Buck TM , Kolb AR , Boyd CR , Kleyman TR , Brodsky JL .

DK61296 NIDDK NIH HHS , DK65161 NIDDK NIH HHS , DK79307 NIDDK NIH HHS , GM75061 NIGMS NIH HHS , GM83540 NIGMS NIH HHS , P30 DK072506 NIDDK NIH HHS , R01 DK065161 NIDDK NIH HHS , P30 DK079307 NIDDK NIH HHS , R56 DK065161 NIDDK NIH HHS , F32 GM083540 NIGMS NIH HHS , R01 GM075061 NIGMS NIH HHS , T32 DK061296 NIDDK NIH HHS

	Figure 1. ENaC is ER localized in yeast. (A) Schematic representation of the ENaC subunits demonstrating topology and N-linked glycosylation sites (triangles), which initially reside in the ER lumen. (B) Cell lysates from wild-type yeast expressing C-terminally HA-tagged α-, β-, or γ-ENaC treated with endoglycosidase H (Endo H). A small amount of glycosylated β-ENaC was evident due to incomplete conversion. In all cases, the fastest migrating species corresponds to the predicted size for each unglycosylated subunit. (C) Localization of ENaC by immunofluorescence. Panels left to right show the same fluorescent field for DAPI nuclear staining, Kar2 (yeast BiP), used as an ER marker, and HA antibody for ENaC, respectively.
	Figure 2. The ERAD of ENaC subunits is dependent on both the Hrd1 and Doa10 ubiquitin ligases. Cycloheximide chase reactions were performed as described in Materials and Methods in HRD1/DOA10 (●), hrd1Δ (▴), doa10Δ (▵), or hrd1Δdoa10Δ (○) yeast strains (Pagant et al., 2007) expressing C-terminally HA-tagged α-, β-, or γ-ENaC. Chase reactions were performed at 37°C, lysates were immunoblotted with anti-HA (ENaC) or with anti-Sec61 (as a loading control). Data represent the means of 4–6 experiments, ±SEM.
	Figure 3. ENaC ubiquitination is impaired in yeast strains lacking the E3 ubiquitin ligases Hrd1 and Doa10. (A) Wild type (WT) or the indicated mutant yeast strains expressing either the α- (■) or β- (□) ENaC subunit were processed as described in Materials and Methods, and the level of ubiquitination was assessed. The bar graph represents the means of 5–7 determinations, ±SEM, and the data in the mutant strains were standardized to the amount in the wild-type cells. A typical experimental result is shown in B. (C) Microsomes from wild type (HRD1/DOA10) or the hrd1Δdoa10Δ mutant strain expressing either α- or β-ENaC were prepared and subjected to the in vitro ubiquitination assay as described in Figure S1. The bar graphs represent the means of three (β) or eight (α) determinations, ±SEM.
	Figure 4. The ERAD of ENaC subunits depends on the ER lumenal Hsp40s, Jem1 and Scj1. (A) Cycloheximide chase reactions were performed as described in Materials and Methods in JEM1/SCJ1 (●) and jem1Δscj1Δ (○) yeast strains expressing C-terminally HA-tagged α-, β-, or γ-ENaC or CPY*-3HA. Chase reactions were performed at 37°C and lysates were immunoblotted with anti-HA (ENaC) or with anti-glucose-6-phosphate dehydrogenase (as a loading control) antibodies. Data represent the means of 4–6 experiments, ±SEM. (B) Microsomes from wild type (JEM1/SCJ1) or the jem1Δscj1Δ mutant strain expressing either α- or β-ENaC were prepared and subjected to the in vitro ubiquitination assay as described in Materials and Methods. The bar graphs represent the means of six determinations, ±SEM.
	Figure 5. The ERAD of ENaC is BiP-independent. Cycloheximide chase reactions were performed as described in Materials and Methods in KAR2 (●) and kar2-1 (○) yeast strains expressing C-terminally HA-tagged α-, β-, or γ-ENaC or CPY*-3HA. Chase reactions were performed at 37°C, and lysates were immunoblotted with anti-HA (ENaC) or with anti-glucose-6-phosphate dehydrogenase (as a loading control) antibodies. Data represent the means of 4–6 experiments, ±SEM.
	Figure 6. ENaC current is reduced in a proteasome dependent manner in oocytes coinjected with the human ER lumenal Hsp40s, ERdj3 and ERdj4. (A–C) Amiloride sensitive current was measured 24 h after injection by TEV. Oocytes were injected with α-, β-, and γ-ENaC cRNA (1 ng each) with increasing amounts of either ERdj3 (A) or ERdj4 (B) cRNA. (C) Oocytes were injected with ENaC cRNA as in A and B with either no additional cRNA or 5 ng ERdj3 or ERdj4 cRNA in either the presence (□) or absence (■) of 6 μM MG132. Average baseline current for oocytes expressing ENaC with no additional cRNA was −2.5 μA. (D) ENaC surface expression was assessed as described in Materials and Methods. Oocytes were injected with 1 ng α-, γ-, and β-FLAG ENaC and 5 ng ERdj3 or ERdj4 cRNA. The “No tag” control includes the same amounts of subunits injected but the β-subunit lacks the FLAG tag. In each panel, data are expressed as the means of 17 oocytes from three frogs. Data in D are the means of >35 oocytes from two frogs, ±SEM.

Adams, Interactions between subunits of the human epithelial sodium channel. 1997, Pubmed, Xenbase

Adams, Interactions between subunits of the human epithelial sodium channel. 1997, Pubmed , Xenbase
Adle, Cadmium-mediated rescue from ER-associated degradation induces expression of its exporter. 2009, Pubmed
Ahn, Cloning and functional expression of the mouse epithelial sodium channel. 1999, Pubmed , Xenbase
Ahner, Small heat-shock proteins select deltaF508-CFTR for endoplasmic reticulum-associated degradation. 2007, Pubmed
Asher, Aldosterone-induced increase in the abundance of Na+ channel subunits. 1996, Pubmed , Xenbase
Becker, Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo. 1996, Pubmed
Beggah, Degradation and endoplasmic reticulum retention of unassembled alpha- and beta-subunits of Na,K-ATPase correlate with interaction of BiP. 1996, Pubmed , Xenbase
Bhalla, Mechanisms of ENaC regulation and clinical implications. 2008, Pubmed
Bhamidipati, Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. 2005, Pubmed
Blond-Elguindi, Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. 1993, Pubmed
Brodsky, The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. 1999, Pubmed
Brodsky, Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70. 1993, Pubmed
Buck, The activities and function of molecular chaperones in the endoplasmic reticulum. 2007, Pubmed
Canessa, Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. 1994, Pubmed , Xenbase
Canessa, Membrane topology of the epithelial sodium channel in intact cells. 1994, Pubmed , Xenbase
Carvalho, Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. 2006, Pubmed
Cheng, Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. 1990, Pubmed
Christianson, OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD. 2008, Pubmed
Clerc, Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. 2009, Pubmed
Cormier, EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex. 2009, Pubmed
Coughlan, Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control. 2004, Pubmed
Cox, Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. 1993, Pubmed
Craig, The diverse roles of J-proteins, the obligate Hsp70 co-chaperone. 2006, Pubmed
DeLaBarre, Central pore residues mediate the p97/VCP activity required for ERAD. 2006, Pubmed
Denic, A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. 2006, Pubmed
Dong, ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C. 2008, Pubmed
Ellgaard, Quality control in the endoplasmic reticulum. 2003, Pubmed
Flynn, Peptide-binding specificity of the molecular chaperone BiP. 1991, Pubmed
Fu, Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator. 2003, Pubmed
Gauss, The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. 2006, Pubmed
Gillece, Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase. 1999, Pubmed
Gnann, Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. 2004, Pubmed
Goldfarb, Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels. 2006, Pubmed , Xenbase
Gonzales, Pore architecture and ion sites in acid-sensing ion channels and P2X receptors. 2009, Pubmed
Gupta, Heterologous expression of a mammalian epithelial sodium channel in yeast. 2000, Pubmed
Han, Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1. 2007, Pubmed
Hanwell, Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells. 2002, Pubmed , Xenbase
Harris, Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis. 2008, Pubmed , Xenbase
Hebert, In and out of the ER: protein folding, quality control, degradation, and related human diseases. 2007, Pubmed
Hennessy, Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions. 2005, Pubmed
Hill, Degradation of unassembled Vph1p reveals novel aspects of the yeast ER quality control system. 2000, Pubmed
Hirsch, The ubiquitylation machinery of the endoplasmic reticulum. 2009, Pubmed
Hosokawa, Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP. 2008, Pubmed
Hughey, Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits. 2003, Pubmed , Xenbase
Huyer, Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. 2004, Pubmed
Ismail, Have you HRD? Understanding ERAD is DOAble! 2006, Pubmed
Jasti, Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. 2007, Pubmed
Jentsch, Cdc48 (p97): a "molecular gearbox" in the ubiquitin pathway? 2007, Pubmed
Jin, ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues. 2009, Pubmed
Kabani, Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. 2003, Pubmed
Kashlan, Small heat shock protein alphaA-crystallin regulates epithelial sodium channel expression. 2007, Pubmed , Xenbase
Kim, Yos9p detects and targets misfolded glycoproteins for ER-associated degradation. 2005, Pubmed
Kimata, Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. 2007, Pubmed
Knittler, Molecular chaperones involved in protein degradation in the endoplasmic reticulum: quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum. 1995, Pubmed
Kota, Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD. 2007, Pubmed
Li, Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein. 2000, Pubmed
Lilley, Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane. 2005, Pubmed
Liu, Heat shock response relieves ER stress. 2008, Pubmed
Mall, Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. 2004, Pubmed
Masilamani, Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. 1999, Pubmed
McCracken, Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. 1996, Pubmed
Meacham, The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. 1999, Pubmed
Meacham, The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. 2001, Pubmed
Meerovitch, Proparathyroid hormone-related protein is associated with the chaperone protein BiP and undergoes proteasome-mediated degradation. 1998, Pubmed
Meunier, A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins. 2002, Pubmed
Meusser, ERAD: the long road to destruction. 2005, Pubmed
Mohan, Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells. 2004, Pubmed
Molinari, Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER. 2002, Pubmed
Mori, A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. 1993, Pubmed
Mueller, Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit. 2007, Pubmed
Mumberg, Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. 1995, Pubmed
Nakatsukasa, Dissecting the ER-associated degradation of a misfolded polytopic membrane protein. 2008, Pubmed
Neuber, Ubx2 links the Cdc48 complex to ER-associated protein degradation. 2005, Pubmed
Nishikawa, The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion. 1997, Pubmed
Nishikawa, Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. 2001, Pubmed
Oda, Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. 2006, Pubmed
Okuda-Shimizu, Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp. 2007, Pubmed
Pagant, Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal. 2007, Pubmed
Plemper, Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. 1997, Pubmed
Qiu, The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones. 2006, Pubmed
Quan, Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. 2008, Pubmed
Ravid, Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. 2006, Pubmed
Renard, Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. 1994, Pubmed , Xenbase
Rossier, The epithelial sodium channel (ENaC): new insights into ENaC gating. 2003, Pubmed
Rubenstein, Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. 2000, Pubmed
Rüdiger, Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone. 2001, Pubmed
Rüdiger, Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. 1997, Pubmed
Sato, Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase. 2009, Pubmed
Schlenstedt, A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s. 1995, Pubmed
Schmitz, In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER. 1995, Pubmed
Shen, Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress. 2002, Pubmed
Shen, ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates. 2005, Pubmed
Sikorski, A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. 1989, Pubmed
Silberstein, A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum. 1998, Pubmed
Skowronek, The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP. 1998, Pubmed
Snyder, Membrane topology of the amiloride-sensitive epithelial sodium channel. 1994, Pubmed , Xenbase
Snyder, Gating induces a conformational change in the outer vestibule of ENaC. 2000, Pubmed , Xenbase
Snyder, Minireview: regulation of epithelial Na+ channel trafficking. 2005, Pubmed
Staub, Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. 1997, Pubmed , Xenbase
Stirling, Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. 1992, Pubmed
Szathmary, Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. 2005, Pubmed
Valentijn, Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes. 1998, Pubmed , Xenbase
Vashist, Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. 2004, Pubmed
Vembar, One step at a time: endoplasmic reticulum-associated degradation. 2008, Pubmed
Walsh, The J-protein family: modulating protein assembly, disassembly and translocation. 2004, Pubmed
Wang, Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. 2006, Pubmed
Weisz, Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions. 2000, Pubmed , Xenbase
Yan, Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels. 2005, Pubmed
Ye, Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. 2005, Pubmed
Youker, Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast. 2004, Pubmed
Yu, HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells. 2000, Pubmed
Yu, Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3. 2005, Pubmed
Zerangue, A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. 1999, Pubmed , Xenbase
Zhang, Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. 2001, Pubmed