Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1

The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.

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Systematic cysteine-crosslinking in native membranes establishes the transmembrane architecture in Ire1 clusters

10.1101/772772 ◽

2019 ◽

Author(s):

Kristina Väth ◽

Roberto Covino ◽

John Reinhard ◽

Gerhard Hummer ◽

Robert Ernst

Keyword(s):

Lipid Bilayer ◽

Secretory Proteins ◽

Transmembrane Helices ◽

Unfolded Protein ◽

Proteotoxic Stress ◽

Stress Sensors ◽

Obesity And Diabetes ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

AbstractThe endoplasmic reticulum (ER) is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Stress sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for the maintenance of ER homeostasis. More recently, it became clear that aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR with important implications in obesity and diabetes. How the distinct signals from lipid bilayer stress and proteotoxic stress are processed by the highly conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine crosslinking experiments to establish the transmembrane architecture of signaling-active clusters in native membranes. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration and that this configuration is independent of the primary cause for ER stress. Based on these findings, we propose that different forms of stress converge in a single, signaling-active conformation of Ire1.

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ER homeostasis and autophagy

Essays in Biochemistry ◽

10.1042/ebc20170092 ◽

2017 ◽

Vol 61 (6) ◽

pp. 625-635 ◽

Cited By ~ 69

Author(s):

Matthew Smith ◽

Simon Wilkinson

Keyword(s):

Secretory Proteins ◽

Unfolded Protein ◽

Effector Pathway ◽

Autophagy Pathway ◽

The Unfolded Protein Response ◽

And Function ◽

Protein Response ◽

Er Homeostasis ◽

Effector Mechanisms

The endoplasmic reticulum (ER) is a key site for lipid biosynthesis and folding of nascent transmembrane and secretory proteins. These processes are maintained by careful homeostatic control of the environment within the ER lumen. Signalling sensors within the ER detect perturbations within the lumen (ER stress) and employ downstream signalling cascades that engage effector mechanisms to restore homeostasis. The most studied signalling mechanism that the ER employs is the unfolded protein response (UPR), which is known to increase a number of effector mechanisms, including autophagy. In this chapter, we will discuss the emerging role of autophagy as a UPR effector pathway. We will focus on the recently discovered selective autophagy pathway for ER, ER-phagy, with particular emphasis on the structure and function of known mammalian ER-phagy receptors, namely FAM134B, SEC62, RTN3 and CCPG1. Finally, we conclude with our view of where the future of this field can lead our understanding of the involvement of ER-phagy in ER homeostasis.

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Endoplasmic reticulum stress in biological processing and disease

Journal of Investigative Medicine ◽

10.1136/jim-2020-001570 ◽

2021 ◽

Vol 69 (2) ◽

pp. 309-315

Author(s):

Ali Riza Koksal ◽

George Nicholas Verne ◽

QiQi Zhou

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Chronic Diseases ◽

Unfolded Proteins ◽

Unfolded Protein ◽

Inflammatory Bowel ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis ◽

Multiple Signaling

The ability of translated cellular proteins to perform their functions requires their proper folding after synthesis. The endoplasmic reticulum (ER) is responsible for coordinating protein folding and maturation. Infections, genetic mutations, environmental factors and many other conditions can lead to challenges to the ER known as ER stress. Altering ER homeostasis results in accumulation of misfolded or unfolded proteins. To eliminate this problem, a response is initiated by the cell called the unfolded protein response (UPR), which involves multiple signaling pathways. Prolonged ER stress or a dysregulated UPR can lead to premature apoptosis and an exaggerated inflammatory response. Following these discoveries, ER stress was shown to be related to several chronic diseases, such as diabetes mellitus, neurodegenerative disorders, fatty liver disease and inflammatory bowel disease that have not yet been clearly demonstrated pathophysiologically. Here, we review the field and present up-to-date information on the relationship between biological processing, ER stress, UPR, and several chronic diseases.

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Stressed to death – mechanisms of ER stress-induced cell death

Biological Chemistry ◽

10.1515/hsz-2013-0174 ◽

2014 ◽

Vol 395 (1) ◽

pp. 1-13 ◽

Cited By ~ 107

Author(s):

Natalia Sovolyova ◽

Sandra Healy ◽

Afshin Samali ◽

Susan E. Logue

Keyword(s):

Cell Death ◽

Er Stress ◽

Post Translational Modification ◽

Unfolded Proteins ◽

Unfolded Protein ◽

Cell Death Pathway ◽

The Unfolded Protein Response ◽

And Function ◽

Protein Response ◽

Er Homeostasis

Abstract The endoplasmic reticulum (ER) is a highly dynamic organelle of fundamental importance present in all eukaryotic cells. The majority of synthesized structural and secreted proteins undergo post-translational modification, folding and oligomerization in the ER lumen, enabling proteins to carry out their physiological functions. Therefore, maintenance of ER homeostasis and function is imperative for proper cellular function. Physiological and pathological conditions can disturb ER homeostasis and thus negatively impact upon protein folding, resulting in an accumulation of unfolded proteins. Examples include hypoxia, hypo- and hyperglycemia, acidosis, and fluxes in calcium levels. Increased levels of unfolded/misfolded proteins within the ER lumen triggers a condition commonly referred to as ‘ER stress’. To combat ER stress, cells have evolved a highly conserved adaptive stress response referred to as the unfolded protein response (UPR). UPR signaling affords the cell a ‘window of opportunity’ for stress resolution however, if prolonged or excessive the UPR is insufficient and ER stress-induced cell death ensues. This review discusses the role of ER stress sensors IRE1, PERK and ATF6, describing their role in ER stress-induced death signaling with specific emphasis placed upon the importance of the intrinsic cell death pathway and Bcl-2 family regulation.

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Two Distinctly Localized P-Type ATPases Collaborate to Maintain Organelle Homeostasis Required for Glycoprotein Processing and Quality Control

Molecular Biology of the Cell ◽

10.1091/mbc.02-06-0090 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3955-3966 ◽

Cited By ~ 61

Author(s):

Shilpa Vashist ◽

Christian G. Frank ◽

Claude A. Jakob ◽

Davis T.W. Ng

Keyword(s):

Quality Control ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Ion Homeostasis ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Coa Reductase ◽

Protein Response ◽

P Type ◽

Er Homeostasis

Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1(control of HMG-CoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role.COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca2+/Mn2+pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside.

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The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

Molecular Biology of the Cell ◽

10.1091/mbc.8.9.1805 ◽

1997 ◽

Vol 8 (9) ◽

pp. 1805-1814 ◽

Cited By ~ 277

Author(s):

J S Cox ◽

R E Chapman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Lipid Synthesis ◽

Secretory Proteins ◽

Endoplasmic Reticulum Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

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Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast

Science Signaling ◽

10.1126/scisignal.aaz4401 ◽

2021 ◽

Vol 14 (684) ◽

pp. eaaz4401

Author(s):

Chandrima Ghosh ◽

Jagadeesh Kumar Uppala ◽

Leena Sathe ◽

Charlotte I. Hammond ◽

Ashish Anshu ◽

...

Keyword(s):

Unfolded Protein Response ◽

Protein Kinases ◽

Cellular Stress ◽

Mrna Processing ◽

Mrna Splicing ◽

Cellular Stress Response ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)–resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3′ untranslated region (3′UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.

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The Role of the ER-Induced UPR Pathway and the Efficacy of Its Inhibitors and Inducers in the Inhibition of Tumor Progression

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/5729710 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 13

Author(s):

Anna Walczak ◽

Kinga Gradzik ◽

Jacek Kabzinski ◽

Karolina Przybylowska-Sygut ◽

Ireneusz Majsterek

Keyword(s):

Er Stress ◽

Molecular Mechanisms ◽

Unfolded Proteins ◽

Cell Protection ◽

Protection Mechanism ◽

Unfolded Protein ◽

Er Stress Response ◽

A Cell ◽

The Unfolded Protein Response ◽

Protein Response

Cancer is the second most frequent cause of death worldwide. It is considered to be one of the most dangerous diseases, and there is still no effective treatment for many types of cancer. Since cancerous cells have a high proliferation rate, it is pivotal for their proper functioning to have the well-functioning protein machinery. Correct protein processing and folding are crucial to maintain tumor homeostasis. Endoplasmic reticulum (ER) stress is one of the leading factors that cause disturbances in these processes. It is induced by impaired function of the ER and accumulation of unfolded proteins. Induction of ER stress affects many molecular pathways that cause the unfolded protein response (UPR). This is the way in which cells can adapt to the new conditions, but when ER stress cannot be resolved, the UPR induces cell death. The molecular mechanisms of this double-edged sword process are involved in the transition of the UPR either in a cell protection mechanism or in apoptosis. However, this process remains poorly understood but seems to be crucial in the treatment of many diseases that are related to ER stress. Hence, understanding the ER stress response, especially in the aspect of pathological consequences of UPR, has the potential to allow us to develop novel therapies and new diagnostic and prognostic markers for cancer.

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ER stress causes widespread protein aggregation and prion formation

The Journal of Cell Biology ◽

10.1083/jcb.201612165 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2295-2304 ◽

Cited By ~ 28

Author(s):

Norfadilah Hamdan ◽

Paraskevi Kritsiligkou ◽

Chris M. Grant

Keyword(s):

Protein Aggregation ◽

Er Stress ◽

Secretory Pathway ◽

Cellular Protein ◽

Protein Homeostasis ◽

Er Quality Control ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

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XBP1

The Journal of Cell Biology ◽

10.1083/jcb.200406136 ◽

2004 ◽

Vol 167 (1) ◽

pp. 35-41 ◽

Cited By ~ 431

Author(s):

Rungtawan Sriburi ◽

Suzanne Jackowski ◽

Kazutoshi Mori ◽

Joseph W. Brewer

Keyword(s):

Active Form ◽

Membrane Phospholipids ◽

Unfolded Protein ◽

Xbp1 Mrna ◽

Phosphatidylcholine Biosynthesis ◽

Rough Er ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis ◽

Area And Volume

When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

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