Enzymatic blockade of the ubiquitin-proteasome pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.     

Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein-class I heavy chain (EGFP-HC) chimeric molecule to study its dislocation and degradation in US2- and US11-expressing cells. The EGFP-HC fusion is stable in control cells, but is degraded rapidly in US2- or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP-HC molecules in US2- and US11-expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse-chase analysis and subcellular fractionation show that EGFP-HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.     

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.     

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 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.     

A glycosylated type I membrane protein becomes cytosolic when peptide: N-glycanase is compromised

The human cytomegalovirus-encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N-glycanase-type activity. siRNA molecules designed to inhibit the expression of the light chain, beta(2)-microglobulin, block the dislocation of Class I MHC molecules, which implies that US2-dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N-glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2-expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA-treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N-linked type I membrane glycoprotein in the cytosol. N-glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.     

TRAM1 Participates in Human Cytomegalovirus US2- and US11-mediated Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein

The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic reticulum (ER) quality control to facilitate the destruction of major histocompatibility complex class I heavy chains. The class I heavy chains are dislocated from the ER to the cytosol, where they are deglycosylated and subsequently degraded by the proteasome. We examined the role of TRAM1 (translocating chain-associated membrane protein-1) in the dislocation of class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein initially characterized for its role in processing nascent polypeptides. Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the wild type US2 and US11 proteins as well as deglycosylated and polyubiquitinated class I degradation intermediates. In studies using US2- and US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy chains. Strikingly, increased levels of glycosylated heavy chains were observed in TRAM1 knockdown cells when compared with control cells in a pulse-chase experiment. In fact, US11-mediated class I dislocation was more sensitive to the lack of TRAM1 than US2. These results provide further evidence that these viral proteins may utilize distinct complexes to facilitate class I dislocation. For example, US11-mediated class I heavy chain degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is critical for US2-induced class I destabilization. In addition, TRAM1 can complex with the dislocation factors Derlin-1 and signal peptide peptidase. Collectively, the data support a model in which TRAM1 functions as a cofactor to promote efficient US2- and US11-dependent dislocation of major histocompatibility complex class I heavy chains.HCMV² can down-regulate cell surface expression of the immunologically important molecule major histocompatibility complex class I to avoid immune detection by cytotoxic T cells (1, 2). More specifically, the HCMV US2 and US11 gene products alone can target the ER-localized major histocompatibility complex class I heavy chains for extraction across the ER membrane by a process referred to as dislocation or retrograde translocation. The N-linked glycan is then removed upon exposure to the cytosol by N-glycanase (3), followed by proteasomal destruction (4, 5). The HCMV US2 and US11 proteins utilize the ER quality control process to eliminate class I heavy cells in a similar manner as misfolded or damaged ER proteins (e.g. genetic mutants of α₁-antitrypsin (6) and the cystic fibrosis transmembrane conductance regulator protein (7)) are targeted for degradation (8). Hence, analysis of US2- and US11-mediated destruction of class I heavy chains provides an excellent system to delineate viral protein function as well as the ER quality control process.ER and cytosolic proteins are required for US2- and US11-mediated dislocation/degradation of class I heavy chains. Some of these proteins have also been identified in the processing of aberrant ER polypeptides. The ER chaperones calnexin, calreticulin, and BiP have been implicated in US2-mediated class I destruction (9) as well as in the removal of some misfolded ER proteins (10). The ubiquitination machinery also participates in the extraction of class I heavy chains as ubiquitinated heavy chains are observed prior to dislocation (11, 12). For misfolded ER degradation substrates, ubiquitin conjugation enzymes (e.g. Ubc6p and Ubc7p/Cue1p) and ubiquitin ligases Hrd1p/Der3p, Doa10p, and Ubc1p have been implicated in the dislocation reaction (8). Interestingly, the ER membrane protein Derlin-1 along with SEL1L are involved in US11-mediated class I heavy chain degradation (13-15), whereas SPP is critical for US2-induced class I destabilization (16). The ubiquitinated substrates are dislocated by the AAA-ATPase complex composed of p97-Ufd1-Npl4 (17) while docked to the ER through its interaction with VIMP (14) followed by proteasome destruction. The inhibition of the proteasome causes the accumulation of deglycosylated class I heavy chain intermediate in US2 and US11 cells, allowing the dislocation and degradation reactions to be studied as separate processes (4, 5).Despite the identification of some cellular proteins that assist US2- and US11-mediated class I dislocation, the dislocation pore and accessory factors that mediate the efficient extraction of class I through the bilayer have yet to be completely defined. The current study explores the role of TRAM1 (translocating chain-associated membrane protein-1) in US2- and US11-mediated class I dislocation. TRAM1 is an ER-resident multispanning membrane protein that can mediate the lateral movement of select signal peptides and transmembrane segments from the translocon into the membrane bilayer (18), a property that makes it uniquely qualified to participate in the dislocation of a membrane protein. TRAM1 has been cross-linked to signal peptides as well as transmembrane domains of nascent polypeptides during the early stages of protein processing (19-25). Interestingly, unlike the Sec61 complex and the signal recognition particle receptor, TRAM1 is not essential for the translocation of all membrane proteins into the ER (20, 21). Hence, TRAM1 may utilize its ability to engage hydrophobic domains to assist in the efficient dislocation of membrane proteins. In fact, association and TRAM1 knockdown studies demonstrate that TRAM1 participates in US2- and US11-mediated dislocation of class I heavy chains. Collectively, our data suggest for the first time that TRAM1 plays a role in the dislocation of a membrane glycoprotein.     

Distinct steps in dislocation of luminal endoplasmic reticulum-associated degradation substrates: roles of endoplamic reticulum-bound p97/Cdc48p and proteasome

Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ER-bound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

Membrane-specific, host-derived factors are required for US2- and US11-mediated degradation of major histocompatibility complex class I molecules.

Human cytomegalovirus encodes two glycoproteins, US2 and US11, that target major histocompatibility complex (MHC) class I heavy chains for proteasomal degradation. We have developed a mRNA-dependent cell-free system that recapitulates US2- and US11-mediated degradation of MHC class I heavy chains. Microsomes support the degradation of MHC class I heavy chains in the presence of US2 or US11 in a cytosol-dependent manner. In vitro, the glycosylated heavy chain is exported from the microsomes. A deglycosylated breakdown intermediate of the heavy chain identical to that generated in intact cells accumulates in soluble form in the presence of proteasome inhibitors. Microsomes derived from the U373 astrocytoma cell line are far more effective than canine-derived membranes in supporting this US2- or US11-dependent reaction. In contrast, the HIV-encoded Vpu membrane protein can cause the destruction of CD4 from either human- or canine-derived membranes. Using the in vitro system, we show that a truncation mutant of US2 that lacks the cytosolic domain is unable to catalyze degradation, whereas a similar truncation of US11 continues to catalyze degradation of class I heavy chains. Therefore, US2 requires both transmembrane and cytosolic interactions to trigger dislocation of heavy chains, whereas US11 relies on the transmembrane domain to target heavy chains. US2 and US11 thus utilize different targeting mechanisms for class I degradation.     

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 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.     

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.     

The glycoprotein gp48 of murine cytomegalovirusL proteasome-dependent cytosolic dislocation and degradation.

Degradation of misfolded or unassembled proteins that are co-translationally inserted into the endoplasmic reticulum involves the cytosolic proteasome system. Different principles may exist for the export of proteins into the cytosol for proteasomal degradation. Here we studied the degradation pathway of the viral glycoprotein gp48, a type I transmembrane protein, encoded by the m06 gene of murine cytomegalovirus. In cells stably transfected with the cytomegalovirus m06 gene or infected with the virus itself, two populations of gp48 can be distinguished that have different fates. Complexes of gp48 and the major histocompatibility complex (MHC) class I molecule, are transported to the lysosome for degradation. Unassembled gp48 is degraded by the cytosolic proteasome. Proteasomal inhibitors stabilize the unassembled gp48 in its core-glycosylated and membrane-associated form in the endoplasmic reticulum (ER)-Golgi intermediate compartment. This implicates that both endoplasmic reticulum and ER-Golgi intermediate compartment export gp48 and that degradation is coupled to a functional proteasome. Analysis of gp48 mutants revealed that the cytosolic part of gp48 was not responsible for the proteasome-dependent substrate transport out of the ER-Golgi intermediate compartment. Thus an indirect interaction between the proteasome and its substrate has to be discussed.     

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.     

Tyrosinase degradation via two pathways during reverse translocation to the cytosol.

Previous studies established that after inhibition of proteasome activity, tyrosinase could be detected in the cytosol after initial translation in the endoplasmic reticulum (ER), with a molecular weight consistent with that of a full-length, deglycosylated polypeptide. Here we show that most of these molecules are glycosylated, but have been proteolyzed at the carboxyl terminus by a protease that is insensitive to proteasome inhibitors. We also demonstrate the inhibitor-dependent accumulation of a membrane species that appears structurally homologous to the glycosylated and partially proteolyzed cytosolic form. Under some circumstances, cytosolic tyrosinase that had been deglycosylated and not proteolyzed prior to proteasomal degradation could also be detected. The presence of cytosolic tyrosinase was dependent upon glycosylation of the molecule during synthesis in the ER. These results suggest the existence of at least two alternative pathways for degradation of tyrosinase in the cytosol.     

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

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

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

The polytopic endoplasmic reticulum (ER)–localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.     

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

The human cytomegalovirus (HCMV) protein US2 hijacks the endoplasmic reticulum (ER)-associated degradation machinery to dispose of MHC class I heavy chain (HC) at the ER. This process requires retrotranslocation of newly synthesized HC molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been elusive. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.     

Dislocation of Type I Membrane Proteins from the ER to the Cytosol Is Sensitive to Changes in Redox Potential

The human cytomegalovirus (HCMV) gene products US2 and US11 dislocate major histocompatibility class I heavy chains from the ER and target them for proteasomal degradation in the cytosol. The dislocation reaction is inhibited by agents that affect intracellular redox potential and/or free thiol status, such as diamide and N-ethylmaleimide. Subcellular fractionation experiments indicate that this inhibition occurs at the stage of discharge from the ER into the cytosol. The T cell receptor α (TCR α) chain is also degraded by a similar set of reactions, yet in a manner independent of virally encoded gene products. Diamide and N-ethylmaleimide likewise inhibit the dislocation of the full-length TCR α chain from the ER, as well as a truncated, mutant version of TCR α chain that lacks cysteine residues. Cytosolic destruction of glycosylated, ER-resident type I membrane proteins, therefore, requires maintenance of a proper redox potential for the initial step of removal of the substrate from the ER environment.