ER-associated degradation in protein quality control and cellular regulation

The ER-associated degradation (ERAD) pathway directs ubiquitin-mediated degradation of a variety of ER-associated misfolded and normal proteins. Recent studies have delineated the molecular machinery responsible for protein ubiquitination and highlighted mechanistic questions surrounding the recognition, extraction and proteasomal destruction of the diverse array of ERAD substrates. Consideration of separate lines of work on this versatile pathway now indicate that despite its central role as an avenue of cellular quality control, ERAD is also harnessed for feedback regulation of sterol synthesis, and most likely numerous other cellular processes. These studies give ERAD a larger role in cellular function, and imply that cellular quality-control pathways could be widely employed in both natural and pharmaceutical control of individual proteins.     

A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation

The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER-associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing alpha-mannosidase-like protein (EDEM) is similar to class I alpha1,2-mannosidases involved in N-glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded alpha1-antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N-glycan dependent manner.     

ER protein quality control and proteasome-mediated protein degradation

A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system. In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER. Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome. Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones.     

Quality control: ER-associated degradation: Protein quality control and beyond

Even with the assistance of many cellular factors, a significant fraction of newly synthesized proteins ends up misfolded. Cells evolved protein quality control systems to ensure that these potentially toxic species are detected and eliminated. The best characterized of these pathways, the ER-associated protein degradation (ERAD), monitors the folding of membrane and secretory proteins whose biogenesis takes place in the endoplasmic reticulum (ER). There is also increasing evidence that ERAD controls other ER-related functions through regulated degradation of certain folded ER proteins, further highlighting the role of ERAD in cellular homeostasis.Newly synthesized membrane and secreted proteins enter the ER in an unfolded state through a protein-conducting channel named the translocon (Rapoport, 2007). In the ER, a myriad of chaperones and modifying enzymes assist their membrane integration and folding. In many cases folding involves post-translational modifications, such as glycosylation or disulfide bond formation (Braakman and Hebert, 2013). At this stage many proteins are also assembled into multisubunit complexes with defined stoichiometries. As newly synthesized proteins reach a native conformation, they leave the ER to perform their function elsewhere; either along the secretory pathway or outside of the cell.Despite all the resources dedicated to protein folding, a significant fraction of newly synthesized polypeptides entering the ER fails to acquire a native conformation (Hartl and Hayer-Hartl, 2009). The degree of misfolding of these proteins varies considerably and can have several causes such as mutations, substoichiometric amounts of a binding partner, or merely a shortage of chaperone availability. In most cases, the misfolded molecules are retained in the ER and eventually become substrates of the ER-associated protein degradation (ERAD), a collection of quality-control mechanisms that clears the ER from these potentially harmful species. Inactivation of ERAD results in the accumulation of misfolded proteins in the lumen and membrane of the ER, a condition known as ER stress that is common to several diseases (Walter and Ron, 2011). For this reason, ERAD plays a key role in ER homeostasis across eukaryotes. Genetic ablation of a number of ERAD components leads to embryonic lethality in mice, also highlighting the importance of this process in cellular and organismal homeostasis (Yagishita et al., 2005; Francisco et al., 2010; Eura et al., 2012). Whether this essential function of ERAD during mouse development is due to its role in the degradation of misfolded proteins remains to be determined.Certain folded, perfectly active proteins are also targeted by ERAD. However, their degradation is highly regulated and only occurs in the presence of a specific signal. The best-characterized regulated substrate is the 3-hydroxy-3-methylglutaryl acetyl-coenzyme-A reductase (HMGR), a key enzyme in sterol biosynthesis (Gil et al., 1985; Hampton et al., 1996; Bays et al., 2001a; Song et al., 2005). Both in yeast and in mammals, HMGR degradation by ERAD is part of a feedback inhibition system critical for sterol homeostasis. Interestingly, another enzyme of the sterol biosynthetic pathway, squalene monooxygenase (Erg1 in yeast and SQLE in mammals), was recently identified as a regulated ERAD substrate (Foresti et al., 2013). The degradation of Erg1/SQLE by ERAD is again part of a feedback inhibition system to prevent the accumulation of intermediate sterol metabolites, which are toxic for cells (Foresti et al., 2013). Recent evidence shows that regulation of the synthesis of sterols and other sterol-derived metabolites by ERAD is also present in plants (Doblas et al., 2013; Pollier et al., 2013). This evolutionarily conserved role of ERAD in sterol regulation might have been one of its primordial functions.The ERAD machinery is also exploited by certain viruses to degrade host proteins thereby escaping immune surveillance. Well characterized examples are the degradation of newly synthesized major histocompatibility complex class I (MHC I) heavy chain (Wiertz et al., 1996a) or CD4 molecules by the human cytomegalovirus or the immunodeficiency virus (HIV; Fujita et al., 1997; Schubert et al., 1998), respectively. Moreover, some bacterial toxins, such as cholera, and viruses, like simian virus 40 (SV40), travel to the ER retrogradely through the secretory pathway. At the ER these toxins and viruses exploit ERAD components to reach the cytosol, where ultimately they will act (Tsai et al., 2001; Schelhaas et al., 2007; Bernardi et al., 2008).Finally, ERAD components are also involved in the turnover of several soluble proteins in the cytoplasm and the nucleus of cells (Swanson et al., 2001; Ravid et al., 2006; Yamasaki et al., 2007). Most of these cases, however, involve only a subset of the ERAD steps and components. In sum, although a complete repertoire of substrates is not available, it is clear that misfolded proteins are not the exclusive clients of ERAD.

ERAD, linking ER quality control to cytoplasmic protein degradation

The earliest evidence for protein quality control at the ER came from observations that unassembled subunits of the T cell receptor were rapidly degraded in the cells (Lippincott-Schwartz et al., 1988). This degradation occurred independently of lysosomal proteases, leading to the proposal that the ER itself would house some uncharacterized proteolytic activity toward misfolded proteins. Then a landmark study in yeast showed that the degradation of a short-lived misfolded ER membrane protein was blocked in cells lacking Ubc6, a component of the ubiquitin conjugation machinery (Sommer and Jentsch, 1993). The ubiquitin system mediates the covalent attachment of ubiquitin, a small 76–amino acid protein, to target proteins in the cytoplasm by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes (Pickart, 2001). Ubiquitin-modified proteins are then recognized and degraded by the proteasome. The involvement of the ubiquitin–proteasome system in ER protein quality control was confirmed by studies on the degradation of mutant and wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a large membrane protein with a complicated folding process (Jensen et al., 1995; Ward et al., 1995). Inhibition of proteasome function led to accumulation of CFTR molecules, and interestingly, a significant fraction of these was detected as ubiquitin conjugates (Jensen et al., 1995; Ward et al., 1995). Soon after, it became clear that a similar mechanism could also account for the degradation of luminal misfolded proteins such as CPY*, a mutant version of the yeast vacuolar carboxypeptidase Y and a prototype ERAD substrate (Hiller et al., 1996). Together, these papers demonstrated that aberrant proteins in the lumen and membrane of the ER are degraded in the cytoplasm where the components of the ubiquitin–proteasome system reside.

Ubiquitin ligase complexes: The hubs in ERAD

Subsequent genetic and biochemical studies, primarily in budding yeast but also in mammalian cells, identified many ERAD components and led to a general understanding of the organization of the pathway. An important realization was that the “one-size-fits-all” model does not apply to ERAD and that this pathway encompasses multiple branches with distinct specificity for different classes of misfolded proteins (Taxis et al., 2003; Vashist and Ng, 2004; Carvalho et al., 2006; Bernasconi et al., 2010; Christianson et al., 2012). However, irrespective of the branch, the same sequence of events leads to the degradation of all ERAD substrates (Fig. 1 A). The first step is the recognition of a substrate in the crowded ER environment. Then the substrate is transported across the ER lipid bilayer back into the cytoplasm, a step known as retrotranslocation. On the cytosolic side of the ER membrane, the substrate is ubiquitinated by a membrane-associated ubiquitin ligase (or E3 ligase). Subsequently, the ubiquitinated substrate is extracted from the membrane in an ATP-dependent manner and released in the cytoplasm for degradation by the proteasome. The execution of these steps is coordinated by a membrane-embedded protein complex named after the E3 ligase at its core. The canonical E3 ligases involved in ERAD are themselves multispanning membrane proteins, in which the RING (really interesting new gene) domain responsible for the ligase activity is in the cytoplasm. These E3 ligase complexes are best characterized in yeast (Fig. 1 B and Swanson et al., 2001) and Hrd1 (Bordallo et al., 1998; Bays et al., 2001a) assemble into the Doa10 and the Hrd1 complexes, respectively, each responsible for the degradation of a class of ERAD substrates (Carvalho et al., 2006).Open in a separate windowFigure 1.The different steps and branches in ERAD. (A) The events defining the ERAD of a generic luminal substrate with a misfolded domain (red star). Substrate recognition, retrotranslocation, and ubiquitination are coordinated by a membrane-embedded E3 ligase complex. Ubiquitin is depicted as small circles. (B) The E3 ligase complexes involved in ERAD in S. cerevisiae and their substrate specificities. ER proteins with a misfolded domain in the cytoplasm (ERAD-C substrates) are degraded via the Doa10 complex. Proteins with luminal (ERAD-L) or intramembrane (ERAD-M) misfolded domains are degraded via the Hrd1 complex. Misfolded domains on proteins are indicated by a red star. The Cdc48 cofactors Npl4 and Ufd1 are depicted as N and U, respectively.

Table 1.

Components of the yeast E3 ligase complexes and their mammalian counterparts

Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.     

Autophagy: an ER protein quality control process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome-dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both alpha1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.     

Human HRD1 protects against ER stress-induced apoptosis through ER-associated degradation

Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.     

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.     

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.     

ERdj4 protein is a soluble endoplasmic reticulum (ER) DnaJ family protein that interacts with ER-associated degradation machinery

Protein localization within cells regulates accessibility for interactions with co-factors and substrates. The endoplasmic reticulum (ER) BiP co-factor ERdj4 is up-regulated by ER stress and has been implicated in ER-associated degradation (ERAD) of multiple unfolded secretory proteins. Several other ERdj family members tend to interact selectively with nascent proteins, presumably because those ERdj proteins associate with the Sec61 translocon that facilitates entry of nascent proteins into the ER. How ERdj4 selects and targets terminally misfolded proteins for destruction remains poorly understood. In this study, we determined properties of ERdj4 that might aid in this function. ERdj4 was reported to retain its signal sequence and to be resistant to mild detergent extraction, suggesting that it was an integral membrane protein. However, live cell photobleaching analyses of GFP-tagged ERdj4 revealed that the protein exhibits diffusion coefficients uncommonly high for an ER integral membrane protein and more similar to the mobility of a soluble luminal protein. Biochemical characterization established that the ERdj4 signal sequence is cleaved to yield a soluble protein. Importantly, we found that both endogenous and overexpressed ERdj4 associate with the integral membrane protein, Derlin-1. Our findings now directly link ERdj4 to the ERAD machinery and suggest a model in which ERjd4 could help recruit clients from throughout the ER to ERAD sites.     

A novel ER J-protein DNAJB12 accelerates ER-associated degradation of membrane proteins including CFTR

Cytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membrane-spanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.     

A regulatory link between ER-associated protein degradation and the unfolded-protein response

Ubiquitin conjugation during endoplasmic-reticulum-associated degradation (ERAD) depends on the activity of Ubc7. Here we show that Ubc1 acts as a further ubiquitin-conjugating enzyme in this pathway. Absence of both enzymes results in marked stabilization of an ERAD substrate and induction of the unfolded-protein response (UPR). Furthermore, basic ERAD activity is sufficient to eliminate unfolded proteins under normal conditions. However, when stress is applied, the UPR is required to increase ERAD activity. We thus demonstrate, for the first time, a regulatory loop between ERAD and the UPR, which is essential for normal growth of yeast cells.     

213 Structures and interactions of proteins involved in ER-associated protein degradation

Proteins are translocated into the endoplasmic reticulum (ER) of cells in an unfolded state, and acquire their native conformation in the ER lumen after signal peptide cleavage. ER-associated degradation (ERAD) of folding-incompetent protein chains is mediated by the protein complexes residing in the ER membrane. We study the architecture and function of one of these, the HRD complex assembled around the E3 ubiquitin ligase Hrd1. The recognition of ERAD substrates is linked to the maturation of their carbohydrate structures. The HRD complex-associated lectin Yos9 is involved in ERAD substrate recognition by binding carbohydrates through its mannose-6-phosphate receptor homology (MRH) domain. We have determined the crystal structure of a central domain of Yos9, adjacent to the MRH domain, which was previously annotated as interaction region with the HRD subunit Hrd3 (Hanna et al., 2012). We find that this domain does not support Hrd3 association which we map to the N-terminal half of Yos9 instead. In contrast, the domain has a function in Yos9 dimerization as seen in the crystal structure, in various solution experiments and as supported by mutagenesis of dimer interface residues. The dimerization of the ER-luminal Yos9, in conjunction with studies of the cytosolic domain of the HRD component Usa1 (Horn et al., 2009) and other biochemical data thus supports a model of a HRD complex that exists and functions as a dimer or a higher multimer. The delivery of ubiquitinated ERAD substrates to the proteasome is mediated by the cytosolic AAA ATPase Cdc48 (p97 in mammalian cells). The p97 (VCP) serves a wide variety of cellular functions in addition to its role in ERAD, including organelle membrane fusion, mitosis, DNA repair, and apoptosis. These different functions are linked to the binding of adaptor proteins to p97, many of which contain ubiquitin regulatory X (UBX) domains. One of these adaptors, ASPL (alveolar soft part sarcoma locus), uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers leading to partial inactivation of the AAA ATPase. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA ATPase has been demonstrated. This observation has far reaching implications for AAA ATPase-regulated processes.     

ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together

A protein-degradation pathway associated with the endoplasmic reticulum (ER) can selectively remove polypeptides from the secretory pathway. The mechanisms of this ER-associated protein degradation were obscure, but recent studies using both yeast and mammalian cells have indicated that substrates for degradation are targeted to the cytosol where proteolysis is catalysed by the proteasome. The degradation process is now known to comprise at least three distinct events: first, recognition of a polypeptide for degradation; second, efflux of this substrate from the ER to the cytosol; and, finally, degradation by the proteasome. This review summarizes recent advances in understanding how each of these steps is achieved.     

The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway

The hepatitis B virus precore protein is closely related to the nucleocapsid core protein but is processed distinctly in the cell and plays a different role in the viral cycle. Precore is addressed to the endoplasmic reticulum (ER) through a signal peptide, and the form present in the ER is the P22 protein. P22 is then cleaved in its C-terminal part to be secreted as HBe antigen. In addition, a cytosolic form of 22 kDa less characterized has been observed. Precore gene was shown to be implicated in viral persistence, but until now, the actual protein species involved has not been determined. Our work focuses on the cytosolic form of precore. Using human cells expressing precore and a convenient fractionation assay, we demonstrated that the cytosolic form is identical to the ER form and retrotransported in the cytoplasm through the ER-associated degradation pathway. This cellular machinery translocates misfolded proteins to the cytoplasm, where they are ubiquitinated on lysine residues and degraded by proteasome. We showed that precore escapes proteasome due to its low lysine content and accumulates in the cytosol. The role of this retrotransport was investigated. In the presence of precore, we found a specific redistribution of the Grp78/BiP chaperone protein to cytosol and demonstrated a specific interaction between precore and Grp78/BiP. Altogether, these data support the idea that the hepatitis B virus develops a strategy to take advantage of the ER-associated degradation pathway, allowing distinct subcellular localization and probably distinct roles for the viral precore protein.