Fig. 5. Substrate-facing PPP1R15A mutations interfere with eIF2α^P dephosphorylation.

a, Coomassie-stained SDS–PAGE PhosTag gels of eIF2α following dephosphorylation in vitro with PPP1R15A holophosphatases. Concentrations of the binary PPP1R15A–PP1A and G-actin are indicated, as are the genotypes of the PPP1R15A component. Shown is a representative of experiments reproduced at least three times. b, As in a but in the absence of G-actin. c, Display of the best fit of three independent experiments in panels a and b ±95% confidence intervals of the k_cat/K_m of the indicated PPP1R15A–PP1A pairs in reactions lacking (apo) and containing (holo) G-actin. d, Two-dimensional flow cytometry plots of untreated and thapsigargin (Tg)-treated CHOP::GFP transgenic CHO-K1 cells transfected with mCherry alone or full-length (wild type or mutant) mouse PPP1R15A fused to mCherry at its C termini (as in Fig. 2e). e, Box (25th and 75th percentile) and whiskers plots (cut-off at the 99th percentile) of the CHOP::GFP signal in untreated and thapsigargin-treated cells from the gray region in d (cell numbers shown in d). f, Individual data points and mean ± s.d. of the ISR attenuation factor of the indicated subset of PPP1R15A::mCherry fusions from replicate experiments as in d (P values for two-tailed t-test comparisons shown, n = 3 independent experiments). g, Quantitation of stress granules in sodium arsenite-treated cells expressing the proteins as in d (all data, mean ± s.d., P values for two-tailed t-test comparisons shown, n = 8 biological replicates for mCherry, 16 for WT, 10 for W575 and 11 for R584A). Results are representative of experiments reproduced three times. h, Time-dependent change in the BLI signal arising from phosphorylated eIF2α^P and nonphosphorylated eIF2α⁰ immobilized on a BLI probe and introduced in a 4 µM solution of a ternary complexes (TC, PP1A^D64A–PPP1R15A–G-actin), binary complexes of the same lacking G-actin (BC) or G-actin alone. At 900 s into the association phase the probe was shifted to a solution lacking proteins, to record the dissociation phase. i, BLI signal arising from immobilized eIF2α^P and ternary complexes of PP1A^D64A, G-actin and wild type or the indicated mutant PPP1R15A (as in h). Shown are representative traces of experiments reproduced at least three times. Source data for panels a–c and e–i are available online.

Source data