Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.     

The Png1-Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.     

A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation

Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal-lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTA(P250A) is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.     

N‐glycosylation Does Not Affect the Catalytic Activity of Ricin A Chain but Stimulates Cytotoxicity by Promoting Its Transport Out of the Endoplasmic Reticulum

Ricin A chain (RTA) depurinates the α‐sarcin/ricin loop after it undergoes retrograde trafficking to the cytosol. The structural features of RTA involved in intracellular transport are not known. To explore this, we fused enhanced green fluorescent protein (EGFP) to precursor (preRTA‐EGFP), containing a 35‐residue leader, and mature RTA (matRTA‐EGFP). Both were enzymatically active and toxic in Saccharomyces cerevisiae. PreRTA‐EGFP was localized in the endoplasmic reticulum (ER) initially and was subsequently transported to the vacuole, whereas matRTA‐EGFP remained in the cytosol, indicating that ER localization is a prerequisite for vacuole transport. When the two glycosylation sites in RTA were mutated, the mature form was fully active and toxic, suggesting that the mutations do not affect catalytic activity. However, nonglycosylated preRTA‐EGFP had reduced toxicity, depurination and delayed vacuole transport, indicating that N‐glycosylation affects transport of RTA out of the ER. Point mutations in the C‐terminal hydrophobic region restricted RTA to the ER and eliminated toxicity and depurination, indicating that this sequence is critical for ER exit. These results demonstrate that N‐glycosylation and the C‐terminal hydrophobic region stimulate the toxicity of RTA by promoting ER export. The timing of depurination coincided with the timing of vacuole transport, suggesting that RTA may enter the cytosol during vacuole transport.     

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated.     

Lectin-like ERAD players in ER and cytosol

Protein quality control in the endoplasmic reticulum (ER) is an elaborate process conserved from yeast to mammals, ensuring that only newly synthesized proteins with correct conformations in the ER are sorted further into the secretory pathway. It is well known that high-mannose type N-glycans are involved in protein-folding events. In the quality control process, proteins that fail to achieve proper folding or proper assembly are degraded in a process known as ER-associated degradation (ERAD). The ERAD pathway comprises multiple steps including substrate recognition and targeting to the retro-translocation machinery, retrotranslocation from the ER into the cytosol, and proteasomal degradation through ubiquitination. Recent studies have documented the important roles of sugar-recognition (lectin-type) molecules for trimmed high-mannose type N-glycans and glycosidases in the ERAD pathways in both ER and cytosol. In this review, we discuss a fundamental system that monitors glycoprotein folding in the ER and the unique roles of the sugar-recognizing ubiquitin ligase and peptide:N-glycanase (PNGase) in the cytosolic ERAD pathway.     

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

BACE457 is a recently identified pancreatic isoform of human beta-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.     

Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.     

An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein

The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("Gp(alpha)F") is stable, a non-glycosylated version of the same protein ("p(alpha)F") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.     

Folding-competent and Folding-defective Forms of Ricin A Chain Have Different Fates after Retrotranslocation from the Endoplasmic Reticulum

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA_Δ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA_Δ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.     

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.     

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.     

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation.     

Pkc1p modifies CPY* degradation in the ERAD pathway

The process of endoplasmic reticulum-associated degradation (ERAD) involved in the degradation of misfolded N-linked glycoproteins utilizes Cdc48p which extracts misfolded glycoproteins from the lumen to the cytosol to present them for deglycosylation and degradation. Pkc1p has been identified as a component of the ERAD pathway, because deletion of the pkc1 gene impairs ERAD and causes accumulation of CPY* in the lumen of the ER, most probably because of the mislocalization of Cdc48p. In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein.     

Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD)

ER-associated degradation (ERAD) is a component of the protein quality control system, ensuring that aberrant polypeptides cannot transit through the secretory pathway. This is accomplished by a complex sequence of events in which unwanted proteins are selected in the ER and exported to the cytosol for degradation by the proteasome. Given that protein quality control can be essential for cell survival, it is not surprising that ERAD is linked to numerous disease states. Here we review the molecular mechanisms of ERAD, its role in metabolic regulation and biomedical implications, and the unanswered questions regarding this process.     

The Grp170 nucleotide exchange factor executes a key role during ERAD of cellular misfolded clients

When a protein misfolds in the endoplasmic reticulum (ER), it retrotranslocates to the cytosol and is degraded by the proteasome via a pathway called ER-associated degradation (ERAD). To initiate ERAD, ADP-BiP is often recruited to the misfolded client, rendering it soluble and translocation competent. How the misfolded client is subsequently released from BiP so that it undergoes retrotranslocation, however, remains enigmatic. Here we demonstrate that the ER-resident nucleotide exchange factor (NEF) Grp170 plays an important role during ERAD of the misfolded glycosylated client null Hong Kong (NHK). As a NEF, Grp170 triggers nucleotide exchange of BiP to generate ATP-BiP. ATP-BiP disengages from NHK, enabling it to retrotranslocate to the cytosol. We demonstrate that Grp170 binds to Sel1L, an adapter of the transmembrane Hrd1 E3 ubiquitin ligase postulated to be the retrotranslocon, and links this interaction to Grp170’s function during ERAD. More broadly, Grp170 also promotes degradation of the nonglycosylated transthyretin (TTR) D18G misfolded client. Our findings thus establish a general function of Grp170 during ERAD and suggest that positioning this client-release factor at the retrotranslocation site may afford a mechanism to couple client release from BiP and retrotranslocation.     

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1 , the gene coding for the plasma membrane H ⁺-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1 -D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1 -D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1 -D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.     

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

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

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.