ERAD of proteins containing aberrant transmembrane domains requires ubiquitination of cytoplasmic lysine residues

Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Whilst both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitination. Ubiquitination of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whilst CD8LUM* continued to be degraded in the absence of cytoplasmic lysines. Cytoplasmic lysines were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain, and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER via a specific ERAD mechanism that depends upon ubiquitination of cytoplasmic lysines.