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PMID: 19458199 Published · ppublish English Journal Article

Dislocation of HMG-CoA reductase and Insig-1, two polytopic endoplasmic reticulum proteins, en route to proteasomal degradation.

Molecular biology of the cell ·Vol. 20 ·No. 14 ·2009-07-00 ·Pages 3330-41

Leichner GS, Avner R, Harats D, Roitelman J

Abstract

The endoplasmic reticulum (ER) glycoprotein HMG-CoA reductase (HMGR) catalyzes the rate-limiting step in sterols biosynthesis. Mammalian HMGR is ubiquitinated and degraded by the proteasome when sterols accumulate in cells, representing the best example for metabolically controlled ER-associated degradation (ERAD). This regulated degradation involves the short-lived ER protein Insig-1. Here, we investigated the dislocation of these ERAD substrates to the cytosol en route to proteasomal degradation. We show that the tagged HMGR membrane region, HMG(350)-HA, the endogenous HMGR, and Insig-1-Myc, all polytopic membrane proteins, dislocate to the cytosol as intact full-length polypeptides. Dislocation of HMG(350)-HA and Insig-1-Myc requires metabolic energy and involves the AAA-ATPase p97/VCP. Sterols stimulate HMG(350)-HA and HMGR release to the cytosol concurrent with removal of their N-glycan by cytosolic peptide:N-glycanase. Sterols neither accelerate dislocation nor stimulate deglycosylation of ubiquitination-defective HMG(350)-HA((K89 + 248R)) mutant. Dislocation of HMG(350)-HA depends on Insig-1-Myc, whose dislocation and degradation are sterol independent. Coimmunoprecipitation experiments demonstrate sterol-stimulated association between HMG(350)-HA and Insig-1-Myc. Sterols do not enhance binding to Insig-1-Myc of HMG(350)-HA mutated in its sterol-sensing domain or of HMG(350)-HA((K89 + 248R)). Wild-type HMG(350)-HA and Insig-1-Myc coimmunoprecipitate from the soluble fraction only when both proteins were coexpressed in the same cell, indicating their encounter before or during dislocation, raising the possibility that they are dislocated as a tightly bound complex.

MeSH Terms
Adenosine Triphosphatases/metabolism Cell Line Cytosol/drug effects,enzymology Endoplasmic Reticulum/drug effects,enzymology Glycosylation/drug effects Humans Hydroxymethylglutaryl CoA Reductases/metabolism Intracellular Membranes/drug effects,enzymology Intracellular Signaling Peptides and Proteins/metabolism Mutation/genetics Nuclear Proteins/metabolism Peptides/metabolism Proteasome Endopeptidase Complex/metabolism Protein Binding/drug effects Protein Processing, Post-Translational/drug effects Protein Transport/drug effects Recombinant Fusion Proteins/metabolism Sterols/pharmacology Thermodynamics Time Factors Ubiquitination/drug effects
Chemicals
Intracellular Signaling Peptides and Proteins Nuclear Proteins Peptides Recombinant Fusion Proteins Sterols Hydroxymethylglutaryl CoA Reductases Proteasome Endopeptidase Complex Adenosine Triphosphatases p97 ATPase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Leichner Gil S
Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Avner Rachel
Harats Dror
Roitelman Joseph
References (73)
73 references, click to expand
  1. Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme.
    Cell. 1985 May;41(1):249-58 PMID: 3995584
  2. Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum.
    J Cell Biol. 1992 Jun;117(5):959-73 PMID: 1374417
  3. Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hydroxycholesterol in UT-1 cells.
    Proc Natl Acad Sci U S A. 1982 Sep;79(17):5205-9 PMID: 6957860
  4. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.
    Cell. 2000 Aug 4;102(3):315-23 PMID: 10975522
  5. Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi.
    Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11235-40 PMID: 10500160
  6. Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake.
    Cell Metab. 2006 Jan;3(1):15-24 PMID: 16399501
  7. Mutation in the lumenal part of the membrane domain of HMG-CoA reductase alters its regulated degradation.
    Biochem Biophys Res Commun. 1995 Jan 5;206(1):186-93 PMID: 7818519
  8. Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for sterol-regulated enzyme degradation.
    J Biol Chem. 2004 Sep 10;279(37):38184-93 PMID: 15247208
  9. Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins.
    Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12753-8 PMID: 12242332
  10. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
    J Biol Chem. 2003 Dec 26;278(52):52479-90 PMID: 14563840
  11. Viral modulation of antigen presentation: manipulation of cellular targets in the ER and beyond.
    Immunol Rev. 2005 Oct;207:126-44 PMID: 16181332
  12. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER.
    Cell. 2002 Aug 23;110(4):489-500 PMID: 12202038
  13. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol.
    Nature. 2001 Dec 6;414(6864):652-6 PMID: 11740563
  14. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation.
    Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11163-8 PMID: 11562482
  15. Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis.
    EMBO J. 2000 Mar 1;19(5):819-30 PMID: 10698924
  16. Cdc48 (p97): a "molecular gearbox" in the ubiquitin pathway?
    Trends Biochem Sci. 2007 Jan;32(1):6-11 PMID: 17142044
  17. Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein.
    J Biol Chem. 2004 Sep 10;279(37):38369-78 PMID: 15252059
  18. Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78.
    J Biol Chem. 2006 Dec 22;281(51):39308-15 PMID: 17043353
  19. Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.
    Mol Cell. 2003 Jan;11(1):25-33 PMID: 12535518
  20. Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.
    J Biol Chem. 1999 Jun 11;274(24):17171-8 PMID: 10358074
  21. Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment.
    Cell. 1996 Jun 28;85(7):1037-46 PMID: 8674110
  22. 5' end of HMG CoA reductase gene contains sequences responsible for cholesterol-mediated inhibition of transcription.
    Cell. 1985 Aug;42(1):203-12 PMID: 3860301
  23. Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.
    J Biol Chem. 1997 Aug 8;272(32):20213-21 PMID: 9242699
  24. The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation).
    Biochem J. 2007 Jun 15;404(3):353-63 PMID: 17521290
  25. The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum.
    Traffic. 2008 Jun;9(6):861-70 PMID: 18315532
  26. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase.
    Mol Cell. 2005 Sep 16;19(6):829-40 PMID: 16168377
  27. The alpha chain of the T cell antigen receptor is degraded in the cytosol.
    Immunity. 1997 Jul;7(1):113-22 PMID: 9252124
  28. Distinct steps in dislocation of luminal endoplasmic reticulum-associated degradation substrates: roles of endoplamic reticulum-bound p97/Cdc48p and proteasome.
    J Biol Chem. 2004 Feb 6;279(6):3980-9 PMID: 14607830
  29. Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.
    J Biol Chem. 1992 Dec 15;267(35):25264-73 PMID: 1460026
  30. Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation.
    J Lipid Res. 2007 Feb;48(2):318-27 PMID: 17090658
  31. The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.
    J Biol Chem. 1988 May 15;263(14):6836-41 PMID: 2834394
  32. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.
    Methods Enzymol. 1983;98:241-60 PMID: 6321901
  33. The Png1-Rad23 complex regulates glycoprotein turnover.
    J Cell Biol. 2006 Jan 16;172(2):211-9 PMID: 16401726
  34. 3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides.
    Proc Natl Acad Sci U S A. 1983 Dec;80(23):7165-9 PMID: 6580634
  35. Role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation.
    EMBO Rep. 2002 Dec;3(12):1222-7 PMID: 12446567
  36. N-linked glycosylation does not impair proteasomal degradation but affects class I major histocompatibility complex presentation.
    J Biol Chem. 2008 Jan 4;283(1):244-254 PMID: 17951257
  37. Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol-sensing domain.
    J Biol Chem. 1998 Jul 3;273(27):17243-50 PMID: 9642295
  38. Membrane topology of human insig-1, a protein regulator of lipid synthesis.
    J Biol Chem. 2004 Feb 27;279(9):8487-96 PMID: 14660594
  39. Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
    Gene. 1989 Apr 15;77(1):51-9 PMID: 2744487
  40. In vitro analysis of Hrd1p-mediated retrotranslocation of its multispanning membrane substrate 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase.
    J Biol Chem. 2009 May 29;284(22):14710-22 PMID: 19324879
  41. Cytoplasmic peptide:N-glycanase (PNGase) in eukaryotic cells: occurrence, primary structure, and potential functions.
    FASEB J. 2002 May;16(7):635-41 PMID: 11978727
  42. Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.
    Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16672-7 PMID: 12482938
  43. Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2.
    J Biol Chem. 2005 Jul 1;280(26):25242-9 PMID: 15866869
  44. Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells.
    J Biol Chem. 2002 Dec 6;277(49):47358-65 PMID: 12351637
  45. The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum.
    J Biol Chem. 1990 Dec 15;265(35):22004-10 PMID: 2254343
  46. Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.
    J Cell Biol. 1987 Jun;104(6):1693-704 PMID: 3584246
  47. The sterol-sensing domain: multiple families, a unique role?
    Trends Genet. 2002 Apr;18(4):193-201 PMID: 11932020
  48. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.
    J Clin Invest. 2002 May;109(9):1125-31 PMID: 11994399
  49. A direct role for sterol regulatory element binding protein in activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene.
    J Biol Chem. 1996 May 24;271(21):12247-53 PMID: 8647822
  50. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.
    Cell. 1996 Nov 1;87(3):415-26 PMID: 8898195
  51. The activities and function of molecular chaperones in the endoplasmic reticulum.
    Semin Cell Dev Biol. 2007 Dec;18(6):751-61 PMID: 17964199
  52. Ufd1 is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase.
    Cell Metab. 2007 Aug;6(2):115-28 PMID: 17681147
  53. Proteolytic activation of sterol regulatory element-binding protein induced by cellular stress through depletion of Insig-1.
    J Biol Chem. 2004 Oct 22;279(43):45257-65 PMID: 15304479
  54. Regulation of the mevalonate pathway.
    Nature. 1990 Feb 1;343(6257):425-30 PMID: 1967820
  55. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.
    J Cell Biol. 2003 Jul 7;162(1):71-84 PMID: 12847084
  56. Inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin.
    J Biol Chem. 1983 Jul 25;258(14):8547-9 PMID: 6553055
  57. Dislocation of type I membrane proteins from the ER to the cytosol is sensitive to changes in redox potential.
    J Cell Biol. 1998 Jul 27;142(2):365-76 PMID: 9679137
  58. Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol.
    Mol Cell Biol. 2005 Oct;25(20):8844-53 PMID: 16199864
  59. The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum.
    Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14422-7 PMID: 11724934
  60. Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation.
    Semin Cell Dev Biol. 2007 Dec;18(6):770-9 PMID: 17950636
  61. Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum.
    J Biol Chem. 1985 Jan 10;260(1):522-30 PMID: 3965461
  62. Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.
    Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12848-53 PMID: 9789003
  63. Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes.
    J Biol Chem. 1983 Jun 25;258(12):7272-5 PMID: 6863245
  64. The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor.
    Proc Natl Acad Sci U S A. 2006 May 30;103(22):8348-53 PMID: 16709668
  65. Biogenesis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an integral glycoprotein of the endoplasmic reticulum.
    Proc Natl Acad Sci U S A. 1984 Mar;81(6):1674-8 PMID: 6584901
  66. The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
    J Biol Chem. 2000 Nov 17;275(46):35840-7 PMID: 10964918
  67. The role of p97/Cdc48p in endoplasmic reticulum-associated degradation: from the immune system to yeast.
    Curr Top Microbiol Immunol. 2005;300:95-125 PMID: 16573238
  68. Ubiquitination is essential for human cytomegalovirus US11-mediated dislocation of MHC class I molecules from the endoplasmic reticulum to the cytosol.
    Biochem J. 2001 Sep 1;358(Pt 2):369-77 PMID: 11513735
  69. Dissecting the ER-associated degradation of a misfolded polytopic membrane protein.
    Cell. 2008 Jan 11;132(1):101-12 PMID: 18191224
  70. Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides.
    J Biol Chem. 2007 Sep 14;282(37):26845-26856 PMID: 17650499
  71. Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover.
    Chem Biol. 2004 Dec;11(12):1677-87 PMID: 15610852
  72. Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation.
    J Cell Biol. 2001 May 28;153(5):1061-70 PMID: 11381090
  73. A proteasomal ATPase contributes to dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates.
    J Biol Chem. 2008 Mar 14;283(11):7166-75 PMID: 18174173
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1939-4586
DOI
10.1091/mbc.e08-09-0953
Published
2009-07-00
Epub
2009-00-20
Pages
3330-41
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC2710830
Subset
IM
External Links
PubMed source DOI: 10.1091/mbc.e08-09-0953 PMC Full Text View journal details
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