title: Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome.
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.
authors.affiliation: Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany.
authors.forename: R K
authors.initials: RK
authors.lastname: Plemper
authors.forename: R
authors.initials: R
authors.lastname: Egner
authors.forename: K
authors.initials: K
authors.lastname: Kuchler
authors.forename: D H
authors.initials: DH
authors.lastname: Wolf
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016296:Mutagenesis; D046988:Proteasome Endopeptidase Complex; D000069816:SEC Translocation Channels; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D005656:Fungal Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C092438:PDR5 protein, S cerevisiae; D000069816:SEC Translocation Channels; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex