The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct

Abstract

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 cne1⌬ and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-␣ 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-␣ 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.

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

“…This result was unexpected given that A1PiZ is an ERAD substrate in both yeast (McCracken and Kruse, 1993;Werner et al, 1996;Brodsky et al, 1999) and human . Because vacuole morphology is largely unperturbed in vps30 yeast (Raymond et al, 1992), these data suggest that the A1PiZ degradation defect does not arise from gross ultrastructural perturbations.…”

Genetic Evidence for a Role of BiP/Kar2 That Regulates Ire1 in Response to Accumulation of Unfolded Proteins

“…For instance, refolding of carboxypeptidase Y (CPY), after denaturation by dithiothreitol (DTT), was impaired in some kar2 mutant strains (Simons et al, 1995). In addition, other reports implicate Kar2 involvement in ER-associated protein degradation (ERAD;Plemper et al, 1997;Brodsky et al, 1999;Nishikawa et al, 2001).We have proposed that KAR2 is not only a UPR target, but is itself a critical negative regulator of the UPR pathway (Okamura et al, 2000). In the current model, Kar2 associates with Ire1 to repress the activation of Ire1 in nonstressed cells, and in response to accumulation of unfolded proteins in the ER, Kar2 dissociates from Ire1, resulting in dimerization and activation of Ire1.…”

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

“…BiP appears to bind to hydrophobic sequences of polypeptides (Blond-Elguindi et al 1993), thereby preventing premature and incorrect folding. Malfolded proteins, accumulated in the ER lumen, are often eliminated by ERAD; BiP seems to be essential for this process (Brodsky et al 1999). Associations between BiP and retained secretory proteins have often been observed, even as proteinaceous aggregates (Yang et al 1998;Umebayashi et al 1997;Sagt et al 1998).…”

“…During import of proteins into the ER, BiP binds to hydrophobic stretches of the polypeptide (Blond-Elguindi et al 1993) thereby preventing aggregation. Recently it was shown that BiP also facilitates ER-associated degradation (ERAD) (Brodsky et al 1999). This degradation process involves the retrograde transport of malfolded proteins out of the ER.…”