Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2

Peter C W Lee, Navdar Sever, Russell A. Debose-Boyd

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Abstract

Insig-1 and Insig-2, a pair of endoplasmic reticulum (ER) membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to the following two polytopic ER membrane proteins: sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. Here we report the isolation of a new line of mutant Chinese hamster ovary cells, designated SRD-15, deficient in both Insig-1 and Insig-2. The SRD-15 cells were produced by γ-irradiation of Insig-1-deficient SRD-14 cells, followed by selection in high levels of the oxysterol, 25-hydroxycholesterol. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 cells. Sterol regulation of SREBP processing and reductase ubiquitination/degradation is fully restored in SRD-15 cells when they are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results demonstrate an absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells.

Original languageEnglish (US)
Pages (from-to)25242-25249
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number26
DOIs
StatePublished - Jul 1 2005

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Sterol Regulatory Element Binding Proteins
Sterols
Cricetulus
Ovary
Cells
Oxidoreductases
Ubiquitination
Endoplasmic Reticulum
Cholesterol
Degradation
Membrane Proteins
Processing
Endoplasmic Reticulum-Associated Degradation
Feedback control
Animals
Proteins
Plasmids
Transcription Factors
Irradiation
Membranes

ASJC Scopus subject areas

  • Biochemistry

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Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2. / Lee, Peter C W; Sever, Navdar; Debose-Boyd, Russell A.

In: Journal of Biological Chemistry, Vol. 280, No. 26, 01.07.2005, p. 25242-25249.

Research output: Contribution to journalArticle

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