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*

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 cleavage-activating protein (SCAP). The SRD-13A cells were obtained by two rounds of γ-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 NH2-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 suppres-sion 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 protein, and the developmental receptor Patched (4,5,8). Missense mutations at two positions within this domain of SCAP render cleavage of SREBPs insensitive to suppression by sterols (9). The COOHterminal domain of SCAP (ϳ550 amino acids) is hydrophilic and projects into the cytosol. It contains five WD repeats (5), which are sequences of ϳ40 amino acids that are found in many proteins where they mediate protein-protein interactions (10). The COOH-terminal WD repeat domain is the region of SCAP that forms a complex with the COOH-terminal regulatory domain of SREBPs (2).
Proteolytic release of the NH 2 -terminal fragments of SREBPs begins with the action of site 1 protease (S1P), a membrane-bound serine protease that cleaves within the luminal loop, thereby separating the two transmembrane helices (11). S1P is distantly related to the mammalian subtilisin-like proteases furin and the prohormone convertases. It cleaves after the leucine of the sequence Arg-Ser-Val-Leu (RSVL) (12). Extensive, but so far indirect, experiments suggest that formation of the SCAP⅐SREBP complex is essential for the proteolytic cleavage of SREBPs by S1P (3). These experiments were performed with SREBP-2, one of the three isoforms of SREBP found in animal cells. The observations are as follows: 1) COOH-terminal truncations of SREBP-2 prevent formation of a complex with SCAP and abrogate cleavage of the SREBP; 2) overexpression of the COOH-terminal domain of either SREBP or SCAP disrupts the complex between full-length SCAP and SREBP and abolishes cleavage at site 1; and 3) the block can be overcome in both cases by overexpression of full-length SCAP, which restores the full-length SCAP⅐SREBP complex (3).
Cleavage by S1P separates the two transmembrane helices of the SREBPs, but both halves remain bound to the membrane because each retains a single transmembrane helix. The NH 2terminal fragment is released from the membrane by site 2 protease (S2P), which cuts near the junction between the hydrophilic NH 2 -terminal fragment and the first transmembrane helix (12,13). The cleavage site is three residues within the transmembrane helix. After cleavage by S2P, the NH 2 -terminal fragment leaves the membrane with three hydrophobic residues at its COOH terminus. This fragment then enters the nucleus, where it binds to sterol regulatory elements and activates transcription of more than 15 genes, whose products play roles in the biosynthesis and uptake of cholesterol and unsaturated fatty acids (1,14).
In unraveling the SREBP proteolytic pathway, our laboratory has relied on mutant Chinese hamster ovary (CHO) cells with defects in the genes encoding S2P and S1P (13,15). The S2P-deficient cell line was derived by mutagenesis followed by selection for cholesterol auxotrophy (16). The selection scheme is based on the method of Chang and Chang (17), who showed that cholesterol auxotrophs are resistant to the polyene antibiotic amphotericin, which kills cells by forming complexes with cholesterol in the plasma membrane. Prior to amphotericin treatment, the mutagenized CHO cells are incubated briefly in the absence of exogenous cholesterol and in the presence of a low concentration of LDL. During this period, wildtype cells activate cholesterol synthesis, and they take up LDL via LDL receptors. As a result, they maintain high levels of plasma membrane cholesterol. The auxotrophs are unable to produce cholesterol or to take it up from LDL, and their plasma membranes become relatively depleted of this sterol. Brief treatment with amphotericin kills the wild-type cells but not the auxotrophs. The mutant cells are then rescued by growth in the presence of cholesterol, the unsaturated fatty acid oleate, and a low concentration of mevalonate to supply nonsterol products (13,15).
When this selection procedure was first employed, all of the mutants recovered from the selection had defects in the gene encoding S2P, which resides on the X chromosome. We attributed this bias to the fact that wild-type CHO cells have only one functional copy of the S2P gene. This problem was overcome when we cloned the gene for S2P by complementation (13) and used transfection techniques to produce a line of CHO cells, designated CHO/pS2P, with multiple expressed copies of the S2P cDNA (15). To isolate cholesterol auxotrophs with recessive defects at loci other than S2P, we employed a double mutagenesis protocol. The CHO/pS2P cells were first mutagenized with ␥-irradiation, and potential heterozygotes were identified by incubation with fluorescently labeled LDL. The cells with low levels of LDL receptor expression were isolated with a fluorescence-activated cell sorter and subjected to a second round of ␥-irradiation. Potential homozygotes were then selected for cholesterol auxotrophy using the amphotericin resistance protocol described above. This procedure yielded 12 cell lines that were auxotrophic for cholesterol. One of these, designated SRD-12B, had a defect in S1P, and these cells were used for the cloning of the S1P cDNA by complementation (11). The 11 remaining cholesterol-auxotrophic cell lines were not further characterized.
In the current studies, we characterize one of the 11 remaining amphotericin-resistant CHO cell lines. This cell line, designated SRD-13A, was found to have two defective copies of the gene encoding SCAP. As a result of the SCAP deficiency, the SRD-13A cells are unable to cleave SREBPs at site 1, and they are therefore cholesterol auxotrophs. The experiments with the SRD-13A cells provide formal genetic proof that the site 1 cleavage reaction requires SCAP.
Cultured Cells-The cell lines and culture medium used are described in Table I. Cells were maintained in monolayer culture at 37°C in a 9% CO 2 incubator. CHO-7 cells are a subline of CHO-K1 cells selected for growth in lipoprotein-deficient serum (22). These cells were maintained in medium A supplemented with 5% (v/v) fetal calf lipoprotein-deficient serum. M19 cells are previously described mutant CHO cells auxotrophic for cholesterol, mevalonate, and unsaturated fatty acid (16), due to a deficiency of S2P (13). SRD-12B cells are previously described mutant CHO cells auxotrophic for cholesterol, mevalonate, and unsaturated fatty acid, due to a deficiency of S1P (11). They were isolated by an amphotericin B resistance protocol (15) and maintained in medium B. CHO/pS2P cells are CHO-7 cells expressing extra copies of a cDNA encoding S2P. Stock cultures of CHO/pS2P cells were maintained in medium A supplemented with 5% fetal calf lipoprotein-deficient serum, 2 M compactin, and 500 g/ml G418.
Isolation of Amphotericin B-resistant Cells Deficient in SCAP-SRD-13A cells were isolated in the same experiment that yielded the SRD-12B cells (15). Briefly, CHO/pS2P cells were subjected to ␥-irradiation mutagenesis. These cells were then grown for several days, incubated with fluorescent r-(PMCA oleate)LDL, and subjected to fluorescenceactivated cell sorting. The sorted population of cells showing a reduced uptake of fluorescent LDL was grown to confluence, remutagenized with ␥-irradiation, replated, and subjected to multiple rounds of selection with amphotericin B as described previously (15). Of the 100 dishes plated following the second round of mutagenesis, 12 contained colonies, ranging from 1 to 5 colonies/dish. Each colony was replated and subjected to additional rounds of amphotericin B selection. Cells from a single colony from one dish were cloned by limiting dilution, and one of the resulting clones was designated SRD-13A. The SRD-13A cells were maintained in medium B at 37°C and were subjected weekly to the amphotericin B selection protocol as described (15).
SREBP Processing in Transfected Cells-Cells were transfected using the Fugene 6 reagent (Roche Molecular Biochemicals). On day 0, cells were set up in 60-mm dishes at the following densities: CHO-7/ pS2P, 300,000 cells; SRD-12B, 400,000 cells; and SRD-13A, 600,000 cells. On day 1, the cells were transfected with 4 g of DNA/dish using a ratio of 12 l of Fugene to 4 g of DNA in medium A (without antibiotics) in a final volume of 0.2 ml. Fugene was diluted in medium A (without antibiotics) and incubated for 5 min at room temperature prior to being added dropwise to the DNA solution. This mixture was then further incubated for 15-30 min at room temperature. Plates were washed one time with 2 ml of medium A supplemented with 5% fetal calf serum and refed with 3 ml of the same medium. The Fugene/DNA mixture (0.2 ml) was then added to each dish. Cells were incubated at 37°C for 16 -24 h. On day 2, cells were washed one time with phosphate-buffered saline (PBS) and refed with either medium C or medium D. After incubation for 16 h at 37°C, the cells received N-acetylleucinyl-leucinyl-norleucinal (ALLN) at a final concentration of 25 g/ ml. After incubation for 1 h, the cells were harvested and fractionated into nuclear extract and 10 5 ϫ g membrane pellet fractions as described previously (23).
Complementation Assay Using Transient Heterokaryon Cell Fusion-This assay was performed as described previously (15). Briefly, on day 0 the cells to be tested were mixed and plated at 3.5 ϫ 10 5 cells/well in 12-well plates and fused in the presence of polyethylene glycol. On day 2, the fused cells were trypsinized, fed, and replated onto coverslips. On day 3, the cells were fed fluorescent r-(PMCA oleate)LDL at 10 g of protein/ml in either medium C (inducing) or medium D (suppressing). After 12 h, the cells were washed, refed medium B, fixed 4 -6 h later, mounted on a slide, and then viewed by fluorescence microscopy.
Metabolic Assays-The incorporation of [ 14 C]pyruvate into cellular sterols and fatty acids (18), the incorporation of [ 14 C]oleate into cellular cholesteryl esters and triglycerides (19), and the proteolytic degradation of 125 I-LDL (19) were measured in cell monolayers as described in Refs. 18 and 19. The protein content of cell extracts was determined by the method of Lowry et al. (27).
PCR on high molecular weight genomic DNA from parental CHO/ pS2P cells (0.8 g) and SRD-13A cells (0.5 g) was performed in 25-l reactions using the following primer pair: 5Ј-CATCGGTATCTCCCTG-GCACAAAA-3Ј and 5Ј-GAGTCCTGGCAGCAAGTCGGTCAC-3Ј. The PCR products were cloned and sequenced as described above. Table I lists the cell lines and culture media that were used in these studies. Fig. 1 shows the growth pattern of CHO/pS2P cells, which express multiple copies of the S2P cDNA, and two mutant lines designated SRD-12B and SRD-13A, which were derived from CHO/pS2P cells by two steps of mutagenesis followed by selection for low expression of LDL receptors and for cholesterol auxotrophy. Details of the two-step selection scheme are given in Ref. 15 and under "Experimental Procedures." The SRD-12B cells were shown previously to harbor two mutant S1P genes, and their cholesterol auxotrophy can be overcome by transfection of a cDNA encoding S1P (11). The SRD-13A cells were not previously characterized. As shown in Fig. 1, the parental CHO/pS2P cells grow well in lipoproteindeficient medium, which is low in cholesterol. The SRD-12B and SRD-13A cells grow normally only when the lipoproteindeficient medium is supplemented with a mixture of cholesterol, mevalonate, and oleate, all of which are products of SREBP-dependent genes. Fig. 2 shows a cell fusion assay designed to determine whether the SRD-13A cells have defects in the genes encoding S2P or S1P. In the upper panels, mixed cultures of SRD-12B and SRD-13A cells were incubated with or without the fusogen PEG, after which they were incubated with LDL that contains a fluorescent derivative of cholesterol esterified with oleate (r-(PMCA oleate)LDL). In the absence of PEG, there was no visible PMCA oleate uptake due to the absence of LDL receptors (Fig. 2B). In the PEG-fused cells, LDL uptake was readily apparent (Fig. 2D). These data indicate that the defect in the SRD-13A cells does not reside in the gene encoding S1P. The cell fusion assay was validated by control experiments showing that homotypic fusions of SRD-13A cells with themselves did not lead to enhanced LDL uptake (data not shown). To determine whether the defect in the SRD-13A cells lies in the gene encoding S2P, we performed a similar fusion experiment with mixtures of SRD-13A cells and M-19 cells, which lack the gene for S2P. Again the fused cells showed LDL uptake (Fig. 3H), confirming that the SRD-13A cells were not defective in the S2P gene.

RESULTS
To study lipid synthesis in the SRD-13A cells, we incubated the cells for 24 h in the absence of cholesterol, and we then incubated them with [ 14 C]pyruvate (Table II). The SRD-13A  (28).
They indicate that nuclear SREBPs are absolutely required for cholesterol synthesis, but they are only partially required for fatty acid synthesis in CHO cells.
As expected, the SRD-12B and SRD-13A cells showed marked reductions in LDL receptor activity, as determined by measurement of the high affinity degradation of 125 I-LDL (Table III). The cells also showed a marked reduction in the ability of LDL to stimulate incorporation of [ 14 C]oleate into cholesteryl [ 14 C]oleate (Table IV). The cells did show an increase in synthesis of cholesteryl [ 14 C]oleate when stimulated with a mixture of 25-hydroxycholesterol and cholesterol, indicating that the failure to respond to LDL was a result of the LDL receptor deficiency. The incorporation of [ 14 C]oleate into [ 14 C]triglycerides was essentially normal in the SRD-12B and SRD-13A cells (Table IV).
To pinpoint the biochemical defect in the SRD-13A cells, we incubated the cells in the absence or presence of sterols. Nuclear and membrane extracts were subjected to SDS-PAGE and blotted with antibodies against various components of the processing system (Fig. 3). The SRD-13A cells were compared with the S1P-deficient SRD-12B cells and the parental CHO/pS2P cells. As shown in

. Growth of parental and mutant CHO cells in the presence and absence of nutrients.
On day 0, cells were plated at 4 ϫ 10 4 cells/25-mm well in medium B. On day 1, the cells were washed once with PBS and refed every 2-3 days with one of the following media: medium B containing 5% fetal calf serum, cholesterol, mevalonate, and oleate (left column) or medium A supplemented with 5% fetal calf lipoprotein-deficient serum (right column). On day 14, the cells were washed, fixed in 95% ethanol, and stained with crystal violet.

FIG. 2. Complementation of deficient LDL receptor activity by PEGmediated fusion of mutants auxotrophic for cholesterol.
SRD-12B cholesterol-auxotrophic mutants or M19 cholesterol-auxotrophic mutants were mixed with SRD-13A cholesterol-auxotrophic mutants and incubated in the absence or presence of PEG as described under "Experimental Procedures." The fused cells were incubated for 16 h with 10 g of protein/ml of fluorescent r-(PMCA oleate)LDL in medium C as described previously (15). amounts of SCAP (Fig. 3B) that were not affected by the absence or presence of sterols (lanes 1 and 2). The SRD-12B cells had reduced amounts of SCAP when incubated in the absence of sterols (lane 3), and the amount rose when sterols were present (lane 4). The SRD-13A cells lacked detectable SCAP when incubated either in the absence or presence of sterols ( lanes 5 and 6).
The CHO/pS2P and SRD-13A cells had normal amounts of S1P as visualized by blotting with anti-S1P (Fig. 3B, middle  blot). Moreover, both cell lines had a similar distribution of the three processed forms of S1P (A, B, and C) (26). As expected, the SRD-12B cells lacked all immunodetectable S1P (Fig. 3B,   middle blot, lanes 3 and 4). The bottom blot of Fig. 3B is a control that was incubated with an antibody against an irrelevant ER protein (Grp78). The blot shows that all of the membrane fractions contained equal amounts of the two proteins (Grp94 and Grp78) that react with this antibody.
To determine whether the SCAP deficiency is responsible for the failure of SRD-13A cells to cleave SREBPs, we transfected SRD-13A cells with expression vectors encoding HSV-tagged SREBP-1a (Fig. 4A) or SREBP-2 ( Fig. 4B) with increasing amounts of a plasmid encoding SCAP (Fig. 4). The cells were incubated in the absence or presence of sterols, and the amounts of membrane-bound and nuclear SREBPs were measured. As expected, in the absence of SCAP there was no nuclear SREBP (lane 1). As the amount of SCAP plasmid increased, there was a corresponding increase in the amounts of nuclear SREBP-1a and SREBP-2 in the cells incubated in the absence of sterols (lanes 3, 5, and 7). When sterols were present, the amounts of the nuclear SREBPs were reduced (lanes 4, 6, and  8). Thus, expression of SCAP restores sterol-regulated cleavage of SREBPs in the SRD-13A cells.
If the SCAP cDNA restores site 1 cleavage in SRD-13A cells, then it should also restore growth in the absence of cholesterol. The experiment of Fig. 5 indicates that this was the case.
The experiment of Fig. 6 was designed to determine whether permanent restoration of SCAP expression would restore sterol-regulated processing of endogenous SREBP-2 in SRD-13A

FIG. 3. Sterol-regulated processing of endogenous SREBP-1 and SREBP-2 (A) and immunoblot analysis of endogenous SCAP and S1P (B) in parental CHO cells and mutants auxotrophic for cholesterol.
On day 0, the cells were set up in 100-mm dishes in medium B at the following densities: CHO/pS2P, 5 ϫ 10 5 cells; SRD-12B, 4 ϫ 10 5 cells; and SRD-13A, 8 ϫ 10 5 cells. On day 2, the cells were washed twice with prewarmed medium A and then switched to inducing medium C (Ϫ Sterols) or suppressing medium D (ϩ Sterols). After incubation for 16 h at 37°C, the dishes received ALLN at a final concentration of 25 g/ml. After incubation for 1 h, the cells were harvested and fractionated as described previously (23). A, aliquots of the membrane (40 g of protein/lane), and nuclear extract fractions (60 g protein/lane) were subjected to SDS-PAGE. Immunoblot analysis was carried out with 5 g/ml of either monoclonal IgG-2A4 for SREBP-1 or monoclonal IgG-7D4 for SREBP-2. The filters were exposed to film for 40 s at room temperature. B, aliquots of the membrane fractions (20 g) were subjected to SDS-PAGE and immunoblot analysis with 5 g/ml R139, a rabbit polyclonal antibody directed against SCAP (top panel); a 1:250 dilution of U1683, a rabbit polyclonal antiserum directed against S1P (middle panel); or 1 g/ml anti-Grp78, a monoclonal antibody directed against rat Grp78 (bottom panel). Filters were exposed for less than 1 s at room temperature.

Receptor-mediated degradation of 125 I-LDL in parental CHO/pS2P cells and mutant cells auxotrophic for cholesterol
Cells were set up as described in the legend to Table II. On day 1, the cells were washed with PBS and switched to medium A supplemented with 5% fetal calf lipoprotein-deficient serum, 50 M compactin, and 50 M sodium mevalonate. On day 2, the cells were switched to 2 ml of Dulbecco's modified Eagle's medium (without glutamine) containing 2 mg/ml bovine serum albumin and 15 g of protein/ml of 125 I-LDL (129 cpm/ng of protein) in the absence or presence of 600 g of protein/ml of unlabeled LDL. After incubation for 5 h at 37°C, high affinity degradation of LDL was determined by subtracting the values for nonspecific degradation (with unlabeled LDL) from those for total degradation (without unlabeled LDL). Each value is the mean of triplicate incubations. cells. For this purpose, we obtained three independent clones of SRD-13A cells that were permanently transfected with pTK-SCAP and had regained the ability to grow in the absence of cholesterol (see Fig. 6). The three cloned cell lines are designated A, B, and C. To induce SREBP processing, we incubated the cells overnight in suppressing medium that contained a mixture of 25-hydroxycholesterol plus cholesterol. The cells were then washed and switched to fresh medium with or without cyclodextrin, an agent that efficiently removes sterols from cells (29). The medium also contained ALLN, which inhibits the degradation of the nuclear form of SREBPs (30). As controls, we studied the parental CHO/pS2P cells and the SCAP-deficient SRD-13A cells. In the presence of cyclodextrin, nuclear On day 2, the cells were switched to inducing medium C (Ϫ Sterols) or suppressing medium D (ϩ Sterols) and incubated for 16 h as described in Fig. 3. Aliquots of membrane and nuclear extract fractions (20 g of protein/lane) were subjected to SDS-PAGE and immunoblot analysis with either 0.2 g/ml of IgG-HSV-Tag TM or 5 g/ml R139, a rabbit polyclonal antibody directed against SCAP. The filters were exposed to film at room temperature for 1-2 s.

FIG. 5. Restoration of growth of SRD-13A cells by transfection with pTK-SCAP.
On day 0, cells were plated at 6 ϫ 10 5 cells/60-mm dish in medium B. On day 1, the cells were transfected with either 4 g/dish of empty vector or 0.25 g/dish of pTK SCAP together with 3.75 g/dish of empty vector as described under "Experimental Procedures." On day 2, the cells were washed once with PBS and refed with either medium B containing cholesterol, mevalonate, and oleate or medium A supplemented with 5% fetal calf lipoprotein-deficient serum. Cells were refed every 2-3 days. On day 14, the cells were washed, fixed in 95% ethanol, and stained with crystal violet.  Fig.  6 were obtained by limiting dilution and designated SRD-13A/ pSCAP-A, -B, and -C. On day 0, cells were set up in 100-mm dishes in medium B at the following densities: CHO/pS2P, 4 ϫ 10 5 cells/dish; SRD-13A, 7 ϫ 10 5 cells/dish; SRD-13A/pSCAP-A, -B, and -C, 5 ϫ 10 5 cells/dish. On day 2, the cells were washed twice in prewarmed medium A and switched to suppressing medium D (ϩ Sterols). After incubation for 16 h at 37°C, the dishes were washed once with PBS and refed with either medium C containing 25 g/ml ALLN in the absence (Ϫ CD) or presence (ϩ CD) of 1% (w/v) cyclodextrin. After incubation for 1.5 h, the cells were harvested and fractionated as described under "Experimental Procedures." Aliquots of membranes (50 g/lane) and nuclear extract fractions (70 g/lane) were subjected to SDS-PAGE, and immunoblot analysis was carried out with 5 g/ml of monoclonal IgG-7D4 for SREBP-2. Filters were exposed for 30 s (nuclear extracts) and 4 s (membranes) at room temperature. Additional aliquots of membranes (25 g/lane) were also subjected to SDS-PAGE and analyzed by immunoblotting with 5 g/ml R139, a rabbit polyclonal antibody directed against SCAP. The filters were exposed for 1 s at room temperature. extracts from the CHO/pS2P cells contained the nuclear form of SREBP-2 as determined by immunoblotting (Fig. 6, lane 1). The nuclear protein was not detected when cyclodextrin was omitted (lane 2). As expected, the SRD-13A cells lacked nuclear SREBP-2 under either condition (lanes 3 and 4). All three of the permanently transfected SCAP-expressing SRD-13A cells showed normal amounts of nuclear SREBP-2, and this was abolished when cyclodextrin was omitted (lanes 5-10). The upper panel of Fig. 6 also shows that restoration of SCAP expression restored normal levels of the precursor form of SREBP-2 in the membrane fraction of SRD-13A cells. The bottom panel of Fig.  6 confirms that SCAP expression was restored in lines A-C as determined by blotting with anti-SCAP. We next conducted a series of experiments designed to assess the nature of the genetic defect that leads to SCAP deficiency in the SRD-13A cells. Fig. 7 shows a Northern blot, comparing the amounts of SCAP mRNA in the CHO/pS2P cells, the SRD-12B cells, and the SRD-13A cells. Quantification by exposing the filters to a Fujix BAS1000 Bio-Imaging Analyzer revealed that the amount of SCAP mRNA was reduced by 55% in the SRD-13A cells when compared with the other two cell lines. The mRNA that remained in the SRD-13A cells was approximately the same size as the SCAP mRNA in the CHO/pS2P cells (ϳ4.2 kilobases).
To determine whether the residual SCAP mRNA in SRD-13A cells could encode a functional SCAP protein, we amplified the mRNA by reverse transcriptase-PCR, inserted the amplified fragments into a cloning vector, and sequenced nine independent clones. All of the clones showed a 14-bp deletion that disrupted the reading frame at codon 133, leading to an altered sequence of amino acids that terminates at position 160 (Fig.  8A). If this truncated protein were produced, it would contain only one of the eight membrane-spanning helices, and it would lack both the sterol-sensing domain and the WD repeat region (Fig. 8A). Such a protein could not be functional.
To determine the cause of the 14-bp deletion in the SCAP mRNA, we subjected the relevant region of genomic DNA to PCR, and we sequenced 16 clones derived from wild-type cells and SRD-13A cells. All of the SRD-13A sequences had a single point mutation that changed a C to a G. This creates a new splice site that is 14 bp upstream from the normal splice site (Fig. 8B). The resulting mRNA has a deletion of 14 bp. Apparently, the new splice site is used exclusively, because all of the cellular mRNA shows the 14-bp deletion.
The fact that all of the genomic clones had the same point FIG. 7. Northern blot analysis of total RNA from parental CHO cells and mutants auxotrophic for cholesterol. Total RNA was prepared from the indicated cell lines using RNAStat60 (TelTest B) according to the manufacturer's instructions. Aliquots of total RNA (20 g/lane) were subjected to electrophoresis in a denaturing (formaldehyde) gel (32) and transferred to a Hybond Nϩ membrane (Amersham Pharmacia Biotech) by capillary blotting. Replicate blots were probed with one of the following 32 P-labeled probes: 2.8-kilobase fragment from the 5Ј-end of hamster SCAP (4) or 1.2-kilobase cDNA encoding rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (33). Hybridization was carried out for 60 min at 65°C in RapidHyb buffer (Amersham Pharmacia Biotech) at 1 ϫ 10 6 cpm/ml. The filters for SCAP and GAPDH were exposed at Ϫ70°C for 14 h and 20 min, respectively. The mutant sequence was determined by reverse transcriptase PCR analysis and sequencing of nine independent clones as described under "Experimental Procedures." B, sequence of genomic DNA. The sequence of the genomic DNA from the region encoding the deleted portion of the transcript from mutant SRD-13A cells in A was determined from PCR-generated clones. Sixteen clones from the wild-type and mutant cells were sequenced. The mutation resulting from the C to G change in codon 132 (denoted by an asterisk) creates a new consensus splice donor site in the SRD-13A DNA (34), utilization of which yields a transcript with the observed 14-bp deletion shown in A. mutation suggests that the SRD-13A cells are either homozygous for the point mutation shown in Fig. 8, or else they have a deletion in the second copy of the gene that removes at least this region. Genomic Southern blots failed to show abnormal restriction fragments in the SRD-13A cells (data not shown). Thus, if there is a deletion in the second copy of the SCAP gene, the deletion may be large enough to encompass the whole gene plus substantial amounts of flanking DNA. DISCUSSION The current data provide compelling genetic evidence that SCAP is absolutely required in order for the site 1 protease to cleave SREBPs. The mutant SRD-13A cells, which fail to produce functional SCAP mRNA, are unable to cleave endogenous SREBPs (Fig. 3) or SREBPs that have been overexpressed as a result of transfection (Fig. 4). Cleavage is restored when wildtype SCAP is expressed as a result of transient or stable transfection ( Fig. 4 and 6, respectively). As a result of the failure to cleave SREBPs, the SRD-13A cells fail to synthesize normal amounts of cholesterol, and they require cholesterol, oleate, and mevalonate for growth. The auxotrophies are abolished when SCAP expression is restored (Fig. 5).
An unexpected finding was the low levels of SREBP precursors in the SRD-13A cells (Figs. 3 and 6). This phenomenon appears to be secondary to the SCAP deficiency, since the levels of SREBP precursors were restored to normal in the three lines of SRD-13A cells that were permanently transfected with pTK-SCAP (Fig. 6).
Co-immunoprecipitation experiments indicate the vast majority of SREBPs in CHO cells are present in a complex with SCAP (2). If the COOH terminus of SREBPs cannot form a complex with SCAP, the proteins may be rapidly degraded. Such degradation must occur through a pathway that does not release the NH 2 -terminal fragments of SREBPs into the cytoplasm.
Interestingly, we did not observe a deficiency of SREBP-2 when the precursor was produced in SRD-13A cells as a result of transient transfection with the pTK-SREBP-2 cDNA (Fig. 4). It is possible that overexpression of SREBP-2 saturates the degradative process, allowing the precursor to accumulate. Resolution of this issue will require further studies of the synthesis and turnover of SREBPs in the SRD-13A cells.
The mechanism by which SCAP facilitates S1P activity is not yet known. Previous data provide evidence that the SCAP⅐SREBP complex remains sequestered in the ER when cells are overloaded with sterols. When sterols are depleted, the carbohydrate chains of SCAP become resistant to endoglycosidase H, suggesting that the SCAP⅐SREBP complex has moved to the Golgi (31). It is possible that SREBP must leave the ER in order to be cleaved by S1P and that the function of SCAP is to control this movement according to the sterol content of the cell. The availability of the SCAP-deficient SRD-13A cells should permit testing of this hypothesis by transfection of wild-type and mutant SCAP in an attempt to correlate site 1 cleavage with the ability of mutant forms of SCAP to leave the ER.