Unfolded Protein Response Of The Endoplasmic Reticulum Research Articles

SAT074 Induction Of Insulin Hypersecretion Uncovers Distinctions Between Adaptive And Maladaptive Endoplasmic Reticulum Stress Response In Beta Cells

Abstract Disclosure: G. Roy: None. K. Rodrigues dos Santos: None. M.B. Kwakye: None. Z. Tan: None. T.S. Johnson: None. M.A. Kalwat: None. Pancreatic islet β-cells release insulin to maintain glucose homeostasis. β-cells must translate, package, and secrete large amounts of insulin. During this process the unfolded protein response of the endoplasmic reticulum (UPRER) is induced to maintain these functions. However, stimuli that force β-cell to secrete insulin at enhanced rates and for prolonged durations risk inducing the terminal UPRER and eventual apoptosis. In a chemical screen for insulin secretion modulators, we discovered SW016789 which caused hypersecretion of insulin and led to a transient induction of the UPRER, but not apoptosis. In contrast, SERCA2 ER Ca2+ pump inhibitor thapsigargin induces the terminal UPRER. We hypothesized that SW016789 can be used as a tool compound to discover genes involved in β-cell adaptation to hypersecretion-induced stress. We performed time course transcriptomics in MIN6 β-cells exposed to SW016789 (5 µM) or thapsigargin (100 nM) from 0-24 h. Unbiased analyses using a Dirichlet process Gaussian process (DPGP) method revealed clusters of genes temporally co-regulated and the genes within these clusters were distinct between SW016789 and thapsigargin treatments. In particular, after 6 h of SW016789-induced hypersecretion we found a highly induced cluster of genes (SW cluster 3) enriched in adaptive UPRER factors (e.g. Manf). Conversely, most of the thapsigargin-induced genes clustered at 24 h and were enriched for terminal UPRER factors (e.g. Txnip). Pathway analysis of SW cluster 3 indicated that genes involved in in regulation of mRNA methylation and ER-associated degradation were also induced by SW016789 sooner and with greater amplitude than by thapsigargin, suggesting distinct differences in the handling of protein translation and degradation. From the SW cluster 3 genes we selected proteins known to be ER-associated or secreted and generated stable transgenic or CRISPR knockout MIN6 β-cell lines for each. Our data suggest altered expression of these factors may impair glucose-stimulated insulin secretion responses and alter cell viability in presence or absence of ER stressors including cytokines, thapsigargin, and tunicamycin. In conclusion, we have successfully shown that pharmacological induction of insulin hypersecretion can induce a distinct transcriptional outcome from that of canonically-induced UPRER and that we can take advantage of this property to discover new β-cell regulatory pathways and targets. We envision this dataset as a resource for the secretory biology and islet biology communities. Presentation: Saturday, June 17, 2023

Lipid homeostasis is essential for a maximal ER stress response.

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identified let-767, a putative hydroxysteroid dehydrogenase in Caenorhabditis elegans, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.

IRE-1 endoribonuclease activity declines early in C. elegans adulthood and is not rescued by reduced reproduction.

The proteome of a cell helps to define its functional specialization. Most proteins must be translated and properly folded to ensure their biological function, but with aging, animals lose their ability to maintain a correctly folded proteome. This leads to the accumulation of protein aggregates, decreased stress resistance, and the onset of age-related disorders. The unfolded protein response of the endoplasmic reticulum (UPRER) is a central protein quality control mechanism, the function of which is known to decline with age. Here, we show that age-related UPRER decline in Caenorhabditis elegans occurs at the onset of the reproductive period and is caused by a failure in IRE-1 endoribonuclease activities, affecting both the splicing of xbp-1 mRNA and regulated Ire1 dependent decay (RIDD) activity. Animals with a defect in germline development, previously shown to rescue the transcriptional activity of other stress responses during aging, do not show restored UPRER activation with age. This underlines the mechanistic difference between age-associated loss of UPRER activation and that of other stress responses in this system, and uncouples reproductive status from the activity of somatic maintenance pathways. These observations may aid in the development of strategies that aim to overcome the proteostasis decline observed with aging.

Characterization of the mitochondrial unfolded protein response in human coronary artery endothelial cells

Abstract Background The unfolded protein response of the endoplasmic reticulum (UPRER) is highly activated in cardiovascular diseases and aims to restore homeostasis by improving protein folding. An analogue cytoprotective mitochondrial unfolded protein response (UPRMito) regulated by an orthologue of the activating transcription factor 5 (ATF5) was described in model organism c. elegans. However, in humans it is not clear if there is a distinct UPRMito, and if there is an activation of an UPRMito during stress induced by cardiovascular diseases. The endothelium is subjected to high levels of mitochondrial stress, e.g. in atherosclerosis. Purpose To provide a systematic characterization of the UPRMito in human coronary artery endothelial cells (HCAEC). Methods and results HCAEC were treated with Nicotinamide Riboside (NR), a NAD+ precursor that activates UPRMito without inducing cellular stress. NR treatment did not induce toxicity and improved cell viability at a concentration of 100μM NR. Quantitative PCR confirmed a dose-dependent upregulation of UPRMito markers. Additionally, we measured expression of ATF4, which is discussed to regulate an integrated stress response in mammals by activating both the UPRMito and UPRER. ATF4 upregulation upon NR treatment was more pronounced than ATF5 upregulation. Analysis of GTEX-Data showed that ATF5 is almost exclusively expressed in liver tissue, while ATF4 shows ubiquitous expression. Among 54 tissues, aortic tissue and coronary arterial tissue are among the five tissues with the highest ATF4 expression. Analysis of RNA-seq-data of human carotid atherosclerosis revealed that the two transcription factors are differentially regulated. ATF4 is downregulated in unstable plaque, while ATF5 is upregulated. Moreover, ATF4 is in both conditions highly more abundant than ATF5. Accordingly, endothelial cells incubated under atherosclerotic conditions (oxLDL + high glucose) exhibited a ATF4 down- and ATF5 upregulation. Genes previously described to be involved in the UPRMito exhibited a similar expression pattern as ATF4. To further elucidate these findings, we will perform transcriptomic analyses of HCAEC treated with atherosclerotic stimuli (IL-1β, oxLDL), UPRER inductors (Thapsigargin, Tunicamycin) and stressors of mitochondrial proteostasis (Oligomycin, MitoBloCK-6), to analyze, if ATF5- or ATF4-related pathways are dysregulated. Specific inhibitors and UPRMito activators will be used to investigate the significance of ATF4 and ATF5 on endothelial cell function (proliferation, migration, apoptosis, monocyte adhesion). Conclusion The UPRMito is dysregulated during the pathogenesis of atherosclerosis in HCAEC. Further findings are required to elucidate, if the UPRMito is activated separately and specifically by ATF5 or as part of an integrated cellular stress response by ATF4. A deeper understanding of these stress responses is crucial for the identification of novel therapeutic targets in atherosclerosis. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): University hospital Bonn

UPRmt and coordinated UPRER in type 2 diabetes.

The mitochondrial unfolded protein response (UPRmt) is a molecular mechanism that maintains mitochondrial proteostasis under stress and is closely related to various metabolic diseases, such as type 2 diabetes (T2D). Similarly, the unfolded protein response of the endoplasmic reticulum (UPRER) is responsible for maintaining proteomic stability in the endoplasmic reticulum (ER). Since the mitochondria and endoplasmic reticulum are the primary centers of energy metabolism and protein synthesis in cells, respectively, a synergistic mechanism must exist between UPRmt and UPRER to cooperatively resist stresses such as hyperglycemia in T2D. Increasing evidence suggests that the protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) signaling pathway is likely an important node for coordinating UPRmt and UPRER. The PERK pathway is activated in both UPRmt and UPRER, and its downstream molecules perform important functions. In this review, we discuss the mechanisms of UPRmt, UPRER and their crosstalk in T2D.

Hijacking Cellular Stress Responses to Promote Lifespan.

Organisms are constantly exposed to stress both from the external environment and internally within the cell. To maintain cellular homeostasis under different environmental and physiological conditions, cell have adapted various stress response signaling pathways, such as the heat shock response (HSR), unfolded protein responses of the mitochondria (UPRMT), and the unfolded protein response of the endoplasmic reticulum (UPRER). As cells grow older, all cellular stress responses have been shown to deteriorate, which is a major cause for the physiological consequences of aging and the development of numerous age-associated diseases. In contrast, elevated stress responses are often associated with lifespan extension and amelioration of degenerative diseases in different model organisms, including C. elegans. Activating cellular stress response pathways could be considered as an effective intervention to alleviate the burden of aging by restoring function of essential damage-clearing machinery, including the ubiquitin-proteosome system, chaperones, and autophagy. Here, we provide an overview of newly emerging concepts of these stress response pathways in healthy aging and longevity with a focus on the model organism, C. elegans.

DNA damage promotes ER stress resistance through elevation of unsaturated phosphatidylcholine in Caenorhabditis elegans

DNA damage triggers the cellular adaptive response to arrest proliferation and repair DNA damage; when damage is too severe to be repaired, apoptosis is initiated to prevent the spread of genomic insults. However, how cells endure DNA damage to maintain cell function remains largely unexplored. By using Caenorhabditis elegans as a model, we report that DNA damage elicits cell maintenance programs, including the unfolded protein response of the endoplasmic reticulum (UPRER). Mechanistically, sublethal DNA damage unexpectedly suppresses apoptotic genes in C. elegans, which in turn increases the activity of the inositol-requiring enzyme 1/X-box binding protein 1 (IRE-1/XBP-1) branch of the UPRER by elevating unsaturated phosphatidylcholine. In addition, UPRER activation requires silencing of the lipid regulator skinhead-1 (SKN-1). DNA damage suppresses SKN-1 activity to increase unsaturated phosphatidylcholine and activate UPRER. These findings reveal the UPRER activation as an organismal adaptive response that is important to maintain cell function during DNA damage.

Phenolic Compounds Reduce the Fat Content in Caenorhabditis elegans by Affecting Lipogenesis, Lipolysis, and Different Stress Responses.

Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds (myricetin, kaempferol, naringin, hesperidin, apigenin, luteolin, resveratrol, curcumin, and epicatechin) and phenolic acids (p-coumaric, ellagic, ferulic, gallic, and vanillic acids) regulating C. elegans fat accumulation. Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress, and aging, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPRER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, aging, and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.

Beyond the cell factory: Homeostatic regulation of and by the UPRER.

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis, and immunity.

Measurements of Physiological Stress Responses in C. Elegans.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Taking the stress out of life

Life-Span Extension In the model organism Caenorhabditis elegans , a roundworm, it has been shown that neurons can communicate proteostasis to the periphery to affect aging. Frakes et al. have now identified astrocytelike glial cells that also act as central regulators of systemic protein homeostasis and aging (see the Perspective by Miklas and Brunet). They found that the life span of C. elegans can be extended by expression of a constitutively active version of the transcription factor XBP-1s, which mediates the unfolded protein response of the endoplasmic reticulum (UPRER), in a specific subset of glial cells. Glial XBP-1s initiates induction of the UPRER in distal intestinal cells, which makes the worms more resistant to chronic ER stress. Neuropeptide signaling was required for glial-mediated longevity and induction of the peripheral UPRER, suggesting a distinct mechanism from that initiated by neuronal XBP-1s. Thus, in this animal model of aging, a mere four cells can control organismal physiology and aging Science , this issue p. [436][1]; see also p. [365][2] [1]: /lookup/doi/10.1126/science.aaz6896 [2]: /lookup/doi/10.1126/science.aba4474

Four glial cells regulate ER stress resistance and longevity via neuropeptide signaling in C. elegans

The ability of the nervous system to sense cellular stress and coordinate protein homeostasis is essential for organismal health. Unfortunately, stress responses that mitigate disturbances in proteostasis, such as the unfolded protein response of the endoplasmic reticulum (UPRER), become defunct with age. In this work, we expressed the constitutively active UPRER transcription factor, XBP-1s, in a subset of astrocyte-like glia, which extended the life span in Caenorhabditis elegans Glial XBP-1s initiated a robust cell nonautonomous activation of the UPRER in distal cells and rendered animals more resistant to protein aggregation and chronic ER stress. Mutants deficient in neuropeptide processing and secretion suppressed glial cell nonautonomous induction of the UPRER and life-span extension. Thus, astrocyte-like glial cells play a role in regulating organismal ER stress resistance and longevity.

Proteostasis In The Endoplasmic Reticulum: Road to Cure.

The endoplasmic reticulum (ER) is an interconnected organelle that is responsible for the biosynthesis, folding, maturation, stabilization, and trafficking of transmembrane and secretory proteins. Therefore, cells evolve protein quality-control equipment of the ER to ensure protein homeostasis, also termed proteostasis. However, disruption in the folding capacity of the ER caused by a large variety of pathophysiological insults leads to the accumulation of unfolded or misfolded proteins in this organelle, known as ER stress. Upon ER stress, unfolded protein response (UPR) of the ER is activated, integrates ER stress signals, and transduces the integrated signals to relive ER stress, thereby leading to the re-establishment of proteostasis. Intriguingly, severe and persistent ER stress and the subsequently sustained unfolded protein response (UPR) are closely associated with tumor development, angiogenesis, aggressiveness, immunosuppression, and therapeutic response of cancer. Additionally, the UPR interconnects various processes in and around the tumor microenvironment. Therefore, it has begun to be delineated that pharmacologically and genetically manipulating strategies directed to target the UPR of the ER might exhibit positive clinical outcome in cancer. In the present review, we summarize recent advances in our understanding of the UPR of the ER and the UPR of the ER–mitochondria interconnection. We also highlight new insights into how the UPR of the ER in response to pathophysiological perturbations is implicated in the pathogenesis of cancer. We provide the concept to target the UPR of the ER, eventually discussing the potential of therapeutic interventions for targeting the UPR of the ER for cancer treatment.

Neuronal XBP-1 Activates Intestinal Lysosomes to Improve Proteostasis in C. elegans

SummaryThe unfolded protein response of the endoplasmic reticulum (UPRER) is a crucial mediator of secretory pathway homeostasis. Expression of the spliced and active form of the UPRER transcription factor XBP-1, XBP-1s, in the nervous system triggers activation of the UPRER in the intestine of Caenorhabditis elegans (C. elegans) through release of a secreted signal, leading to increased longevity. We find that expression of XBP-1s in the neurons or intestine of the worm strikingly improves proteostasis in multiple tissues, through increased clearance of toxic proteins. To identify the mechanisms behind this enhanced proteostasis, we conducted intestine-specific RNA-seq analysis to identify genes upregulated in the intestine when XBP-1s is expressed in neurons. This revealed that neuronal XBP-1s increases the expression of genes involved in lysosome function. Lysosomes in the intestine of animals expressing neuronal XBP-1s are more acidic, and lysosomal protease activity is higher. Moreover, intestinal lysosome function is necessary for enhanced lifespan and proteostasis. These findings suggest that activation of the UPRER in the intestine through neuronal signaling can increase the activity of lysosomes, leading to extended longevity and improved proteostasis across tissues.

Unfolded Protein Response of the Endoplasmic Reticulum in Tumor Progression and Immunogenicity.

The endoplasmic reticulum (ER) is a pivotal regulator of folding, quality control, trafficking, and targeting of secreted and transmembrane proteins, and accordingly, eukaryotic cells have evolved specialized machinery to ensure that the ER enables these proteins to acquire adequate folding and maturation in the presence of intrinsic and extrinsic insults. This adaptive capacity of the ER to intrinsic and extrinsic perturbations is important for maintaining protein homeostasis, which is termed proteostasis. Failure in adaptation to these perturbations leads to accumulation of misfolded or unassembled proteins in the ER, which is termed ER stress, resulting in the activation of unfolded protein response (UPR) of the ER and the execution of ER-associated degradation (ERAD) to restore homeostasis. Furthermore, both of the two axes play key roles in the control of tumor progression, inflammation, immunity, and aging. Therefore, understanding UPR of the ER and subsequent ERAD will provide new insights into the pathogenesis of many human diseases and contribute to therapeutic intervention in these diseases.

PEK-1 is crucial for hormesis induced by inhibition of the IRE-1/XBP-1 pathway in the Caenorhabditis elegans mev-1 mutant

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes an imbalance of proteostasis and is related to many pathological conditions. In answer to this ER stress cells activate a network of three integrated signaling pathways consolidated as the unfolded protein response of the ER (UPRER), which is also present in the stress-sensitive Caenorhabditis elegans mutant mev-1. Whereas inhibition of one of those pathways by RNA-interference (RNAi) versus xbp-1 results in reduced survival of mev-1 nematodes under heat stress, additional knockdown of the xbp-1 splicing activator ire-1 results in a PEK-1-dependent hormetic response. In contrast, increased survival under ire-1/xbp-1 double RNAi was found to be independent of the presence of HSP-4, an UPRER-specific chaperone, as evidenced under ire-1/xbp-1/hsp-4 triple knockdown conditions. Moreover, ire-1/xbp-1 double-RNAi significantly increased chymotrypsin-like proteasomal activity, which was completely blocked under additional RNAi versus pek-1.In conclusion, we identified PEK-1 as a mediator of hormesis in the mev-1 mutant of C. elegans which is induced by simultaneous inhibition of XBP-1 and its splicing activator IRE-1 and mediated through activation of the proteasome.

PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress.

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan.

Membrane lipids and the endoplasmic reticulum unfolded protein response: An interesting relationship.

The unfolded protein response of the endoplasmic reticulum (UPRER) is a conserved signaling circuit that ensures ER protein homeostasis (proteostasis). In the UPRER of higher eukaryotes, multiple sensors cooperatively perceive proteostatic disturbances in the ER lumen and induce downstream adaptive changes. Besides direct proteotoxic insults, altered lipid profiles can also lead to UPRER activation, evidently because abnormal lipid composition impairs protein folding. However, 2 recent studies propose an alternative mechanism of UPRER sensor activation. In one report, UPRER activation occurred in cells expressing UPRER sensors lacking the very domains that sense unfolded proteins; the other study found that Caenorhabditis elegans worms displayed UPRER activation without apparent proteostatic imbalance in the ER lumen. Collectively, these studies suggest that lipid disequilibrium-activated UPRER is not strictly accompanied by compromised ER proteostasis and hint at a lipid membrane-monitoring role of the UPRER. These discoveries raise several important questions: does the UPRER monitor and maintain homeostasis of the ER membrane and/or its lipids? In turn, does the UPRER initiate downstream regulatory events that specifically alleviate lipid or proteostatic imbalance? And what is the physiological significance of proteostasis-independent UPRER activation? In this commentary, we will discuss these issues and highlight the utility of C. elegans as an in vivo model to study lipid disequilibrium-induced UPRER and related pathways.

Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation.

The Unfolded Protein Response of the endoplasmic reticulum (UPRER) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. Here we show that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. We find that ISCs require activation of the UPRER for regenerative responses, but that a tissue-wide increase in ER stress triggers ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. Our results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium.

Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo.

The Mediator is a conserved transcriptional coregulator complex required for eukaryotic gene expression. In Caenorhabditis elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress responses. mdt-15 loss of function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that worms with mutated or depleted mdt-15 (mdt-15 worms) exhibit decreased membrane phospholipid desaturation, especially in phosphatidylcholine. Accordingly, mdt-15 worms exhibit disturbed endoplasmic reticulum (ER) homeostasis, as indicated by a constitutively activated ER unfolded protein response (UPR(ER)). Activation of this stress response is only partially the consequence of reduced membrane lipid desaturation, implicating other mdt-15-regulated processes in maintaining ER homeostasis. Interestingly, mdt-15 inactivation or depletion of the lipid metabolism enzymes stearoyl-CoA-desaturases (SCD) and S-adenosyl methionine synthetase (sams-1) activates the UPR(ER) without promoting misfolded protein aggregates. Moreover, these worms exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with mutations in UPR(ER) genes, which cause protein misfolding. Therefore, the constitutively activated UPR(ER) in mdt-15, SCD, and sams-1 worms is not the consequence of proteotoxic stress but likely is the direct result of changes in ER membrane fluidity and composition. Together, our data suggest that the UPR(ER) is induced directly upon membrane disequilibrium and thus monitors altered ER homeostasis.