Figure 5. ATP – but not ADP – primes and facilitates the release of BiP from IRE1 LD.

a, Experimental design for three-component in vitro UPR FRET assay^22,32 used to measure the inhibition of CFP-IRE1 – YFP-BiP complex by misfolded protein in the presence and absence of nucleotides. b-d, Bar graph showing FRET signal observed upon addition of ADP (b), ATP (c) or AMP-PNP (d). In each case, FRET inhibition due to dissociation of the complex was solely dependent on binding of misfolded protein C_H1. Data are mean ± s.d., n=9 independent experiments. e-h, FRET signal inhibition (%) as a function of the C_H1 concentration in the absence and presence of different nucleotides: ADP (e), ATP (f), APO (g) and AMP-PNP (h). Data were fitted to exponential curve (two phase association) and the IC₅₀ (equivalent to half-life) for each phase was deduced. The IC₅₀ measurement was then used to calculate the Ki³². The ATP bound BiP-IRE1 LD complex required 21-fold less misfolded protein to facilitate full dissociation of complex than the ADP bound state, suggesting a priming effect towards misfolded protein on ATP addition. Data are mean ± s.d., n = 9 independent experiments. Source Data are available online. The IC₅₀ and inhibition constant values for both low and high concentration phases in the absence and presence of different nucleotides are in Supplementary Table 7.