Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP^Ufd1–Npl4 promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate αTCR. This favors the model where the Ufd1–Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.     

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

alpha-Chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin 1 fails to induce accumulation of alphaTCR despite inducing accumulation of alpha1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of alpha1-antitrypsin, it induces an increase in levels of alphaTCR. RNAi of VCP induces preferential accumulation of alphaTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high-mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they cannot be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.     

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.     

Characterization of WDR20: A new regulator of the ERAD machinery

ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.     

Sequential Actions of the AAA-ATPase Valosin-containing Protein (VCP)/p97 and the Proteasome 19 S Regulatory Particle in Sterol-accelerated,Endoplasmic Reticulum (ER)-associated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.     

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1

Subtilase cytotoxin (SubAB) is a AB₅ type toxin produced by Shiga-toxigenic Escherichia coli , which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α). The phosphorylation of PERK and eIF2α was maximal at 30–60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.     

Interaction between Salt-inducible Kinase 2 (SIK2) and p97/Valosin-containing Protein (VCP) Regulates Endoplasmic Reticulum (ER)-associated Protein Degradation in Mammalian Cells

Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.     

BRSK2 is a valosin-containing protein (VCP)-interacting protein that affects VCP functioning in endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated protein degradation (ERAD) removes improperly-folded proteins from the ER membrane into the cytosol where they undergo proteasomal degradation. Valosin-containing protein (VCP)/p97 mediates in the extraction of ERAD substrates from the ER. BRSK2 (also known as SAD-A), a serine/threonine kinase of the AMP-activated protein kinase family affected VCP/p97 activity in ERAD. In addition, BRSK2 interacted with VCP/p97 via three of the four functional domains of VCP/p97. Immunofluorescence demonstrated that BRSK2 and VCP/p97 were co-localized and also that knockdown of endogenous BRSK2 induced increased levels of CD3δ, a substrate in ERAD for VCP/p97. Thus, BRSK2 might affect the activity of VCP/p97 in ERAD.     

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man₉GlcNAc₂ is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.     

Ufd1-Npl4 is a negative regulator of cholera toxin retrotranslocation

The A1 chain of the cholera toxin (CT) undergoes retrotranslocation to the cytosol across the endoplasmic reticulum (ER) membrane by hijacking ER-associated degradation (ERAD). In the cytosol the CT A1 chain stimulates adenylyl cyclase. The VCP(Ufd1-Npl4) complex mediates retrotranslocation of emerging ER proteins. While one group reported that VCP is required for CT retrotranslocation, another group concluded the opposite. We show that VCP is dispensable for CT retrotranslocation, however RNAi of either Ufd1 or Npl4 induces an increase in adenylyl cyclase activity induced by CT. RNAi of VCP, Ufd1 or Npl4 did not affect adenylyl cyclase activity induced by forskolin. These findings are coherent with our previous report showing that depletion of Ufd1-Npl4 accelerates ERAD of reporter substrates. To integrate contradictory results we propose a new model, where Ufd1-Npl4 is a negative regulator of retrotranslocation, delaying the retrotranslocation of ERAD substrates independently of its association with VCP.     

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.     

BiP prevents rod opsin aggregation

Mutations in rod opsin-the light-sensitive protein of rod cells-cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT-green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP-expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.     

PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the ER membrane kinases PERK and IRE1 leading to the unfolded protein response (UPR). We show here that UPR activation triggers PERK and IRE1 segregation from BiP and their sorting with misfolded proteins to the ER-derived quality control compartment (ERQC), a pericentriolar compartment that we had identified previously. PERK phosphorylates translation factor eIF2alpha, which then accumulates on the cytosolic side of the ERQC. Dominant negative PERK or eIF2alpha(S51A) mutants prevent the compartmentalization, whereas eIF2alpha(S51D) mutant, which mimics constitutive phosphorylation, promotes it. This suggests a feedback loop where eIF2alpha phosphorylation causes pericentriolar concentration at the ERQC, which in turn amplifies the UPR. ER-associated degradation (ERAD) is an UPR-dependent process; we also find that ERAD components (Sec61beta, HRD1, p97/VCP, ubiquitin) are recruited to the ERQC, making it a likely site for retrotranslocation. In addition, we show that autophagy, suggested to play a role in elimination of aggregated proteins, is unrelated to protein accumulation in the ERQC.     

Glycosylation-independent ERAD pathway serves as a backup system under ER stress

During endoplasmic reticulum (ER)–associated degradation (ERAD), terminally misfolded proteins are retrotranslocated from the ER to the cytosol and degraded by the ubiquitin-proteasome system. Misfolded glycoproteins are recognized by calnexin and transferred to EDEM1, followed by the ER disulfide reductase ERdj5 and the BiP complex. The mechanisms involved in ERAD of nonglycoproteins, however, are poorly understood. Here we show that nonglycoprotein substrates are captured by BiP and then transferred to ERdj5 without going through the calnexin/EDEM1 pathway; after cleavage of disulfide bonds by ERdj5, the nonglycoproteins are transferred to the ERAD scaffold protein SEL1L by the aid of BiP for dislocation into the cytosol. When glucose trimming of the N-glycan groups of the substrates is inhibited, glycoproteins are also targeted to the nonglycoprotein ERAD pathway. These results indicate that two distinct pathways for ERAD of glycoproteins and nonglycoproteins exist in mammalian cells, and these pathways are interchangeable under ER stress conditions.     

The Unfolded Protein Response Transducer ATF6 Represents a Novel Transmembrane-type Endoplasmic Reticulum-associated Degradation Substrate Requiring Both Mannose Trimming and SEL1L Protein

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.     

AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.     

Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum chaperone BiP

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB₅ cytotoxins produced by Shiga toxigenic Escherichia coli . Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.