Extended Data Fig. 7: Processing of full-length DELE1 into S-DELE1 is mediated by catalytically active OMA1.
From: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol
a, Data from Fig. 1a, with statistical significance of mutation enrichment assessed and visualized in the same way (two-sided Fisher’s exact test, Padj < 0.05). Known mitochondrial proteases44,45 are highlighted in red. XRCC6BP1 is also known as ATP23. b, Wild-type HAP1 cells and clonal OMA1 knockout cells were transfected with DELE1-HA, treated with CCCP and assayed by immunoblotting. c, HeLa cells of the stated genotypes were transfected with DELE1-HA and pre-treated with the indicated protease inhibitors for 5 h, followed by the addition of CCCP for 2 h. The fates of DELE1, OMA1 and OPA1 were monitored by immunoblotting. DCI, 3,4-dichloroisocoumarin; pepstat. A, pepstatin A; o-phen, o-phenanthroline. d, Quantification of c. The fraction of S-DELE1 (versus L-DELE1) is indicated. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons correction and represents the significance relative to the wild type plus CCCP condition (mean ± s.d. of n = 3 independent experiments). e, HeLa cells transiently exposed to an sgRNA directed against OMA1 were co-transfected with the indicated cDNAs and cleavage of DELE1–Flag–mNeon (FmN) after 2 h of CCCP treatment was monitored by immunoblotting (one representative experiment shown of two independent experiments). f, Workflow of the in vitro assay for DELE1 fate upon mitochondrial depolarization using mitochondria purified from wild-type or OMA1-deficient 293T cells that were transiently transfected with DELE1-HA. g, h, Purity analysis of isolated mitochondria using electron microscopy (g) and immunoblotting (h) (one representative experiment shown of two independent experiments for each). For immunoblotting, around 1% of the mitochondrial and 0.2% of the cytosolic fraction was used. The electron micrographs show a high proportion and purity of mitochondria, only slight contaminations of membrane fragments or other cell organelles and the complete absence of intact cells within the mitochondrial suspensions. Scale bars, 5 μm (left) or 1 μm (right). i–k, Isolated mitochondria from g, h were treated in vitro with FCCP and analysed for DELE1 processing. i, Mitochondria build up a membrane potential that remains stable for one hour (demonstrated by constant low fluorescence). FCCP (500 nM) was used to induce dissipation of the mitochondrial membrane potential, yielding an increase in fluorescence. j, After one hour of FCCP treatment, the mitochondria and supernatant were separated by centrifugation and the protein levels in both compartments were analysed by immunoblotting (top, long exposure; bottom, short exposure for DELE1–HA) k, The amount of S-DELE1 in the supernatants was quantified and compared to the wild type plus DMSO condition using two-way ANOVA with Tukey’s multiple comparisons correction. In i, k, data are mean ± s.d. of n = 5 independent experiments; j shows one representative experiment of n = 5 independent experiments, of which the purity analysis shown in g, h was performed for two independent experiments.
