Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system

Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alpha. In addition, pro-alpha factor photocrosslinked derlin-1, but not Sec61alpha. Thus, derlin-1 appears to be involved in pro-alpha factor retrotranslocation.     

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p.

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.     

Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY

Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD). CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex. However, although the functions of some of the components of this complex are known, the nature of the protein dislocation channel remains obscure. Sec61p has been suggested as an obvious candidate because of its role as a protein-conducting channel through which polypeptides are initially translocated into the ER. However, it has not yet been possible to functionally dissect any role for Sec61p in dislocation from its essential function in translocation. By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.     

The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron

Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates.     

Inhibition of p97-dependent protein degradation by Eeyarestatin I

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.     

The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.     

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.     

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.     

Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway.

We have investigated the degradation of subunits of the trimeric Sec61p complex, a key component of the protein translocation apparatus of the ER membrane. A mutant form of Sec6lp and one of the two associated proteins (Sss1p) are selectively degraded, while the third constituent of the complex (Sbh1p) is stable. Our results demonstrate that the proteolysis of the multispanning membrane protein Sec61p is mediated by the ubiquitin-proteasome pathway, since it requires polyubiquitination, the presence of a membrane-bound (Ubc6) and a soluble (Ubc7) ubiquitin-conjugating enzyme and a functional proteasome. The process is proposed to be specific for unassembled Sec61p and Sss1p. Thus, our results suggest that one pathway of ER degradation of abnormal or unassembled membrane proteins is initiated at the cytoplasmic side of 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.     

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process.The endoplasmic reticulum (ER)²-associated degradation (ERAD) pathway mediates the destruction of numerous integral membrane or lumenal ER-localized proteins (1, 2). ERAD functions mainly in the disposal of misfolded or unassembled proteins but also participates in the physiological regulation of some normal residents of the organelle. This ER-localized degradation pathway has been implicated in a wide variety of normal and pathophysiological processes, including sterol synthesis (3, 4), rheumatoid arthritis (5), fungal differentiation (6), cystic fibrosis (7, 8), and several neurodegenerative diseases (9). Accordingly, there is great impetus to understand the molecular mechanisms that mediate this broadly important route of protein degradation.ERAD proceeds by the ubiquitin-proteasome pathway, by which an ER-localized substrate is covalently modified by the addition of multiple copies of 7.6-kDa ubiquitin to form a multiubiquitin chain that is recognized by the cytosolic 26S proteasome (10, 11). Ubiquitin is added to the substrate by the successive action of three enzymes. The E1 ubiquitin-activating enzyme uses ATP to covalently add ubiquitin to an E2 ubiquitin-conjugating (UBC) enzyme. Ubiquitin is then transferred from the charged E2 to the substrate or the growing ubiquitin chain by the action of an E3 ubiquitin ligase, resulting in a substrate-attached multiubiquitin chain that is recognized by the proteasome, leading to degradation of the ubiquitinated substrate. This is a skeletal picture; in most cases, ancillary factors participate in substrate recognition and transfer of the ubiquitinated substrate to the proteasome (12–14).ERAD substrates are either sequestered in the lumen or embedded in the ER membrane with lumenal portions. Thus, a critical step in the ERAD pathway involves transfer of the ERAD substrate to the cytosol for proteasomal degradation by a process referred to as retrotranslocation or dislocation (15). Retrotranslocation requires the hexameric AAA-ATPase called Cdc48p in yeast and p97 in mammals, and it is thought that a protein channel mediates the movement of substrates across the ER membrane. Channel candidates include the derlins (16, 17), the Sec61p anterograde channel (18, 19), or the multispanning domains of the ER ligases themselves (18–20).The yeast HRD pathway mediates ERAD of numerous misfolded ER proteins and the physiologically regulated degradation of the Hmg2p isozyme of HMG-CoA reductase, an 8-transmembrane span (8-spanning) integral membrane protein critical for sterol synthesis (3). The integral membrane ER ligase Hrd1p, in conjunction with Hrd3p, is responsible for ubiquitination of Hmg2p. Efficient delivery of ubiquitinated Hmg2p to the proteasome requires the Cdc48p-Ufd1p-Npl4p complex presumably by promoting retrotranslocation of ER-embedded Hmg2p.Due to the requirement for retrotranslocation in all ERAD pathways we have adapted our in vitro assay of Hrd1p-mediated ubiquitination of the normally degraded fusion Hmg2p-GFP to study this ER removal step in ERAD. We have reconstituted Hrd1p-mediated ubiquitination and retrotranslocation of Hmg2p-GFP in vitro (21, 22). We have now directly demonstrated that the entire 8-spanning Hmg2p-GFP protein is removed from the membrane by this process, remaining intact yet soluble after retrotranslocation. The dislocation of intact Hmg2p-GFP required both Cdc48p and hydrolysis of the β–γ bond of ATP. The Ubx2p adaptor protein functioned in a manner consistent with its proposed role in Cdc48p anchoring to the ER. Surprisingly, the Dsk2p/Rad23p proteasomal coupling factors were also required for retrotranslocation. Neither derlins nor Sec61p were implicated in Hmg2p-GFP retrotranslocation by our assay. Furthermore, an engineered substrate based on HMG-CoA reductase underwent ERAD in the complete absence of Hrd1p or Doa10p and in vitro, full-length retrotranslocation, both indicating that the large transmembrane domains of either of these ERAD E3 ligases were not required for membrane extraction. Taken together, these studies define a core set of proteins that can mediate recognition and retrotranslocation of the HRD substrate Hmg2p-GFP and will allow mechanistic analysis along all points of the ERAD pathway.     

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here, we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.     

Endoplasmic reticulum-associated degradation of ricin A chain has unique and plant-specific features

Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.     

Role of HERP and a HERP-related Protein in HRD1-dependent Protein Degradation at the Endoplasmic Reticulum

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2.     

AAA-ATPase p97/Cdc48p, a Cytosolic Chaperone Required for Endoplasmic Reticulum-Associated Protein Degradation

Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones. Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response. We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

Endoplasmic reticulum-associated degradation-induced dissociation of class II invariant chain complexes containing a glycosylation-deficient form of p41

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.     

Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p

Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.     

The engagement of Sec61p in the ER dislocation process

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.