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.

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