Membrane Topology and Function of Der3/Hrd1p as a Ubiquitin-Protein Ligase (E3) Involved in Endoplasmic Reticulum Degradation

doi:10.1074/jbc.M008608200

Membrane Topology and Function of Der3/Hrd1p as a Ubiquitin-Protein Ligase (E3) Involved in Endoplasmic Reticulum Degradation*

Peter M. Deak and
Dieter H. Wolf‡

From the Institut für Biochemie der Universität Stuttgart, 70569 Stuttgart, Germany

Abstract

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.

Footnotes

↵* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, the Deutsch-Israelische Projektkooperation, Bonn, and the Fonds der Chemischen Industrie, Frankfurt.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.
↵‡ To whom correspondence should be addressed: Institut für Biochemie der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany. Tel.: 49-711-685-4390; Fax: 49-711-685-4392; E-mail: dieter.wolf@po.uni-stuttgart.de.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M008608200
Abbreviations:

ER

endoplasmic reticulum

CPY

carboxypeptidase yscY

CPY*

mutated, malfolded CPY

PCR

polymerase chain reaction

GST

glutathioneS-transferase

5-FOA

5-fluoroorotic acid

HA

hemagglutinin

Nub and Cub

amino- and carboxyl-halves of ubiquitin
- Received September 20, 2000.
- Revision received December 21, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.