The Lhs1/GRP170 Chaperones Facilitate the Endoplasmic Reticulum-associated Degradation of the Epithelial Sodium Channel*

Background: The epithelial sodium channel (ENaC) is a substrate for the endoplasmic reticulum associated degradation (ERAD) quality control system. Results: The chaperone Lhs1/GRP170 selects the nonglycosylated form of the α subunit for ERAD. Conclusion: This study is the first to show a role for Lhs1/GRP170 in ERAD substrate selection. Significance: Mutations in ENaC are associated with human disease; therefore, Lhs1/GRP170, as a modulator of ENaC expression, may be a target for new therapeutic agents. The epithelial sodium channel, ENaC, plays a critical role in maintaining salt and water homeostasis, and not surprisingly defects in ENaC function are associated with disease. Like many other membrane-spanning proteins, this trimeric protein complex folds and assembles inefficiently in the endoplasmic reticulum (ER), which results in a substantial percentage of the channel being targeted for ER-associated degradation (ERAD). Because the spectrum of factors that facilitates the degradation of ENaC is incomplete, we developed yeast expression systems for each ENaC subunit. We discovered that a conserved Hsp70-like chaperone, Lhs1, is required for maximal turnover of the ENaC α subunit. By expressing Lhs1 ATP binding mutants, we also found that the nucleotide exchange properties of this chaperone are dispensable for ENaC degradation. Consistent with the precipitation of an Lhs1-αENaC complex, Lhs1 holdase activity was instead most likely required to support the ERAD of αENaC. Moreover, a complex containing the mammalian Lhs1 homolog GRP170 and αENaC co-precipitated, and GRP170 also facilitated ENaC degradation in human, HEK293 cells, and in a Xenopus oocyte expression system. In both yeast and higher cell types, the effect of Lhs1 on the ERAD of αENaC was selective for the unglycosylated form of the protein. These data establish the first evidence that Lhs1/Grp170 chaperones can act as mediators of ERAD substrate selection.

Endoplasmic reticulum (ER) 3 -associated degradation (ERAD) targets defective secreted and integral membrane proteins for destruction (1)(2)(3). After being selected for ERAD, substrates are delivered to the cytosolic proteasome. ERAD substrates include proteins containing mutations that lead to folding defects, proteins that fail to assemble with specific partners, and proteins whose activities are regulated by degradation. Because protein folding is inherently error-prone, even a significant percentage of some wild-type proteins is also targeted for ERAD.
Proteins that transit through the secretory pathway play essential roles in cellular homeostasis, so it is not surprising that many prominent diseases are also linked to ERAD substrates. Some of these diseases include cystic fibrosis (the cystic fibrosis transmembrane conductance regulator (CFTR)), antitrypsin deficiency (␣1-antitrypsin Z), nephrogenic diabetes insipidus (AQP2), and atherosclerosis (apolipoprotein B) (4). The epithelial Na ϩ channel (ENaC) is another protein that folds inefficiently in the ER and is associated with several diseases.
ENaC is a heterotrimeric protein composed of ␣, ␤, and ␥ subunits that is required for the reabsorption of sodium in the kidney collecting duct. Each subunit contains two transmembrane domains, a large extracellular loop, and a short N and C terminus that reside within the cytoplasm (5). It is generally assumed that the trimeric holochannel assembles in the ER before it traffics through the secretory pathway and functions at the apical membrane of several epithelia. Here, ENaC plays a critical role in the regulation of blood pressure and extracellular fluid balance within the body, and not surprisingly ENaC levels and activity are highly regulated (6 -9). Gain of function mutations in ENaC that prolong its residency at the plasma membrane result in high blood pressure associated with Liddle's syn-drome (10). In turn, loss of function mutations result in low blood pressure, salt-wasting, and pseudohypoaldosteronism type I (11).
The regulation and trafficking itinerary of ENaC at the cell surface have been extensively studied. In contrast, the early steps during ENaC biogenesis and quality control are not well characterized. However, it is clear that a significant proportion of each subunit is targeted for ERAD either when expressed alone or when coexpressed with the other subunits (12)(13)(14). These data are consistent with the notion that the ENaC subunits and holochannel are inherently unstable.
The ERAD pathway can be divided into distinct steps (3,15,16). First, proteins are recognized and targeted for degradation by molecular chaperones and chaperone-like lectins (17,18). Second, ERAD substrates are ubiquitinated and retrotranslocated from the ER to the cytosol. A specialized set of E2 (ubiquitin conjugating) and E3 (ubiquitin ligase) enzymes modifies ERAD substrates (19), which facilitates interaction with a AAA-ATPase, Cdc48-p97, and its ubiquitin binding partners. The Cdc48-p97 complex then drives the ATP-dependent retrotranslocation of substrate into the cytoplasm (20 -23). Finally, the cytosolic 26 S proteasome complex degrades ubiquitinated ERAD substrates, most likely with the aid of associated factors (24).
The molecular chaperone network housed within the ER not only helps select ERAD substrates but directly facilitates protein folding. Like other Hsp70s, the ER lumenal Hsp70, known as BiP (or Kar2 in yeast), recognizes substrates when bound to ATP. BiP/Kar2 specificity and function depend on cochaperones that assist in substrate binding, ATP hydrolysis (which traps the substrate), and nucleotide exchange (which helps release the substrate). One class of BiP/Kar2 cochaperones is the J-domain containing Hsp40s, which stimulate ATP hydrolysis. In yeast, the ER lumenal Hsp40s Jem1 and Scj1 function redundantly during BiP/Kar2-dependent ERAD substrate selection (25), and in mammals a select group of ER Hsp40s, the ERdjs, probably acts similarly (26 -28). Hsp40s can also bind substrates independent of Hsp70 and may deliver these polypeptides to Hsp70s (29 -32). To release bound substrates, nucleotide exchange factors (NEFs) stimulate the release of ADP, thus allowing the Hsp70 cycle to restart upon ATP binding. In yeast, there are two ER lumenal NEFs that act on BiP, Lhs1 and Sil1, and the mammalian homologs are known as GRP170 and SIL1, respectively. Lhs1 and Sil1 act as BiP cochaperones during protein translocation, or import, into the ER (33)(34)(35)(36)(37). However, there is no evidence linking the function of any of the ER NEFs to ERAD. This may be because Lhs1 and Sil1 are functionally redundant, and because the deletion of both Lhs1 and Sil1 is lethal in yeast (36) the role of these chaperones in ERAD cannot easily be investigated. Alternatively, the modest increase in the overall chaperone cycle facilitated by NEFs, in contrast to the profound stimulation by Hsp40s (38,39), may indicate that their function is dispensable for ERAD.
To better define the pathway by which ENaC is targeted for ERAD, we established a yeast expression system for each subunit and as expected found that degradation was ubiquitin ligase, Cdc48, and proteasome-dependent; in addition, we discovered that cytoplasmic small heat shock proteins (Hsps) facil-itated the degradation of the ENaC subunits in both yeast and in a Xenopus oocyte expression system (40,41). By further employment of the yeast ENaC expression system, we then found that the ER lumenal Hsp40s, Jem1 and Scj1, help target all three ENaC subunits for degradation, which was again confirmed in the oocyte expression system. Specifically, overexpression of the human Hsp40 homologs, ERdj3 and ERdj4, augmented the proteasome dependent degradation of ENaC (40).
In this study we show for the first time a requirement for an ER lumenal NEF for ERAD. We discovered that yeast lacking Lhs1 exhibit a degradation defect for the ENaC ␣ subunit. By examining the effect of an Lhs1 mutant on the turnover of ␣ENaC, we found that NEF activity was dispensable, consistent with previous data that ␣ENaC degradation was BiP/Kar2-independent (40) and suggesting that the chaperone "holdase" activity (i.e. the ability to prevent aggregation of a misfolded protein) is vital. Importantly, the effect of this chaperone on the stability of the ENaC ␣ subunit was conserved; when levels of the Lhs1 homolog, GRP170, were modulated in either Xenopus oocytes or in HEK293 cells, ␣ENaC turnover was impacted. Moreover, both Lhs1 and GRP170 co-precipitated with the ENaC ␣ subunit in yeast and mammalian cells, respectively. These data provide further support for the notion that the ERAD of ENaC exhibits unique requirements and provide the first demonstration that the Lhs1/Grp170 family of chaperones is involved in ERAD.

EXPERIMENTAL PROCEDURES
Yeast Strains and Growth Conditions-Yeast strains were propagated at 26°C using established methods, and media preparation and transformation were performed as published unless otherwise noted (42).
Yeast growth assays were performed as follows. The ⌬lhs1 yeast strain was transformed with pRS313-LHS1, pRS313-lhs1D26A, or pRS313 (see below) as a vector control. Yeast cultures were grown overnight in selective media to log phase, serially diluted, and pinned onto selective media. The plates were incubated at 26°C.
Mammalian Cell Growth and Transfection Conditions-HEK-293T cells were cultured at 37°C in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells were analyzed 24 h after transfection.
Plasmid Construction and Molecular Techniques-The constitutive expression of a C-terminal HA epitope tagged form of the ENaC subunits (40) or CPY* (43) was previously described. The CFTR expression plasmid was also previously described (44). pRS313-LHS1 and pRS313-lhs1D26A expression plasmids were generously supplied by the Stirling laboratory (45). A higher level of Lhs1 expression was achieved by inserting the Lhs1 coding sequence into plasmid, pRS423GPD (46), to generate pRS423GPD-myc-LHS1. In brief, the LHS1 gene was amplified and epitope-tagged by PCR using pRS313-LHS1 as a template, and the PCR product was digested with BamHI and SalI and ligated into the same sites in pRS423GPD.
To express non-glycosylated forms of the ENaC subunits, a pRS426GPD-⌬G␣ENaC-HA construct was created by PCR amplification of pBluescript (SKϪ)-⌬G␣ENaC (Kleyman laboratory) that lacked N-linked glycosylation sites, and the product was digested with EcoRI and ClaI and ligated into the corresponding sites in pRS426GPD (46). pRS426GPD-⌬G␤ENaC-HA and pRS426GPD-⌬G␥ENaC-HA were constructed using PCR overlap extension (47) to mutate the Asn residue (AAT or AAC) within each of the glycosylation consensus sites (Asn-X-Ser/Thr) to Gln (CAG). Five consensus sites were mutated to create ⌬G␥ENaC, and 13 sites were mutated to create ⌬G␤ENaC. pRS426GPD-␤ENaC-HA and pRS426GPD-␥ENaC-HA were used as template DNAs, and the final PCR products were digested with EcoRI and BamHI for ⌬G␤ENaC-HA or SpeI and HindIII for ⌬G␥ENaC-HA and ligated into the appropriate sites in pRS426GPD (46).
The pcDNA3.1-␣ENaC-HA mammalian expression vector was described previously (48) and a pcDNA3.1-GRP170 mammalian expression plasmid was created by PCR using pBacPAK-His1 GRP170 (49) as a template (a gift from the Subjeck laboratory, Roswell Park Medical Institute). PCR products were digested with XbaI and BamHI and ligated into the same sites in pCDNA3.1(Ϫ) (Invitrogen). All constructs generated for this study were confirmed by restriction digest and DNA sequence analysis. Primer sequences are available upon request.
Protein Degradation and Other Biochemical Assays-Cycloheximide chase analyses of the ENaC subunits, CPY* and CFTR, in yeast were performed as previously described (40). Cell lysates from chase samples were generated using alkaline lysis followed by trichloroacetic acid precipitation (50), and proteins were immediately resolved by SDS-PAGE before Western blot analysis. The ENaC subunits, CPY* and CFTR, were detected using anti-HA-HRP (clone 3F10; Roche Applied Science) at a dilution of 1:5000. Western blots were also probed with anti-glucose-6-phosphate dehydrogenase (G6P; Sigma) antisera, which served as a loading control. The G6P primary antibody was detected with a donkey horseradish peroxidase conjugated anti-rabbit IgG secondary (GE Healthcare). The Western blot signals were imaged using enhanced chemiluminescence (Pierce) and visualized on a Kodak 440CF Image Station. Quantitation was carried out using the imager-associated Kodak 1D software (Roche Applied Science). p values for all quantitative experiments were calculated using Student's t test.
For cycloheximide chase studies in HEK293 cells, cells transiently expressing the ENaC ␣ subunit were assayed 24 h posttransfection. To stop protein translation, cells were placed in fresh high glucose DMEM containing 10% FBS, L-glutamine, and cycloheximide (Sigma) at a final concentration of 100 g/ml. The cells were chased in a 37°C 5% CO 2 incubator for the indicated times. The cycloheximide-containing medium was aspirated; cells were washed twice with ice-cold Dulbecco's phosphate-buffered saline, scraped, collected, and isolated by centrifugation at 1000 ϫ g for 5 min. Cell pellets were lysed using detergent solution (50 mM Tris-HCl, pH 8.5, 1% Nonidet P-40, 0.4% sodium deoxycholate, and 62.5 mM EDTA supplemented with one Roche Applied Science mini EDTA-free complete protease inhibitor tablet per 7 ml of buffer, 1 mM PMSF, and 1 mM pepstatin (Sigma)). The samples were then incubated on ice for 20 min and centrifuged at 16,000 ϫ g for 5 min to remove insoluble material. Protein concentrations were determined using the Bradford assay (Bio-Rad). For samples subjected to SDS-PAGE, lysates were denatured in Laemmli buffer for 20 min at room temperature or heated to 90°C for 5 min, loaded onto 10% polyacrylamide gels, and resolved by SDS-PAGE as described previously (51). Other antibodies used for this study include anti-(mammalian) Sec61 (AbCam), and anti-GRP170 (a kind gift from the Hendershot Laboratory, St. Jude Children's Research Hospital).
For digestion of proteins from yeast with endoglycosidase H (EndoH; New England Biolabs), samples were prepared as described above except before SDS-PAGE, EndoH was added according to the manufacturer's instructions (Roche Applied Science) for 3 h at 37°C. For deglycosylation studies using mammalian cell lysates, 100 g of lysate diluted to a total of 300 l was precleared with 50 l of Sepharose 6B (Sigma) by endover-end rotation for 2 h at 4°C. Precleared lysate was added to 20 l of anti-HA-agarose (Sigma) and rotated end-over-end at 4°C overnight. The samples were centrifuged at low speed, and the beads were washed 4 times in 1 ml of ice-cold PBS. The immunoprecipitated proteins were then eluted with glycoprotein denaturing buffer (New England Biolabs) by incubating the beads at 90°C for 2 min. G5 buffer and Endoglycosidase H were added to the eluate (10 g of lysate), and the sample was incubated at 37°C for 3 h.
For peptide-N-glycosidase F digestions, immunoprecipitated proteins were eluted with glycoprotein denaturing buffer by incubating the beads at 90°C for 2 min. G7 Buffer, Nonidet P-40, and peptide-N-glycosidase F (New England Biolabs) were added to the eluate, and the sample was incubated at 37°C for 1 h. A total of 10 g of lysate was used.
To separate yeast cellular membrane and cytosolic fractions, a total of ϳ1 A 600 of cells in log phase were pelleted in a microcentrifuge. The pellet was resuspended in 200 l of lysis buffer (20 mM HEPES, pH 7.4, 0.1 M sorbitol, 50 mM potassium acetate, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 2 g/ml leupeptin, 0.1 g/ml pepstatin A), and cells were disrupted by the addition of glass beads and agitation on a Vortex mixer 4 times for 1 min with incubations of 1 min on ice in between each step. After the supernatant was removed, unbroken cells were pelleted by cen-Lhs1 Selects ␣ENaC for ERAD trifugation at 3000 rpm for 3 min in a microcentrifuge, and the supernatant was retained. An aliquot was removed for Western blot analysis for total (T) protein, and the remaining lysate was centrifuged at 13,000 rpm for 20 min. An aliquot of the supernatant was saved (S1), and the pellet (P1) was resuspended in 100 l of lysis buffer. A second 13,000 rpm 20 min spin was carried-out to obtain a second supernatant fraction (S2) and pellet fraction (P2). Aliquots from each fraction were mixed with SDS sample buffer, and proteins were resolved by SDS-PAGE and Western blotting as described above. For the integral membrane protein control, anti-Sec61 antiserum (52) was used.
Lhs1 and Grp170 coimmunoprecipitation assays were performed in yeast and in ␣ENaC transfected HEK293 cells, respectively. In yeast, ⌬lhs1 yeast were transformed with pRS423GPD-myc-LHS1 and either pRS426GPD-␣ENaC-HA or a vector control, pRS316. A 25-ml overnight culture of each strain was grown to log phase (A 600 ϭ 1.0), and cells were pelleted and resuspended in 500 l of lysis buffer (150 mM NaCl 50 mM Tris, pH 7.5, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 2 g/ml leupeptin, 0.1 g/ml pepstatin A) and then disrupted by the addition of glass beads and agitation on a Vortex mixer 4 times for 1 min with incubations of 1 min on ice in between each step. The cell debris was removed from the resulting lysate by centrifugation at 8000 rpm for 8 min. The lysate was then incubated with 18 l of anti-HA affinity matrix (Roche Applied Science) for 3 h at 4°C on a rotator. The affinity beads were washed 3 times with lysis buffer and resuspended in SDS sample buffer, heated at 70°C for 10 min, and subject to analysis by SDS-PAGE and Western blotting. Western blots were probed with either anti-HA antibody (Roche Applied Science) or anti-Lhs1 antibody (a kind gift from the E. Craig laboratory, University of Wisconsin) and detected as described above.
To detect co-immunoprecipitation of HA-tagged ␣ENaC with GRP170 in HEK293 cells, 100 g of lysate diluted to a total volume of 300 l was precleared with 30 l of protein A/Gagarose slurry (Calbiochem) by end-over-end rotation overnight at 4°C. A 1-l aliquot of GRP170 antibody was added to the precleared lysate with 30 l of fresh protein A/G beads, and the solution was rotated end-over-end at room temperature for 1 h. The samples were centrifuged at low speed, and the beads were washed 4 times in 1 ml of PBS containing 0.1% Triton X-100 and 100 mM KCl. Immunoprecipitated proteins were eluted by incubating the beads at room temperature for 20 min in SDS sample buffer. For reciprocal co-immunoprecipitation of GRP170 with ␣ENaC-HA, a total of 200 g of lysate diluted to 300 l was precleared with 50 l of Sepharose 6B (Sigma) by end-over-end rotation overnight at 4°C. Precleared lysate was added to 30 l of anti-HA-agarose (Sigma) and rotated endover-end at room temperature for 1 h. The samples were centrifuged at low speed, and the beads were washed 4 times in 1 ml of PBS containing 0.1% Triton X-100 and 100 mM KCl. Immunoprecipitated proteins were eluted by incubating the beads at room temperature for 20 min with SDS sample buffer.
Functional Analysis of ␣ENaC Channel Activity in Xenopus Oocytes-␣ENaC function was analyzed in Xenopus oocytes using two-electrode voltage clamp (41). The cDNA encoding the murine ␣ENaC subunit was previously described (40), and the cDNA encoding GRP170 from pGEM4Z-GRP170 was made by PCR amplifying GRP170 from pBacPAK-His1-GRP170 (49). The PCR product was digested with BamHI and SacI and ligated into pGEM4Z (Promega). For ␣ENaC the cRNA was prepared from linearized DNA templates using T3 RNA polymerase according to the manufacturer's instructions (Ambion). For GRP170 the cRNA was prepared from linearized DNA using T7 polymerase according to manufacturer's instructions (Promega). Stage V and VI oocytes were injected with cRNAs encoding ␣ENaC (1 or 5 ng) and 5 ng of GRP170 cRNA.
After injection the oocytes were incubated at 18°C in modified Barth's saline (15 mM HEPES, pH 7.4, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3 , 0.3 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , 10 g/ml sodium penicillin, 10 g/ml streptomycin sulfate, and 100 g/ml gentamycin sulfate). A two-electrode voltage clamp assay was performed 48 h after injection using a DigiData 1320A interface and a GeneClamp 500B voltage clamp amplifier (Axon Instruments). Data acquisition and analyses were performed using pClamp software, Version 8.2 (Axon Instruments). Glass pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc.) with a Micropipette Puller (Sutter Instrument Co.) and had a resistance of 0.3-5 megaohms when filled with 3 M KCl and inserted into the bath solution. During the recording oocytes were placed in a recording chamber (Automate Scientific) and perfused continuously at a flow rate of 3 ml/min with bath solution (10 mM HEPES, pH 7.4, 110 mM NaCl, 2 mM KCl, 2 mM CaCl 2 ), and subsequently with this solution supplemented with 10 M amiloride.

Lhs1
Facilitates the Degradation of ␣ENaC-By developing and then employing a yeast expression system for each ENaC subunit, we showed that the ERAD of the ENaC subunits does not require the ER lumenal Hsp70, BiP/Kar2, but depends on two ER lumenal Hsp40s, Jem1 and Scj1 (40). This result was unexpected because the requirement for Hsp40s during ERAD is usually accompanied by Hsp70 dependence (25,26,(53)(54)(55)(56)(57). Because ENaC degradation was BiP/Kar2P-independent, we hypothesized that ENaC degradation might be facilitated by another Hsp70-like molecule in the ER, Lhs1. Lhs1 is an ER resident, ϳ116-kDa glycoprotein that contains an ER retention signal. The mammalian homolog, GRP170, is also an ER retained glycoprotein (58,59). Lhs1/GRP170 have an extended loop domain (60) and exhibit holdase activity (45,61), which prevents the aggregation of protein substrates until they are delivered to other chaperone systems. Lhs1/GRP170 are also NEFs (35,45,59,62,63) and triggers nucleotide exchange by affecting the Hsp70 structure in a very similar manner to another class of NEFs, the Hsp110s, (62) but in a mechanistically distinct fashion from Sil1 (63).
To test whether Lhs1 affects ENaC subunit stability, cycloheximide chase experiments were performed in either a WT yeast strain or a ⌬lhs1 strain expressing ␣, ␤, or ␥ENaC over a 90-min time period (Fig. 1A). As observed previously (40), the turnover of the ␣ subunit was more extensive than the ␤ or ␥ subunits in the WT strain (40). As hypothesized, we found that ␣ENaC degradation was significantly less efficient in the ⌬lhs1 strain in comparison to the WT strain. In contrast, the degradation of the ␤and ␥ENaC subunits was unaffected. To confirm that the Lhs1 protein was absent in the ⌬lhs1 strain, a Western blot was performed on cell lysates from LHS1 and ⌬lhs1 yeast, and the blot was probed with anti-Lhs1 antisera (Fig. 1B). The Lhs1 antibody detected a prominent species at the expected molecular weight in the LHS1 strain that was absent in the ⌬lhs1 strain, thus confirming that the Lhs1 gene was deleted.
To determine whether Lhs1 only affects ␣ENaC or plays a more general role in selecting misfolded proteins for degradation, we assayed the degradation of two other well characterized ERAD substrates, the inefficiently folded transmembrane protein CFTR and the misfolded soluble protein, CPY*; yeast expression systems for both of these ERAD substrates have been used extensively (50, 54, 64 -67). Deletion of Lhs1 had no effect on the degradation of either protein (Fig. 2, A and B). These results are also consistent with a pulse-chase degradation assay showing that the deletion of Lhs1 had essentially no effect on CPY* degradation (25).
The best-characterized role for Lhs1 is as a NEF for BiP/Kar2 (35,45,63). To determine whether the degradation defect is Lhs1-specific we assayed ␣ENaC degradation in yeast deleted for the gene that encodes another ER lumenal NEF, Sil1. Lhs1 and Sil1 exhibit overlapping functions, and both catalyze nucleotide exchange on BiP/Kar2 and facilitate protein translocation (36). In fact, Sil1 is the only gene that rescues the synthetic lethal effect of an ⌬lhs1⌬ire1 strain, which besides lacking Lhs1 is unable to mount an unfolded protein response. However, we found that ␣ENaC degradation was not significantly altered in a ⌬sil1 strain when compared with WT yeast (Fig. 2C). In con-trast, the Weissman laboratory observed a minor effect on the ERAD of CPY* when a pulse-chase assay was performed in a ⌬sil1 strain (68). Together, these data suggest that Lhs1 and Sil1 play unique roles in ER physiology. The data are also consistent with the lack of structural similarity between these chaperones and with the fact that select functions are associated with only one of the two NEFS (i.e. only Lhs1 exhibits holdase activity (45)).
Nonglycosylated ␣ENaC Is Preferentially Stabilized in the Absence of Lhs1-Close inspection of ␣ENaC degradation in ⌬lhs1 yeast suggested that a lower molecular weight band was preferentially stabilized (see the Fig. 1A gel). ␣ENaC contains six consensus sites for N-linked glycosylation (5, 69) that are modified with variable efficiencies in divergent systems (40, 70 -72). To confirm that the lower molecular weight band represents the unglycosylated protein rather than a truncated form of the protein, we treated samples from both WT and ⌬lhs1 yeast expressing ␣ENaC with EndoH to remove N-linked oligosaccharides. EndoH treatment collapsed the higher molecular weight species to a single lower molecular weight species in both the WT and ⌬lhs1 samples (Fig. 3A). This result confirms that the preferentially stabilized protein is unglycosylated and is not a truncated or proteolytically cleaved form of ␣ENaC. We compared the relative ratio of these two forms of ␣ENaC in WT and ⌬lhs1 strains at time 0 (from Fig. 1A) and found that significantly less unglycosylated material was present in WT strains (Fig. 3B). To confirm these data, the Lhs1 mutant was transformed with either an empty vector or with an Lhs1 expression vector. Lhs1 reduced the proportion of unglycosylated ␣ENaC compared with the empty vector (Fig. 3C, compare the ratio of glycosylated to unglycosylated protein in the ϩLHS1 versus ϩvector conditions), suggesting Lhs1 specifically facilitates the degradation of unglycosylated ␣ENaC.
The addition of N-linked sugars occurs during and after protein translocation into the ER lumen (73)(74)(75). Because Lhs1 aids in the translocation of some proteins (33,34,36), we investigated whether the unglycosylated protein observed in the ⌬lhs1 strain was untranslocated, which would leave a portion of the protein in the cytosol and inaccessible to the glycosylation machinery. To rule this scenario out, we isolated supernatant and pellet (ER membrane containing) fractions from WT and ⌬lhs1 yeast cell extracts and immunoblotted for ␣ENaC (HA), a cytosolic protein, glucose-6-phosphate dehydrogenase (G6P), or an integral ER membrane protein, Sec61 (Fig. 3D). All three proteins were present in the total cell lysates (T), whereas only the soluble, cytosolic protein, G6P, was present in the superna-tant (S1) fraction. Sec61 and ␣ENaC were present in the pellet fraction (P1) in both WT and ⌬lhs1 strains. After the initial spin the supernatant (S1) was removed, and the pellet was resuspended in buffer and subject to a second centrifugation. The resulting supernatant fraction (S2) lacked G6P because the cytosol was completely removed with the S1 fraction, whereas the integral membrane proteins ␣ENaC and Sec61 were observed in the P2 fractions. Importantly, we observed no differences between the residence of ␣ENaC in the WT and ⌬lhs1 strains. These results suggest that ␣ENaC is both efficiently translocated and membrane-associated. We also examined whether the conformation of ␣ENaC was significantly altered when Lhs1 was deleted, which might occur if the protein was inefficiently translocated. We performed a protease digestion assay using increasing concentrations of trypsin. However, we FIGURE 2. The ERAD of CFTR and CPY* is Lhs1-independent, and the nucleotide exchange factor Sil1 is dispensable for ␣ENaC degradation. A and B, cycloheximide chase reactions were performed as described under "Experimental Procedures" in LHS1 wild-type (filled circles) or ⌬lhs1 (open circles) yeast strains expressing a C-terminally 3HA epitope-tagged form of CFTR or a C-terminally 3HA-tagged form of CPY*. C, cycloheximide chase reactions were performed as described above in SIL1 (filled circles) or ⌬sil1 (open circles) yeast strains expressing a C-terminally HA epitope tagged form of ␣ENaC. All chase reactions were performed at 37°C, and lysates were immunoblotted with anti-HA antisera (ENaC, CFTR, and CPY*) and with anti-G6P as a loading control. Data represent the means of 4 -7 experiments, ϮS.E. failed to identify any major differences in the ␣ENaC proteolytic patterns when using lysates from the ⌬lhs1 strain in comparison to the WT strain (data not shown).
Based on the data presented above we next wished to investigate if the presence of N-linked sugars altered the Lhs1-dependent degradation of the ENaC subunits. We generated ENaC expression constructs that lacked recognition sites for N-linked glycosylation (⌬G constructs) (Fig. 4A). The Asn within the glycan acceptor consensus sequence, Asn-X-Ser/ Thr, was mutated to a Gln at six sites in ␣ENaC, 13 sites in ␤ENaC, and 5 sites in ␥ENaC. EndoH digestion of all three native constructs collapsed the bands to a single, lower, molecular weight species, which co-migrated with the corresponding ⌬G ENaC construct regardless of whether EndoH was added (Fig. 4B). This confirms that the ⌬G ENaC subunits are not glycosylated.
We then individually transformed the ⌬G ␣, ␤, and ␥ENaC constructs into WT and ⌬lhs1 yeast and assessed degradation as described above. Interestingly, deletion of the glycosylation sites had no measurable effect on the degradation rates of ␣, ␤, or ␥ENaC in WT yeast (compare Fig. 4C, WT, to Fig. 1A, WT, degradation rates) despite data suggesting glycans are important for degradation, folding, and maturation (17,18,76). Deletion of the glycan acceptor sites also had no effect on the Lhs1dependent degradation of the ␤or ␥ENaC subunit, as neither subunit showed any Lhs1 dependence. In contrast, the ERAD of the ⌬G version of ␣ENaC was clearly Lhs1-dependent (Fig. 4C). We compared the quantified signals corresponding to the unglycosylated ␣ENaC species in ⌬lhs1 yeast from Fig. 1A to the ⌬G-␣ENaC signals in WT and ⌬lhs1 cells (Fig. 4C) and found them indistinguishable (Fig. 4D). These data confirm that Lhs1 selectively facilitates the degradation of the unglycosylated form of ␣ENaC.
Lhs1 Nucleotide Exchange Activity Is Dispensable for ␣ENaC Degradation-Lhs1 mutants that are unable to bind ATP no longer associate with or stimulate BiP/Kar2 ATPase activity (45) but are still able to act as holdases, preventing the aggregation of a misfolded protein in vitro. Because the degradation of ␣ENaC is BiP/Kar2-independent (40), we hypothesized that the holdase, but not the NEF activity, is important during the ERAD of ␣ENaC. To examine this hypothesis, ⌬lhs1 yeast expressing ␣ENaC were transformed with a vector engineered to express Lhs1 or Lhs1 with a mutation in the ATP binding domain that prevents NEF activity (Lhs1 D26A; Ref. 45) or with a vector control. We found that both Lhs1 and Lhs1 D26A rescued ␣ENaC degradation, in agreement with our hypothesis (Fig.  5A). It is noteworthy that both Lhs1 and Lhs1 D26A specifically accelerated the degradation of the unglycosylated ␣ENaC species, which was quite stable in the ⌬lhs1 background. This result also supports our previous finding that ␣ENaC degradation is BiP/Kar2-independent (40), as Lhs1 D26A is unable to associate with or stimulate the ATPase activity of BiP/Kar2. Although Lhs1 D26A enhanced ␣ENaC degradation, the expression of this mutant does impair other Lhs1-dependent activities (45). We performed two experiments to confirm that Lhs1 D26A-expressing yeast exhibit the anticipated mutant phenotype. First, we tested whether Lhs1 and Lhs1 D26A could rescue the growth defect of the ⌬lhs1 strain. Growth of ⌬lhs1 yeast expressing Lhs1 D26A was more robust than that of the vector control but less so than ⌬lhs1 yeast expressing Lhs1 (Fig.   FIGURE 3. The unglycosylated form of ␣ENaC accumulates in the absence of Lhs1. A, lysates were prepared from LHS1 WT or ⌬lhs1 yeast expressing a C-terminal HA-tagged form of ␣ENaC and were incubated at 37°C in the presence or absence of EndoH before performing an immunoblot analysis with anti-HA antiserum to detect ENaC. B, the relative percent of unglycosylated protein was quantitated using the zero time point in Fig. 1A (␣ENaC). Data represent the means of four experiments Ϯ S.E.; **, p Ͻ 0.01 C, lysates were prepared from ⌬lhs1 yeast expressing a C-terminal, HA-tagged form of ␣ENaC and a plasmid containing Lhs1 or a vector control and were treated with EndoH and analyzed as above. D, lysates were prepared from WT or ⌬lhs1 yeast expressing a C-terminal HA-tagged form of ␣ENaC and membrane (P1 and P2) and supernatant fractions (S1 and S2) were isolated as described under "Experimental Procedures." These fractions and an aliquot of the total cell lysate (T) were immunoblotted with anti-HA antisera (ENaC) or anti-G6P as a cytosolic control or anti-Sec61 as an integral ER membrane protein control. 5B). Western blots of lysates from these yeast suggested similar levels of Lhs1 expression (Fig. 5B). Second, we tested whether Lhs1 and Lhs1 D26A restored efficient translocation of a model yeast preprotein, prepro-␣-factor, into the ER of ⌬lhs1 yeast (34,35). Lhs1 can rescue this defect of ⌬lhs1 yeast, but Lhs1 D26A cannot (45). Therefore, we transformed ⌬lhs1 yeast with a plasmid that encodes a version of prepro-␣-factor that has had the glycosylation sites mutated and contains a C-terminal HA epitope tag (⌬Gpp␣F-HA) (77) and monitored translocation into the ER by a decrease in molecular weight caused by signal sequence cleavage. In the absence of Lhs1 (Fig. 5C, vector), a significant population of the ⌬Gpp␣F-HA remained untranslocated (upper species), whereas yeast expressing the wild-type version of Lhs1 completely translocated the substrate (i.e. only the faster migrating species is evident). In contrast, the Lhs1 D26A mutant partially rescued the translocation defect, implying that the holdase function of Lhs1 helps facilitate the action of Lhs1 during protein translocation. To some extent, these data are in contrast to previous work in which translocation was suggested to exclusively require Lhs1 NEF activity (36,45). In any event, our results indicate that the Lhs1 D26A version of Lhs1 facilitates ␣ENaC degradation while exhibiting other, expected cellular defects.
We previously reported that two ER lumenal Hsp40s, Jem1 and Scj1, are also required to support maximal rates of ␣ENaC degradation (40). To determine whether Lhs1 functions in the same degradation pathway with these Hsp40s, we constructed a yeast strain in which all three chaperones were deleted (⌬jem1⌬scj1⌬lhs1). We found that ␣ENaC degradation was slower in the ⌬jem1⌬scj1⌬lhs1 (Fig. 6) strain than in either the ⌬lhs1 (Fig. 1A)   90-min time point versus only 27% for the ⌬lhs1 strain and 36% for the ⌬jem1⌬scj1 strain. Because the degradation defect was greater in the triple mutant than in the single or double mutants, we suggest that Lhs1 functions in an independent degradation pathway from Jem1 and Scj1 or that these proteins bind to unique regions in ␣ENaC and maintain the protein in a soluble state before retrotranslocation and degradation. Lhs1 and GRP170 Coprecipitate with ␣ENaC-Our data suggest that Lhs1 selects unglycosylated ␣ENaC for ERAD. Therefore, we tested whether Lhs1 co-precipitates with ␣ENaC in yeast. Yeast expressing ␣ENaC or containing a vector control were lysed, and the protein was precipitated. As shown in Fig.   7A, we found Lhs1 but not an abundant cytoplasmic protein (G6P) in the precipitate.
The mammalian ortholog of Lhs1 is GRP170, which has similarly been shown to act as a NEF for BiP (59). To assess whether GRP170 interacts with ␣ENaC, we performed co-immunoprecipitation experiments in HEK-293T cells. As demonstrated in Fig. 7B, anti-HA-agarose resin immunoprecipitated endogenously expressed GRP170 from lysates of cells expressing HAtagged ␣ENaC. We observed almost no background binding of the chaperone to resin lacking the anti-HA antibody conjugate. We also observed co-immunoprecipitation of GRP170 and ␣ENaC in the reciprocal direction. In these experiments anti-GRP170 antibody specifically immunoprecipitated two lower molecular mass forms of ␣ENaC that migrated at ϳ70 kDa (Fig.  7B). We suspected that these lower molecular weight bands represented the immature, unglycosylated channel based on our yeast experiments. Therefore, we next performed deglycosylation studies. We digested ␣ENaC-HA from whole cell lysates with Endo H (which cleaves high mannose core-N-glycans) or peptide-N-glycosidase F (which removes all types of N-linked oligosaccharides), both of which resulted in the collapse of the anti-HA immunoreactive signal to two bands (Fig.  7C). These bands were identical in molecular weight to the species that selectively co-immunoprecipitated with GRP170. Collectively, these data strongly suggest that ␣ENaC and Lhs1/ GRP170 selectively interact and that this interaction appears to favor the unglycosylated ␣ subunit.
GRP170 Promotes ␣ENaC Degradation in Human and Vertebrate Cells-Because Lhs1 promotes the ERAD of ␣ENaC in yeast, we reasoned that overexpression of GRP170 in human cells should have a similar effect on the channel stability. To test this hypothesis, we cotransfected HEK293 cells with HAtagged ␣ENaC and either a cDNA encoding human GRP170 or an empty vector control. We performed cycloheximide chase yeast strains expressing the indicated protein or containing a vector control were serial-diluted and plated on selective medium and incubated at 26°C. Cells from the plates were resuspended in lysis buffer and lysed, and the total protein was subjected to immunoblot analysis with anti-Lhs1 antisera and anti-G6P as a loading control. C, ⌬lhs1 yeast strains expressing ⌬Gpp␣F-HA and containing a vector engineered for the expression of either wild-type Lhs1 or Lhs1 D26A or a vector control were grown to log phase, and proteins were precipitated as described under "Experimental Procedures" and immunoblotted with anti-HA antisera. assays to monitor ␣ENaC turnover. As predicted, and consistent with stabilization of ␣ENaC in the absence of Lhs1 in yeast, GRP170 overexpression significantly increased the rate of ␣ENaC degradation (Fig. 8). GRP170 overexpression was also evident by Western blot with anti-GRP170 anti-serum. Of note, overexpression of the GRP170 chaperone accelerated the turnover of both core and unglycosylated ␣ENaC. Because GRP170 selectively interacts with unglycosylated ␣ENaC (Fig. 7), these results are consistent with our other data indicating that GRP170 enhances the clearance of unglycosylated ␣ENaC sub-units from the ER. This, in turn, would decrease the number of channels that advance along the biosynthetic pathway, resulting in destabilization of the core-glycosylated ␣ENaC pool.
To confirm that GRP170 facilitates the ERAD of the ␣ subunit of ENaC, we assayed the functional effect of GRP170 overexpression on ␣ENaC. In Xenopus oocytes, the ␣ subunit of ENaC is capable of forming a Na ϩ conducting channel in the absence of the ␤ and ␥ subunits (78), albeit with significantly reduced Na ϩ currents. We previously established that the current mediated by the ␣ subunit correlates inversely with pro-FIGURE 7. Lhs1 and its mammalian homolog, GRP170, co-immunoprecipitate with ␣ENaC in yeast and mammalian cells, respectively. A, right, cell lysates from ⌬lhs1 yeast expressing pRS423GPD myc-LHS1 and C-terminal HA-tagged ␣ENaC or containing a vector control were subject to immunoprecipitation (IP) with anti-HA-agarose resin and immunoblotted for anti-HA (␣ENaC), anti-Lhs1, or anti-G6P to serve as both a loading and an immunoprecipitation control. Left, 5% whole cell lysate inputs for immunoprecipitation were immunoblotted as indicated. B, top left, whole cell lysates from HEK293T cells transfected with either HA-tagged ␣ENaC (ϩ) cDNA or an empty vector (Ϫ) were immunoprecipitated with anti-HA agarose resin and immunoblotted (IB) with anti-GRP170 antibody to detect the co-immunoprecipitation of endogenous GRP170. Bottom left, 10% of the whole cell lysate inputs for the immunoprecipitation were immunoblotted for ␣ENaC-HA or GRP170 with the indicated antibodies. The experiment is representative of four experiments. Top right, whole cell lysates from HEK293T cells transfected with either ␣ENaC-HA (ϩ) cDNA or empty pcDNA3.1 vector (Ϫ) were subjected to immunoprecipitation with polyclonal anti-GRP-170 antibody, and the immunoprecipitates were immunoblotted with anti-HA antibodies. Two lower molecular weight ␣ENaC species that specifically co-immunoprecipitate with the anti-GRP170 antibodies are indicated with brackets. Bottom right, 10% inputs of the whole cell lysates used in the immunoprecipitation were immunoblotted with the indicated antibodies. Data are representative of three experiments. C, deglycosylation of ␣ENaC in lysates from HEK293T cells is shown. Whole cell lysates were prepared from HEK293T cells transiently expressing HA-tagged ␣ENaC and were subject to an immunoprecipitation analysis with anti-HA agarose resin. The immunoprecipitates were then examined in deglycosylation reactions with EndoH or peptide-Nglycosidase F (PNGaseF) as described under "Experimental Procedures." The samples were fractionated by SDS-PAGE, and ␣ENaC subunits were detected with HRP-conjugated anti-HA antibodies. Core-glycosylated ␣ENaC is indicated with an asterisk, and unglycosylated ␣ENaC is indicated with brackets. teasome activity in the oocyte system and thus provides a readout for ERAD; moreover, we used this model to verify that homologs of yeast chaperones function analogously in vertebrate cells (40,41). We injected cRNA for ␣ENaC into Xenopus oocytes with or without GRP170 cRNA and measured amiloride-sensitive Na ϩ (ENaC-specific) current 48 h later. We predicted that if GRP170 promotes the degradation of ␣ENaC, then co-injection of GRP170 cRNA would increase ␣ENaC degradation and decrease the amiloride-sensitive Na ϩ current. Consistent with our prediction, co-injection of GRP170 cRNA reduced the amiloride-sensitive Na ϩ current by ϳ14 -20% when either 1 or 5 ng of cRNA corresponding to ␣ENaC was introduced (Fig. 9). Although the reduction of ␣ENaC current was not as substantial as the results seen in HEK293 and yeast experiments, this may be due to very low currents (ϳ30 -100 nA) produced by expressing the ␣ subunit alone and perhaps the inability of the oocyte ER to translocate substantial amounts of both ␣ENaC and GRP170 into the ER. Nonetheless these data are consistent with results in both the yeast and mammalian cell systems that the chaperones play an important role in ␣ENaC quality control. More broadly, our results provide the first evidence that GRP170 facilitates the degradation of an established ERAD substrate.

DISCUSSION
We present three novel findings in this study. First, members of the Lhs1 family of chaperones play a role in ERAD. Second, the mechanism of Lhs1 action during ERAD is BiP/Kar2-independent and appears to require the chaperones' holdase activity. Third, Lhs1 selectively targets the unglycosylated form of ␣ENaC for degradation, suggesting that Lhs1 exhibits substrate specificity for ERAD substrates that are unglycosylated.
Our discovery that the human Lhs1 ortholog, GRP170, contributes to the ERAD of ␣ENaC in higher cell types once more validates our yeast genetics approach to identifying factors that affect quality control decisions for mammalian ERAD substrates. We have successfully applied our approach to varied substrates including: CFTR (50,54,66,79), apolipoprotein B (80, 81), antitrypsin-Z (82)(83)(84), and the sodium chloride cotransporter (51). In each case yeast model studies have led us to identify conserved, human homologs of chaperones and chaperone-like proteins that impact the ERAD of these substrates in higher eukaryotes.
Previous work demonstrated that Lhs1 helps repair a heatdenatured, synthetic protein substrate in the ER (85). Specifically, yeast lacking Lhs1 were unable to refold Hsp105⌬-␤-lactamase after denaturation, which led to its degradation. Furthermore, Lhs1 precipitated with the heat-denatured protein as well as with pro-CPY, another secretory pathway protein, after cells were heated. However, Lhs1 was dispensable for the folding and secretion of Hsp105⌬-␤-lactamase and pro-CPY under standard cellular conditions. GRP170 has also been shown to coprecipitate with misfolded and incompletely folded proteins (58,86,87). The contribution of Lhs1/GRP170 to the degradation of these substrates was unexplored. These data are consistent with our finding that Lhs1/GRP170 associates with ␣ENaC.
We also found that Lhs1 is not required for the degradation of a variety of other ERAD substrates. A dual role for a chaperone or cochaperone in both preventing the degradation of a misfolded protein and promoting the degradation of another substrate is not without precedent. For example, BiP/Kar2 is required to traffic the yeast secretory protein, CPY (88), but  targets a mutated version of the same protein, CPY*, for ERAD (89,90). Similarly, Hsp90 can act as either a pro-folding or prodegradative chaperone during ERAD, depending on the substrate (see for example Refs. [91][92][93][94]. Perhaps the best-known role of the Lhs1/GRP170 chaperones is that they act as NEFs for BiP/Kar2. The exchange activities of Lhs1 and Sil1, the other NEF in the ER, facilitate the translocation of nascent-secreted proteins into the ER in cooperation with BiP/Kar2 (33)(34)(35)(36)(37). Sil1 lacks sequence similarity with Lhs1/GRP170 but shows some similarity to a cytosolic yeast NEF, Fes1, and a homologous cytoplasmic mammalian NEF, HspBP1 (33,37,95). Unlike Sil1, Lhs1 contains an Hsp70like domain and is 25% identical and shares 50% similarity with Kar2 (85, 96) but is structurally more similar to the cytosolic Hsp110 chaperone family (97). Both Lhs1/GRP170 and the Hsp110 proteins possess an extended loop that separates the ␣-helical region within the substrate binding domain from a series of ␤-sheets (60,98,99). Although the function of this loop is largely uncharacterized, Lhs1/GRP170 triggers nucleotide exchange by affecting the Hsp70 structure in a very similar manner to the Hsp110, Sse1 (62), but in a mechanistically distinct fashion from Sil1 (63). We initially reasoned that Lhs1 NEF activity would be critical for its action during the ERAD of ␣ENaC. This was not the case, as a mutant form of the protein defective in NEF activity and BiP/Kar2 binding completely rescued the ⌬lhs1 degradation defect. These data are consistent with a previous study from our laboratory showing that the degradation of ␣ENaC is BiP/Kar2-independent (40). Thus, Hsp70 cochaperones can exhibit Hsp70 (BiP)-independent activities in cells and can be considered bona fide chaperones. This is also consistent with our conclusion that the Lhs1/ GRP170 holdase activity is instead required for the ERAD of unglycosylated ␣ENaC. Because unglycosylated substrates are aggregation-prone (100, 101), we envision that the Lhs1/ GRP170 holdase activity maintains ␣ENaC in a soluble, retrotranslocation-competent state.
One question that arises from our work is why Lhs1 specifically targets unglycosylated ␣ENaC, but not ␤or ␥ENaC, for ERAD. We hypothesize several non-mutually exclusive sources for these differences. First, although 30 -40% identical, the three subunits are regulated and modified differently (7,102). For example, in the kidney the ␤ and ␥ subunits are constitutively expressed at much higher levels than the ␣ subunit and are subject to degradation by the proteasome (6,13,14) until ␣ subunit expression is induced by aldosterone (103,104). When all three subunits are present, channel assembly and trafficking to the cell surface can occur. In contrast the ␣ subunit can assemble and traffic to the plasma membrane in the absence of the ␤ and ␥ subunits, albeit with low efficiency (78), whereas the orphaned ␤ and ␥ subunits do not form a functional sodium channel and may be retained within the ER. This is most likely evident because ␣ENaC contains a C-terminal ER-exit signal (105), which the other subunits apparently lack. It is possible that the ability to assemble and exit the ER may alter the complement of chaperones involved in the quality control of the ␣ subunit. Second, ␣ subunit folding in yeast is less efficient than the ␤ or ␥ subunits, which may cause it to be recognized by Lhs1 (40). Third, the three subunits exhibit differences in post-trans-lational modification that may be recognized by Lhs1 (7,102). For example, only the ␤ and ␥ subunits are modified by Cys palmitoylation (106).
Our findings further suggest that unique degradation pathways exist for the unglycosylated and glycosylated protein. Importantly, the inefficient glycosylation as well as the degradation differences we observed for the glycosylated versus unglycosylated forms of ␣ENaC are not merely an artifact of the yeast expression system; a study by Snyder et al. (71) found that both the glycosylated and unglycosylated species of ␣ENaC were present in Madin-Darby canine kidney cells and migrated distinctly after centrifugation on a sucrose gradient. These results imply that each species associates with a different cohort of protein partners. Another study by Prince and Welsh (72) found that a mixed population of glycosylated and unglycosylated ␣and ␤ENaC exists in COS-7 cells, and over time the levels of the soluble/glycosylated species decreased as the insoluble-unglycosylated protein increased.
One class of ER chaperones, the lectins, binds to N-linked glycans and assists in both protein folding and degradation pathways in the ER (77,(107)(108)(109)(110)(111). We reasoned that the lectins target the glycosylated protein for degradation but were unable to recognize the unglycosylated protein, making the unglycosylated species dependent on a distinct degradation pathway. Contrary to our predictions, deletion of the ER lectins (calnexin, Yos9, or Mnl1) had no effect on the degradation rate of glycosylated or unglycosylated ␣ENaC (data not shown). Alternatively, it is possible that there is redundancy among the ER lectins and that deletion of multiple lectins may be required to reveal a phenotype. A study by Schmidt and Perlmutter (86) found that GRP170 associates with ␣ 1 -antitrypsin Z, which is similarly an ERAD substrate in both yeast and mammalian cells (82,112,113). Interestingly, the amount of GRP170 associated with ␣ 1 -antitrypsin Z increased in the presence of tunicamycin, which blocks glycosylation. The authors reasoned that this was due to an increase in the amount of GRP170 due to induction of the unfolded protein response. Alternatively, an increase in association with the non-glycosylated protein is consistent with our result that Lhs1/GRP170 is more important for the quality control of unglycosylated ␣ENaC.
Finally, our results add to the growing list of chaperones that act on ENaC during its biogenesis; we and others reported that the small heat shock proteins and ER lumenal Hsp40s contribute to the ERAD of ENaC subunits (40,41), and cytosolic Hsp70 promotes ENaC trafficking to the cell surface, whereas the cytosolic Hsp70s and Hsp40s were dispensable for ENaC ERAD (114,115). Because several ERAD-linked diseases arise from specific mutations and because polymorphisms in the ENaCencoding genes may predispose individuals to other maladies (116), it will be important to continue to define potential therapeutic targets that can be modulated to alter ENaC biogenesis.