Figure 2. The cross-linking of Ire1 via... | Rockefeller University Press

Figure 2.

The cross-linking of Ire1 via single cysteines in microsomes requires CuSO4 and preformed clusters.(A) Cultivation of yeast cells for cysteine cross-linking. 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, the clustering of Ire1 was induced either by DTT (1 h, 2 mM, SCD) or TM (1 h, 1.5 µg/ml, SCD) as indicated. After harvesting, the cells were lysed and used to prepare microsomes. (B) Schematic representation of the cysteine cross-linking with CuSO4. Only microsomes from stressed cells contain clusters of Ire1 clusters that can cross-link via cysteines using CuSO4. (C) Cross-linking of a single-cysteine variant of Ire1 in microsomes. The indicated strains were cultivated in the presence and absence of ER stressors as described in A. 80 OD equivalents of cells were harvested, and microsomes were prepared. 8 µl microsomes (1 mg/ml protein) 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 by the addition of 2 µl 1 M NEM, 2 µl 0.5 M EDTA, and 4 µl membrane sample buffer. The resulting samples were analyzed by SDS-PAGE and immunoblotting using anti-HA antibodies.

The cross-linking of Ire1 via single cysteines in microsomes requires CuSO₄ and preformed clusters. (A) Cultivation of yeast cells for cysteine cross-linking. 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, the clustering of Ire1 was induced either by DTT (1 h, 2 mM, SCD) or TM (1 h, 1.5 µg/ml, SCD) as indicated. After harvesting, the cells were lysed and used to prepare microsomes. (B) Schematic representation of the cysteine cross-linking with CuSO₄. Only microsomes from stressed cells contain clusters of Ire1 clusters that can cross-link via cysteines using CuSO₄. (C) Cross-linking of a single-cysteine variant of Ire1 in microsomes. The indicated strains were cultivated in the presence and absence of ER stressors as described in A. 80 OD equivalents of cells were harvested, and microsomes were prepared. 8 µl microsomes (1 mg/ml protein) 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 by the addition of 2 µl 1 M NEM, 2 µl 0.5 M EDTA, and 4 µl membrane sample buffer. The resulting samples were analyzed by SDS-PAGE and immunoblotting using anti-HA antibodies.