An additional kinase domain mutation displays a sustained HAC1 mRNA-splicing phenotype, and the inability to attenuate HAC1 splicing in Ire1-D828A is more sensitive to ER stress. (A) The D828A mutation partially rescues RNase-inactive Ire1-S840A/S841A/T844A. For each mutant, 0- and 2-h time points are shown. (B) Alignment of amino acid residues in the activation loop surrounding the DFG motif (shown in red). Serines at 840 and 841 and threonine at 844 (shown in blue) become phosphorylated upon UPR activation (Shamu and Walter, 1996; Lee et al., 2008). The conserved phenylalanine (F843) is shown in the purple box. (C) A mutation at the conserved phenylalanine in the activation loop (Ire1-F842A) phenocopies the Ire1-D828A mutant. (D) Ire1-D828A cells are more sensitive to ER stress. Ire1-WT or Ire1-D828A cells were grown to an OD_600nm of 0.25, and then cells were serially diluted by fivefold and spotted on plates containing different amounts of Tm (0, 0.1, and 0.4 µg/ml). Error bars represent standard deviations from three independent experiments. Dros., Drosophila melanogaster.