ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

Abstract Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD), a process requiring their exit from the ER and delivery to cytosolic proteasomes via the key retrotranslocation step. Conventional assays detect fully extracted substrates, overlooking early initiation events. Here, we use an Escherichia coli -derived Biotin Acceptor Peptide (BAP)-biotin ligase BirA system to detect ERAD substrates upon cytosolic exposure. Using the model ERAD substrate H2a, we show that cytosolic BirA selectively biotinylates luminal BAP tags during early cytosolic exposure, enabling detection of substrates engaging the mammalian retrotranslocation machinery independently of subsequent extraction. Combined with high-resolution expansion fluorescence microscopy, this approach reveals active initiation of ERAD at the specialized ER-derived quality control compartment (ERQC). Substrates become transiently exposed to the cytosol while remaining membrane-associated, followed by tight coupling of polyubiquitination, transmembrane segment extraction, deglycosylation and degradation. Functional perturbations indicate that mannose trimming enables initiation, whereas p97 and HRD1 support later extraction and ubiquitination.