title: Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
abstract: Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.
authors.affiliation: From the Departments of Molecular Genetics and.
authors.forename: Lindsey L
authors.initials: LL
authors.lastname: Morris
authors.affiliation: From the Departments of Molecular Genetics and.
authors.forename: Isamu Z
authors.initials: IZ
authors.lastname: Hartman
authors.affiliation: From the Departments of Molecular Genetics and.
authors.forename: Dong-Jae
authors.initials: DJ
authors.lastname: Jun
authors.affiliation: Cell Biology and.
authors.forename: Joachim
authors.initials: J
authors.lastname: Seemann
authors.affiliation: From the Departments of Molecular Genetics and the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046 Russell.DeBose-Boyd@utsouthwestern.edu.
authors.forename: Russell A
authors.initials: RA
authors.lastname: DeBose-Boyd
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics; D013261:Sterols / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D013261:Sterols; D006903:Hydroxymethylglutaryl CoA Reductases; D014357:Trypsin; D008666:Metalloendopeptidases; C114583:m-AAA proteases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases
keywords: Cholesterol Metabolism; Cytosolic Dislocation; ER-associated Degradation; Endoplasmic Reticulum (ER); Endoplasmic Reticulum-associated Protein Degradation (ERAD); Membrane Extraction; Membrane Trafficking; Proteasome