Figure 4.

A and B, Autophosphorylation activity of the C-terminal domain of AtIre1-2. A, Domain structure of Ire1 (top) and of the GST fusions showing the location of the K to A mutation. B, The fusion protein of GST with the C-terminal domain of AtIre1-2 was expressed in E. coli and affinity purified on glutathione-Sepharose. Purified protein incubated with γ-³²P ATP was subjected to SDS-PAGE. The gel was Coomassie stained (CBB) and exposed to x-ray film (autoradiogram). The same fraction was used for an immunoblot analysis with anti-GST antibody (anti-GST). In A and B, lane 1 has the wild-type GST-AtIre1-2 fusion and lane 2 has the mutant GST-AtIre1-2 fusion. There is no ³²P labeling of the fusion polypeptide of the mutant (lane 2 in the autoradiogram).