Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2.
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 gamma-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.
Lee
Peter C W
PC
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
Sever
Navdar
N
Debose-Boyd
Russell A
RA
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006888:Hydroxycholesterols / Q000737:chemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism*; D013347:Subcellular Fractions; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006888:Hydroxycholesterols; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C102196:SREBP cleavage-activating protein; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D025801:Ubiquitin; C007997:25-hydroxycholesterol; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases