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.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates

“…After immunoprecipitation, we observed that the 125 I-ubiquitin signal was equal to or greater than the predicted molecular weight for Chimera A* on the autoradiograph (Figure 6B, arrow), as observed previously for Ste6p* and other substrates (Nakatsukasa et al. , 2008; Buck et al. , 2016).…”

Regulation of the epithelial Na+ channel by paraoxonase-2

“…Subunit-specific interactions have also been shown with ENaC and Lhs1/GRP170, an ER-resident Hsp70-like chaperone protein (33). Lhs1/GRP170 -mediated ENaC degradation was specific for the ␣ subunit expressed alone and was blocked when the other two subunits were present in the channel complex (33,56). Nonetheless, our data show that PON-2 interacts with ENaC subunits in a heterologous expression system and raise the possibility that these interactions occur in the distal nephron and perhaps in other epithelia.…”

Hsp104 facilitates the endoplasmic‐reticulum–associated degradation of disease‐associated and aggregation‐prone substrates

“…Carbonate extraction was conducted on yeast expressing ApoB essentially as described . This procedure has long been used as a means to remove peripherally associated proteins bound to membranes .…”