Substrate Insolubility Dictates Hsp104-Dependent Endoplasmic-Reticulum-Associated Degradation

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species. Misfolded proteins are destroyed in the endoplasmic reticulum by the ERAD pathway. Preston et al. demonstrate that the propensity for misfolded membrane proteins to aggregate dictates their rate of clearance. The Hsp104 chaperone is required to retrotranslocate these substrates and is recruited to the ER membrane when they accumulate.