Differential fates of invertase mutants in the yeast endoplasmic reticulum.

A number of proteins have been identified as substrates for endoplasmic reticulum (ER)-associated protein degradation (ERAD) and we describe here a new model substrate with which to study this process. Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538p levels were low, while Suc2-533p was present in high amounts. Because Suc2-533p and Suc2-538p mRNA levels were comparable, we examined whether Suc2-538p was targeted for degradation. Both mutant polypeptide levels were unaffected in a yeast strain deficient in vacuolar protease activity and, additionally, we showed that Suc2-538p was stabilized in ERAD-deficient strains, demonstrating that Suc2-538p was a substrate for ERAD.

The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct.

Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor.

Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, approximately 30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.

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.