Figure 2

Dimerisation of human IRE1α enhances ribonuclease activity. (A) Native polyacrylamide gel showing that GST-G547 IRE1α (77 kDa) migrates as dimers whereas G547 IRE1α (49.6 kDa) migrates as monomers. (B) Intact protein mass spectra of lambda phosphatase treated GST-G547 IRE1α (grey) and after incubation with Mg/ATP (black). (C) 5’FAM, 3’BHQ-labeled XBP-1 splice site mimic RNA was incubated with increasing concentrations of phosphorylated GST-G547 IRE1α (filled triangles) or dephosphorylated GST-G547 IRE1α (open triangles) for 30 minutes at 30°C. Error bars represent the standard error of the mean of 3 independent experiments. (D) Comparison of extent of cleavage of the labelled XBP1 splice site mimic RNA when incubated with 100 nM of the indicated IRE1α construct, with or without pre-incubation with ATP, for 1 hour at 30°C, error bars show the S.E.M of three independent experiments. (E) Immunoblot of H499 IRE1α after treatment with 250 μM of the crosslinker disuccinimidyl suberate (DSS). Increasing concentrations of either dephosphorylated or autophosphorylated H499 IRE1α were incubated with DSS for 45 minutes before separation by SDS-PAGE and transfer to nitrocellulose membrane. Monomer* indicates the reduced mobility of autophosphorylated H499 IRE1α. (F) Comparison of phosphorylation status of IRE1α immunoprecipitated from human cells and purified IRE1α constructs. Phosphorylation sites of IRE1α were determined by tryptic digest mass spectrometry in protein immunoprecipitated from human cells (Cell IP), autophosphorylated G547 IRE1α (G547) and autophosphorylated H499 IRE1α (H499). Where the site could not be uniquely identified a ‘/’ is used to indicate the possible residues. *T973 discovered in immunoprecipitated IRE1α when chymotrypsin was used for digestion. Only trypsin was used to digest H499 and G547.