Endoplasmic Reticulum Degradation Research Articles

Ricin A Chain Insertion into Endoplasmic Reticulum Membranes Is Triggered by a Temperature Increase to 37 °C

After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 °C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 °C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 °C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

Evaluating Protein Interactions & Organelle Dynamics in Saccharomyces cerevisiae: Spatial Distribution of Molecular Chaperone/Co-Chaperones Evident at a Sub-organelle Level in the Endoplasmic Reticulum

BiP/Kar2 is a member of the Hsp70 family of chaperones that resides in the endoplasmic reticulum (ER) of S. cerevisiae. Biochemical and genetics experiments have demonstrated BiP's association with selective co-chaperones in multiple critical processes of the cell including translocation of protein into the ER, protein folding/maturation, and ER-associated degradation (ERAD). BiP's relative high cellular concentration combined with the low resolution of traditional immunofluorescence techniques has hindered the determination of protein localization effects. We hypothesize that the spatial heterogeneity of chaperones is regulated by co-chaperones, and this heterogeneity serves as a means of dictating cellular functions. To generate physiologically-relevant data of protein dynamics in S. cerevisiae, variants of green fluorescent protein (GFP) coupled with advances in confocal light microscopy techniques have allowed us to track multiple fluorescently-tagged proteins in vivo.Dual expression strains composed of fusion proteins, BiP and co-chaperone Sec63, reveal that a heterogeneous spatial distribution is evident at the sub-organelle level. Secondary confirmation of our results has been performed using immunofluorescence techniques in multiple S. cerevisiae strains. Deconvolution of fixed cell images has allowed us to reconstruct and quantify the co-localization of BiP and Sec63 in three dimensions. We have captured the spatiotemporal effects of protein dynamics in live cells by monitoring ER membrane and luminal proteins, in addition to the nuclear pore complex; confirmed that the ER is continuous through Fluorescence Loss in Photobleaching (FLiP) experiments; and captured rapid diffusion of ER resident proteins. Integration of experimental data and computational design enables us to develop stochastic models of biological systems that accurately reflect spatiotemporal effects of molecular chaperone/co-chaperone interactions.

The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.

ERdj5 Is Required as a Disulfide Reductase for Degradation of Misfolded Proteins in the ER

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.

Cytokine-Induced β-Cell Death Is Independent of Endoplasmic Reticulum Stress Signaling

OBJECTIVE—Cytokines contribute to β-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress–mediated apoptosis has been proposed as a mechanism for β-cell death. We tested whether ER stress was necessary for cytokine-induced β-cell death and also whether ER stress gene activation was present in β-cells of the NOD mouse model of type 1 diabetes.RESEARCH DESIGN AND METHODS—INS-1 β-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin (IL)-1β and γ-interferon (IFN-γ). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression in INS-1 β-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed.RESULTS—Cytokines and palmitate triggered ER stress in β-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK), eukaryotic initiation factor (EIF)2α, and Jun NH2-terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA levels of several ER stress genes were reduced (ATF4, BiP [binding protein], GRP94 [glucose regulated protein 94], p58, and XBP-1 [X-box binding protein 1] splicing) or unchanged (CHOP and Edem1 [ER degradation enhancer, mannosidase α–like 1]).CONCLUSIONS—While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP ER stress–signaling pathway is not necessary for cytokine-induced β-cell death.

Ubiquitin Ligase Hul5 Is Required for Fragment-specific Substrate Degradation in Endoplasmic Reticulum-associated Degradation

To identify new components of the protein quality control and degradation pathway of the endoplasmic reticulum (ER), we performed a growth-based genome-wide screen of about 5000 viable deletion mutants of the yeast Saccharomyces cerevisiae. As substrates we used two misfolded ER membrane proteins, CTL* and Sec61-2L, chimeric derivatives of the classical ER degradation substrates CPY* and Sec61-2. Both substrates contain a cytosolic Leu2 protein fusion, and stabilization of these substrates in ER-associated degradation-deficient strains enables a restored growth of the transformed LEU2-deficient deletion mutants. We identified the strain deleted for the ubiquitin chain elongating ligase Hul5 among the mutant strains with a strong growth phenotype. Here we show that Hul5 is necessary for the degradation of two misfolded ER membrane substrates. Although the degradation of their N-terminal parts is Hul5-independent, the breakdown of their C-terminal fragments requires the ubiquitin chain elongating ligase activity of Hul5. In the absence of Hul5, a truncated form of CTL*myc remains to a large extent embedded in the ER membrane. Hul5 activity promotes the interaction of this truncated CTL*myc with the AAA-ATPase Cdc48, which is known to pull proteins out of the ER membrane. This study unravels the stepwise elimination of the ER membrane-localized CTL*myc substrate. First, N-terminal, lumenal CPY* is transferred to the cytoplasm and degraded by the proteasome. Subsequently, the remaining C-terminal membrane-anchored part requires Hul5 for its effective extraction out of the endoplasmic reticulum and proteasomal degradation.

Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases

Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRDeltaF508, by specifically promoting ubiquitylation of CFTRDeltaF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRDeltaF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRDeltaF508 and catalyzes further polyubiquitylation of CFTRDeltaF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRDeltaF508.

Proteasome degradation of protein C and plasmin inhibitor mutants

Protein C (PC) deficiency and plasmin inhibitor (PI) deficiency are inherited thrombotic and haemorrhagic disorders. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. To examine the necessity of N-linked glycosylation for the proteasomal degradation of PC and PI, PC178 and PC331 mutants treated with tunicamycin and N-glycosylation-lacking mutants, PC92Stop and PI-America were pulse chased. The analysis revealed that the speed of degradation of the tunicamycin-treated PC mutants, PC92Stop and PI-America lacking glycosylation, was slower than that of N-glycosylated mutants. Immunoprecipitation and immunoblot analysis showed that PC178 and PC331 mutants were associated with molecular chaperones, Bip, GRP94, and calreticulin. PI-America was associated with only Bip. Although degradation of mutants was mediated by proteasomes, no association with ubiquitin was detected. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase-like protein (EDEM) accelerated the degradation of N-glycosylated PC. In the absence of autophagy using Atg5-deficient cell lines, the degradation of the PC331 mutant was mildly accelerated but that of PC178, PI-America and PI-Okinawa mutants was not influenced. While the degradation of the PC and PI mutants was facilitated by N-glycosylation moieties, they were ubiquitin-independently degraded by proteasomes, irrespective of the presence or absence of N-glycosylation. Molecular chaperone binding was influenced by the presence of N-glycosylation moieties. When the misfolded or truncated mutant proteins are functionally active, proteasome inhibitors such as bortezomib may have therapeutic potential for treatment of protein deficiencies.

N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation

The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue (local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation.

Endoplasmic reticulum-associated degradation of mutant CFTR requires a guanine nucleotide-sensitive step

Proteasome degradation of endoplasmic reticulum (ER)-misfolded proteins requires retrograde transport from ER to the cytosol. To date, it is not clear whether this event constitutes the exclusive ER degradation process for non-native membrane proteins. Here we describe the role of GTP in the degradation of ΔF508-CFTR and the α subunit of the T-cell receptor (TCRα), representative misfolded ER membrane proteins. Selective intracellular GTP depletion extended the ΔF508-CFTR half-life sixfold, whereas ATP depletion accelerated its turnover and inhibited only 80% of the proteasome activity that was not affected by GTP depletion. AlF4−, a well-known inhibitor of heterotrimeric G proteins, but not of AlF3, delayed the mutant CFTR turnover in vivo, in semi-intact cells and in ER-enriched microsomes, without affecting ER to Golgi cargo transport. ΔF508-CFTR degradation was also inhibited by alkaline stripping of ER-associated membrane proteins. We propose that at the ER, GTP may participate in the disposal of misfolded membrane proteins through activation of heterotrimeric G proteins.

The Cyclophilin-like Domain of Ran-binding Protein-2 Modulates Selectively the Activity of the Ubiquitin-Proteasome System and Protein Biogenesis

The ubiquitin-proteasome system (UPS) plays a critical role in protein degradation. The 19S regulatory particle (RP) of the 26S proteasome mediates the recognition, deubiquitylation, unfolding, and channeling of ubiquitylated substrates to the 20S proteasome. Several subunits of the 19S RP interact with a growing number of factors. The cyclophilin-like domain (CLD) of Ran-binding protein-2 (RanBP2/Nup358) associates specifically with at least one subunit, S1, of the base subcomplex of the 19S RP, but the functional implications of this interaction on the UPS activity are elusive. This study shows the CLD of RanBP2 promotes selectively the accumulation of a subset of reporter substrates of the UPS, such as the ubiquitin (Ub)-fusion yellow fluorescent protein (YFP) degradation substrate, Ub(G76V)-YFP, and the N-end rule substrate, Ub-R-YFP. Conversely, the degradation of endoplasmic reticulum and misfolded proteins, and of those linked to UPS-independent degradation, is not affected by CLD. The selective effect of CLD on the UPS in vivo is independent of, and synergistic with, proteasome inhibitors, and CLD does not affect the intrinsic proteolytic activity of the 20S proteasome. The inhibitory activity of CLD on the UPS resides in a purported SUMO binding motif. We also found two RanBP2 substrates, RanGTPase-activating protein and retinitis pigmentosa GTPase regulator interacting protein-1alpha1, whose steady-state levels are selectively modulated by CLD. Hence, the CLD of RanBP2 acts as a novel auxiliary modulator of the UPS activity; it may contribute to the molecular and subcellular compartmentation of the turnover of properly folded proteins and modulation of the expressivity of several neurological diseases.

In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases

A substantial fraction of eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) membrane. These polypeptides enter cotranslationally in the ER lumen, which contains resident molecular chaperones and folding factors that assist their maturation. Native proteins are released from the ER lumen and are transported through the secretory pathway to their final intra- or extracellular destination. Folding-defective polypeptides are exported across the ER membrane into the cytosol and destroyed. Cellular and organismal homeostasis relies on a balanced activity of the ER folding, quality control, and degradation machineries as shown by the dozens of human diseases related to defective maturation or disposal of individual polypeptides generated in the ER.

Inhibiting Endoplasmic Reticulum (ER)-associated Degradation of Misfolded Yor1p Does Not Permit ER Export Despite the Presence of a Diacidic Sorting Signal

Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the "B-site" cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-DeltaF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-DeltaF by inhibiting its degradation does not permit access of Yor1p-DeltaF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.

Improved maturation of CFTR by an ER export signal

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel in the plasma membrane of several epithelial cells. Maturation of CFTR is inefficient in most cells, with only a fraction of nascent chains being properly folded and transported to the cell surface. The most common mutation in CFTR, CFTR-deltaF508, leads to the genetic disease cystic fibrosis. CFTR-deltaF508 has a temperature-sensitive folding defect and is almost quantitatively degraded in the endoplasmic reticulum (ER). Here we tested whether a strong ER export signal appended to CFTR improves its transport and surface expression. We show that a single valine ER export signal at the C terminus of the cytoplasmic tail of CFTR improves maturation of wild-type CFTR by 2-fold. This conservative mutation interfered with neither plasma membrane localization nor stability of mature CFTR. In contrast, the valine signal was unable to rescue CFTR-deltaF508 from ER-associated degradation. Our finding of improved maturation of CFTR mediated by a valine signal may be of potential use in gene therapy of cystic fibrosis. Moreover, failure of the valine signal to rescue CFTR-deltaF508 from ER degradation indicates that the inability of CFTR-deltaF508 to leave the ER is unlikely to be due to a malfunctioning ER export signal.

The oligomeric state of Derlin-1 is modulated by endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.

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.

Selective Inhibition of Endoplasmic Reticulum-associated Degradation Rescues ΔF508-Cystic Fibrosis Transmembrane Regulator and Suppresses Interleukin-8 Levels: THERAPEUTIC IMPLICATIONS

Selective Inhibition of Endoplasmic Reticulum-associated Degradation Rescues ΔF508-Cystic Fibrosis Transmembrane Regulator and Suppresses Interleukin-8 Levels: THERAPEUTIC IMPLICATIONS

Cysteine String Protein Monitors Late Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.

Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing α-mannosidase–like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.