RECURRENT G-TO-A SUBSTITUTION IN A SINGLE CODON OF SREBP CLEAVAGE-ACTIVATING PROTEIN CAUSES STEROL RESISTANCE IN 3 MUTANT CHINESE-HAMSTER OVARY CELL-LINES

Oxygenated sterols such as 25-hydroxycholesterol kill Chinese hamster ovary cells because they inhibit the proteolytic processing of sterol regulatory element binding proteins (SREBPs), a pair of membrane-bound transcription factors that activate genes controlling cholesterol syn thesis and uptake from lipoproteins, The unprocessed SREBPs remain mem brane-bound, they cannot activate the cholesterol biosynthetic pathway , and the cells die of cholesterol deprivation. Several sterol-resista nt hamster cell lines have been isolated previously by chemical mutage nesis and selection for resistance to killing by 25-hydroxycholesterol . We recently identified the defect in one such cell line (25-RA cells ) as a point mutation in a newly discovered membrane protein of 1276 a mino acids, designated SREBP cleavage-activating protein (SCAP), The m utation in the 25-RA cells resulted from a G-to-A transition in codon 443 of the SCAP gene, changing aspartic acid to asparagine. Wild-type SCAP, when overexpressed by transfection, stimulates the proteolytic p rocessing of both SREBPs, The D443N substitution is an activating muta tion that increases the activity of SCAP and renders it resistant to i nhibition by 25-hydroxycholesterol. We here report the identical G-to- A transition in two additional lines of Chinese hamster ovary cells th at were mutagenized and isolated by a similar protocol, The three muta tions occurred independently as indicated by haplotype analysis of the mutant genes using two intragenic sequence polymorphisms. All three c ell lines were mutagenized with alkylating agents (nitrosoethylurea or ethylmethane sulfonate) that favor Gto-A transitions. Nevertheless, t he finding of the same nucleotide substitution at the same location in all three cell lines indicates that SCAP may be unique in its ability to stimulate SREBP cleavage, and residue 433 is a crucial determinant of the protein's ability to be inhibited by 25-hydroxycholesterol.