title: Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.
abstract: In the kidney, the epithelial sodium channel (ENaC) regulates blood pressure through control of sodium and volume homeostasis, and in the lung, ENaC regulates the volume of airway and alveolar fluids. ENaC is a heterotrimer of homologous α-, β- and γ-subunits, and assembles in the endoplasmic reticulum (ER) before it traffics to and functions at the plasma membrane. Improperly folded or orphaned ENaC subunits are subject to ER quality control and targeted for ER-associated degradation (ERAD). We previously established that a conserved, ER lumenal, molecular chaperone, Lhs1/GRP170, selects αENaC, but not β- or γ-ENaC, for degradation when the ENaC subunits were individually expressed. We now find that when all three subunits are co-expressed, Lhs1-facilitated ERAD was blocked. To determine which domain-domain interactions between the ENaC subunits are critical for chaperone-dependent quality control, we employed a yeast model and expressed chimeric α/βENaC constructs in the context of the ENaC heterotrimer. We discovered that the βENaC transmembrane domain was sufficient to prevent the Lhs1-dependent degradation of the α-subunit in the context of the ENaC heterotrimer. Our work also found that Lhs1 delivers αENaC for proteasome-mediated degradation after the protein has become polyubiquitinated. These data indicate that the Lhs1 chaperone selectively recognizes an immature form of αENaC, one which has failed to correctly assemble with the other channel subunits via its transmembrane domain.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Buck
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Alexa S
authors.initials: AS
authors.lastname: Jordahl
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Yates
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: G Michael
authors.initials: GM
authors.lastname: Preston
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Emily
authors.initials: E
authors.lastname: Cook
authors.affiliation: Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D053503:Epithelial Sodium Channels; D018840:HSP70 Heat-Shock Proteins; C102115:LHS1 protein, S cerevisiae; D050506:Mutant Chimeric Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex
keywords: ENaC; Lhs1/GRP170; endoplasmic reticulum-associated degradation (ERAD); molecular chaperones; ubiquitin–proteasome system