Extended Data Fig. 9: Physical interaction between DELE1 and HRI.
From: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol
a, 293T cells were co-transfected with StrepTagII-HA-HRI and the indicated Flag-mNeon-tagged cDNAs before stimulation with CCCP as indicated. Cells were then lysed and StrepTagII-containing protein complexes were immobilized on Strep-Tactin beads and analysed by immunoblotting along with the input lysate. DELE1FL, full-length DELE1. b, A C-terminal in-frame fusion of a triple Flag tag was introduced into the endogenous EIF2AK1 (HRI) locus of 293T cells using CRISPR. c, Quantification of Fig. 3b. The bar chart shows the relative abundance of the slowly migrating species of HRI (phosphorylated HRI) compared to the faster migrating band. Data were normalized to the empty vector (black) and significance (compared to full-length DELE1 untreated, grey) was assessed using one-way ANOVA with Dunnett’s multiple comparisons correction (mean ± s.d. of n = 3 independent experiments). d, 293T cells were co-transfected with HRI-Flag and DELE1-HA and exposed to CCCP for the denoted amounts of time before immunoblotting. The shift in electrophoretic mobility of HRI upon activation is reversed by treatment of the lysates with alkaline phosphatase. e, 293T cells of the indicated genotypes were co-transfected with HRI-Flag and the specified HA-tagged constructs. After CCCP treatment, lysates were immobilized on anti-Flag beads and analysed by immunoblotting along with the input lysate (one representative experiment shown of four independent experiments). f, Quantification of e (mean ± s.d. of n = 4 independent experiments; two-way ANOVA with Tukey’s multiple comparisons correction). g, HeLa cells were transiently transfected with the indicated constructs, treated with CCCP or DMSO and analysed for subcellular localization of the transfected cDNAs by HA staining and confocal microscopy with cellular structures labelled as indicated. h, DELE1 mutants were transiently transfected into HeLa and 293T cells before exposure to CCCP for 2 h. The processing of DELE1–HA was monitored by immunoblotting. i, HeLa cells were transfected as indicated and analysed for subcellular localization as in g. j, 293T cells were co-transfected with HRI-Flag and the indicated StrepTagII-HA cDNAs. Cells were then lysed and StrepTagII-containing protein complexes were immobilized on Strep-Tactin beads and analysed by immunoblotting along with the input lysate. k, l, Clonal DELE1-knockout HAP1 cells were transiently transfected with the specified cDNAs together with mCherry and induction of CHOPNeon was monitored by flow cytometry as in Fig. 2a. Per genotype, data were normalized to the empty vector and statistical significance (compared to DELE1(∆MTS(N101))) was assessed using one-way ANOVA with Tukey’s multiple comparisons correction (mean ± s.d. of n = 4 independent experiments). m, Wild-type 293T cells were transiently transfected with HRI-Flag and the indicated HA-tagged cDNAs and processed as in e (one representative experiment shown of four independent experiments). n, DELE1-deficient HAP1 CHOPNeon cells were transiently transfected as indicated and analysed by flow cytometry 32 h after transfection. Per genotype, data were normalized to the empty vector (black) and statistical significance (compared to DELE(∆MTS)) was assessed using one-way ANOVA with Tukey’s multiple comparisons correction (mean ± s.d. of n = 3 independent experiments). o, Cell lysates from Fig. 3c were analysed by Coomassie staining before (lysate) and after exposure to GFP-TRAP beads (IP (GFP)). Scale bars, 10 μm (g, i).
