Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein

Abstract

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...

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis

“…CHO-7 cells are a clone of CHO-K1 cells selected for growth in lipoprotein-deficient serum (27); they were maintained in medium A supplemented with 5% lipoprotein-deficient serum. SRD-13A cells are mutant CHO-7 cells deficient in Scap (14); they were maintained in medium B (medium A supplemented with 5% FCS͞5 g/ml cholesterol͞1 mM sodium mevalonate͞20 M sodium oleate).…”

“…Fig. 2A shows an experiment in which plasmids encoding epitope-tagged SREBP-2, Scap, and WT or D205A mutant human Insig-1 were transfected into SRD-13A cells (a line of mutant CHO cells that are deficient in Scap) (14). When SREBP-2 was cotransfected with Scap, the cleaved NH 2 -terminal fragment of SREBP-2 appeared in nuclear extracts.…”

Membrane Topology of Human Insig-1, a Protein Regulator of Lipid Synthesis

“…Human embryonic kidney (HEK)-293 cells were maintained in medium A (Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate). SRD-13A cells are previously described cholesterol and unsaturated fatty acid auxotrophs derived from ␥-irradiated Chinese hamster ovary (CHO) cells (15) and maintained in medium B (a 1:1 mixture of Ham's F-12 medium and DMEM containing 5% fetal calf serum, 5 g/ml cholesterol, 1 mM sodium mevalonate, 20 M sodium oleate, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate).…”

“…On day 1, the cells were transfected with 4 g of DNA per dish by using FuGENE 6 reagent as described (15). After transfection, cells were incubated at 37°C for 12 h. On day 3, the cells were washed once with phosphate-buffered saline (PBS), switched to medium C (a 1:1 mixture of Ham's F-12 medium and DMEM containing 5% newborn calf lipoprotein-deficient serum, 50 M sodium compactin, and 50 M sodium mevalonate) containing 1% (w/v) hydroxypropyl-␤-cyclodextrin.…”

“…(12) and IgG-9E10 against c-Myc epitope (1) have been previously described. All other reagents were obtained from previously reported sources (13)(14)(15)(16).…”