A second mutation in membrane domain of srebp cleavageactivating protein (SCAP) that renders cho cells resistant to sterols

In the presence of oxygenated cholesterol derivatives, mammalian cells die of cholesterol deprivation, owing to suppression of cleavage and release of sterol regulatory element binding proteins (SREBPs) from membranes. SREBPs are membrane-bound proteins that stimulate transcription of genes required for synthesis and uptake of cholesterol. We previously identified three oxysterol-resistant CHO cell lines that harbored the same Asp443Asn mutation in SCAP, a 1276-amino acid (aa) membrane protein that activates cleavage of SREBPs. Here, we report a new SCAP mutation in a fourth sterol-resistant CHO cell line. Like the Asp443Asn mutation, this mutation (Tyr298Cys) enhances the activity of SCAP, and renders cleavage of SREBPs insensitive to inhibition by sterols. Tyr298 and Asp443 are located within a stretch of 163 aa (comprising 5 transmembrane segments, aa 280-444) that resemble the sterol-sensing domain of HMG CoA reductase, an enzyme in the cholesterol synthesis pathway whose degradation is accelerated by sterols. The residue corresponding to Tyr298 in hamster SCAP is conserved in HMG CoA reductase of all animal species. These results substantiate the hypothesis that SCAP regulates cholesterol metabolism by regulating cleavage of SREBPs and point to aa 280-444 as the putative sterol-sensing domain of this protein.