The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate.

The human cytomegalovirus protein, US11, initiates the destruction of MHC class I heavy chains by targeting them for dislocation from the ER to the cytosol and subsequent degradation by the proteasome. We report the development of a permeabilized cell system that recapitulates US11-dependent degradation of class I heavy chains. We have used this system, in combination with experiments in intact cells, to identify and order intermediates in the US11-dependent degradation pathway. We find that heavy chains are ubiquitinated before they are degraded. Ubiquitination of the cytosolic tail of heavy chain is not required for its dislocation and degradation, suggesting that ubiquitination occurs after at least part of the heavy chain has been dislocated from the ER. Thus, ubiquitination of the heavy chain does not appear to be the signal to start dislocation. Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane. Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.     

SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER

Protein quality control in the endoplasmic reticulum (ER) involves recognition of misfolded proteins and dislocation from the ER lumen into the cytosol, followed by proteasomal degradation. Viruses have co-opted this pathway to destroy proteins that are crucial for host defense. Examination of dislocation of class I major histocompatibility complex (MHC) heavy chains (HCs) catalyzed by the human cytomegalovirus (HCMV) immunoevasin US11 uncovered a conserved complex of the mammalian dislocation machinery. We analyze the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process. Perturbation of SEL1L function discriminates between the dislocation pathways used by US11 and US2, which is a second HCMV protein that catalyzes dislocation of class I MHC HCs. Furthermore, reduction of the level of SEL1L by small hairpin RNA (shRNA) inhibits the degradation of a misfolded ribophorin fragment (RI332) independently of the presence of viral accessories. These results allow us to place SEL1L in the broader context of glycoprotein degradation, and imply the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER.     

Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.     

Dislocation of a type I membrane protein requires interactions between membrane-spanning segments within the lipid bilayer

The human cytomegalovirus gene product US11 causes rapid degradation of class I major histocompatibility complex (MHCI) heavy chains by inducing their dislocation from the endoplasmic reticulum (ER) and subsequent degradation by the proteasome. This set of reactions resembles the endogenous cellular quality control pathway that removes misfolded or unassembled proteins from the ER. We show that the transmembrane domain (TMD) of US11 is essential for MHCI heavy chain dislocation, but dispensable for MHCI binding. A Gln residue at position 192 in the US11 TMD is crucial for the ubiquitination and degradation of MHCI heavy chains. Cells that express US11 TMD mutants allow formation of MHCI-beta2m complexes, but their rate of egress from the ER is significantly impaired. Further mutagenesis data are consistent with the presence of an alpha-helical structure in the US11 TMD essential for MHCI heavy chain dislocation. The failure of US11 TMD mutants to catalyze dislocation is a unique instance in which a polar residue in the TMD of a type I membrane protein is required for that protein's function. Targeting of MHCI heavy chains for dislocation by US11 thus requires the formation of interhelical hydrogen bonds within the ER membrane.     

The viral E3 ubiquitin ligase mK3 uses the Derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class I proteins

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.     

Adapter-mediated Substrate Selection for Endoplasmic Reticulum-associated Degradation

During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence.     

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.     

Polyubiquitination is required for US11-dependent movement of MHC class I heavy chain from endoplasmic reticulum into cytosol

The human cytomegalovirus protein US11 induces the dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol for degradation by the proteasome. With the use of a fractionated, permeabilized cell system, we find that US11 activity is needed only in the cell membranes and that additional cytosolic factors are required for heavy chain dislocation. We identify ubiquitin as one of the required cytosolic factors. Cytosol depleted of ubiquitin does not support heavy chain dislocation from the ER, and activity can be restored by adding back purified ubiquitin. Methylated-ubiquitin or a ubiquitin mutant lacking all lysine residues does not substitute for wild-type ubiquitin, suggesting that polyubiquitination is required for US11-dependent dislocation. We propose a new function for ubiquitin in which polyubiquitination prevents the lumenal domain of the MHC class I heavy chain from moving back into the ER lumen. A similar mechanism may be operating in the dislocation of misfolded proteins from the ER in the cellular quality control pathway.     

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.     

The E3 Ubiquitin Ligase gp78 Protects against ER Stress in Zebrafish Liver

Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 （gp78-R2M） renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein （Srebp）-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.     

Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides

Accumulation of improperly folded polypeptides in the endoplasmic reticulum (ER) can trigger a stress response that leads to the export of aberrant proteins into the cytosol and their ultimate proteasomal degradation. Human cytomegalovirus encodes a type I glycoprotein, US11, that binds to nascent MHC class I heavy chain molecules and causes their dislocation from the ER to the cytosol where they are degraded by the proteasome. Examination of US11-mediated class I degradation has identified a host of cellular proteins involved in the dislocation reaction, including the cytosolic AAA ATPase p97, the membrane protein Derlin-1, and the E3 ubiquitin ligase Sel1L. However, the intermediate steps occurring between the initiation of dislocation and full extraction of the misfolded substrate into the cytosol are not known. We demonstrate that US11 itself undergoes ER export and proteasomal degradation and utilize this system to define multiple steps of US11 dislocation. Treatment of US11-expressing cells with proteasome inhibitor resulted in the accumulation of glycosylated and ubiquitinated species as well as a deglycosylated US11 intermediate. Subcellular fractionation of proteasome-inhibited US11 cells demonstrated that deglycosylated intermediates continued to be integrated within the ER membrane, suggesting that the proteasome functions in the latter steps of dislocation. The data supports a model in which US11 is modified with ubiquitin, whereas the transmembrane region is integrated in the ER membrane, and deglycosylation occurs before complete dislocation.     

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.     

Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor alpha. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.     

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 thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation

The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.     

The role of BiP in endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain induced by cytomegalovirus proteins

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.     

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.