Figure S1. Protein levels of... | Rockefeller University Press

Figure S1.

$Protein levels of cysteine-less Ire1 and characterization of its membrane association. (A) Protein levels of cells expressing either IRE13xHA-GFP WT or the cysteine-less (cys-less) variant. The isogenic WT strain BY4741 that does not express a HA-tagged variant of IRE1 was used as a negative control (NC). Stationary overnight cultures were used to inoculate a fresh culture in SCD complete to an OD600 of 0.2 and cultivated until an OD600 of 1 was reached. 0.1 OD equivalents of cell lysates were immunoblotted using anti-HA and anti-Pgk1 antibodies. (B) Subcellular fractionation of exponentially growing cells expressing cysteine-less IRE13xHA-GFP by differential centrifugation at 5,000 ×g and 100,000 ×g. Stationary overnight cultures were used to inoculate a fresh culture in SCD complete to an OD600 of 0.2 and cultivated until an OD600 of 1 was reached. 80 OD600 equivalents were harvested, lysed, and served as total input (T) for microsomal membrane preparation. The total lysate (T), and the individual supernatant (S) and pellet (P) fractions from centrifugation steps at 5,000 ×g (5k) and 100,000 ×g (100k) were analyzed separately by immunoblotting using anti-HA, anti-Pgk1, and anti-Dpm1 antibodies. 0.4 OD equivalents were loaded per lane. (C) Extraction assay of microsomes. Carbonate and urea extraction validate proper membrane integration of cysteine-less IRE13xHA-GFP (cys-less). Samples of each step corresponding to 0.2 OD equivalents were analyzed by immunoblotting using anti-HA, anti-Pgk1, and anti-Dpm1 antibodies. (D) The indicated strains from a stationary culture were used to inoculate fresh culture in SCD to an OD600 of 0.2. After cultivation at 30°C to an OD600 of 0.7, cells were either left untreated or stressed with DTT (1 h, 2 mM, SCD). The level of the cDNA obtained from the spliced and unspliced HAC1 mRNA was amplified and separated by a 2% agarose gel. (E) PDI1 mRNA levels in acutely stressed cells normalized to the fold change of unstressed cells expressing IRE13xHA-GFP WT. Exponentially growing cells of the indicated strains were used to inoculate fresh YPD media to an OD600 of 0.2, cultivated in YPD, and acutely stressed with either 4 mM DTT (left) or 1.0 µg/ml TM (right) for 1 h. The relative level of PDI1 in these cells was analyzed by RT-qPCR and quantitated using the comparative ΔΔCT method using normalization to ACT1 levels. The data were normalized to the PDI1 level in unstressed cells carrying the IRE13xHA-GFP WT construct. All error bars in this figure represent the mean ± SEM. Number of independent experiments: (left) -DTT: WT (n = 6); -DTT: cysteine-less (n = 6); +DTT: WT (n = 5); cysteine-less (n = 6); (right) -TM: WT (n = 5); cysteine-less (n = 6); +TM: WT (n = 5); cysteine-less (n = 3). Significance was tested by an unpaired, two-tailed Student’s t test. *, P < 0.05; **, P < 0.01. Data distribution was assumed to be normal, but this was not formally tested. (F) Cells were cultivated from OD600 of 0.2 to OD600 of 0.7 in SCD medium and then either left untreated or stressed with 2 mM DTT for 1 h. Live cells were mounted on agar slides, and z-stacks were recorded using confocal microscopy. Images show the center plane of indicated channels. DIC, differential interference contrast; rel., relative; TX-100, Triton X-100.$

Protein levels of cysteine-less Ire1 and characterization of its membrane association. (A) Protein levels of cells expressing either IRE1_3xHA-GFP WT or the cysteine-less (cys-less) variant. The isogenic WT strain BY4741 that does not express a HA-tagged variant of IRE1 was used as a negative control (NC). Stationary overnight cultures were used to inoculate a fresh culture in SCD complete to an OD₆₀₀ of 0.2 and cultivated until an OD₆₀₀ of 1 was reached. 0.1 OD equivalents of cell lysates were immunoblotted using anti-HA and anti-Pgk1 antibodies. (B) Subcellular fractionation of exponentially growing cells expressing cysteine-less IRE1_3xHA-GFP by differential centrifugation at 5,000 ×g and 100,000 ×g. Stationary overnight cultures were used to inoculate a fresh culture in SCD complete to an OD₆₀₀ of 0.2 and cultivated until an OD₆₀₀ of 1 was reached. 80 OD₆₀₀ equivalents were harvested, lysed, and served as total input (T) for microsomal membrane preparation. The total lysate (T), and the individual supernatant (S) and pellet (P) fractions from centrifugation steps at 5,000 ×g (5k) and 100,000 ×g (100k) were analyzed separately by immunoblotting using anti-HA, anti-Pgk1, and anti-Dpm1 antibodies. 0.4 OD equivalents were loaded per lane. (C) Extraction assay of microsomes. Carbonate and urea extraction validate proper membrane integration of cysteine-less IRE1_3xHA-GFP (cys-less). Samples of each step corresponding to 0.2 OD equivalents were analyzed by immunoblotting using anti-HA, anti-Pgk1, and anti-Dpm1 antibodies. (D) The indicated strains from a stationary culture were used to inoculate fresh culture in SCD to an OD₆₀₀ of 0.2. After cultivation at 30°C to an OD₆₀₀ of 0.7, cells were either left untreated or stressed with DTT (1 h, 2 mM, SCD). The level of the cDNA obtained from the spliced and unspliced HAC1 mRNA was amplified and separated by a 2% agarose gel. (E) PDI1 mRNA levels in acutely stressed cells normalized to the fold change of unstressed cells expressing IRE1_3xHA-GFP WT. Exponentially growing cells of the indicated strains were used to inoculate fresh YPD media to an OD₆₀₀ of 0.2, cultivated in YPD, and acutely stressed with either 4 mM DTT (left) or 1.0 µg/ml TM (right) for 1 h. The relative level of PDI1 in these cells was analyzed by RT-qPCR and quantitated using the comparative ΔΔCT method using normalization to ACT1 levels. The data were normalized to the PDI1 level in unstressed cells carrying the IRE1_3xHA-GFP WT construct. All error bars in this figure represent the mean ± SEM. Number of independent experiments: (left) -DTT: WT (n = 6); -DTT: cysteine-less (n = 6); +DTT: WT (n = 5); cysteine-less (n = 6); (right) -TM: WT (n = 5); cysteine-less (n = 6); +TM: WT (n = 5); cysteine-less (n = 3). Significance was tested by an unpaired, two-tailed Student’s t test. *, P < 0.05; **, P < 0.01. Data distribution was assumed to be normal, but this was not formally tested. (F) Cells were cultivated from OD₆₀₀ of 0.2 to OD₆₀₀ of 0.7 in SCD medium and then either left untreated or stressed with 2 mM DTT for 1 h. Live cells were mounted on agar slides, and z-stacks were recorded using confocal microscopy. Images show the center plane of indicated channels. DIC, differential interference contrast; rel., relative; TX-100, Triton X-100.