG-3′) and Cas9 were expressed in HeLa cells using transient transfection of the px330 expression vector. Guide RNA targeting Slc25a33 (5′-CGCGTCTTAATGACTTCTAG-3′) and Cas9 were expressed in WT and Yme1l^−/− MEFs using transient transfection of the px459 expression vector before puromycin selection. Individual clones were validated by immunoblotting and genomic sequencing. Unedited WT and Yme1l^−/− MEF clones were used as controls. HeLa cells expressing mouse SLC25A33–MycFlag were generated by lentiviral transduction and selected using 1 µg ml⁻¹ puromycin. Lentiviral particles were generated by transfecting HEK293T cells with pLVXpuro–SLC25A33–MycFlag using Lenti-X Packaging Single Shots (Takara) and the viral supernatant was collected after 48 h. WT and Yme1l^−/− MEFs expressing HA-MITO were generated by retroviral transduction and selected using 10 µg ml⁻¹ of blasticidin. Retrovirus was generated by transfecting HEK293T cells with pMXs–3XHA–EGFP–OMP25 (HA-MITO; a gift from David M. Sabatini, Addgene, catalogue no. 83356) using GeneJuice (Merck) and the viral supernatant was collected after 48 h. Primary MEFs were derived from WT and Tfam^+/− animals and immortalized by transfection with the SV40 large T antigen. Immortalized WT and Bax^−/−Bak^−/− MEFs were a kind gift from Richard Youle (NINDS).