Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols

We report the isolation and characterization of a new line of mutant Chinese hamster ovary cells, designated SRD-5, that are resistant to 25HC, a potent suppressor of cleavage of sterol regulatory element-binding proteins (SREBPs) in mammalian cells. In SRD-5 cells, SREBPs are cleaved constitutively, generating transcriptionally active nuclear SREBP even in the presence of sterols. Sequence analysis of SREBP cleavage-activating protein (SCAP) transcripts from SRD-5 cells revealed the presence of a mutation in one SCAP allele that results in substitution of a conserved Leu by Phe at amino acid 315 within the sterol-sensing domain. Sterols fail to inhibit the packaging of SREBP/SCAP(L315F) complexes into budding vesicles in vitro. Sterols also fail to induce binding of SCAP(L315F) to insig-1 or insig-2, two proteins that function in the sterol-mediated retention of SREBP/SCAP complexes in the endoplasmic reticulum. Similar findings were observed for SCAP(D443N) and SCAP(Y298C), both of which cause a sterol-resistant phenotype. Thus, three different point mutations, each within the sterol-sensing domain of SCAP, prevent sterol-induced binding of SCAP to insig proteins and abolish feedback regulation of SREBP processing by sterols.