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

doi:10.1074/jbc.274.6.3453

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

From the ^‡Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 and the ^¶Department of Biology, University of Nevada, Reno, Nevada 89557

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 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.

Footnotes

↵* This work was supported by Grants MCB-9506002 and MCB-9722889 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Recipient of a Junior Faculty Achievement Award from the American Cancer Society. To whom correspondence should be addressed: Dept. of Biological Sciences, Univ. of Pittsburgh, 267 Crawford Hall, Pittsburgh, PA 15260. Tel.: 412-624-4831; Fax: 412-624-4759; E-mail:jbrodsky+{at}pitt.edu.
↵‖ Supported by a Research Experience for Undergraduates Award from the National Science Foundation.
↵2 J. L. Goeckeler and J. L. Brodsky, manuscript in preparation.
↵3 Jannatipour, M., Callejo, M. Parodi, A. J., Armstrong, J., and Rokeach, L. A. (1998) Biochemistry 37,17253–17261
↵4 J. L. Brodsky, unpublished observations.
Abbreviations:

ER

endoplasmic reticulum

ERAD

ER-associated protein degradation

pαF

pro-α factor

ppαF

prepro-α factor.
- Received July 23, 1998.
- Revision received October 26, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.