We describe a line of mutant Chinese hamster ovary cells, designated SRD-13A, that cannot cleave sterol regulatory element-binding proteins (SREBPs) at site 1, due to mutations in the gene encoding SREBP cleavageactivating protein (SCAP). The SRD-13A cells were obtained by two rounds of ␥-irradiation followed first by selection for a deficiency of low density lipoprotein receptors and second for cholesterol auxotrophy. In the SRD-13A cells, the only detectable SCAP allele encodes a truncated nonfunctional protein. In the absence of SCAP, the site 1 protease fails to cleave SREBPs, and their transcriptionally active NH 2 -terminal fragments cannot enter the nucleus. As a result, the cells manifest a marked reduction in the synthesis of cholesterol and its uptake from low density lipoproteins. The SRD-13A cells grow only when cholesterol is added to the culture medium. SREBP cleavage is restored and the cholesterol requirement is abolished when SRD-13A cells are transfected with expression vectors encoding SCAP. These results provide formal proof that SCAP is essential for the cleavage of SREBPs at site 1. Sterol regulatory element-binding proteins (SREBPs)1 are membrane-bound transcription factors whose active fragments are released from membranes by controlled proteolysis in order to activate the synthesis of cholesterol and unsaturated fatty acids and their uptake from plasma lipoprotein (1). SREBP cleavage-activating protein (SCAP) is a membrane-bound regulatory protein that forms a complex with SREBPs and facilitates proteolytic release of the active fragments (2, 3). SCAP contains a sterol-sensing domain that allows sterols to suppress SREBP cleavage, thereby mediating feedback suppression of lipid synthesis and uptake (4, 5).The SREBPs are tripartite proteins that are embedded in membranes of the endoplasmic reticulum (ER) and nuclear envelope in a hairpin orientation (1). The transcriptionally active NH 2 -terminal segment of ϳ480 amino acids projects into the cytosol. This segment contains a basic helix-loop-helixleucine zipper sequence that allows the protein to dimerize, to bind DNA, and to recruit transcriptional coactivators (6, 7). The NH 2 -terminal segment is followed by a 90-amino acid membrane attachment segment that consists of two membrane-spanning helices separated by a short hydrophilic loop of 31 amino acids that projects into the lumen of the ER and nuclear envelope. The third segment of the SREBP comprises ϳ590 amino acids that project into the cytosol, where they form a complex with SCAP. This segment has been called the regulatory domain (1).SCAP is a polytopic membrane protein of 1276 amino acids that has two distinct domains. The NH 2 -terminal domain of ϳ730 amino acids consists of eight transmembrane helices separated by hydrophilic loops (5). Transmembrane helices 2-6 have been designated the sterol-sensing domain (5). This domain resembles sequences in three other proteins that are postulated to interact with sterols: 3-hydroxy-3-methylglutaryl CoA reductase, the Niemann-Pick C1 pr...