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The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.

Buck TM, Kolb AR, Boyd CR, Kleyman TR, Brodsky JL - Mol. Biol. Cell (2010)

Bottom Line: Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD).We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells.Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
The epithelial sodium channel (ENaC) is composed of a single copy of an alpha-, beta-, and gamma-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

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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.
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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.

Mentions: To assay whether the extent of stabilization correlated with the degree of ubiquitination—and thus that the effect of deleting the ligases was direct—we examined α- and β-subunit ubiquitination in vivo. To this end, the α- and β-subunits were immunoprecipitated from wild type and from hrd1Δ and hrd1Δdoa10Δ mutant yeast, and an anti-ubiquitin antibody was used in a subsequent Western blot analysis. The results presented in Figure 3, A and B, indicate that subunit ubiquitination was significantly decreased in yeast lacking Hrd1 or both Hrd1 and Doa10. These data are consistent with a requirement for either Hrd1 or Doa10, as observed in Figure 2, and indicate that the effects of deleting the ligases are direct.


The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.

Buck TM, Kolb AR, Boyd CR, Kleyman TR, Brodsky JL - Mol. Biol. Cell (2010)

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.
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Related In: Results  -  Collection

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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.
Mentions: To assay whether the extent of stabilization correlated with the degree of ubiquitination—and thus that the effect of deleting the ligases was direct—we examined α- and β-subunit ubiquitination in vivo. To this end, the α- and β-subunits were immunoprecipitated from wild type and from hrd1Δ and hrd1Δdoa10Δ mutant yeast, and an anti-ubiquitin antibody was used in a subsequent Western blot analysis. The results presented in Figure 3, A and B, indicate that subunit ubiquitination was significantly decreased in yeast lacking Hrd1 or both Hrd1 and Doa10. These data are consistent with a requirement for either Hrd1 or Doa10, as observed in Figure 2, and indicate that the effects of deleting the ligases are direct.

Bottom Line: Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD).We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells.Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
The epithelial sodium channel (ENaC) is composed of a single copy of an alpha-, beta-, and gamma-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

Show MeSH
Related in: MedlinePlus