ER stress induces ER membrane expansion. (A) Sec63-GFP cells (SSY139) untreated or treated with DTT or tunicamycin for 2 h. Both ER stressors induce expansion of the peripheral ER and appearance of cytoplasmic ER extensions (arrows). (B) Quantification of ER expansion from images obtained as in A. The IE was determined as described in Materials and methods. Statistical significance compared with t = 0 of P ≤ 10⁻² (*) and P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM. (C) Cytoplasmic ER extensions induced by ER stress exclude the reticulon Rtn1. Sec63-GFP Rtn1-Cherry cells (SSY421) untreated or treated with DTT for 2 h. The cytoplasmic extensions in DTT-treated cells (arrows) are almost devoid of Rtn1. (D) Cytoplasmic ER extensions induced by ER stress are large membrane sheets. Electron micrographs of wild-type cells (SSY139) treated with DTT for 2 h. (top) Low magnification images of cells with cytoplasmic ER extensions (arrows) are shown. (bottom) Sequential 50-nm sections are shown at a higher magnification corresponding to the boxed area (top). The 0-nm image is the same as that shown in the top panel. The ER membrane is traced in blue. The ER sheet shown extends for at least 350 nm in the z direction. M, mitochondrion; N, nucleus; V, vacuole. Bars: (A and C) 2 µm; (D) 250 nm.

Proper ER membrane expansion requires UPR signaling. (A) Wild-type, hac1Δ, and ire1Δ cells expressing Sec63-GFP (SSY139, SSY161, and SSY467) treated with DTT for 2 h. Expansion of the peripheral ER is impaired in UPR mutants, and aberrant cytoplasmic ER patches form at the cell periphery and adjacent to the nucleus (arrows). (B) Quantification of ER expansion from images was obtained as in A. Statistical significance compared with the wild type at the same time point of P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM. (C) Sec63-GFP Rtn1-Cherry hac1Δ cells (SSY429) untreated or treated with DTT for 2 h. The cytoplasmic ER patches in DTT-treated cells contain both Sec63 and Rtn1. (D) Cytoplasmic ER patches induced by ER stress in hac1Δ cells are tangles of smooth ER. Electron micrographs of hac1Δ cells (SSY161) treated with DTT for 2 h. (top) Low magnification images of hac1Δ cells with cytoplasmic ER patches (arrows), which are found either at the cell periphery or close to the nucleus, are shown. (bottom) Sequential 50-nm sections are shown at a higher magnification corresponding to the boxed area (top). The 0-nm image is the same as shown in the top panel. The ER tangle shown consists of numerous randomly arranged ribosome-free elements. N, nucleus; V, vacuole. Bars: (A and C) 2 µm; (D) 250 nm.

Proper ER membrane expansion requires the Ino2/4 complex. (A) ER stress increases the levels of lipid synthesis enzymes. Western blot of HA tag from Opi3-HA–expressing wild-type, hac1Δ, and ino2Δ cells (SSY477, SSY485, and SSY484) treated with DTT for up to 3 h. Pgk1 served as a loading control. (B) Quantification of Opi3 levels normalized to Pgk1 from Western blots obtained as in A. Error bars indicate SEM from three independent experiments. (C) ER expansion requires both Ino2 and Ino4. Sec63-GFP–expressing wild-type, ino2Δ, and ino4Δ cells (SSY139, SSY369, and SSY460) treated with DTT for 2 h. (D) Quantification of ER expansion from images obtained as in C. Statistical significance compared with wild-type cells of P ≤ 10⁻⁴ (**) and P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM. (E) The requirement for Ino2 and Ino4 is bypassed in lipid-containing medium. Wild-type, ino2Δ, and ino4Δ cells (SSY139, SSY369, and SSY460) treated with DTT for 2 h in YPD medium. Normal ER expansion is observed in all three strains. (F) Quantification of ER expansion from images obtained as in E. Neither ino2Δ nor ino4Δ cells show statistically significant differences compared with wild-type cells. Expansion in wild-type cells is less pronounced here because of the lower effectiveness of DTT in YPD than in SC medium. Error bars indicate SEM. Bars, 2 µm.

ER membrane expansion is driven by Ino2/4 activity. (A) Schematic depiction of the negative regulation of Ino2/4 by Opi1. (B) Activation of Ino2/4 results in constitutive ER expansion. Untreated wild-type, opi1Δ, and ino2(L119A) cells expressing Sec63-GFP (SSY433, SSY290, and SSY400). Bar, 2 µm. (C) Quantification of ER expansion from images obtained as in A. Statistical significance compared with wild-type cells of P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM. (D) Activation of Ino2/4 produces expanded ER morphologically similar to that generated after ER stress. Electron micrographs of untreated opi1Δ cells (SSY290) are shown. (left) A low magnification image is shown. (right) Sequential 50-nm sections are shown at a higher magnification corresponding to the boxed area. The 0-nm image is the same as that shown in the low magnification image. The ER sheet shown extends for at least 350 nm in the z direction. LD, lipid droplet; V, vacuole. Bars, 500 nm.

ER membrane expansion can be uncoupled from the UPR. (A) ER stress induces the levels of ER protein folding enzymes. Western blot of Kar2, Lhs1, and Pdi1 from wild-type cells (SSY139) treated with DTT for up to 4 h. Pgk1 served as a loading control. (B) Quantification of Western blotting shown in A with values normalized to Pgk1. (C) The levels of ER protein–folding enzymes are unchanged by activation of Ino2/4. Western blot of Kar2, Lhs1, and Pdi1 from untreated wild-type, opi1Δ, and ino2(L119A)-expressing cells (SSY433, SSY290, and SSY400). (D) Quantification of Western blots obtained as in C with values normalized to Pgk1. Error bars indicate SEM from three independent experiments. (E) ER expansion by activation of Ino2/4 can occur independently of UPR signaling. Untreated hac1Δ, hac1Δ opi1Δ, and hac1Δ ino2(L119A) cells expressing Sec63-GFP (SSY434, SSY364, and SSY441). Bar, 2 µm. (F) Quantification of ER expansion from images obtained as in E. Statistical significance compared with hac1Δ cells of P ≤ 10⁻⁴ (**) and P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM.

ER membrane expansion alleviates ER stress. (A) Deletion of OPI1 increases resistance of hac1Δ cells to ER stress. Tunicamycin sensitivity of wild-type, hac1Δ, opi1Δ, and hac1Δ opi1Δ cells (SSY139, SSY161, SSY290, and SSY364) as assessed by plating dilution series of cells onto solid SC medium containing 0.05 µg/ml tunicamycin. Series represent fivefold dilutions from one step to the next. (B) Overexpression of Ino2 and especially ino2(L119A) increases resistance of hac1Δ cells to ER stress. Tunicamycin sensitivity of wild-type, hac1Δ, hac1Δ ino2Δ pINO2, and hac1Δ ino2Δ pino2(L119A) cells (SSY433, SSY434, SSY439, and SSY441) assessed as in A except that leucine was omitted from the plates. (C) ino2 mutants show increased sensitivity to ER stress. Tunicamycin sensitivity of wild-type and ino2Δ cells (SSY139 and SSY430) assessed as in A. (D) ino2 mutants depend on HAC1 to overcome even mild ER stress. Tunicamycin sensitivity of wild-type, hac1Δ, ino2Δ, and hac1Δ ino2Δ cells (SSY139, SSY161, SSY369, and SSY430) assessed as in A. (E) Untreated ino2 mutants show chaperone induction indicative of constitutive UPR signaling. Western blot of Kar2, Lhs1, and Pdi1 from untreated wild-type and ino2Δ cells (SSY139 and SSY369) grown for 24 h in lipid-free SC medium. Pgk1 served as a loading control. Numbers indicate fold induction in ino2 mutants compared with wild type and normalized to Pgk1.

ER membrane expansion alleviates stress as a result of increased ER size rather than altered ER shape. (A) Rtn1 levels are unchanged by ER stress or activation of Ino2/4. Western blot of Cherry tag from Rtn1-Cherry–expressing wild-type cells treated with DTT for up to 4 h and from untreated Rtn1-Cherry–expressing wild-type, opi1Δ, and ino2(L119A) cells (SSY421, SSY478, and SSY473). Pgk1 served as a loading control. (B) Quantification of Western blots shown in A with values normalized to Pgk1. (C) Overexpression of Rtn1 converts sheets (arrows) into tubules (arrowheads). Untreated wild-type and opi1Δ cells expressing dsRed-HDEL and Rtn1-GFP were used to mark the entire ER lumen and ER tubules, respectively, and carrying an empty vector or an expression plasmid encoding untagged Rtn1 (SSY523, SSY531, and SSY532). (D) ER expansion is unaffected by Rtn1 overexpression. Quantification of ER expansion from images of untreated wild-type, opi1Δ, and Rtn1-overexpressing opi1Δ cells (SSY523, SSY531, and SSY532). Statistical significance compared with wild-type cells of P ≤ 10⁻⁶ (***) is shown. Error bars indicate SEM. (E) Rtn1 overexpression does not affect sensitivity to ER stress. Tunicamycin sensitivity of wild-type, hac1Δ, and hac1Δ opi1Δ cells carrying empty vectors and hac1Δ opi1Δ cells carrying an expression plasmid encoding untagged Rtn1 (SSY510, SSY533, SSY535, and SSY536) as assessed by plating dilution series of cells onto solid SC medium (without uracil) containing 0.05 µg/ml tunicamycin. Series represent fivefold dilutions from one step to the next. Bar, 2 µm.