Details

Autor(en) / Beteiligte

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

Titel

Isolation of Sterol-resistant Chinese Hamster Ovary Cells with Genetic Deficiencies in Both Insig-1 and Insig-2

Ist Teil von

• The Journal of biological chemistry, 2005-07, Vol.280 (26), p.25242-25249

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2005

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.