Network biology analysis of P23H rhodopsin interactome identifies protein and mRNA quality control mechanisms.

Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from RhoP23H/P23H mice and compared them with protein interaction partners of wild-type rhodopsin from Rho+/+ mice. We identified 286 proteins associated with P23H rhodopsin and 276 proteins associated with wild-type rhodopsin. 113 proteins were shared between wild-type and mutant rhodopsin protein interactomes. In the P23H rhodopsin protein interactome, we saw loss of phototransduction, retinal cycle, and rhodopsin protein trafficking proteins but gain of ubiquitin-related proteins when compared with the wild-type rhodopsin protein interactome. In the P23H rhodopsin protein interactome, we saw enrichment of gene ontology terms related to ER-associated protein degradation, ER stress, and translation. Protein-protein interaction network analysis revealed that translational and ribosomal quality control proteins were significant regulators in the P23H rhodopsin protein interactome. The protein partners identified in our study may provide new insights into how photoreceptors recognize and clear mutant rhodopsin, offering possible novel targets involved in retinal degeneration pathogenesis.

ATF6 is essential for human cone photoreceptor development.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

Multiexon deletion alleles of ATF6 linked to achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.

Pathomechanisms of ATF6-Associated Cone Photoreceptor Diseases.

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response (UPR). In response to endoplasmic reticulum (ER) stress, ATF6 is transported from the ER to the Golgi apparatus where it is cleaved by intramembrane proteolysis, releasing its cytosolic fragment. The cleaved ATF6 fragment, which is a basic leucine zipper (bZip) transcription factor, translocates to the nucleus and upregulates the expression of ER protein-folding chaperones and enzymes. Mutations in ATF6 cause heritable forms of cone photoreceptor dysfunction diseases. These mutations include missense, nonsense, splice site, and deletion or duplication changes found across the entire ATF6. To date, there are 11 ATF6 mutations reported, and we classified them into three classes based on their functional defects that interrupt distinct steps in the ATF6 signaling pathway.

ER stress and unfolded protein response in ocular health and disease.

The human eye is the organ that is able to react to light in order to provide sharp three-dimensional and colored images. Unfortunately, the health of the eye can be impacted by various stimuli that can lead to vision loss, such as environmental changes, genetic mutations, or aging. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling have been detected in many diverse ocular diseases, and chemical and genetic approaches to modulate ER stress and specific UPR regulatory molecules have shown beneficial effects in animal models of eye disease. This review highlights specific eye diseases associated with ER stress and UPR activity, based on a recent symposia exploring this theme.

The unfolded protein response regulator ATF6 promotes mesodermal differentiation.

ATF6 encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6ß results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with ATF6 mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.

A 45 nm Stacked CMOS Image Sensor Process Technology for Submicron Pixel.

A submicron pixel's light and dark performance were studied by experiment and simulation. An advanced node technology incorporated with a stacked CMOS image sensor (CIS) is promising in that it may enhance performance. In this work, we demonstrated a low dark current of 3.2 e-/s at 60 °C, an ultra-low read noise of 0.90 e-·rms, a high full well capacity (FWC) of 4100 e-, and blooming of 0.5% in 0.9 µm pixels with a pixel supply voltage of 2.8 V. In addition, the simulation study result of 0.8 µm pixels is discussed.

Autosomal recessive cone-rod dystrophy can be caused by mutations in the ATF6 gene.

Inherited retinal dystrophies (IRDs) are clinically and genetically highly heterogeneous, making clinical diagnosis difficult. The advances in high-throughput sequencing (ie, panel, exome and genome sequencing) have proven highly effective on defining the molecular basis of these disorders by identifying the underlying variants in the respective gene. Here we report two siblings affected by an IRD phenotype and a novel homozygous c.1691A>G (p.(Asp564Gly)) ATF6 (activating transcription factor 6A) missense substitution identified by whole exome sequencing analysis. The pathogenicity of the variant was confirmed by functional analyses done on patients' fibroblasts and on recombinant p.(Asp564Gly) protein. The ATF6Asp564Gly/Asp564Gly variant shows impaired production of the ATF6 cleaved transcriptional activator domain in response to endoplasmic reticulum stress. Detailed phenotypic examination revealed extinguished cone responses but also decreased rod responses together with the ability to discriminate some colours suggestive rather for cone-rod dystrophy than achromatopsia.

Achromatopsia mutations target sequential steps of ATF6 activation.

Achromatopsia is an autosomal recessive disorder characterized by cone photoreceptor dysfunction. We recently identified activating transcription factor 6 (ATF6) as a genetic cause of achromatopsia. ATF6 is a key regulator of the unfolded protein response. In response to endoplasmic reticulum (ER) stress, ATF6 migrates from the ER to Golgi to undergo regulated intramembrane proteolysis to release a cytosolic domain containing a basic leucine zipper (bZIP) transcriptional activator. The cleaved ATF6 fragment migrates to the nucleus to transcriptionally up-regulate protein-folding enzymes and chaperones. ATF6 mutations in patients with achromatopsia include missense, nonsense, splice site, and single-nucleotide deletion or duplication changes found across the entire gene. Here, we comprehensively tested the function of achromatopsia-associated ATF6 mutations and found that they group into three distinct molecular pathomechanisms: class 1 ATF6 mutants show impaired ER-to-Golgi trafficking and diminished regulated intramembrane proteolysis and transcriptional activity; class 2 ATF6 mutants bear the entire ATF6 cytosolic domain with fully intact transcriptional activity and constitutive induction of downstream target genes, even in the absence of ER stress; and class 3 ATF6 mutants have complete loss of transcriptional activity because of absent or defective bZIP domains. Primary fibroblasts from patients with class 1 or class 3 ATF6 mutations show increased cell death in response to ER stress. Our findings reveal that human ATF6 mutations interrupt distinct sequential steps of the ATF6 activation mechanism. We suggest that increased susceptibility to ER stress-induced damage during retinal development underlies the pathology of achromatopsia in patients with ATF6 mutations.

Endoplasmic reticulum stress in human photoreceptor diseases.

Photoreceptors are specialized sensory neurons essential for light detection in the human eye. Photoreceptor cell dysfunction and death cause vision loss in many eye diseases such as retinitis pigmentosa and achromatopsia. Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling have been implicated in the development and pathology of heritable forms of retinitis pigmentosa and achromatopsia. We review the role of ER stress and UPR in retinitis pigmentosa arising from misfolded rhodopsins (RHO) and in achromatopsia arising from genetic mutations in Activating Transcription Factor 6 (ATF6). This article is part of a Special Issue entitled SI:ER stress.

Ablation of Chop Transiently Enhances Photoreceptor Survival but Does Not Prevent Retinal Degeneration in Transgenic Mice Expressing Human P23H Rhodopsin.

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse.

PURPOSE: Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. METHODS: We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. RESULTS: We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. CONCLUSIONS: Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.

Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6(-/-) mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype.

Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death.

Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection.

Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

The effects of IRE1, ATF6, and PERK signaling on adRP-linked rhodopsins.

Many mutations in rhodopsin gene linked to retinitis pigmentosa (RP) cause rhodopsin misfolding. Rod photoreceptor cells expressing misfolded rhodopsin eventually die. Identifying mechanisms to prevent rhodopsin misfolding or to remove irreparably misfolded rhodopsin could provide new therapeutic strategies. IRE1, ATF6, and PERK signaling pathways, collectively called the unfolded protein response (UPR), regulate the functions of endoplasmic reticulum, responsible for accurate folding of membrane proteins such as rhodopsin. We used chemical and genetic approaches to selectively activate IRE1, ATF6, or PERK signaling pathways one at a time and analyzed their effects on mutant rhodopsin linked to RP. We found that both artificial IRE1 and ATF6 signaling promoted the degradation of mutant rhodopsin with lesser effects on wild-type rhodopsin. Furthermore, IRE1 and ATF6 signaling preferentially reduced levels of aggregated rhodopsins. By contrast, PERK signaling reduced levels of wild-type and mutant rhodopsin. These studies indicate that activation of either IRE1, ATF6, or PERK prevents mutant rhodopsin from accumulating in the cells. In addition, activation of IRE1 or ATF6 can selectively remove aggregated or mutant rhodopsin from the cells and may be useful in treating RP associated with rhodopsin protein misfolding.

De novo prion aggregates trigger autophagy in skeletal muscle.

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Induction of endoplasmic reticulum stress genes, BiP and chop, in genetic and environmental models of retinal degeneration.

PURPOSE: Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes. METHODS: Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses. RESULTS: S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss. CONCLUSIONS: Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Selective activation of ATF6 and PERK endoplasmic reticulum stress signaling pathways prevent mutant rhodopsin accumulation.

PURPOSE: Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding. METHODS: To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins. RESULTS: ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell. CONCLUSIONS: This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin.

Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.