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.
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 gamma-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.
Sever
Navdar
N
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
Lee
Peter C W
PC
Song
Bao-Liang
BL
Rawson
Robert B
RB
Debose-Boyd
Russell A
RA
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins; D004164:Diphosphonates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000235:genetics*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism; D013347:Subcellular Fractions; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004164:Diphosphonates; D005227:Fatty Acids; C108626:INSIG1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C100355:SR 12813; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D025801:Ubiquitin; D004247:DNA; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases