Substrate ubiquitination retains misfolded membrane proteins in the endoplasmic reticulum for degradation.

To maintain secretory pathway fidelity, misfolded proteins are commonly retained in the endoplasmic reticulum (ER) and selected for ER-associated degradation (ERAD). Soluble misfolded proteins use ER chaperones for retention, but the machinery that restricts aberrant membrane proteins to the ER is unclear. In fact, some misfolded membrane proteins escape the ER and traffic to the lysosome/vacuole. To this end, we describe a model substrate, SZ, that contains an ER export signal but is also targeted for ERAD. We observe decreased ER retention when chaperone-dependent SZ ubiquitination is compromised. In addition, appending a linear tetra-ubiquitin motif onto SZ overrides ER export. By screening known ubiquitin-binding proteins, we then positively correlate SZ retention with Ubx2 binding. Deletion of Ubx2 also inhibits the retention of another misfolded membrane protein. Our results indicate that polyubiquitination is sufficient to retain misfolded membrane proteins in the ER prior to ERAD.