Fig. 6. Analysis of additional amino acid substitutions at and around S103 in Ire1. (A) Amino acid sequences of the mutant alleles used in this assay. The mutated residues are in bold. Note that all alleles carry the core mutation. (B,C) KMY1015 (ire1 null mutant) cells carrying both the UPRE-lacZ reporter pCZY1 and a mutant allele of pRS315-Ire1-HA were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 4 hours, and subjected to -galactosidase assay. Each value is the mean ± s.d. for three independent clones and was normalized to that of the Tun+ wild-type Ire1-HA control, which was set at 100. (D) KMY1015 (ire1 null mutant) cells carrying the indicated mutant alleles of pRS315-Ire1-HA were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and total RNA was analyzed by northern blotting using the HAC1 probe. See Fig. 3 legend for further details.