Figure 5.
The impact of mutations in the TMH and the AH of Ire1 on its functionality and cross-linking propensity.(A) The ER stress resistance of cells expressing the F544A variant of IRE13xHA-GFP containing the native cysteine 552 was determined. Stationary overnight cultures of the indicated strains were used to inoculate a fresh culture minimal medium to an OD600 of 0.2. After cultivation for 5 to 7 h at 30°C, the cells were diluted in 96-well plates to an OD600 = 0.01 with prewarmed minimal medium and cultivated in the presence of the indicated concentrations of DTT for 18 h at 30°C. The density of the resulting culture was determined using the OD600. Number of experiments including technical replicates: ΔIRE1 (n = 12 from two colonies); cysteine-less (n = 12 from two colonies); F544A/C552 (n = 12 from four colonies). (B) The impact of the F544A mutation on Ire1 degree of cross-linking via cysteine 552 was analyzed. The indicated strains were subjected to the cross-linking procedure as outlined in Fig. 3 E. Data for the C552 variant are identical to the data in Fig. 3 F. Number of experiments from DTT-stressed cells including technical duplicates: C552 (n = 11 from 6 colonies); F531R/C552 (n = 4 from two colonies). Number of experiments from TM-stressed cells including technical duplicates: C552 (n = 8 from four colonies); F544A/C552 (n = 4 from two colonies). (C) ER stress resistance of indicated cells including a single-cysteine variant (C552) of IRE13xHA-GFP with an AH-disrupting F531R mutation was determined. The cells were cultivated and treated as in A. The data for ΔIRE1 and cysteine-less IRE1 are identical to the data in A. Number of experiments including technical triplicates for F531R/C552 (n = 9 from three colonies). (D) The impact of the AH-disrupting F531R mutation on Ire1 cross-linking via cysteine 552 was determined. The indicated strains were subjected to the same cross-linking procedure used for Fig. 3 E. Data for the C552 single-cysteine variant are identical to the data in Fig. 3 F. The immunoblot for the C552 single-cysteine variant is identical to that in B. Number of experiments including technical duplicates for DTT-stressed F531R/C552 cells (n = 4 from two colonies) and TM-stressed F531R/C552 cells (n = 4 from two colonies). All data are represented as the mean ± SEM derived from at least three independent experiments. Significance was tested by an unpaired, two-tailed Student’s t test. *, P < 0.05. Data distribution was assumed to be normal, but this was not formally tested.

The impact of mutations in the TMH and the AH of Ire1 on its functionality and cross-linking propensity. (A) The ER stress resistance of cells expressing the F544A variant of IRE13xHA-GFP containing the native cysteine 552 was determined. Stationary overnight cultures of the indicated strains were used to inoculate a fresh culture minimal medium to an OD600 of 0.2. After cultivation for 5 to 7 h at 30°C, the cells were diluted in 96-well plates to an OD600 = 0.01 with prewarmed minimal medium and cultivated in the presence of the indicated concentrations of DTT for 18 h at 30°C. The density of the resulting culture was determined using the OD600. Number of experiments including technical replicates: ΔIRE1 (n = 12 from two colonies); cysteine-less (n = 12 from two colonies); F544A/C552 (n = 12 from four colonies). (B) The impact of the F544A mutation on Ire1 degree of cross-linking via cysteine 552 was analyzed. The indicated strains were subjected to the cross-linking procedure as outlined in Fig. 3 E. Data for the C552 variant are identical to the data in Fig. 3 F. Number of experiments from DTT-stressed cells including technical duplicates: C552 (n = 11 from 6 colonies); F531R/C552 (n = 4 from two colonies). Number of experiments from TM-stressed cells including technical duplicates: C552 (n = 8 from four colonies); F544A/C552 (n = 4 from two colonies). (C) ER stress resistance of indicated cells including a single-cysteine variant (C552) of IRE13xHA-GFP with an AH-disrupting F531R mutation was determined. The cells were cultivated and treated as in A. The data for ΔIRE1 and cysteine-less IRE1 are identical to the data in A. Number of experiments including technical triplicates for F531R/C552 (n = 9 from three colonies). (D) The impact of the AH-disrupting F531R mutation on Ire1 cross-linking via cysteine 552 was determined. The indicated strains were subjected to the same cross-linking procedure used for Fig. 3 E. Data for the C552 single-cysteine variant are identical to the data in Fig. 3 F. The immunoblot for the C552 single-cysteine variant is identical to that in B. Number of experiments including technical duplicates for DTT-stressed F531R/C552 cells (n = 4 from two colonies) and TM-stressed F531R/C552 cells (n = 4 from two colonies). All data are represented as the mean ± SEM derived from at least three independent experiments. Significance was tested by an unpaired, two-tailed Student’s t test. *, P < 0.05. Data distribution was assumed to be normal, but this was not formally tested.

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