Increased mitochondrial respiration promotes survival from endoplasmic reticulum stress

Abstract

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O₂ consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O₂ consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

Fig. 1

Cells without Ire1 or calcineurin have increased sensitivity to ER stress. a ER stress impairs cell growth: cnb1∆ and ire1∆ cells have impaired growth on tunicamycin, DTT, or the misfolded protein CPY*. Serial dilutions of cells were spotted on plates. b Cell death is induced by ER stress. cnb1∆ and ire1∆ cells have increased susceptibility to ER stress-induced death, as determined by colony plating assay after treating mid-log cultures with 0.5 µg/mL tunicamycin for 5 h. Viable cells expressed as a percentage of untreated cells +/− standard error of the mean (SEM); n = 3. c ER stress-induced death is associated with ROS accumulation. Wild-type, cnb1∆ and ire1∆ cells treated with tunicamycin (0.5 µg/mL) for 5 h were stained with DHE. Fluorescence was imaged at identical exposure times for all samples, and quantitated by counting cell numbers displaying DHE staining throughout the entire cell out of 50 cells using ImageJ software; n = 3.

Fig. 2

ER stress-induced death is caused by mitochondrial ROS. a ER stress-induced death is rescued in rho0 cells and by addition of the proton ionophore CCCP. Exponentially growing cells were treated with 0.5 µg/ml tunicamycin with or without CCCP (10 µM) for 5 h. Cell viability was then determined as in Fig. 1b legend, expressed as a percentage of untreated cells +/− SEM, n = 3. b ROS accumulation is rescued in rho0 cells and by addition of the protonophore CCCP. ROS was assayed by staining with DHE as in Fig. 1c legend; n = 3+/− SEM. Fluorescence images from cells +/− tunicamycin were imaged for the same exposure time and adjusted using identical Photoshop settings. c Impaired growth of cnb1 on tunicamycin is rescued in cnb1 rho0 cells. Serially diluted cells were spotted onto plates with and without tunicamycin (0.25 µg/mL). d ER stress-induced death (left panel) and ROS (right panel) rescued by overexpression of mitochondrial catalase. Cells transformed with pMET-CTA1 were induced to express catalase after washing with water and resuspending in methionine-free SC medium. Tunicamycin (0.5 µg/mL) was then added for 5 h before measuring cell viability and ROS as in Fig. 1b, c legends +/− SEM, n = 3.

Fig. 3

ER stress induces mitochondrial response. a O₂ consumption increases in response to ER stress in wild-type but not cnb1∆ or ire1∆ cells. Cells were treated with 0.5 µg/mL tunicamycin or 1 mM DTT for 2 h before measuring O₂ consumption expressed as pmol/ml/s/OD₆₀₀+/− SEM, n = 3. b ETC protein levels are increased by overexpression of HAP4 (top left panel) and SAK1 overexpression (bottom left panel). Lysates from exponentially growing wild-type and cnb1∆ cells +/− HAP4 overexpression were assayed by Western blot for ETC components, Cox2 and ATP synthase, and mitochondrial outer membrane protein VDAC1/porin. Right panel, schematic indicating signaling pathways regulating mitochondrial biogenesis. c O₂ consumption is increased upon overexpression of HAP4 or SAK1. O₂ consumption is increased in cells overexpressing HAP4 or SAK1. d Overexpression of HAP4 or SAK1 suppresses death of cnb1∆ and ire1∆ cells after 5 h tunicamycin treatment. Top panel, cell viability was assayed as in Fig. 1b legend. Bottom panel, HAP4 overexpression relieves impaired growth of cnb1∆ cells on tunicamycin. Serial dilutions of cells were spotted on SC-uracil plates with 0.2 µg/mL tunicamycin. e ALA to promote heme synthesis increases O₂ consumption (left panel) and rescues viability (right panel) of ire1∆ and cnb1∆ cells during ER stress. Cells were grown overnight in SC medium supplemented with ALA to mid-log phase. Viability was determined after 5 h treatment with tunicamycin (0.5 µg/mL) as described in Fig. 1b legend.

Fig. 4

Extracellular Ca²⁺ exacerbates ER stress in cnb1∆ cells but alleviates tunicamycin sensitivity in ire1∆ cells. a Serial dilutions of cells were spotted on YPD plates with or without 0.2 µg/mL tunicamycin with or without 200 mM CaCl₂. b Calcium influx promotes survival in response to ER stress. ER stress-induced ROS accumulation (right panel) and death (left panel) are increased in wild-type cells in extracellular EGTA and in cch1∆ cells. Cells were grown overnight in SC supplemented with 10 µM EGTA, treated with 0.5 µg/mL tunicamycin for 5 h, and stained with DHE to visualize ROS; quantitation was as described in Fig. 1c legend. Viability was assayed and quantitated as described in Fig. 1b legend. c, d Mitochondrial membrane potential (MMP) responds to Ca²⁺ flux. Exponentially growing cells were stained with TMRM (5 nM, nonquenching mode) before and after 2 h treatment with 0.5 µg/ml tunicamycin. Wild-type cells in SC medium plus EGTA (10 µM) were grown overnight prior to tunicamycin treatment. Wild-type, cnb1∆ and ire1∆ cells transformed with pMET17-CTA1 were induced to express mitochondrial catalase by washing with water and resuspending in methionine-free medium. Cells were then further incubated for 2 h with tunicamycin (0.5 µg/mL). Fluorescence images were taken at identical exposure times and brightness and contrast were adjusted using identical Photoshop settings.

Fig. 5

O₂ consumption and COX activity are impaired in cnb1∆ and ire1∆ cells. a Left panel, O₂ consumption by cells grown overnight to mid-log phase in FK506 (2 µg/mL) were compared with that of wild-type and cnb1∆ cells. Right panel, COX activity measured in isolated mitochondria as oxidation of cytochrome c. b O₂ consumption in ire1∆ cells is not entirely attributable to oxidative phosphorylation. O₂ consumption was measured in wild-type, cnb1∆, and ire1∆ cells. Cells were treated with TET (10 mM) to inhibit ATP synthase; O₂ consumption in wild-type and cnb1∆, but not ire1∆ cells falls to 0. Addition of CCCP (10 µM) induces O₂ consumption to return to maximal. c Reduced ETC activity in ire1∆ cells. Left panel, O₂ consumption by oxidative phosphorylation (TET-inhibitable) in wild-type, cnb1∆ and ire1∆ cells. Right panel, COX activity was measured in isolated mitochondria. Wild-type cells were grown to mid-log phase overnight in SC with 3% glycerol for comparison with strains grown in SC with 2% glucose.

Fig. 6

High copy suppressors of ER stress-induced death of cnb1∆ cells. a, b Upon overexpression on 2 µ plasmids, three mitochondrial genes, AIM19, MRM1, and MRX9 rescue cnb1∆ and ire1∆ cells from ER stress-induced death and ROS. Viability and ROS after 5 h incubation with 0.5 µg/mL tunicamycin was determined as described in Fig. 1b, c legends. The results are expressed as a percent of untreated cells. c Overexpression of Aim19, Mrm1, and Mrx9 increase O₂ consumption in wild-type, ire1∆, and cnb1∆ cells. d High copy suppressors of ER stress-induced death increase ETC component levels. Western blot of wild-type, cnb1∆, and ire1∆ cells with and without high copy suppressors.

Fig. 7

ER stress induces ROS and death in PCCL3 cultured cells. a Calcineurin protects against ROS production. Cells were co-treated with FK506 (5 µM) and tunicamycin (100 ng/mL) for 2 days. Cells were then co-stained with DHE and Mitotracker green to detect ROS and mitochondria, respectively. b ER stress-induced cytotoxicity (top panel) and ROS accumulation (bottom panel) are exacerbated by calcineurin inhibition and Ire1 knockdown (kd). siRNAs were added to cells for 2 days before incubation with tunicamycin (100 ng/mL) for another 2 days before cytotoxicity was assayed. The cytotoxic effect of FK506 was determined after 2 days incubation with tunicamycin (100 ng/mL) with or without FK506 (5 µM). Cytotoxicity was assayed as leakage of LDH. n = 3 experiments; error bars are SEM. *p < 0.05. ROS accumulation was assayed by DHR123 fluorescence staining, as described in Methods. c Mitochondrial hyperpolarization is induced by tunicamycin (100 ng/mL). MMP, as measured by TMRM fluorescence, is increased by 1 h after tunicamycin treatment; this ER stress response is abrogated by FK506-mediated inhibition of calcineurin. d Tunicamycin-induced cytotoxicity (top panel) is dependent on mitochondrial ROS, and is suppressed by anti-oxidants (N-acetyl cysteine, 5 mM), heme (10 µM), increased heme synthesis (ALA, 300 µg/ml), increased O₂ flux (induced by CCCP, 10 nM), and increased mitochondrial respiration (induced by AICAR, 0.5 mM). n = 3 experiments +/− SEM. *p < 0.05, **p < 0.01. Bottom panel, ROS accumulation was assayed by DHR123 fluorescence, as described in Methods. e AICAR increases O₂ consumption. PCCL3 cells were treated with AICAR (0.5 mM) for 1 day, and then trypsinized, washed, and placed in a oxygraph for O₂ consumption measurements, as described previously [62]. f Left panel, Calcineurin is necessary for tunicamycin-induced phosphorylation of eIF2α. Cells were treated with tunicamycin +/− FK506 (5 µM) for 2 days, as in a, and phosphorylated eIF2α and total eIF2α protein were assayed by Western blot. Right panel, Cell lysate from experiment shown in the left panel was blotted with anti-PERK antibody. Arrowhead, in tunicamycin-treated samples, phosphorylated PERK is seen as an extensive smear.

Fig. 8

Schematic model of ER stress-induced stress. Details described in text. Cell survival: protein misfolding in the ER is sensed by Ire1 that dimerizes to induce increased transcriptional activation in the nucleus; Ire1 activity is necessary for survival of ER stress. In response to ER stress, MMP and O₂ consumption are increased [by Ca²⁺ influx into mitochondria [3]]. Activation of calcineurin is a requisite event for cell survival [this paper and [31]]. Cell death: protein misfolding in the ER cannot induce a transcriptional response without Ire1. Ca²⁺ flux is abnormal without calcineurin [9]. Genetic evidence suggests Ca²⁺ homeostasis is also dysregulated in ire1∆ cells. In both cnb1∆ and ire1∆ cells, MMP and O₂ consumption fail to respond to ER stress, ROS is accumulated and death ensues (Figs. 1 and 3).