Figure 3.
Systematic cross-linking of cysteines in the TMH region of Ire1 reveals a specific configuration during ER stress.(A) Primary structure of ER-luminal AH of Ire1 and the short TMH. Almost every residue of the short TMH (shown in bold) was substituted individually by cysteine for the cysteine cross-linking strategy. (B) Helical wheel representation of Ire1’s TMH (Ire1540-552). (C) The level of the spliced HAC1 mRNA was determined from the indicated strains by RT-qPCR for either unstressed cells or cells stressed with 2 mM DTT for 1 h (for details, see E). The data are normalized to the level of the spliced HAC1 mRNA in stressed cells with a tagged, WT variant of Ire1. Number of independent experiments with technical duplicates for +DTT condition: WT: n = 5; cysteine-less: n = 6; E540C: n = 6; T541C: n = 3; G542C: n = 6; V543C: n = 9; F544C: n = 9; L545C: n = 3; L546C: n = 9; L547C: n = 3; F548C: n = 3; L549C: n = 6; I550C: n = 3; F551C: n = 5; C552: n = 3. Number of experiments with technical duplicates for the unstressed, -DTT condition: WT: n = 6; cysteine-less: n = 5; E540C: n = 6; T541C: n = 3; G542C: n = 6; V543C: n = 9; F544C: n = 9; L545C: n = 3; L546C: n = 9; L547C: n = 3; F548C: n = 3; L549C: n = 6; I550C: n = 3; F551C: n = 6; C552: n = 3. (D) The level of the spliced HAC1 mRNA was determined from the indicated strains by qPCR using stressed (inositol-depleted) and unstressed cells. The data are normalized to the level of the spliced HAC1 mRNA splicing caused by 2 mM DTT, as determined in C. Number of independent experiments with technical duplicates for the condition of inositol depletion: WT: n = 3; cysteine-less: n = 3; E540C: n = 3; G542C: n = 3; F544C: n = 3; L546C: n = 3; L547C: n = 3; L549C: n = 3. Number of independent experiments with technical duplicates for the unstressed condition: WT: n = 3; cysteine-less: n = 3; E540C: n = 3; G542C: n = 3; F544C: n = 3; L546C: n = 3; L547C: n = 3; L549C: n = 3. (E) A culture in SCD medium was inoculated with stationary cells to an OD600 of 0.2. After cultivation at 30°C to an OD600 of 0.7, Ire1 clustering was induced by either DTT (1 h, 2 mM, SCD) or TM (1 h, 1.5 µg/ml, SCD). 8 µl microsomes (1 mg/ml protein) from unstressed (no) and stressed cells was mixed with 2 µl of 50 mM CuSO4, and the sample was incubated on ice for 5 min to catalyze cysteine cross-linking. The reaction was stopped, and the sample was analyzed by SDS-PAGE and immunoblotting using anti-HA antibodies. (F) Quantification of cysteine cross-linking of the indicated variants of Ire1 in microsomes isolated cells stressed by DTT, TM, or inositol depletion. Cells were cultivated and treated as described in E. For inositol depletion, a culture was inoculated with exponentially growing cells to an OD600 of 0.5 and cultivated for 3 h at 30°C in inositol-free medium (a representative immunoblot after cross-linking is shown in Fig. S3 B). The percentage of cross-linked species was determined by densitometry. Data are represented as the mean ± SEM of at least three independent experiments. IB, immunoblot.

Systematic cross-linking of cysteines in the TMH region of Ire1 reveals a specific configuration during ER stress. (A) Primary structure of ER-luminal AH of Ire1 and the short TMH. Almost every residue of the short TMH (shown in bold) was substituted individually by cysteine for the cysteine cross-linking strategy. (B) Helical wheel representation of Ire1’s TMH (Ire1540-552). (C) The level of the spliced HAC1 mRNA was determined from the indicated strains by RT-qPCR for either unstressed cells or cells stressed with 2 mM DTT for 1 h (for details, see E). The data are normalized to the level of the spliced HAC1 mRNA in stressed cells with a tagged, WT variant of Ire1. Number of independent experiments with technical duplicates for +DTT condition: WT: n = 5; cysteine-less: n = 6; E540C: n = 6; T541C: n = 3; G542C: n = 6; V543C: n = 9; F544C: n = 9; L545C: n = 3; L546C: n = 9; L547C: n = 3; F548C: n = 3; L549C: n = 6; I550C: n = 3; F551C: n = 5; C552: n = 3. Number of experiments with technical duplicates for the unstressed, -DTT condition: WT: n = 6; cysteine-less: n = 5; E540C: n = 6; T541C: n = 3; G542C: n = 6; V543C: n = 9; F544C: n = 9; L545C: n = 3; L546C: n = 9; L547C: n = 3; F548C: n = 3; L549C: n = 6; I550C: n = 3; F551C: n = 6; C552: n = 3. (D) The level of the spliced HAC1 mRNA was determined from the indicated strains by qPCR using stressed (inositol-depleted) and unstressed cells. The data are normalized to the level of the spliced HAC1 mRNA splicing caused by 2 mM DTT, as determined in C. Number of independent experiments with technical duplicates for the condition of inositol depletion: WT: n = 3; cysteine-less: n = 3; E540C: n = 3; G542C: n = 3; F544C: n = 3; L546C: n = 3; L547C: n = 3; L549C: n = 3. Number of independent experiments with technical duplicates for the unstressed condition: WT: n = 3; cysteine-less: n = 3; E540C: n = 3; G542C: n = 3; F544C: n = 3; L546C: n = 3; L547C: n = 3; L549C: n = 3. (E) A culture in SCD medium was inoculated with stationary cells to an OD600 of 0.2. After cultivation at 30°C to an OD600 of 0.7, Ire1 clustering was induced by either DTT (1 h, 2 mM, SCD) or TM (1 h, 1.5 µg/ml, SCD). 8 µl microsomes (1 mg/ml protein) from unstressed (no) and stressed cells was mixed with 2 µl of 50 mM CuSO4, and the sample was incubated on ice for 5 min to catalyze cysteine cross-linking. The reaction was stopped, and the sample was analyzed by SDS-PAGE and immunoblotting using anti-HA antibodies. (F) Quantification of cysteine cross-linking of the indicated variants of Ire1 in microsomes isolated cells stressed by DTT, TM, or inositol depletion. Cells were cultivated and treated as described in E. For inositol depletion, a culture was inoculated with exponentially growing cells to an OD600 of 0.5 and cultivated for 3 h at 30°C in inositol-free medium (a representative immunoblot after cross-linking is shown in Fig. S3 B). The percentage of cross-linked species was determined by densitometry. Data are represented as the mean ± SEM of at least three independent experiments. IB, immunoblot.

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