Endoplasmic Reticulum Degradation of a Mutated ATP-binding Cassette Transporter Pdr5 Proceeds in a Concerted Action of Sec61 and the Proteasome*
- From the ‡Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany and the ¶Department of Molecular Genetics, University and Biocenter of Vienna, A-1030 Vienna, Austria
Abstract
Degradation of misfolded or tightly regulated proteins in the endoplasmic reticulum (ER) is performed by the cytosolic ubiquitin-proteasome system and therefore requires their prior transport back to the cytosol. Here, we report on the extraction and degradation mechanism of a polytopic membrane protein. Rapid proteasomal degradation of a mutated form of the ATP-binding cassette transporter Pdr5 retained in the ER is initialized at the lumenal face of the ER membrane. Using different antibodies directed against the cytosolic tails or a lumenal loop of the transmembrane protein, it could be demonstrated that the turnover of Pdr5* demands the concerted action of both the Sec61 translocon and the ubiquitin-proteasome system. We observed a stabilization of the entire molecule within the ER membrane in yeast mutants characterized by a reduced translocation capacity or by functionally attenuated proteasomes. Moreover, no degradation intermediates were detected in any of the mutants that impede degradation of Pdr5*. Therefore, initial steps are rate-limiting for cleavage and mutations that impede downstream events prevent initiation of the process.
Our data suggest that ER degradation is a mechanistically highly integrated process, requiring the combined operation of components of the degradation system acting at the lumenal face of the ER membrane, the Sec61 translocon, and the ubiquitin-proteasome system.
Footnotes
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↵* This work was supported by the Bundesministerium für Forschung und Technologie (Bonn, Germany), Zentrales Schwerpunktprojekt Bioverfahrenstechnik (Stuttgart, Germany), the Deutsche Forschungsgemeinschaft (Bonn, Germany), the Fonds der Chemischen Industrie (Frankfurt, Germany), and a grant from the Austrian Science Foundation (to K. K.).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.
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↵§ These authors contributed equally to this work.
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↵‖ These authors are the senior authors of the paper.
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↵** To whom correspondence may be addressed. Tel.: 43-1-4277-61807; Fax: 43-1-4277-9618; E-mail: kaku{at}novsrv-mol.mol.univie.ac.at.
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↵‡ To whom correspondence may be addressed. Tel.: 49-711-6854390; Fax: 49-711-6854392; E-mail: dieter.wolf{at}po.uni-stuttgart.de.
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↵2 R. K. Plemper, R. Egner, K. Kuchler, and D. H. Wolf, unpublished data.
- Abbreviations:
- ER
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endoplasmic reticulum
- GFP
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green fluorescent protein
- ABC
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ATP-binding cassette
- pdr
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pleiotropic drug resistance
- CHAPS
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3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- HMG-CoA reductase
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3-hydroxy-3-methylglutaryl CoA reductase
- HA
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influenza hemagglutinin protein.
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- Received July 24, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
