Figure S2. Validation of a covalent,... | Rockefeller University Press

Figure S2.

Validation of a covalent, reversible cross-linking of Ire1 homodimers via disulfide bridges. (A) A cross-linking experiment using CuSO4 was performed with microsomes prepared from cells expressing a HA-tagged variant of Ire1 from endogenous locus (IRE13xHA-GFP) and a Flag-tagged variant (IRE13xFlag-GFP) from a CEN-based plasmid. A yeast culture in selective medium without leucine was inoculated to an OD600 of 0.2 from a stationary overnight culture and cultivated at 30°C until an OD600 of 0.7 was reached. The cells were either stressed with 2 mM DTT or left untreated and were further cultivated for 1 h. 80 OD600 equivalents from these cultures were harvested by centrifugation. Microsomal membranes were isolated by differential centrifugation. Microsomes prepared from cells expressing only one of the two tagged variants of Ire1 served as controls. Both constructs contained a single cysteine in the TMH region at the position 552 (C552). After incubation of the microsomes with 10 mM CuSO4 on ice for 5 min, the cross-linking reaction was stopped by the addition of NEM in a final concentration of 111 mM and EDTA in a final concentration of 50 mM. The microsomes were then solubilized using 2% Triton X-100 and subjected to an IP using anti-Flag beads. Both the input and IP samples were analyzed by immunoblotting using anti-Flag and anti-HA antibodies. (B) The reversibility of the cysteine-mediated cross-link was validated using the indicated F544C variant of Ire13xHA-GFP. The cross-link was induced by CuSO4 in microsomes prepared from cells stressed with TM as described in Fig. 3. The cross-link was reverted by treating the sample with 90 mM DTT and incubating at 70° and 95° as indicated. The monomeric and dimeric species of Ire1 are indicated by symbols.

Validation of a covalent, reversible cross-linking of Ire1 homodimers via disulfide bridges. (A) A cross-linking experiment using CuSO₄ was performed with microsomes prepared from cells expressing a HA-tagged variant of Ire1 from endogenous locus (IRE1_3xHA-GFP) and a Flag-tagged variant (IRE1_3xFlag-GFP) from a CEN-based plasmid. A yeast culture in selective medium without leucine was inoculated to an OD₆₀₀ of 0.2 from a stationary overnight culture and cultivated at 30°C until an OD₆₀₀ of 0.7 was reached. The cells were either stressed with 2 mM DTT or left untreated and were further cultivated for 1 h. 80 OD₆₀₀ equivalents from these cultures were harvested by centrifugation. Microsomal membranes were isolated by differential centrifugation. Microsomes prepared from cells expressing only one of the two tagged variants of Ire1 served as controls. Both constructs contained a single cysteine in the TMH region at the position 552 (C552). After incubation of the microsomes with 10 mM CuSO₄ on ice for 5 min, the cross-linking reaction was stopped by the addition of NEM in a final concentration of 111 mM and EDTA in a final concentration of 50 mM. The microsomes were then solubilized using 2% Triton X-100 and subjected to an IP using anti-Flag beads. Both the input and IP samples were analyzed by immunoblotting using anti-Flag and anti-HA antibodies. (B) The reversibility of the cysteine-mediated cross-link was validated using the indicated F544C variant of Ire13xHA-GFP. The cross-link was induced by CuSO₄ in microsomes prepared from cells stressed with TM as described in Fig. 3. The cross-link was reverted by treating the sample with 90 mM DTT and incubating at 70° and 95° as indicated. The monomeric and dimeric species of Ire1 are indicated by symbols.