Home LiteratureArticle Details
PMID: 19815544 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Regulated endoplasmic reticulum-associated degradation of a polytopic protein: p97 recruits proteasomes to Insig-1 before extraction from membranes.

The Journal of biological chemistry ·Vol. 284 ·No. 50 ·2009-12-11 ·Pages 34889-900

Ikeda Y, Demartino GN, Brown MS, Lee JN, Goldstein JL, Ye J

Abstract

Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes.

MeSH Terms
Adenosine Triphosphatases/genetics,metabolism Amino Acid Sequence Amino Acid Substitution Animals Cell Line Cell Membrane/metabolism Cholesterol/metabolism Endoplasmic Reticulum/metabolism Fatty Acids/chemistry,metabolism Humans Intracellular Signaling Peptides and Proteins/genetics,metabolism Membrane Proteins/genetics,metabolism Molecular Sequence Data Nuclear Proteins/genetics,metabolism Proteasome Endopeptidase Complex/metabolism Protein Subunits/genetics,metabolism RNA Interference Receptors, Autocrine Motility Factor Receptors, Cytokine/genetics,metabolism Sequence Alignment Ubiquitin-Protein Ligases/genetics,metabolism Ubiquitination
Chemicals
Fatty Acids INSIG1 protein, human INSIG2 protein, human Intracellular Signaling Peptides and Proteins Membrane Proteins Nuclear Proteins Protein Subunits Receptors, Cytokine Cholesterol AMFR protein, human Receptors, Autocrine Motility Factor Ubiquitin-Protein Ligases Proteasome Endopeptidase Complex Adenosine Triphosphatases p97 ATPase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Ikeda Yukio
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
Demartino George N
Brown Michael S
Lee Joon No
Goldstein Joseph L
Ye Jin
References (40)
40 references, click to expand
  1. A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum.
    Nature. 2007 Jul 26;448(7152):435-8 PMID: 17653186
  2. Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines.
    Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):4958-63 PMID: 16549805
  3. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4.
    EMBO J. 2002 Nov 1;21(21):5645-52 PMID: 12411482
  4. Protein quality control as a strategy for cellular regulation: lessons from ubiquitin-mediated regulation of the sterol pathway.
    Chem Rev. 2009 Apr;109(4):1561-74 PMID: 19243134
  5. 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
  6. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome.
    Mol Biol Cell. 2008 Mar;19(3):1072-82 PMID: 18162577
  7. ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome.
    Mol Cell. 2006 Oct 6;24(1):39-50 PMID: 17018291
  8. Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different mechanisms, both involving SCAP and Insigs.
    J Biol Chem. 2004 Dec 10;279(50):52772-80 PMID: 15452130
  9. 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
  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. Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.
    Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11487-92 PMID: 8876162
  12. Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins.
    Biochemistry. 2009 Mar 24;48(11):2538-49 PMID: 19182904
  13. Isolation of mutant cells lacking Insig-1 through selection with SR-12813, an agent that stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
    J Biol Chem. 2004 Oct 8;279(41):43136-47 PMID: 15247248
  14. Analysis of Drosophila 26 S proteasome using RNA interference.
    J Biol Chem. 2002 Feb 22;277(8):6188-97 PMID: 11739392
  15. 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
  16. 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
  17. 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
  18. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.
    Methods Enzymol. 1983;98:241-60 PMID: 6321901
  19. Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane.
    J Biol Chem. 2003 Sep 12;278(37):34774-82 PMID: 12813030
  20. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.
    Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12027-32 PMID: 14512514
  21. Membrane topology of human insig-1, a protein regulator of lipid synthesis.
    J Biol Chem. 2004 Feb 27;279(9):8487-96 PMID: 14660594
  22. p97: The cell's molecular purgatory?
    Mol Cell. 2006 Jun 23;22(6):713-717 PMID: 16793541
  23. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase.
    J Biol Chem. 1978 Feb 25;253(4):1121-8 PMID: 624722
  24. Protein sensors for membrane sterols.
    Cell. 2006 Jan 13;124(1):35-46 PMID: 16413480
  25. One step at a time: endoplasmic reticulum-associated degradation.
    Nat Rev Mol Cell Biol. 2008 Dec;9(12):944-57 PMID: 19002207
  26. 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
  27. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.
    Cell. 1997 May 2;89(3):331-40 PMID: 9150132
  28. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.
    Cell. 1984 Nov;39(1):27-38 PMID: 6091915
  29. Proteasome-associated proteins: regulation of a proteolytic machine.
    Biol Chem. 2005 Aug;386(8):725-37 PMID: 16201867
  30. Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome.
    Mol Cell. 2008 Nov 7;32(3):415-25 PMID: 18995839
  31. Unsaturated fatty acids inhibit proteasomal degradation of Insig-1 at a postubiquitination step.
    J Biol Chem. 2008 Nov 28;283(48):33772-83 PMID: 18835813
  32. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.
    Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6511-8 PMID: 17428920
  33. 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
  34. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.
    Cell. 1996 Nov 1;87(3):415-26 PMID: 8898195
  35. Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells.
    J Biol Chem. 2001 Feb 9;276(6):4365-72 PMID: 11085986
  36. Regulation of the mevalonate pathway.
    Nature. 1990 Feb 1;343(6257):425-30 PMID: 1967820
  37. 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
  38. Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein.
    J Biol Chem. 1999 Oct 1;274(40):28549-56 PMID: 10497220
  39. Proteasomes: machines for all reasons.
    Cell. 2007 May 18;129(4):659-62 PMID: 17512401
  40. Recognition and processing of ubiquitin-protein conjugates by the proteasome.
    Annu Rev Biochem. 2009;78:477-513 PMID: 19489727
Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
1083-351X
DOI
10.1074/jbc.M109.044875
Published
2009-12-11
Epub
2009-00-08
Pages
34889-900
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2787351
Subset
IM
Grants
NHLBI NIH HHS · P01 HL020948 · United States
NIDDK NIH HHS · R01 DK046181 · United States
NHLBI NIH HHS · 2P01HL20948 · United States
NIDDK NIH HHS · R01DK46181 · United States
External Links
PubMed source DOI: 10.1074/jbc.M109.044875 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2