Fig. 5. Sorting of DELE1 in the context of mitochondrial stress is instructed by cis elements.

a Schematic illustrating DELE1 and the deletions made in its MTS (mitochondria targeting sequence). b HeLa WT and ΔOMA1 cells were transiently transfected with the MTS variants illustrated in (a), treated with CCCP as indicated and processing of DELE1 was analyzed by immunoblotting. c HeLa ΔOMA1 cells stably expressing the MTS variants illustrated in (a) were treated with CCCP for 2 h and localization of DELE1 was analyzed by confocal microscopy. Scale bars, 10 μm. Nuclei (DAPI, blue), mitochondria (TRAP1, pink), DELE1 (HA, green). d 293T ∆DELE1 cells were transiently transfected with the DELE1 variants in (a), treated with OM for 4 h, and assayed for DELE1 cleavage by immunoblotting. e 293T ∆DELE1 cells were transfected with chimeric constructs in which either only the DELE1 presequence or the entire DELE1 MTS was replaced by the matrix-targeting signal of Su9 (see Supplementary Fig. 5b). Cells were treated with CCCP or OM for 4 h and assayed by immunoblotting. f Mitochondria isolated from 293T DELE1^HA ΔOMA1 cells treated with DMSO or OM for 4 h were processed and analyzed as in Fig. 2l. g DELE1-deficient 293T cells were transfected with full-length DELE1 or DELE1 lacking the identified hydrophobic sequence, treated with OM for 4 h and analyzed by immunoblotting.