Misfolded Proteins In Endoplasmic Reticulum Research Articles

The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes

Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.

Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.

The IRE1α pathway in glomerular diseases: The unfolded protein response and beyond.

Endoplasmic reticulum (ER) function is vital for protein homeostasis ("proteostasis"). Protein misfolding in the ER of podocytes (glomerular visceral epithelial cells) is an important contributor to the pathogenesis of human glomerular diseases. ER protein misfolding causes ER stress and activates a compensatory signaling network called the unfolded protein response (UPR). Disruption of the UPR, in particular deletion of the UPR transducer, inositol-requiring enzyme 1α (IRE1α) in mouse podocytes leads to podocyte injury and albuminuria in aging, and exacerbates injury in glomerulonephritis. The UPR may interact in a coordinated manner with autophagy to relieve protein misfolding and its consequences. Recent studies have identified novel downstream targets of IRE1α, which provide new mechanistic insights into proteostatic pathways. Novel pathways of IRE1α signaling involve reticulophagy, mitochondria, metabolism, vesicular trafficking, microRNAs, and others. Mechanism-based therapies for glomerulopathies are limited, and development of non-invasive ER stress biomarkers, as well as targeting ER stress with pharmacological compounds may represent a therapeutic opportunity for preventing or attenuating progression of chronic kidney disease.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the tunicamycin transporter (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Disposing of misfolded ER proteins: A troubled substrate's way out of the ER

Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Those that fail must be recognized and eliminated to maintain ER homeostasis. Two cellular mechanisms have been identified to rid the ER of terminally unfolded, misfolded, and aggregated proteins. ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. ER-phagy has been more recently described and caters to larger, more complex proteins and protein aggregates that are not readily handled by ERAD. This pathway has unique upstream components and relies on the same downstream effectors of autophagy used in other cellular processes to deliver clients to lysosomes for degradation. In this review, we describe the main elements of ERQC, ERAD, and ER-phagy and focus on recent advances in these fields.

Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2.

The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an 'anti-Warburg' effect.

Crystal structure of the Schizosaccharomyces pombe U7BR E2-binding region in complex with Ubc7.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2-E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Å resolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.

Amino Acid Biosynthesis Regulation during Endoplasmic Reticulum Stress Is Coupled to Protein Expression Demands.

SummaryThe endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a complex cellular response to ER protein misfolding that involves transcriptional regulatory branches and a PERK-mediated translational regulatory branch.Here we revealed that amino acid biosynthesis regulation is coupled to protein synthesis demands during ER stress. Specifically, we demonstrated that the UPR leads to PERK-dependent induction in the biosynthesis of specific amino acids, and to upregulation of their corresponding tRNA synthetases. Furthermore, we found that sequences of UPR-upregulated proteins are significantly enriched with these UPR-induced amino acids. Interestingly, whereas the UPR leads to repression of ER target proteins, we showed that secreted proteins tended to escape this repression and were highly enriched for the UPR-induced amino acids.Our results unravel coordination between amino acid supply, namely, biosynthesis and tRNA loading, and demand from UPR-induced proteins under ER stress, thus revealing an additional regulatory layer of protein synthesis.

Laminar Flow Inhibits ER Stress-Induced Endothelial Apoptosis through PI3K/Akt-Dependent Signaling Pathway.

Atherosclerosis preferentially involves in prone area of low and disturbed blood flow while steady and high levels of laminar blood flow are relatively protected from atherosclerosis. Disturbed flow induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). ER stress is caused under stress that disturbs the processing and folding of proteins resulting in the accumulation of misfolded proteins in the ER and activation of the UPR. Prolonged or severe UPR leads to activate apoptotic signaling. Recent studies have indicated that disturbed flow significantly up-regulated p-ATF6α, p-IRE1α, and its target spliced XBP-1. However, the role of laminar flow in ER stress-mediated endothelial apoptosis has not been reported yet. The present study thus investigated the role of laminar flow in ER stress-dependent endothelial cell death. The results demonstrated that laminar flow protects ER stress-induced cleavage forms of PARP-1 and caspase-3. Also, laminar flow inhibits ER stress-induced p-eIF2α, ATF4, CHOP, spliced XBP-1, ATF6 and JNK pathway; these effects are abrogated by pharmacological inhibition of PI3K with wortmannin. Finally, nitric oxide affects thapsigargin-induced cell death in response to laminar flow but not UPR. Taken together, these findings indicate that laminar flow inhibits UPR and ER stress-induced endothelial cell death via PI3K/Akt pathway.

“Mallostery”—ligand-dependent protein misfolding enables physiological regulation by ERAD

HMG-CoA reductase (HMGR) undergoes regulated degradation as part of feedback control of the sterol pathway. In yeast, the stability of the HMGR isozyme Hmg2 is controlled by the 20-carbon isoprenoid geranylgeranyl pyrophosphate (GGPP). Increasing GGPP levels cause more efficient degradation by the HMG-CoA reductase degradation (HRD) pathway, allowing for feedback regulation of HMGR. The HRD pathway is critical for the endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded ER proteins. Here, we have explored GGPP's role in HRD-dependent Hmg2 degradation. We found that GGPP potently regulates Hmg2 levels in vivo and causes reversible Hmg2 misfolding at nanomolar concentrations in vitro These GGPP-mediated effects were absent in several stabilized or nonregulated Hmg2 mutants. Consistent with its high potency, GGPP's effects were highly specific such that other structurally related molecules were ineffective in altering Hmg2 structure. For instance, two closely related GGPP analogues, 2F-GGPP and GGSPP, were completely inactive at all concentrations tested. Furthermore, GGSPP antagonized GGPP's effects in vivo and in vitro Chemical chaperones reversed GGPP's effects on Hmg2 structure and degradation, suggesting that GGPP causes selective Hmg2 misfolding. These results indicate that GGPP functions in a manner similar to an allosteric ligand, causing Hmg2 misfolding through interaction with a reversible, specific binding site. Consistent with this, the Hmg2 protein formed multimers, typical of allosteric proteins. We propose that this "allosteric misfolding," or mallostery, observed here for Hmg2 may be a widely used tactic of biological regulation with potential for development of therapeutic small molecules that induce selective misfolding.

New Insights into the Physiological Role of Endoplasmic Reticulum-Associated Degradation

Many human diseases are associated with mutations causing protein misfolding and aggregation in the endoplasmic reticulum (ER). ER-associated degradation (ERAD) is a principal quality-control mechanism responsible for targeting misfolded ER proteins for cytosolic degradation. However, despite years of effort, the physiological role of ERAD in vivo remains largely unknown. Several recent studies have reported intriguing phenotypes of mice deficient for ERAD function in specific cell types. These studies highlight that mammalian ERAD has been designed to perform a wide-range of cell-type-specific functions in vivo in a substrate-dependent manner.

Lunapark Is a Component of a Ubiquitin Ligase Complex Localized to the Endoplasmic Reticulum Three-way Junctions

The endoplasmic reticulum (ER) network comprises sheets and tubules that are connected by dynamic three-way junctions. Lunapark (Lnp) localizes to and stabilizes ER three-way junctions by antagonizing the small GTPase Atlastin, but how Lnp shapes the ER network is unclear. Here, we used an affinity purification approach and mass spectrometry to identify Lnp as an interacting partner of the ER protein quality control ubiquitin ligase gp78. Accordingly, Lnp purified from mammalian cells has a ubiquitin ligase activity in vitro Intriguingly, biochemical analyses show that this activity can be attributed not only to associated ubiquitin ligase, but also to an intrinsic ubiquitin ligase activity borne by Lnp itself. This activity is contained in the N-terminal 45 amino acids of Lnp although this segment does not share homology to any known ubiquitin ligase motifs. Despite its interaction with gp78, Lnp does not seem to have a broad function in degradation of misfolded ER proteins. On the other hand, the N-terminal ubiquitin ligase-bearing motif is required for the ER three-way junction localization of Lnp. Our study identifies a new type of ubiquitin ligase and reveals a potential link between ubiquitin and ER morphology regulation.

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis.

In mutant INS gene-induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.

Gp78, an E3 ubiquitin ligase acts as a gatekeeper suppressing nonalcoholic steatohepatitis (NASH) and liver cancer.

Nonalcoholic steatohepatitis (NASH) is related to metabolic dysregulation and the perturbation of endoplasmic reticulum (ER) homeostasis that frequently develops into hepatocellular carcinoma (HCC). Gp78 is E3 ligase, which regulates endoplasmic reticulum-associated degradation (ERAD) by ubiquitinylation of misfolded ER proteins. Here, we report that upon ageing (12 months), gp78-/- mice developed obesity, recapitulating age-related human NASH. Liver histology of gp78-/- mice revealed typical steatosis, hepatic inflammation and fibrosis, followed by progression to hepatocellular tumors. Acute ER stress revealed that loss of gp78 results in up regulation of unfolded protein response (UPR) pathways and SREBP-1 regulating de novo lipogenesis, responsible for fatty liver. Tissue array of human hepatocellular carcinoma (HCC) demonstrated that the expression of gp78 was inversely correlated with clinical grades of cancer. Here, we have described the generation of the first preclinical experimental model system which spontaneously develops age-related NASH and HCC, linking ERAD to hepatosteatosis, cirrhosis, and cancer. It suggests that gp78 is a regulator of normal liver homeostasis and a tumor suppressor in human liver.

The role of EDEM2 compared with EDEM1 in ricin transport from the endoplasmic reticulum to the cytosol

EDEM1 [ER (endoplasmic reticulum)-degradation-enhancing α-mannosidase I-like protein 1] and EDEM2 are crucial regulators of ERAD (ER-associated degradation) that extracts non-native glycoproteins from the calnexin chaperone system. Ricin is a potent plant cytotoxin composed of an A-chain (RTA) connected by a disulfide bond to a cell-binding lectin B-chain (RTB). After endocytic uptake, the toxin is transported retrogradely to the ER, where the enzymatically active RTA is translocated to the cytosol in a similar manner as misfolded ER proteins. This transport is promoted by EDEM1. In the present study we report that EDEM2 is also involved in ricin retrotranslocation out of the ER. However, the role of EDEM1 and EDEM2in ricin transport to the cytosol seems to differ. EDEM2 promotes ricin retrotranslocation irrespectively of ER translocon accessibility; moreover, co-immunoprecipitation and pull-down studies revealed that more ricin can interact with EDEM2 in comparison with EDEM1. On the other hand, interactions of both lectins with RTA are dependent on the structure of the RTA. Thus our data display a newly discovered role for EDEM2. Moreover, analysis of the involvement of EDEM1 and EDEM2 in ricin retrotranslocation to the cytosol may provide crucial information about general mechanisms of the recognition of ERAD substrates in the ER.

Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: Cause and consequence

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR.

Transport of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II molecules

Nascent MHC class II molecules are associated with the invariant chain and are transported to the endolysosomal pathway, where MHC class II molecules acquire peptide antigens. On the other hand, misfolded endoplasmic reticulum (ER) proteins are generally degraded in the cells and are neither expressed on the cell surface nor secreted. Here, we found that MHC class II molecules associate with some misfolded ER proteins via the peptide-binding groove in competition with invariant chain. The misfolded proteins associated with MHC class II molecules are transported intact to the cell surface without processing to peptides. Furthermore, these complexes efficiently stimulate antigen-specific B cells. These findings reveal that MHC class II molecules function as a chaperone for the cell surface expression of misfolded ER proteins. In addition, we suggest that MHC class II molecules present not only peptides but also intact host-cell-derived proteins on the cell surface. These findings provide new insights into the function of MHC class II molecules.

Endoplasmic Reticulum-Dependent Redox Reactions Control Endoplasmic Reticulum-Associated Degradation and Pathogen Entry

Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host-pathogen interactions. Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions.

Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets.