Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein

• Published in: Biochemical journal Vol. 474; no. 3; p. 357
• Main Authors: Buck, Teresa M, Jordahl, Alexa S, Yates, Megan E, Preston, G Michael, Cook, Emily, Kleyman, Thomas R, Brodsky, Jeffrey L
• Format: Journal Article
• Language: English
• Published: England 01.02.2017
• Subjects: Amino Acid Sequence; Cell Membrane - metabolism; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum-Associated Degradation; Epithelial Sodium Channels - chemistry; Epithelial Sodium Channels - genetics; Epithelial Sodium Channels - metabolism; Gene Expression; HSP70 Heat-Shock Proteins - chemistry; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; Humans; Mutant Chimeric Proteins - chemistry; Mutant Chimeric Proteins - genetics; Mutant Chimeric Proteins - metabolism; Proteasome Endopeptidase Complex - metabolism; Protein Domains; Protein Folding; Protein Multimerization; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Proteolysis; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Ubiquitination; ubiquitin–proteasome system; Lhs1/GRP170; ENaC; molecular chaperones; endoplasmic reticulum-associated degradation (ERAD)
• DOI: 10.1042/BCJ20160760

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.