Ardythe A. McCracken

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 cne1Δ and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-α factor is defective inkar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in thekar2 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-α 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.

ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together.

A protein-degradation pathway associated with the endoplasmic reticulum (ER) can selectively remove polypeptides from the secretory pathway. The mechanisms of this ER-associated protein degradation were obscure, but recent studies using both yeast and mammalian cells have indicated that substrates for degradation are targeted to the cytosol where proteolysis is catalysed by the proteasome. The degradation process is now known to comprise at least three distinct events: first, recognition of a polypeptide for degradation; second, efflux of this substrate from the ER to the cytosol; and, finally, degradation by the proteasome. This review summarizes recent advances in understanding how each of these steps is achieved.

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro‐translocated to the cytoplasm and degraded by the 26S proteasome via ER‐associated degradation (ERAD). To begin to resolve the requirements for the retro‐translocation and degradation steps during ERAD, a cell‐free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER‐derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild‐type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP‐dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.

Autophagy: an ER protein quality control process.

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome–dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both a1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control. Addendum to: Characterization of an ERAD Gene as VPS30/ATG6 Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble A1PiZ and Another for Aggregates of A1PiZ K.B. Kruse, J.L. Brodsky and A.A. McCracken Mol Biol Cell 2005; In press.

Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation

In the eukaryotic cell, a protein quality control process termed endoplasmic reticulum-associated degradation (ERAD) rids the ER of aberrant proteins and unassembled components of protein complexes that fail to reach a transport-competent state. To identify novel genes required for ERAD, we devised a rapid immunoassay to screen yeast lacking uncharacterized open reading frames that were known targets of the unfolded protein response (UPR), a cellular response that is induced when aberrant proteins accumulate in the ER. Six genes required for the efficient degradation of the Z variant of theα 1-proteinase inhibitor (A1PiZ), a known substrate for ERAD, were identified, and analysis of other ERAD substrates in the six A1PiZ-degradation-deficient (add) mutants suggested diverse requirements for the Add proteins in ERAD. Finally, we report on bioinformatic analyses of the new Add proteins, which will lead to testable models to elucidate their activities.

A molecular portrait of the response to unfolded proteins

Using DNA microarrays, 381 genes have been found to be induced in response to unfolded proteins. The identity of the previously characterized 208 of these, and further experiments, have revealed new details on the scope of the unfolded protein response and its connection to the degradation of proteins at the endoplasmic reticulum.

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.

Endoplasmic Reticulum-Associated Degradation and Protein Quality Control

Approximately one-third of all polypeptides synthesized in eukaryotes are targeted to the endoplasmic reticulum (ER), and once associated with this compartment they are chemically modified. The folding status of the resulting nascent proteins is then surveyed by molecular chaperones and lectins. To clear the ER of dead-end products, proteins that fail quality control are routed to the cytosol and degraded via ER-associated degradation (ERAD). Although many ERAD-requiring factors have been identified and a basic understanding of this pathway has been achieved, numerous questions remain on the mechanisms that lead to the selection and delivery of ERAD substrates.

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. Copyright