Reconstitution of Endoplasmic Reticulum-Associated Degradation Using Yeast Membranes and Cytosol

The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprisingly, then, the ER lumen houses a high concentration of molecular chaperones, factors that facilitate protein folding. However, if misfolded secreted proteins arise they may be selected and proteolyzed. This process, which removes potentially toxic proteins from the cell, has been termed ER-associated degradation (ERAD). Surprisingly, ERAD substrates are not degraded within the ER after being selected but are retrotranslocated to the cytoplasm and destroyed by the 26S proteasome. Thus, the ERAD pathway comprises substrate selection, substrate export from the ER, and substrate degradation, and each step in this pathway has been elucidated in part through an in vitro ERAD assay using reagents prepared from a model eukaryote, the yeast Saccharomyces cerevisiae . This chapter describes this in vitro ERAD assay in detail, and special considerations when performing the assay are noted.