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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

1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês

10.1074/jbc.274.40.28549

ISSN

1083-351X

Autores

Robert B. Rawson, Russell DeBose-Boyd, Joseph L. Goldstein, Michael S. Brown,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

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. 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) 1The abbreviations used are:SREBPsterol regulatory element-binding proteinSCAPSREBP cleavage-activating proteinALLNN-acetyl-leucinyl-leucinyl-norleucinalCHOChinese hamster ovaryERendoplasmic reticulumHSVherpes simplex virusPBSphosphate-buffered salinePCRpolymerase chain reactionPEGpolyethylene glycolLDLlow density lipoproteinPMCA oleate3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3β-yl oleater-(PMCA oleate)LDLLDL reconstituted with PMCA oleateS1Psite 1 proteaseS2Psite 2 proteasebpbase pair 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 (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). 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 (2Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 3Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). SCAP contains a sterol-sensing domain that allows sterols to suppress SREBP cleavage, thereby mediating feedback suppression of lipid synthesis and uptake (4Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 5Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). sterol regulatory element-binding protein SREBP cleavage-activating protein N-acetyl-leucinyl-leucinyl-norleucinal Chinese hamster ovary endoplasmic reticulum herpes simplex virus phosphate-buffered saline polymerase chain reaction polyethylene glycol low density lipoprotein 3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3β-yl oleate LDL reconstituted with PMCA oleate site 1 protease site 2 protease base pair The SREBPs are tripartite proteins that are embedded in membranes of the endoplasmic reticulum (ER) and nuclear envelope in a hairpin orientation (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). The transcriptionally active NH2-terminal segment of ∼480 amino acids projects into the cytosol. This segment contains a basic helix-loop-helix-leucine zipper sequence that allows the protein to dimerize, to bind DNA, and to recruit transcriptional coactivators (6Naar A.M. Beaurang P.A. Robinson K.M. Oliner J.D. Avizonis D. Scheek S. Zwicker J. Kadonaga J.T. Tjian R. Genes Dev. 1998; 12: 3020-3031Crossref PubMed Scopus (173) Google Scholar, 7Dooley K.A. Bennett M.K. Osborne T.F. J. Biol. Chem. 1999; 274: 5285-5291Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The NH2-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 ∼590 amino acids that project into the cytosol, where they form a complex with SCAP. This segment has been called the regulatory domain (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). SCAP is a polytopic membrane protein of 1276 amino acids that has two distinct domains. The NH2-terminal domain of ∼730 amino acids consists of eight transmembrane helices separated by hydrophilic loops (5Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Transmembrane helices 2–6 have been designated the sterol-sensing domain (5Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). 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 (4Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 5Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 8Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Science. 1997; 277: 232-235Crossref PubMed Scopus (699) Google Scholar). Missense mutations at two positions within this domain of SCAP render cleavage of SREBPs insensitive to suppression by sterols (9Nohturfft A. Hua X. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13709-13714Crossref PubMed Scopus (57) Google Scholar). The COOH-terminal domain of SCAP (∼550 amino acids) is hydrophilic and projects into the cytosol. It contains five WD repeats (5Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), which are sequences of ∼40 amino acids that are found in many proteins where they mediate protein-protein interactions (10Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). The COOH-terminal WD repeat domain is the region of SCAP that forms a complex with the COOH-terminal regulatory domain of SREBPs (2Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Proteolytic release of the NH2-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 (11Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). 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) (12Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Extensive, but so far indirect, experiments suggest that formation of the SCAP·SREBP complex is essential for the proteolytic cleavage of SREBPs by S1P (3Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). 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 SCAP·SREBP complex (3Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). 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 NH2-terminal fragment is released from the membrane by site 2 protease (S2P), which cuts near the junction between the hydrophilic NH2-terminal fragment and the first transmembrane helix (12Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The cleavage site is three residues within the transmembrane helix. After cleavage by S2P, the NH2-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 (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar,14Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (274) Google Scholar). 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 (13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The S2P-deficient cell line was derived by mutagenesis followed by selection for cholesterol auxotrophy (16Hasan M.T. Chang C.C.Y. Chang T.Y. Somatic Cell Mol. Genet. 1994; 20: 183-194Crossref PubMed Scopus (52) Google Scholar). The selection scheme is based on the method of Chang and Chang (17Chang T.-Y. Chang C.C.Y. Biochemistry. 1982; 21: 5316-5323Crossref PubMed Scopus (26) Google Scholar), 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, wild-type 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 (13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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 (13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) and used transfection techniques to produce a line of CHO cells, designated CHO/pS2P, with multiple expressed copies of the S2P cDNA (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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 γ-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 γ-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 (11Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). 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. Lipoprotein-deficient serum (d > 1.215 g/ml), human LDL (d = 1.019–1.063 g/ml), sodium mevalonate, sodium oleate, and sodium compactin were prepared as described (18Brown M.S. Faust J.R. Goldstein J.L. Kaneko I. Endo A. J. Biol. Chem. 1978; 253: 1121-1128Abstract Full Text PDF PubMed Google Scholar, 19Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). We obtained amphotericin B from Sigma-Aldrich; polyethylene glycol (PEG) 1500 from Roche Molecular Biochemicals (catalogue no. 783641); hydroxypropyl β-cyclodextrin from Cyclodextrin Technologies Development, Inc. (Gainesville, FL); sterols from Steraloids, Inc.; and [1-14C]oleic acid (51 mCi/mmol) and [2-14C]pyruvate, sodium salt (16 mCi/mM) from NEN Life Science Products. LDL was radiolabeled with125I as described (19Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). LDL labeled with 3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate, a fluorescent analogue of cholesteryl ester) was prepared as described (20Krieger M. Smith L.C. Anderson R.G.W. Goldstein J.L. Kao Y.J. Pownall H.J. Gotto A.M.J. Brown M.S. J. Supramol. Struct. 1979; l0: 467-478Crossref Scopus (88) Google Scholar). The following expression plasmids driven by the thymidine kinase (pTK) promoter were previously described: pTK-HSV-BP-1, encoding HSV-tagged human SREBP-1; pTK-HSV-BP-2, encoding HSV-tagged human SREBP-2 (21Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar); pTK-SCAP, encoding wild-type hamster SCAP; and pCMV-HSV-S2P, encoding HSV-tagged human S2P (13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The cell lines and culture medium used are described in Table I. Cells were maintained in monolayer culture at 37 °C in a 9% CO2 incubator. CHO-7 cells are a subline of CHO-K1 cells selected for growth in lipoprotein-deficient serum (22Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar). 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 (16Hasan M.T. Chang C.C.Y. Chang T.Y. Somatic Cell Mol. Genet. 1994; 20: 183-194Crossref PubMed Scopus (52) Google Scholar), due to a deficiency of S2P (13Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.-Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). SRD-12B cells are previously described mutant CHO cells auxotrophic for cholesterol, mevalonate, and unsaturated fatty acid, due to a deficiency of S1P (11Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). They were isolated by an amphotericin B resistance protocol (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) 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 μmcompactin, and 500 μg/ml G418.Table ICell lines and culture medium usedCell lineDescriptionSourceCHO-K1Parental cells for all lines used in this study.ATCC No. CRL-9618CHO-7Subline of CHO-K1 cells selected for growth in lipoprotein-deficient serum.Metherall et al. (22Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar)CHO/pS2PCHO-7 cells transfected with plasmid encoding HSV-tagged human S2P.Rawsonet al. (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar)M19CHO-K1 cells deficient in S2P. Auxotrophic for cholesterol, mevalonate, and unsaturated fatty acids.Hasan et al. (16Hasan M.T. Chang C.C.Y. Chang T.Y. Somatic Cell Mol. Genet. 1994; 20: 183-194Crossref PubMed Scopus (52) Google Scholar)SRD-12BMutant CHO/pS2P cells deficient in S1P. Auxotrophic for cholesterol, mevalonate, and unsaturated fatty acids.Rawson et al. (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar); Sakaiet al. (11Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar)SRD-13AMutant CHO/pS2P cells deficient in SCAP. Auxotrophic for cholesterol, mevalonate, and unsaturated fatty acids.This studyCulture mediumSupplemental componentsMedium A1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate.Medium BMedium A, 5% (v/v) fetal calf serum, 5 μg/ml cholesterol, 1 mmsodium mevalonate, 20 μm sodium oleateMedium C (inducing medium)aMedium C and medium D contain ethanol at a final concentration of 0.2% (v/v).Medium A, 5% (v/v) fetal calf lipoprotein-deficient serum, 50 μm sodium compactin, 50 μm sodium mevalonateMedium D (suppressing medium)aMedium C and medium D contain ethanol at a final concentration of 0.2% (v/v).Medium C, 1 μg/ml 15-hydroxycholesterol, 10 μg/ml cholesterola Medium C and medium D contain ethanol at a final concentration of 0.2% (v/v). Open table in a new tab SRD-13A cells were isolated in the same experiment that yielded the SRD-12B cells (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, CHO/pS2P cells were subjected to γ-irradiation mutagenesis. These cells were then grown for several days, incubated with fluorescent r-(PMCA oleate)LDL, and subjected to fluorescence-activated cell sorting. The sorted population of cells showing a reduced uptake of fluorescent LDL was grown to confluence, remutagenized with γ-irradiation, replated, and subjected to multiple rounds of selection with amphotericin B as described previously (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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 (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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 μg of DNA/dish using a ratio of 12 μl of Fugene to 4 μg 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-acetyl-leucinyl-leucinyl-norleucinal (ALLN) at a final concentration of 25 μg/ml. After incubation for 1 h, the cells were harvested and fractionated into nuclear extract and 105 × g membrane pellet fractions as described previously (23Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). This assay was performed as described previously (15Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, on day 0 the cells to be tested were mixed and plated at 3.5 × 105 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 μg 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. Immunoblot analysis of endogenous hamster SREBP-1 and -2 was carried out as described previously (21Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) with mouse monoclonal antibodies against the NH2-terminal domains of SREBP-1 (IgG-2A4) (24Sato R. Yang J. Wang X. Evans M.J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1994; 269: 17267-17273Abstract Full Text PDF PubMed Google Scholar) and SREBP-2 (IgG-7D4) (25Yang J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 12152-12161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), respectively. SCAP was detected with rabbit polyclonal antibody R139 (2Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). S1P was detected with rabbit polyclonal antibody U1683 (26Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Grp78 and Grp94 were detected with anti-Grp78, a monoclonal antibody raised against the KSKDEL epitope of rat Grp78 (StressGen). Epitope-tagged proteins were detected with IgG-HSV-TagTM, a monoclonal antibody directed against the glycoprotein D epitope of Herpes Simplex virus (Novagen, Inc). The antibodies were visualized with peroxidase-conjugated, affinity-purified donkey anti-mouse or anti-rabbit IgG (Jackson Immunoresearch Laboratories) using the Enhanced Chemiluminescence Western blotting detection kit (Amersham Pharmacia Biotech). 8% SDS gels were calibrated with prestained molecular weight markers (Bio-Rad). Blots were exposed to Kodak X-OmatTM Blue XB-1 film at room temperature. The incorporation of [14C]pyruvate into cellular sterols and fatty acids (18Brown M.S. Faust J.R. Goldstein J.L. Kaneko I. Endo A. J. Biol. Chem. 1978; 253: 1121-1128Abstract Full Text PDF PubMed Google Scholar), the incorporation of [14C]oleate into cellular cholesteryl esters and triglycerides (19Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar), and the proteolytic degradation of 125I-LDL (19Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar) were measured in cell monolayers as described in Refs. 18Brown M.S. Faust J.R. Goldstein J.L. Kaneko I. Endo A. J. Biol. Chem. 1978; 253: 1121-1128Abstract Full Text PDF PubMed Google Scholar and 19Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar. The protein content of cell extracts was determined by the method of Lowry et al.(27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from parental CHO/pS2P cells and the mutant SRD-13A cells using the RNA STAT-60 reagent (Tel-Test "B") following the manufacturer's protocol. Reverse transcriptase PCR was performed using 5 μg of total RNA as template with the SuperScript Preamplification System (Life Technologies, Inc.). First-strand cDNA synthesis was primed with the following hamster SCAP-specific oligonucleotide at 0.1 μm: 5′-TATTACAGTCAGGAGACAACAGT-3′. PCRs were performed using the Advantage cDNA PCR kit (CLONTECH) with 1 μl of the first-strand product per 25-μl PCR. The following primer pairs were employed to amplify five overlapping fragments of the hamster SCAP transcript: pair 1, 5′-TTAGCTGAGGATGACCCTGACTGAAAG-3′ and 5′-ACCCAATAACCACCACCAGGTATGG-3′; pair 2, 5′-TGTTTGGTGTGCCTGGGAAGTACAG-3′ and 5′-TTTAGGAGCGTCAGGTGGGAAGATGG-3′; pair 3, 5′-AACTTGCGGCTCCCCAAAAGAC-3′ and 5′-TGGTGTCGATTAAGCAGGTGAGGTC-3′; pair 4, 5′-ACTCAGGAGAACTGGGAAAGGCTGTC-3′ and 5′-TCTCAGGGCTGTGATGGGTTTTTG-3′; pair 5, 5′-TATCTGGAGCTTGGAGCTGCAAGG-3′ and 5′-TTGTATGGTCAACCTGCCCTCAGTC-3′. PCR products were cloned using the TA Cloning Kit (Invitrogen) according to the manufacturer's protocol, and multiple clones from each PCR reaction were sequenced in their entirety. Sequences were assembled using Seqman assembly software package (DNA Star, Inc., Madison, WI). PCR on high molecular weight genomic DNA from parental CHO/pS2P cells (0.8 μg) and SRD-13A cells (0.5 μg) was performed in 25-μl reactions usin

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