Assays for protein retrotranslocation in ERAD.

Elimination of misfolded proteins by endoplasmic reticulum (ER)-associated protein degradation (ERAD) ensures that proteins proceeding through the secretory pathway are correctly folded and processed, which is critical to minimize ER stress. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated misfolded substrates are extracted from the ER and degraded by the cytosolic 26S proteasome. Despite being integral to ERAD, the retrotranslocation process has been largely obscure. Recently, an explosion of discoveries has provided key mechanistic insights into this novel route of protein transport. These advances were facilitated by the development of in vitro and in vivo assays that utilize components from the yeast Saccharomyces cerevisiae. The assays permit detailed study of the distinct steps in ERAD-linked retrotranslocation, including ubiquitination of selected ERAD substrates, substrate removal from the ER, maintenance of cytosolic substrate solubility in the cytosol, and substrate degradation. Here we provide detailed protocols for these assays that pertain to work on retrotranslocation of integral membrane proteins (ERAD-M substrates), with the expectation that these approaches can be adapted for many related biochemical processes.