Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection

Secretory and membrane proteins attain their native structure in the endoplasmic reticulum (ER). Folding-defective polypeptides are selected for degradation by processes collectively defined as ER-associated degradation (ERAD). Enhanced ERAD activity may interfere with protein biogenesis by inappropriately targeting not-yet-native protein folding intermediates for disposal. The regulation of ERAD is therefore crucial to maintain cellular proteostasis. At steady-state, select ERAD regulators are constitutively removed from the ER in a series of processes collectively defined as ERAD tuning. This sets the ERAD activity at levels that do not interfere with completion of ongoing folding programs. Our latest work highlights a crucial, autophagy-independent role of nonlipidated LC3 (LC3-I) as part of a membrane-bound receptor that insures the vesicle-mediated clearance of at least two ERAD regulators from the ER, EDEM1 and OS9. This pathway is hijacked by coronaviruses (CoV), and silencing of LC3 substantially inhibits viral replication.     

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.     

Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities

EDEM1 is a crucial regulator of endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts non-native glycopolypeptides from the calnexin chaperone system. Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. We report that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-I-coated vesicles and is rapidly degraded. The rapid turnover of EDEM1 is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides. We define as ERAD tuning the mechanisms operating in the mammalian ER at steady state to offer kinetic advantage to folding over disposal of unstructured nascent chains by selective and rapid degradation of ERAD regulators.     

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.     

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.     

Characterization of WDR20: A new regulator of the ERAD machinery

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

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of?folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.     

Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD

The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.     

Ubiquitin-Dependent Intramembrane Rhomboid Protease Promotes ERAD of Membrane Proteins

The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of?membrane proteins. Here we show that the evolutionary conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by?a shared mechanism.     

OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.     

The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation)

Over one-third of all newly synthesized polypeptides in eukaryotes interact with or insert into the membrane or the lumenal space of the ER (endoplasmic reticulum), an event that is essential for the subsequent folding, post-translational modification, assembly and targeting of these proteins. Consequently, the ER houses a large number of factors that catalyse protein maturation, but, in the event that maturation is aborted or inefficient, the resulting aberrant proteins may be selected for ERAD (ER-associated degradation). Many of the factors that augment protein biogenesis in the ER and that mediate ERAD substrate selection are molecular chaperones, some of which are heat- and/or stress-inducible and are thus known as Hsps (heat-shock proteins). But, regardless of whether they are constitutively expressed or are inducible, it has been assumed that all molecular chaperones function identically. As presented in this review, this assumption may be false. Instead, a growing body of evidence suggests that a chaperone might be involved in either folding or degrading a given substrate that transits through the ER. A deeper appreciation of this fact is critical because (i) the destruction of some ERAD substrates results in specific diseases, and (ii) altered ERAD efficiency might predispose individuals to metabolic disorders. Moreover, a growing number of chaperone-modulating drugs are being developed to treat maladies that arise from the synthesis of a unique mutant protein; therefore it is critical to understand how altering the activity of a single chaperone will affect the quality control of other nascent proteins that enter the ER.     

ERAD: the long road to destruction

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) eliminates misfolded or unassembled proteins from the ER. ERAD targets are selected by a quality control system within the ER lumen and are ultimately destroyed by the cytoplasmic ubiquitin-proteasome system (UPS). The spatial separation between substrate selection and degradation in ERAD requires substrate transport from the ER to the cytoplasm by a process termed dislocation. In this review, we will summarize advances in various aspects of ERAD and discuss new findings on how substrate dislocation is achieved.     

Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD

The yeast endoplasmic reticulum (ER) membrane-localized chaperone Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the plasma membrane. In the absence of Shr3, AAPs specifically accumulate in the ER, where despite the correct insertion of their 12 transmembrane segments (TMSs), they aggregate forming large molecular weight complexes. We show that Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with and maintains the first five TMSs in a conformation that can posttranslationally assemble with the remaining seven TMSs. We also show that Doa10- and Hrd1-dependent ER-associated degradation (ERAD) pathways redundantly degrade AAP aggregates. In combination, doa10Delta hrd1Delta mutations stabilize AAP aggregates and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independent of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis.     

When worlds collide: IP3 receptors and the ERAD pathway

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca²⁺ store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP₃) receptors are activated, since in addition to acting as release channels for stored ER Ca²⁺, IP₃ receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP₃ receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP₃ receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP₃ receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP₃ receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca²⁺ stores to IP₃ and may protect cells against deleterious effects of over-activation of Ca²⁺ signaling pathways.     

Dfm1 Forms Distinct Complexes with Cdc48 and the ER Ubiquitin Ligases and Is Required for ERAD

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin‐proteasome system. This process is called ER‐associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY^* (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C‐terminally hemagglutinin (HA)₃‐tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1‐HA₃ to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non‐tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD‐L ligase Hrd1/Der3 and Der1 as well as the ERAD‐C ligase Doa10. In addition, ERAD of Ste6^*‐HA₃ was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA‐ATPase Cdc48 in a strain lacking the Cdc48 membrane‐recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.     

A novel role for N-glycans in the ERAD system

The endoplasmic reticulum (ER) provides a quality-control system for newly synthesized secretory and membrane proteins. Any improperly folded or incompletely assembled oligomers are retained in the ER, and they are retro-translocated into the cytosol when misfolding persists, where they are destroyed by the proteasome through ubiquitylation. This disposal process is called ER-associated degradation (ERAD). Although much is known about the fate of ERAD substrates near the point of degradation, little information is available about how these proteins are recognized, retained, and targeted for translocation and ubiquitylation machinery. Recent studies indicate that N-linked oligosaccharides attached to nascent proteins function as tags for several processes of a quality-control system, such as individual steps of ER-retention, selection for ERAD substrates, and ubiquitylation. In this review, I describe recent advances in the molecular basis of the ERAD system, particularly those mediated by N-glycan recognition molecules.     

Yos9, a control protein for misfolded glycosylated and non-glycosylated proteins in ERAD

The endoplasmic reticulum (ER) is responsible for folding and delivery of secretory proteins to their site of action. One major modification proteins undergo in this organelle is N-glycosylation. Proteins that cannot fold properly will be directed to a process known as endoplasmic reticulum associated degradation (ERAD). Processing of N-glycans generates a signal for ERAD. The lectin Yos9 recognizes the N-glycan signal of misfolded proteins and acts as a gatekeeper for the delivery of these substrates to the cytoplasm for degradation. Presence of Yos9 accelerates degradation of the glycosylated model ERAD substrate CPY?. Here we show that Yos9 has also a control function in degradation of the unglycosylated ERAD substrate CPY?0000. It decelerates its degradation rate.