Figure 6.
Cross-linking occurs within and across dimers of Ire1.(A) Indicated variants of an IRE1 knock-in construct (Halbleib et al., 2017) were cultivated and stressed with 2 mM DTT for 1 h as described in Fig. 1 D. A refined, automated counting of Ire1-containing clusters was performed as described in the Online supplemental material. Data from Fig. 1 D were reanalyzed and pooled with new data to yield the following: WT (n = 19 fields of view/441 cells); T226A/F247A (IF1; n = 6/154); W426A (IF2; n = 10/329). All data are represented as the mean ± SEM. Significance was tested by an unpaired, two-tailed Student’s t test. **, P < 0.01; ***, P < 0.001. (B) Clustering in DTT-stressed cells was studied by confocal microscopy of the indicated single-cysteine variants with either an intact or disrupted IF2 (orange). Representative images from at least six independent fields of view are shown. For a quantitative analysis, see Fig. S6 B. (C) ER stress resistance of indicated cells was studied in rich medium containing different concentrations of DTT. Data from independent experiments for E540C/IF2 (n = 4), T541C/IF2 (n = 5), and F544C/IF2 (n = 6; IF2 indicates the W426A mutation) are plotted in orange. Reference datasets for ΔIRE1 (n = 6), Ire13xHA-GFP WT (n = 6 from two individual colonies), and the IF2 variant (n = 12 from two individual colonies) are plotted in gray, black, and blue, respectively. All data are represented as the mean ± SEM. (D) The level of the spliced HAC1 mRNA was determined from the indicated strains by RT-qPCR after treating the cells with 2 mM DTT for the indicated times. The data are normalized to the level of the spliced HAC1 mRNA in cysteine-less Ire1 after 1 h of treatment and derive from three independent colonies with experimental duplicates (n = 6). (E) The level of the spliced HAC1 mRNA was determined for the indicated strains cultivated under inositol-depletion conditions. The data are normalized to the level of the spliced HAC1 mRNA splicing caused by 2 mM DTT, as determined in D. The data derive from three independent colonies with experimental duplicates (n = 6). (F) The impact of the IF2-disrupting W426A mutation on cross-linking via the indicated single cysteines was determined. Single- and double-mutant strains were subjected to the cross-linking procedure as in Fig. 3 E. Data for the single mutant variants are replotted from Fig. 3 F. All data are represented as the mean ± SEM. Number of experiments including technical duplicates for DTT-stressed cells: E540C (n = 8 from four colonies), T541C (n = 8 from four colonies), F544C (n = 14 from seven colonies; identical to data in Fig. 3 F), E540C/IF2 (n = 4 from two colonies), T541C/IF2 (n = 4 from two colonies), and for F544C/IF2 (n = 6 from three colonies). Number of experiments including technical duplicates for TM-stressed cells: E540C (n = 8 from four colonies), T541C (n = 8 from four colonies), F544C (n = 10 from five colonies; identical to data in Fig. 3 F), E540C/IF2 (n = 4 from two colonies), T541C/IF2 (n = 4 from two colonies), and F544C/IF2 (n = 6 from three colonies). 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. (G) Hypothetical model for Ire1’s exquisite sensitivity. The membrane-based oligomerization of Ire1 (blue) and unrelated single-pass membrane proteins (black) leads to the coalescence of deformed membrane regions (green). In the case of Ire1, a larger portion of the deformed membrane region can be shared upon dimerization due to the ellipsoid membrane “footprint” and an association via the longer edge of deformation (parallel to the major axis of the ellipse). According to this model, this maximizes the sensitivity of Ire1 to aberrant membrane properties when compared with unrelated single-pass membrane proteins, which can merge only a relatively small portion of their circular membrane “footprint” upon dimerization.

Cross-linking occurs within and across dimers of Ire1. (A) Indicated variants of an IRE1 knock-in construct (Halbleib et al., 2017) were cultivated and stressed with 2 mM DTT for 1 h as described in Fig. 1 D. A refined, automated counting of Ire1-containing clusters was performed as described in the Online supplemental material. Data from Fig. 1 D were reanalyzed and pooled with new data to yield the following: WT (n = 19 fields of view/441 cells); T226A/F247A (IF1; n = 6/154); W426A (IF2; n = 10/329). All data are represented as the mean ± SEM. Significance was tested by an unpaired, two-tailed Student’s t test. **, P < 0.01; ***, P < 0.001. (B) Clustering in DTT-stressed cells was studied by confocal microscopy of the indicated single-cysteine variants with either an intact or disrupted IF2 (orange). Representative images from at least six independent fields of view are shown. For a quantitative analysis, see Fig. S6 B. (C) ER stress resistance of indicated cells was studied in rich medium containing different concentrations of DTT. Data from independent experiments for E540C/IF2 (n = 4), T541C/IF2 (n = 5), and F544C/IF2 (n = 6; IF2 indicates the W426A mutation) are plotted in orange. Reference datasets for ΔIRE1 (n = 6), Ire13xHA-GFP WT (n = 6 from two individual colonies), and the IF2 variant (n = 12 from two individual colonies) are plotted in gray, black, and blue, respectively. All data are represented as the mean ± SEM. (D) The level of the spliced HAC1 mRNA was determined from the indicated strains by RT-qPCR after treating the cells with 2 mM DTT for the indicated times. The data are normalized to the level of the spliced HAC1 mRNA in cysteine-less Ire1 after 1 h of treatment and derive from three independent colonies with experimental duplicates (n = 6). (E) The level of the spliced HAC1 mRNA was determined for the indicated strains cultivated under inositol-depletion conditions. The data are normalized to the level of the spliced HAC1 mRNA splicing caused by 2 mM DTT, as determined in D. The data derive from three independent colonies with experimental duplicates (n = 6). (F) The impact of the IF2-disrupting W426A mutation on cross-linking via the indicated single cysteines was determined. Single- and double-mutant strains were subjected to the cross-linking procedure as in Fig. 3 E. Data for the single mutant variants are replotted from Fig. 3 F. All data are represented as the mean ± SEM. Number of experiments including technical duplicates for DTT-stressed cells: E540C (n = 8 from four colonies), T541C (n = 8 from four colonies), F544C (n = 14 from seven colonies; identical to data in Fig. 3 F), E540C/IF2 (n = 4 from two colonies), T541C/IF2 (n = 4 from two colonies), and for F544C/IF2 (n = 6 from three colonies). Number of experiments including technical duplicates for TM-stressed cells: E540C (n = 8 from four colonies), T541C (n = 8 from four colonies), F544C (n = 10 from five colonies; identical to data in Fig. 3 F), E540C/IF2 (n = 4 from two colonies), T541C/IF2 (n = 4 from two colonies), and F544C/IF2 (n = 6 from three colonies). 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. (G) Hypothetical model for Ire1’s exquisite sensitivity. The membrane-based oligomerization of Ire1 (blue) and unrelated single-pass membrane proteins (black) leads to the coalescence of deformed membrane regions (green). In the case of Ire1, a larger portion of the deformed membrane region can be shared upon dimerization due to the ellipsoid membrane “footprint” and an association via the longer edge of deformation (parallel to the major axis of the ellipse). According to this model, this maximizes the sensitivity of Ire1 to aberrant membrane properties when compared with unrelated single-pass membrane proteins, which can merge only a relatively small portion of their circular membrane “footprint” upon dimerization.

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