Disrupting ER-luminal interfaces for dimerization (IF1) and oligomerization (IF2) of Ire1 impairs cellular ER-stress resistance and the formation of Ire1 clusters. (A) The resistance to DTT of cells expressing the IF1 (T226A/F247A) or IF2 (W426A) variants of IRE1 with all native cysteines was analyzed in rich medium. The indicated cells were cultivated and treated as in Fig. 5, A and C. Data for ΔIRE1 (gray squares) and IRE1 WT knock-in construct with all native cysteines (black circles) are plotted as a reference. WT (n = 6) and IF2 (n = 12) data were from two individual colonies. IF1 (n = 4) and ΔIRE1 (n = 6) data are from a single colony. (B) The percentage of cluster-containing cells was determined for the indicated strains cultivated in SCD medium and stressed with 2 mM DTT for 1 h. We re-used the raw microscopic data from Fig. S3 F for E540C (n = 15 fields of view/374 cells), T541C (n = 9/181), and F544C (n = 19/440), re-analyzed them as described in the Materials and methods, and pooled those with additional data for E540C (n = 7/281), T541 (n = 6/172), and F544C (n = 6/213). We also studied the clustering of E540C/IF2 (n = 7/98), T541C/IF2 (n = 6/150), and F544C/IF2 (n = 6/208). Microscopic images were analyzed using a customized CellProfiler pipeline. The percentage of cluster-containing cells with single cysteine variants of Ire1 is significantly different from any of the cells where the ER-luminal IF2 was disrupted by mutation (W426A). The data are represented as the mean ± SEM. Significance was tested using a Kolmogorov–Smirnov test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).