Cysteine-less Ire1 expressed from its endogenous locus is functional. (A) Schematic representations of the IRE1_3xHA-GFP construct indicating the position of cysteine residues and topology. All 12 cysteines of Ire1 and C48^yeGFP of yeGFP were substituted to serine to generate a cysteine-less variant. C70^yeGFP remains in the final construct. Two cysteines in the signal sequence of Ire1 are removed upon ER translocation. (B) Resistance of the indicated strains to prolonged ER stress. Stationary overnight cultures of the indicated strains were used to inoculate a fresh culture in full or minimal media to an OD₆₀₀ of 0.2. After cultivation for 5–7 h at 30°C, the cells were diluted with prewarmed full or minimal media to an OD₆₀₀ of 0.01. Cells were cultivated for 18 h at 30°C in the indicated media and stressed with DTT. The density of the resulting culture was determined using the OD₆₂₀ or OD₆₀₀. Number of experiments in SCD (left): ΔIRE1, WT (n = 20), and cysteine-less (n = 12, each from four individual colonies). Number of experiments in YPD (right): ΔIRE1 (n = 14 from three colonies), cysteine-less (n = 12 from four colonies), and WT (n = 9 from three colonies). (C) The relative level of the spliced HAC1 mRNA was determined by RT-qPCR in unstressed and acutely stressed cells. Exponentially growing cells of the indicated strains were used to inoculate a fresh culture in YPD medium to an OD₆₀₀ of 0.2. After cultivation to an OD₆₀₀ of 0.7, the cells were stressed for 1 h with either 4 mM DTT (left) or 1.0 µg/ml TM (right). The data were normalized to the level of the spliced HAC1 mRNA in DTT-stressed cells with the IRE1_3xHA-GFP WT construct. Number of experiments (left): WT -DTT: n = 4; WT +DTT: n = 6; cysteine-less -DTT: n = 6; cysteine-less +DTT: n = 5. Number of experiments (right): WT -TM: n = 4; WT +TM: n = 5; cysteine-less -TM: n = 6; cysteine-less +TM: n = 4. (D) Cells were cultivated from OD₆₀₀ of 0.2 to OD₆₀₀ of 0.7 in SCD medium and then either left untreated or stressed with 2 mM DTT for 1 h. Live cells were mounted on agar slides, and z-stacks were recorded using confocal microscopy. Cells and clusters of Ire1 were automatically detected and quantified. All data are represented as the mean ± SEM of three independent experiments. WT -DTT: (n = 6 fields of view/172 cells); WT +DTT: (n = 13/302); cysteine-less -DTT: (n = 6/209); cysteine-less +DTT: (n = 12/326). Significance was tested by an unpaired, two-tailed Student’s t test (data distribution was assumed to be normal, but this was not formally tested), except for C, which was analyzed using a Kolmogorov–Smirnov test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.