Fig. 5: FICD’s TPR domain is essential for AMPylation of ATP-bound BiP.

a Fluorescence and Coomassie gel-images of an in vitro AMPylation assay, utilising ATP(FAM) as the AMPylation co-substrate, in the presence of excess product trap (Trap(ox), to discourage BiP-AMP(FAM) deAMPylation¹⁰). dFICD, dimeric FICD; mFICD, monomeric FICD^L258D. Gels from a representative experiment are shown with the initial rates (mean ± 95% confidence interval (CI)) of BiP-AMPylation (in relative fluorescent units/s), normalised to the rate of mFICD-mediated BiP-AMPylation, from n = 4 independent experiments. Note, the lack of correlation between FICD (cis)auto-AMPylation and BiP substrate AMPylation. See Supplementary Fig. 6c. b Native-PAGE immunoblot analysis of the accumulation of AMPylated (B-form) BiP in CHO cells lacking endogenous FICD transfected with FICD variants, as indicated. Major, non-AMPylated BiP species (A, II and III) are noted. Right, quantification of AMPylated B-form BiP from n = 3 independent experiments (mean ± SD). c Histograms of the FACS signal of an XBP1::Turquoise UPR reporter in FICD^−/− CHO cells expressing the indicated FICD derivatives. Note the bimodal distribution of the fluorescent signal in FICD-transfected cells. Quantification of the fraction of cells that are stressed, as well as the median FACS signal of the low and high stressed cell populations are shown from n = 4 independent experiments (mean values ± SD). Bars and datapoints are (colour-)coded according to the histogram legend. Source data are provided as a Source Data file.