Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.
Endoplasmic Reticulum-Associated Degradation , Membrane Proteins , Ubiquitin-Protein Ligases , Humans , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA-Binding Proteins , Endoplasmic Reticulum/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , HeLa Cells , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Binding , Protein Domains , Protein Folding , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination
