Degradation of a Mutant Secretory Protein, α1-Antitrypsin Z, in the Endoplasmic Reticulum Requires Proteasome Activity

doi:10.1074/jbc.271.37.22791

Degradation of a Mutant Secretory Protein, α₁-Antitrypsin Z, in the Endoplasmic Reticulum Requires Proteasome Activity*

From the Departments of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine, Division of Gastroenterology and Nutrition, Children's Hospital, St. Louis, Missouri 63110 and
^‡ The Kitasato Institute, Tokyo 108, Japan

^§ To whom correspondence should be addressed:
Dept. of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine, St. Louis Children's Hospital, One Children's Place, St. Louis, MO 63110.
Tel.: 314-454-6033; Fax: 314-454-4218; E-mail: perlmutter{at}a1.kids.wustl.edu.

Abstract

Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRΔF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein α₁-antitrypsin (α₁-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of α₁-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of α1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRΔF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, α₁-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal α₁-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with α₁-ATZ.

Footnotes

↵* This work was supported by Grants HL37784-DP and DK 01379-JT from the National Institutes of Health and by the March of Dimes. 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.
↵¹ The abbreviations used are:

α₁-AT

α₁-antitrypsin

Endo H

endoglycosidase H

DTT

dithiothreitol

ATPγS

adenosine 5′-O-(thiotriphosphate)

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic

CST

castanospermine.
↵²J. H. Teckman, D. Qu, and D. H. Perlmutter, unpublished observation.
- Received June 11, 1996.