No abstract

Until recently, the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized proteases. We now show that the ER-associated degradation (ERAD) of two mutant proteins that accumulate in the ER lumen is inhibited in a proteasome-defective yeast strain and when cytosol from this mutant is used in an in vitro assay. In addition, ERAD is limited in vitro in the presence of the proteasome inhibitors, 3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD substrate is exported from ER-derived microsomes, and the accumulation of exported substrate is 2-fold greater when proteasome mutant cytosol is used in place of wild-type cytosol. We conclude that lumenal ERAD substrates are exported from the yeast ER to the cytoplasm for degradation by the proteasome complex.Quality control of newly synthesized proteins in the endoplasmic reticulum (ER) ensures that only correctly folded, processed, and completely assembled proteins exit this compartment for further transport through the secretory pathway. Most proteins that fail to reach this transport competent state are degraded (1-7). The ER-associated protein degradation pathway (ERAD) is highly selective for specific unassembled and͞or aberrant proteins, while the majority of ER resident and secreted proteins are quite stable. Evidence that the ER chaperone calnexin has a role in ERAD indicates that molecular chaperones might be required for this remarkable substrate selectivity (8).Although previous studies suggested that ERAD involves unidentified proteases localized in the ER, more recent evidence indicates that ERAD may require cytosolic proteases. Studies of ERAD in vitro suggest that degradation of aberrant ER-lumenal proteins occurs in the cytoplasm (8), and there is also evidence that the degradation of ER-retained forms of the cystic fibrosis transmembrane conductance regulator is ubiquitin-and proteasome-dependent (9, 10). In addition, cytomegalovirus-induced down-regulation of major histocompatibility complex class I molecules involves the rapid transport of the unassembled major histocompatibility complex I heavy chains from the ER to the cytoplasm for degradation by the proteasome complex (11). In this report we show that the cytosolic proteasome complex is the proteolytic component for the ER-associated degradation of two ER-lumenal proteins in yeast. Furthermore, we provide evidence that indicates ERAD substrates are exported to the cytosol for degradation by the proteasome. MATERIALS AND METHODSMaterials and Strains. Strains used were: RSY607 (Mat␣, pep4::URA3), provided by R. Schekman, University of California, Berkeley; AB122 (Mata, prc1-407, prb1-1122, pep4-3, leu2, ura3-52), provided by A. Brake, University of California, San Francisco; pre 1-1 pre 2-2 proteasome mutant and isogenic wild-type strain, provided by D. H. Wolf (12, 13); ⌬ubc6 ubiquitin-conjugating enzyme mutant and isogenic wild-type strains, provided by S. Jentsch (14); and ubc4-⌬1 u...