Abstract
The endoplasmic reticulum membrane proteins Insig-1 and Insig-2 limit cholesterol synthesis, in part through their sterol-dependent binding to sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). This binding prevents proteolytic processing of SREBPs, membrane-bound transcription factors that enhance cholesterol synthesis. We report here the characterization of mutant Chinese hamster ovary (CHO) cells, designated SRD-19, that are resistant to 25-hydroxycholesterol, a potent inhibitor of SREBP processing. SRD-19 cells were produced by mutagenesis of Insig-1-deficient SRD-14 cells, followed by selection in high levels of 25-hydroxycholesterol. 25-Hydroxycholesterol fails to suppress SREBP processing in SRD-19, even though they express normal levels of Insig-2. The number of copies of the gene encoding SCAP was found to be increased by 4-fold in SRD-19 cells compared with wild-type CHO cells, leading to the overproduction of SCAP mRNA and protein. Our data indicate that overproduced SCAP saturates the remaining Insig-2 in SRD-19 cells, thus explaining their resistance to 25-hydroxycholesterol. Consistent with this conclusion, regulated SREBP processing is restored in SRD-19 cells upon transfection of plasmids encoding either Insig-1 or Insig-2. These results highlight the importance of SCAP/Insig ratios in normal sterol-regulated processing of SREBPs in cultured cells.
Highlights
The endoplasmic reticulum membrane proteins Insig-1 and Insig-2 limit cholesterol synthesis, in part through their sterol-dependent binding to sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP)
We found that SRD-19 cells survived culture in medium that contained up to 2.5 mM 25-hydroxycholesterol; similar results were obtained with SRD-15 cells and SRD-1 cells
The current data describe the characterization of SRD19 cells, a line of mutant Chinese hamster ovary (CHO) cells that were produced by mutagenesis of Insig-1-deficient SRD-14 cells [19], followed by selection for growth in 25-hydroxycholesterol
Summary
The endoplasmic reticulum membrane proteins Insig-1 and Insig-2 limit cholesterol synthesis, in part through their sterol-dependent binding to sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Sterol-induced retention of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) in the endoplasmic reticulum (ER) and sterolaccelerated degradation of ER-bound HMG-CoA reductase constitute two mechanisms by which animal cells limit the synthesis of cholesterol. Both mechanisms are mediated by sterol-regulated binding of SCAP and reductase to polytopic ER membrane proteins called Insig-1 and Insig-2. Point mutations within the sterol-sensing domains of SCAP and reductase prevent their association with Insigs, thereby abrogating sterol-mediated ER retention of SCAP-SREBP and sterol-induced ubiquitination/ degradation of reductase [4, 15,16,17]
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