title: Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.
abstract: The polytopic endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046.
authors.forename: Rania
authors.initials: R
authors.lastname: Elsabrouty
authors.forename: Youngah
authors.initials: Y
authors.lastname: Jo
authors.forename: Tammy T
authors.initials: TT
authors.lastname: Dinh
authors.forename: Russell A
authors.initials: RA
authors.lastname: DeBose-Boyd
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D013261:Sterols / Q000494:pharmacology*; D054875:Ubiquitination / Q000187:drug effects; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D006888:Hydroxycholesterols; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D013261:Sterols; C007997:25-hydroxycholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C000614180:Vcp protein, rat
references.citation: Curr Opin Cell Biol. 2011 Aug;23(4):464-75
references.pmid: 21664808
references.citation: J Biol Chem. 2004 Jul 2;279(27):28798-806
references.pmid: 15090540
references.citation: Nat Cell Biol. 2005 Aug;7(8):766-72
references.pmid: 16056268
references.citation: J Cell Biol. 1992 Jun;117(5):959-73
references.pmid: 1374417
references.citation: Curr Pharm Des. 2001 Mar;7(4):231-59
references.pmid: 11254888
references.citation: Cell. 2006 Jan 13;124(1):35-46
references.pmid: 16413480
references.citation: J Biol Chem. 1983 Jul 10;258(13):8450-5
references.pmid: 6688077
references.citation: J Lipid Res. 1980 Jul;21(5):505-17
references.pmid: 6995544
references.citation: Nature. 2004 Jun 24;429(6994):834-40
references.pmid: 15215855
references.citation: Mol Biol Cell. 2009 Jul;20(14):3330-41
references.pmid: 19458199
references.citation: Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20503-8
references.pmid: 22143767
references.citation: Nature. 1990 Feb 1;343(6257):425-30
references.pmid: 1967820
references.citation: J Biol Chem. 1985 Jan 10;260(1):522-30
references.pmid: 3965461
references.citation: Mol Endocrinol. 2000 Jan;14(1):27-39
references.pmid: 10628745
references.citation: Mol Cell. 2003 Jan;11(1):25-33
references.pmid: 12535518
references.citation: Proc Natl Acad Sci U S A. 1987 Apr;84(7):1829-33
references.pmid: 3031653
references.citation: Methods Enzymol. 1983;98:241-60
references.pmid: 6321901
references.citation: Nat Cell Biol. 2012 Feb 02;14(2):117-23
references.pmid: 22298039
references.citation: J Biol Chem. 2009 May 29;284(22):14710-22
references.pmid: 19324879
references.citation: J Biol Chem. 2010 Jun 18;285(25):19288-98
references.pmid: 20406816
references.citation: Nature. 2004 Jun 24;429(6994):841-7
references.pmid: 15215856
references.citation: Cell. 1984 Nov;39(1):27-38
references.pmid: 6091915
references.citation: Trends Cell Biol. 2012 Jan;22(1):22-32
references.pmid: 22055166
references.citation: J Biol Chem. 2003 Dec 26;278(52):52479-90
references.pmid: 14563840
references.citation: J Biol Chem. 2004 Oct 8;279(41):43136-47
references.pmid: 15247248
references.citation: PLoS Biol. 2011 Mar;8(3):e1000605
references.pmid: 21468303
references.citation: Anal Biochem. 2006 Jun 1;353(1):153-5
references.pmid: 16643835
references.citation: J Biol Chem. 2004 Feb 20;279(8):6465-73
references.pmid: 14627708