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.     

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.     

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.     

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.     

Search and destroy: ER quality control and ER-associated protein degradation

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms.The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

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.     

Dissecting the ER-associated degradation of a misfolded polytopic membrane protein

It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p(*), a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p(*) association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.     

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.     

A conserved role of Caenorhabditis elegans CDC-48 in ER-associated protein degradation

Protein degradation mediated by the ubiquitin/proteasome system is essential for the elimination of misfolded proteins from the endoplasmic reticulum (ER) to adapt to ER stress. It has been reported that the AAA ATPase p97/VCP/CDC48 is required in this pathway for protein dislocation across the ER membrane and subsequent ubiquitin dependent degradation by the 26S proteasome in the cytosol. Throughout ER-associated protein degradation, p97 cooperates with a binary Ufd1/Npl4-complex. In Caenorhabditis elegans two homologs of p97, designated CDC-48.1 and CDC-48.2, exist. Our results indicate that both p97 homologs interact with UFD-1/NPL-4 in a similar CDC-48(UFD-1/NPL-4) complex. RNAi mediated depletion of the corresponding genes induces ER stress resulting in hypersensitivity to conditions which induce increased levels of unfolded proteins in the ER lumen. Together, these data suggest an evolutionarily conserved retro-translocation machinery at the endoplasmic reticulum.     

Ubx2 links the Cdc48 complex to ER-associated protein degradation

Endoplasmic reticulum (ER)-associated protein degradation requires the dislocation of selected substrates from the ER to the cytosol for proteolysis via the ubiquitin-proteasome system. The AAA ATPase Cdc48 (known as p97 or VCP in mammals) has a crucial, but poorly understood role in this transport step. Here, we show that Ubx2 (Sel1) mediates interaction of the Cdc48 complex with the ER membrane-bound ubiquitin ligases Hrd1 (Der3) and Doa10. The membrane protein Ubx2 contains a UBX domain that interacts with Cdc48 and an additional UBA domain. Absence of Ubx2 abrogates breakdown of ER proteins but also that of a cytosolic protein, which is ubiquitinated by Doa10. Intriguingly, our results suggest that recruitment of Cdc48 by Ubx2 is essential for turnover of both ER and non-ER substrates, whereas the UBA domain of Ubx2 is specifically required for ER proteins only. Thus, a complex comprising the AAA ATPase, a ubiquitin ligase and the recruitment factor Ubx2 has a central role in ER-associated proteolysis.     

Vpu-mediated degradation of CD4 reconstituted in yeast reveals mechanistic differences to cellular ER-associated protein degradation

In HIV infected cells, the plasma membrane protein CD4 is removed from the secretory pathway by proteasomal digestion. This crucial step of viral infection occurs at the endoplasmic reticulum and is triggered by the HIV encoded protein Vpu. Here we show that this process can be recapitulated in baker's yeast. The analysis in the yeast system revealed that Vpu-induced breakdown of CD4 occurs independently of the cellular ER-associated protein degradation system. Moreover, our system allows direct comparison between Vpu-mediated turnover and cellular ER-associated protein degradation of CD4. This analysis suggests fundamental mechanistic differences between both pathways: Vpu-induced turnover strictly relies on ubiquitination of CD4 at cytosolic lysine residues prior to export of the substrate from the membrane. In contrast, the cellular ER-associated protein degradation pathway can transport ER-lumenal parts of CD4 into the cytoplasm before ubiquitination and extraction of the membrane anchor.     

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.     

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.     

Membrane proteins involved in pollen-pistil interactions.

Self-incompatibility (SI) is a widespread mechanism in angiosperms which prevents self-fertilization. This mechanism relies on cell-cell interactions between pollen and pistil. Among the different SI systems that have been reported, two have been particularly investigated: the gametophytic system of Solanaceae and the sporophytic system of Brassicaceae. In these two families, although the molecular bases of SI response are different, secreted and/or membrane-anchored proteins are required for self-pollen rejection. Interestingly, these proteins exhibit two functions: recognition and a catalytic activity. In this review article, we present recent advances which permit a better understanding of how these proteins control the male/female recognition event associated with the SI response.     

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways

About 40% of the eukaryotic cell’s proteins are inserted co- or post-translationally in the endoplasmic reticulum (ER), where they attain the native structure under the assistance of resident molecular chaperones and folding enzymes. Subsequently, these proteins are secreted from cells or are transported to their sites of function at the plasma membrane or in organelles of the secretory and endocytic compartments. Polypeptides that are not delivered within the ER (mis-localized proteins, MLPs) are rapidly destroyed by cytosolic proteasomes, with intervention of the membrane protease ZMPSTE24 if they remained trapped in the SEC61 translocation machinery. Proteins that enter the ER, but fail to attain the native structure are rapidly degraded to prevent toxic accumulation of aberrant gene products. The ER does not contain degradative devices and the majority of misfolded proteins generated in this biosynthetic compartment are dislocated across the membrane for degradation by cytosolic 26S proteasomes by mechanisms and pathways collectively defined as ER-associated degradation (ERAD). Proteins that do not engage ERAD factors, that enter aggregates or polymers, are too large, display chimico/physical features that prevent dislocation across the ER membrane (ERAD-resistant misfolded proteins) are delivered to endo-lysosome for clearance, by mechanisms and pathways collectively defined as ER-to-lysosomes-associated degradation (ERLAD). Emerging evidences lead us to propose ERLAD as an umbrella term that includes the autophagic and non-autophagic pathways activated and engaged by ERAD-resistant misfolded proteins generated in the ER for delivery to degradative endo-lysosomes.     

Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco- acceptor peptide to compete for core carbohydrates, or by using a radio- labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro- alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.