Figure S3.
Functionality of cysteine mutants and their cross-linking potential in lipid bilayer stress conditions. (A) The resistance to ER stress was investigated for the indicated yeast strains. Stationary overnight cultures of the indicated yeast strains were used to inoculate a fresh culture in full or minimal media to an OD600 of 0.2. After cultivation for 5–7 h at 30°C, the cells were diluted with fresh minimal media to an OD600 of 0.1. Cells were cultivated for 18 h at 30°C and stressed with DTT. The density of the resulting culture was determined using the OD620 or OD600. The error bars represent the mean ± SEM of at least two independent clones. Number of experiments to yield OD620: ΔIRE1 (n = 20; identical to Fig. 1 B); cysteine-less (n = 12; identical to Fig. 1 B); E540C to L546C (n = 6; triplicates from two individual colonies); and C552 (n = 12; triplicates from two individual colonies). Number of experiments to yield OD600: ΔIRE1 (n = 10; replicates from three individual colonies); cysteine-less (n = 22; replicates from four individual colonies); L547C, L549C, I550C, and F551C (n = 6; triplicates from two individual colonies); and F548C (n = 5; replicates from two individual colonies). (B) The indicated strains were cultivated and treated as described in Fig. 3, C and D, using conditions of proteotoxic (+DD) and lipid bilayer stress (-ino), respectively. The level of the cDNA obtained from the spliced and unspliced HAC1 mRNA was amplified and separated by a 2% agarose gel using a DNA ladder as size marker (M). (C) Protein levels of cells expressing different IRE13xHA-GFP variants. The lysates of exponentially growing cells were immunoblotted using anti-HA and anti-Pgk1 antibodies. (D) Cross-linking of single cysteine variants of Ire1 in microsomes derived from cells grown in lipid bilayer stress conditions. Exponentially growing cells in SCD complete media were washed and used to inoculate a fresh culture in SCD complete to an OD600 of 0.5. To induce lipid bilayer stress, the cells were washed and then cultivated in pre-warmed SCD complete without inositol medium for 3 h. 80 OD equivalents were harvested and used for microsomal membrane preparation. CuSO4-induced cross-link was performed by incubating 8 µl of microsomes with 2 µl of 50 mM CuSO4 for 5 min on ice. After stopping the reaction with NEM and EDTA, samples were subjected to SDS-PAGE with a subsequent immunoblotting with anti-HA antibody. Notably, all samples subjected to SDS-PAGE underwent a cross-linking procedure. Differences in specific and unspecific cross-linking may falsely suggest differences in loading. (E) Cells were cultivated to the early exponential phase in SCD and either treated with 2 mM DTT for 1 h or left untreated. Representative images (maximum projections of z-stacks) recorded by confocal microscopy. (F) The percentage of cluster-containing cells was determined for stressed (2 mM DTT, 1 h) and unstressed cells using a custom-made CellProfiler pipeline. The percentage of cluster-containing cells with the cysteine-less variant of Ire1 is not significantly different from any of the cells with single-cysteine variants. All data for the WT and cysteine-less variant are identical to the data from Fig. 1 C and plotted as a reference. For stressed cells: E540C (n = 12 fields of view/359 cells); T541C (n = 10/206); G542C (n = 6/124); V543C (n = 8/223); F544C (n = 19/439); L545C (n = 12/181); L546C (n = 14/399); L547C (n = 8/203); F548C (n = 10/279); L549C (n = 9/212); I550C (n = 8/232); F551C (n = 5/152); and C552 (n = 7/188). For unstressed cells: E540C (n = 4 fields of view/130 cells); T541C (n = 7/153); G542C (n = 7/188); V543C (n = 4/121); F544C (n = 4/108); L545C (n = 5/101); L546C (n = 3/111); L547C (n = 4/106); F548C (n = 5/143); L549C (n = 4/103); I550C (n = 5/165); F551C (n = 4/108); and C552 (n = 3/90). (G) The area of the detected clusters in the z-projection was determined and plotted. It was 49.9 px for the WT variant, 42.6 px for the cysteine-less variant, and ranged from a minimum of 37.2 px (G542C) to maximum of 48.9 px (V543C) for the single-cysteine variants. The integrated fluorescent intensity of detected clusters was 0.074 (arbitrary units) for the WT, 0.059 for the cysteine-less construct, and ranged from a minimum of 0.046 for the F548C variant to a maximum of 0.059 for the L547C variant. Significance was tested using a Kolmogorov–Smirnov test (*, P < 0.05). The segmented and analyzed number of clusters for each construct was as follows: WT: n = 395 (raw data in Fig. 1 D); cysteine-less: n = 211 (raw data in Fig. 1 D); E540C: n = 215; T541C: n = 158; G542C: n = 95; V543C: n = 101; F544C: n = 224; L545C: n = 131; L546C: n = 191; L547: n = 121; F548C: n = 127; L549C: n = 168; I550C: n = 121; F551C: n = 113; and C552: n = 75. px, pixels.

Functionality of cysteine mutants and their cross-linking potential in lipid bilayer stress conditions. (A) The resistance to ER stress was investigated for the indicated yeast strains. Stationary overnight cultures of the indicated yeast strains were used to inoculate a fresh culture in full or minimal media to an OD600 of 0.2. After cultivation for 5–7 h at 30°C, the cells were diluted with fresh minimal media to an OD600 of 0.1. Cells were cultivated for 18 h at 30°C and stressed with DTT. The density of the resulting culture was determined using the OD620 or OD600. The error bars represent the mean ± SEM of at least two independent clones. Number of experiments to yield OD620: ΔIRE1 (n = 20; identical to Fig. 1 B); cysteine-less (n = 12; identical to Fig. 1 B); E540C to L546C (n = 6; triplicates from two individual colonies); and C552 (n = 12; triplicates from two individual colonies). Number of experiments to yield OD600: ΔIRE1 (n = 10; replicates from three individual colonies); cysteine-less (n = 22; replicates from four individual colonies); L547C, L549C, I550C, and F551C (n = 6; triplicates from two individual colonies); and F548C (n = 5; replicates from two individual colonies). (B) The indicated strains were cultivated and treated as described in Fig. 3, C and D, using conditions of proteotoxic (+DD) and lipid bilayer stress (-ino), respectively. The level of the cDNA obtained from the spliced and unspliced HAC1 mRNA was amplified and separated by a 2% agarose gel using a DNA ladder as size marker (M). (C) Protein levels of cells expressing different IRE13xHA-GFP variants. The lysates of exponentially growing cells were immunoblotted using anti-HA and anti-Pgk1 antibodies. (D) Cross-linking of single cysteine variants of Ire1 in microsomes derived from cells grown in lipid bilayer stress conditions. Exponentially growing cells in SCD complete media were washed and used to inoculate a fresh culture in SCD complete to an OD600 of 0.5. To induce lipid bilayer stress, the cells were washed and then cultivated in pre-warmed SCD complete without inositol medium for 3 h. 80 OD equivalents were harvested and used for microsomal membrane preparation. CuSO4-induced cross-link was performed by incubating 8 µl of microsomes with 2 µl of 50 mM CuSO4 for 5 min on ice. After stopping the reaction with NEM and EDTA, samples were subjected to SDS-PAGE with a subsequent immunoblotting with anti-HA antibody. Notably, all samples subjected to SDS-PAGE underwent a cross-linking procedure. Differences in specific and unspecific cross-linking may falsely suggest differences in loading. (E) Cells were cultivated to the early exponential phase in SCD and either treated with 2 mM DTT for 1 h or left untreated. Representative images (maximum projections of z-stacks) recorded by confocal microscopy. (F) The percentage of cluster-containing cells was determined for stressed (2 mM DTT, 1 h) and unstressed cells using a custom-made CellProfiler pipeline. The percentage of cluster-containing cells with the cysteine-less variant of Ire1 is not significantly different from any of the cells with single-cysteine variants. All data for the WT and cysteine-less variant are identical to the data from Fig. 1 C and plotted as a reference. For stressed cells: E540C (n = 12 fields of view/359 cells); T541C (n = 10/206); G542C (n = 6/124); V543C (n = 8/223); F544C (n = 19/439); L545C (n = 12/181); L546C (n = 14/399); L547C (n = 8/203); F548C (n = 10/279); L549C (n = 9/212); I550C (n = 8/232); F551C (n = 5/152); and C552 (n = 7/188). For unstressed cells: E540C (n = 4 fields of view/130 cells); T541C (n = 7/153); G542C (n = 7/188); V543C (n = 4/121); F544C (n = 4/108); L545C (n = 5/101); L546C (n = 3/111); L547C (n = 4/106); F548C (n = 5/143); L549C (n = 4/103); I550C (n = 5/165); F551C (n = 4/108); and C552 (n = 3/90). (G) The area of the detected clusters in the z-projection was determined and plotted. It was 49.9 px for the WT variant, 42.6 px for the cysteine-less variant, and ranged from a minimum of 37.2 px (G542C) to maximum of 48.9 px (V543C) for the single-cysteine variants. The integrated fluorescent intensity of detected clusters was 0.074 (arbitrary units) for the WT, 0.059 for the cysteine-less construct, and ranged from a minimum of 0.046 for the F548C variant to a maximum of 0.059 for the L547C variant. Significance was tested using a Kolmogorov–Smirnov test (*, P < 0.05). The segmented and analyzed number of clusters for each construct was as follows: WT: n = 395 (raw data in Fig. 1 D); cysteine-less: n = 211 (raw data in Fig. 1 D); E540C: n = 215; T541C: n = 158; G542C: n = 95; V543C: n = 101; F544C: n = 224; L545C: n = 131; L546C: n = 191; L547: n = 121; F548C: n = 127; L549C: n = 168; I550C: n = 121; F551C: n = 113; and C552: n = 75. px, pixels.

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