Fig. 1. Ire1 mutants and their cellular expression. (A) The luminal domain of wild-type (WT) Ire1 and luminal domain mutants is shown schematically. Ire1 is inactivated by internal 10-a.a. deletions in the shaded region, and subregions I to V were designated based on this observation (Kimata et al., 2004) (subregion I corresponds to a.a. positions 32-111; subregion II, 112-242; subregion III, 243-272; subregion IV, 273-454; and subregion V, 455-524). The I and the V mutations are deletions of T32 to R91 and T463 to N523, respectively. The core mutation is the combination of the I and V mutations. The position of the S103P point mutation is also indicated. (B) Lysates were prepared from KMY1015 (ire1 null mutant) cells carrying pRS315-Ire1-HA (centromeric plasmid for expression of Ire1-HA) or mutant alleles under denaturing conditions, and subjected to anti-HA western blotting with 15 µg total protein per lane. The empty vector control (ire1) carried pRS315 (Sikorski and Hieter, 1989) instead of pRS315-Ire1-HA.