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

Navdar Sever, Peter C W Lee, Bao Liang Song, Robert B. Rawson, Russell A. DeBose-Boyd

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Insig-1 and Insig-2 are membrane proteins of the endoplasmic reticulum that regulate lipid metabolism by the following two actions: 1) sterol-induced binding to 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an action that leads to ubiquitination and degradation of the enzyme; and 2) sterol-induced binding to SREBP cleavage-activating protein, an action that blocks the proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that enhance the synthesis of cholesterol and fatty acids. Here we report the isolation of a new mutant line of Chinese hamster ovary cells, designated SRD-14, in which Insig-1 mRNA and protein are not produced due to a partial deletion of the INSIG-1 gene. The SRD-14 cells were produced by γ-irradiation, followed by selection with the 1,1-bisphosphonate ester SR-12813, which mimics sterols in accelerating reductase degradation but does not block SREBP processing. SRD-14 cells fail to respond to sterols by promoting reductase ubiquitination and degradation. The rate at which sterols suppress SREBP processing is significantly slower in SRD-14 cells than wild type CHO-7 cells. Sterol regulation of reductase degradation and SREBP processing is restored when SRD-14 cells are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results provide formal genetic proof for the essential role of Insig-1 in feedback control of lipid synthesis in cultured cells.

Original languageEnglish (US)
Pages (from-to)43136-43147
Number of pages12
JournalJournal of Biological Chemistry
Volume279
Issue number41
DOIs
StatePublished - Oct 8 2004

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Sterol Regulatory Element Binding Proteins
Cell Separation
Sterols
Oxidoreductases
Degradation
Ubiquitination
Processing
Cells
CHO Cells
Diphosphonates
Cricetulus
Lipid Metabolism
Endoplasmic Reticulum
Feedback control
3-hydroxy-3-methylglutaryl-coenzyme A
SR 12813
Ovary
Cultured Cells
Membrane Proteins
Esters

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Sever, Navdar; Lee, Peter C W; Song, Bao Liang; Rawson, Robert B.; DeBose-Boyd, Russell A.

In: Journal of Biological Chemistry, Vol. 279, No. 41, 08.10.2004, p. 43136-43147.

Research output: Contribution to journalArticle

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