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• W2054264938 abstract "Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP·SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP·SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction. Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP·SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP·SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction. Sterol regulatory element-binding proteins (SREBPs) 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; bp, base pair(s); CHO, Chinese hamster ovary; CMV, cytomegalovirus; ER, endoplasmic reticulum; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HSV, herpes simplex virus; kb, kilobase(s); LDL, low density lipoprotein; PCR, polymerase chain reaction; PSS, prolactin signal sequence; SCAP, SREBP cleavage-activating protein; SRE-1, sterol regulatory element-1; TK, thymidine kinase. are membrane-bound transcription factors that control the synthesis and uptake of cholesterol and fatty acids in animal cells (reviewed in Ref. 1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). SREBPs comprise a family of three proteins, designated SREBP-1a, -1c, and -2, each of ∼1150 amino acids in length, that are bound intrinsically to membranes of the nuclear envelope and endoplasmic reticulum (ER). In sterol-depleted cells, a two-step proteolytic sequence releases the NH2-terminal domains of the SREBPs, which travel to the nucleus where they activate transcription of genes encoding the low density lipoprotein (LDL) receptor; multiple enzymes of the cholesterol biosynthetic pathway, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase; and enzymes of fatty acid biosynthesis, including acetyl-CoA carboxylase and fatty acid synthase. When sterols accumulate in cells, the proteolytic processing of SREBPs is inhibited, the NH2-terminal domains remain bound to membranes, and transcription of the target genes declines. Recent experiments have begun to elucidate the details of the two-step proteolytic process and the mechanism for its regulation by sterols (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 2Sakai 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, 3Duncan 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, 4Sakai 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, 5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A key aspect is the three-domain structure of the SREBP precursors (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). The NH2-terminal domains of the SREBPs are ∼480 amino acids in length and contain a classic basic helix-loop-helix-leucine zipper motif and an acidic transcription activation domain, similar to the ones that are found in numerous transcription factors. The NH2-terminal domain is followed by a membrane anchor domain consisting of two hydrophobic sequences, each of which spans the ER membrane and is separated from the other by a short hydrophilic loop of ∼30 residues. The membrane anchor domain is followed by a long COOH-terminal extension of ∼590 amino acids, which is designated as the regulatory domain. The SREBP precursors are oriented so that the NH2-terminal and COOH-terminal domains face the cytoplasm, and only the short hydrophilic loop projects into the ER lumen (5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In sterol-deprived cells, the proteolytic process is initiated by an enzyme that cleaves the SREBP precursors at Site-1, which is located in the middle of the hydrophilic lumenal loop (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 2Sakai 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, 3Duncan 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). This cleavage separates the NH2-terminal and COOH-terminal domains, but each remains membrane-bound, owing to its transmembrane sequence. At this point a second protease cleaves the NH2-terminal fragment at Site-2 within its membrane-spanning region, liberating the NH2-terminal domain so that it can enter the nucleus (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar,2Sakai 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). The Site-1 cleavage enzyme is directly regulated by sterols; it acts only in sterol-depleted cells, and it is inhibited by sterols. The Site-2 enzyme is not controlled directly by sterols, but it can act only after cleavage at Site-1, and its action is therefore restricted effectively to sterol-depleted cells (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 2Sakai 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). The Site-1 enzyme recognizes the sequence RXXL, which is conserved in all known mammalian and Drosophila SREBPs. The enzyme cleaves after the leucine of this sequence (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 3Duncan 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). In human SREBP-2, cleavage is abolished when the arginine of the RSVL sequence is changed to alanine. Site-1 cleavage also requires the COOH-terminal domain of SREBPs. When this domain is deleted, the Site-1 enzyme will no longer cleave SREBP-2 even though the RSVL sequence is still present (4Sakai 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). For this reason, we refer to the COOH-terminal domain of the SREBPs as the regulatory domain. The activity of the Site-1 cleavage enzyme is proposed to be controlled by a membrane-bound protein called SREBP cleavage-activating protein (SCAP) (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). SCAP consists of two domains: 1) an NH2-terminal membrane anchor of ∼730 amino acids composed of eight putative membrane-spanning segments; and 2) a hydrophilic COOH-terminal segment of ∼546 amino acids that contains at least four “WD repeats.” These repeats, each about 40 residues in length, are found in many proteins that engage in protein-protein interactions (7Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). Like the SREBPs, SCAP is bound to membranes of the ER and nuclear envelope (4Sakai 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). Co-immunoprecipitation assays show that the WD repeat domain of SCAP is bound to the COOH-terminal regulatory domains of the SREBPs (4Sakai 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). Genetic evidence indicates that SCAP is responsible for the sterol regulation of Site-1 cleavage. Thus, a dominantly acting point mutation in SCAP, D443N, was identified as the cause of sterol resistance in several independently derived lines of mutant Chinese hamster ovary (CHO) cells in which Site-1 cleavage was no longer repressed by sterols (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 8Nohturfft 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). Transfection of a cDNA encoding the D443N mutant version of SCAP into wild-type CHO cells reproduced the sterol-resistant phenotype (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). It was hypothesized that SCAP is a required cofactor in the Site-1 cleavage reaction and that sterols abolish the activity of SCAP, thereby halting Site-1 cleavage (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). The D443N mutation renders SCAP resistant to the inhibitory effects of sterols, and hence the cleavage at Site-1 can no longer be down-regulated. The notion that SCAP is part of the sterol-sensing mechanism is supported by the finding that the NH2-terminal membrane attachment domain of SCAP bears significant sequence resemblance to the membrane attachment domain of HMG-CoA reductase (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). The latter domain serves as a sterol sensor, allowing HMG-CoA reductase to be degraded rapidly when the sterol content of the ER rises (9Gil G. Faust J.R. Chin D.J. Goldstein J.L. Brown M.S. Cell. 1985; 41: 249-258Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 10Kumagai H. Chun K.T. Simoni R.D. J. Biol. Chem. 1995; 270: 19107-19113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). A sequence resembling the putative sterol sensor is also present in the protein encoded by the gene defective in Nieman-Pick type C1 disease (11Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie J. Dwyer N.K. Neufeld E.B. Chang T.-Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar). A mutation in this gene prevents cholesterol from being transported normally from the lysosome to the ER. Moreover, the putative sterol sensor motif is present in the transmembrane domain of the morphen receptor Patched (11Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie J. Dwyer N.K. Neufeld E.B. Chang T.-Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar), whose ligand is a signaling protein called Hedgehog that contains cholesterol covalently attached to its COOH terminus (12Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-259Crossref PubMed Scopus (1106) Google Scholar). Thus, four proteins that are postulated to interact with sterols (SCAP, HMG-CoA reductase, Niemann-Pick type C1 protein, and Patched) all bear a similar membranous domain. The D443N mutation, which renders SCAP insensitive to sterols, occurs within this domain (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 8Nohturfft 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 current studies were designed to test the hypothesis that SCAP is necessary for the cleavage of SREBPs at Site-1. For this purpose, we transfected cells with cDNAs encoding truncated proteins bearing the COOH-terminal regulatory domain of one of the SREBPs, namely SREBP-2. We predicted that the truncated proteins would form abortive complexes with endogenous SCAP, thereby preventing it from interacting with full-length SREBP-2. The data show that the truncated forms of SREBP-2 indeed prevent the cleavage of wild-type SREBP-2 and that this inhibition can be overcome by overexpression of SCAP. We also show that Site-1 processing can be inhibited by overexpression of cDNAs encoding truncated proteins containing the WD repeat domain of SCAP, which appears to act by forming abortive complexes with SREBPs, thereby preventing their interaction with full-length SCAP. All of these data support a model in which the interaction of the WD domain of SCAP and the COOH-terminal domain of SREBPs is required in order for SREBPs to be cleaved at Site-1. We obtained monoclonal antibody HSV-Tag™ (IgG1) from Novagen, monoclonal anti-FLAG M2 (IgG2b) from Eastman Kodak Co., and a polyclonal affinity-purified donkey anti-mouse IgG from Jackson Immunoresearch Laboratories. IgG-1C6, a mouse monoclonal antibody directed against the COOH terminus of human SREBP-2 (amino acids 833–1141; Ref. 5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar); IgG-9D5, a mouse monoclonal antibody against hamster SCAP (amino acids 540–707; Ref. 4Sakai 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); and IgG-R139, a rabbit polyclonal antibody against hamster SCAP (amino acids 54–277 and 540–707; Ref. 4Sakai 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), were prepared as described in the indicated reference. Luciferase and β-galactosidase assay kits were obtained from Promega and Stratagene, respectively. Other reagents were obtained from sources as described previously (3Duncan 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, 4Sakai 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, 5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 13Hua 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). All expression vectors were driven by the cytomegalovirus (CMV) promoter-enhancer contained in the pcDNA3 vector (Invitrogen). The structures of all plasmid constructs described below were confirmed by sequencing all ligation joints. The expression vector pCMV-PSS/BP2-(504–1141) encodes a fusion protein consisting of a modified region of the bovine prolactin signal sequence (MDSKGSSQKGSRLLLLLVVSNLLLCQGVVN), followed by a FLAG epitope tag (DYKDDDD), two novel amino acids (VD) encoded by the restriction site for SalI, and amino acids 504–1141 of human SREBP-2. The prolactin signal sequence was modified by the substitution of an asparagine residue at −1 in place of serine, as denoted by the underline. pCMV-PSS/BP2-(504–1141) was constructed as follows. First, a 60-bp fragment encoding the prolactin signal sequence (PSS) followed by the FLAG epitope tag was isolated by KpnI and SalI digestion of a plasmid construct in pBluescript encoding a PSS/FLAG/human thrombin receptor fusion protein (Ref. 14Ishii K. Hein L. Kobilka B. Coughlin S.R. J. Biol. Chem. 1993; 268: 9780-9786Abstract Full Text PDF PubMed Google Scholar, provided by Dr. Shaun R. Coughlin, University of California, San Francisco). The sequence corresponding to amino acids 504–533 of human SREBP-2 (15Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (501) Google Scholar) was amplified by PCR of pTK-HSV-BP2 (13Hua 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 a pair of primers, 5′-GGTACCGTCGACGGAGGGGCCCACGACTCTGACCA-3′ encoding amino acids 504–510 of SREBP-2 preceded by a SalI site and 5′-CTCGAGGAATTCGTCAAACCAGCCCCCAGAACCTG-3′ encoding amino acids 526–533 preceded by an EcoRI site. The PCR fragment was digested with SalI and EcoRI, and the 90-bp fragment was isolated. The above two fragments were cloned into the KpnI-EcoRI sites of pcDNA3 (Invitrogen) to yield intermediate construct I. Second, intermediate construct I was mutagenized by oligonucleotide site-directed mutagenesis to substitute serine for asparagine at amino acid residue −1 of the prolactin signal sequence (denoted by underline, see above) by the method of Kunkelet al. (16Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar) to yield intermediate construct II. Third, intermediate construct II was digested with ApaI to isolate a 6.0-kb backbone fragment, pTK-HSV-BP2 was digested withApaI to isolate a 2.5-kb fragment encoding amino acids 506–1141 of human SREBP-2, and the two fragments were ligated to generate pCMV- PSS/BP2-(504–1141). The expression vector pCMV-PSS/BP2-(504–1141)NSS/NGT encodes a FLAG epitope-tagged prolactin signal sequence/SREBP-2 fusion protein in which serine 515 in the loop region of SREBP-2 was replaced, by site-directed mutagenesis (13Hua 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, 16Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar), with a novel amino acid sequence containing two glycosylation sites, NSSGSSGNGT. Other derivative vectors of pCMV-PSS/BP2 were also constructed by site-directed mutagenesis (13Hua 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, 16Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). The expression vector pCMV-P450-TM/BP2-(555–1141) encodes a fusion protein consisting of amino acids 1–29 of cytochrome P450 2C1 (17Ahn K. Szczesna-Skorupa E. Kemper B. J. Biol. Chem. 1993; 268: 18726-18733Abstract Full Text PDF PubMed Google Scholar), two novel amino acids (ID) encoded by a BspDI restriction site, and amino acids 555–1141 of human SREBP-2. pCMV-P450-TM/BP2-(555–1141) was constructed as follows. A pair of complementary oligonucleotides (top strand) 5′-CTAGCCATGGATCCTGTGGTGGTGCTGGGGCTCTGTCTCTCCTGTTTGCTTCTCCTTTCACTCTGGAAACAGAGCTATGGGGGAGGGAAGCTTAT-3′, which contained 5′ NheI and 3′ BspDI cohesive ends, were annealed as described previously (5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This oligonucleotide encodes the sequence for amino acids 1–29 of cytochrome P450 2C1 (MDPVVVLGLCLSCLLLLSLWKQSYGGGKL) and two novel amino acids (ID). pCMV-HSV-BP2-(555–1141) (4Sakai 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) was digested withNheI and BspDI, and the 90-bp fragment encoding two tandem copies of the HSV epitope was replaced with the amino acids 1–29 of the P450 2C1 sequence. pCMV-P450-TM/SCAP-(731–1276) encodes a fusion protein consisting of amino acids 1–29 of cytochrome P450 2C1, two novel amino acids (RT) encoded by a BsiWI restriction site, and amino acids 731–1276 of hamster SCAP (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). To generate pCMV-P450-TM/SCAP-(731–1276), a PCR fragment was amplified from pC1-GAL (Ref. 17Ahn K. Szczesna-Skorupa E. Kemper B. J. Biol. Chem. 1993; 268: 18726-18733Abstract Full Text PDF PubMed Google Scholar; kindly provided by Byron Kemper, University of Illinois at Urbana-Champaign), a CMV-driven expression vector encoding a hybrid protein in which amino acids 1–29 of P450 2C1 are fused to the NH2 terminus of Escherichia coliβ-galactosidase. (In this paper, pC1-GAL is referred to as pCMV-P450-TM/Gal.) PCR amplification was carried out with the following pair of primers: 5′-CCCACTTGGCAGTACATCAAG-3′ (annealing to a sequence in the CMV promoter) and 5′-ATCGTACGAAGCTTCCCTCCCCCATAGCTCTGTTTCC-3′ (containing codons 20–29 of cytochrome P450 2C1 preceded by aBsiWI restriction site). The PCR fragment was cut withNdeI and BsiWI and used to replace the NdeI-BsiWI fragment of pcDNA3-CB5R-SCAP-(731–1276), a pcDNA3-based intermediate plasmid containing the coding sequence of amino acids 1–7 of human cytochrome B5 reductase (18Tomatsu S. Kobayashi Y. Fukumaki Y. Yubisui T. Orii T. Sakaki Y. Gene (Amst.). 1989; 80: 353-361Crossref PubMed Scopus (84) Google Scholar), followed by a BsiWI restriction site and codons 731–1276 of SCAP. pTK-HSV-BP2, a herpes simplex virus thymidine kinase-driven expression vector encoding human SREBP-2 (13Hua 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), and pCMV-SCAP, a CMV-driven expression vector encoding hamster SCAP (4Sakai 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), were prepared as described in the indicated reference. pCMVβ-gal, a plasmid encoding a CMV promoter-driven β-galactosidase reference gene, was obtained from Stratagene. pSRE-Luc, a luciferase reporter plasmid driven by a promoter consisting of three tandem copies of repeats 2+3 of the LDL receptor promoter (SRE-1) plus the adenovirus E1b TATA box was constructed as described previously (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Monolayers of human embryonic kidney 293 cells were set up on day 0 (4 × 105 cells/60-mm dish) and cultured in 8–9% CO2 at 37 °C in medium A (Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate) supplemented with 10% (v/v) fetal calf serum. On day 2, the cells were transfected with the indicated plasmids using the MBS kit (Stratagene) method as described previously (5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Three h after transfection, the cells were switched to medium B (medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μmcompactin, and 50 μm sodium mevalonate) in the absence or presence of sterols (1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol added in a final concentration of 0.2% ethanol) as indicated in the legends. After incubation for 20 h, the cells received N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 μg/ml, and cells were harvested 3 h later. The pooled cell suspension from two dishes was allowed to swell in hypotonic buffer A (10 mm Hepes-KOH at pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 1 mm sodium EDTA, 1 mm sodium EGTA, 1 mm dithiothreitol, and a mixture of protease inhibitors; Ref. 5Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) for 30 min at 0 °C, passed through a 22.5-gauge needle 30 times, and centrifuged at 1000 × g at 4 °C for 5 min. The 1000 × g pellet was resuspended in 0.1 ml of buffer B (10 mm Hepes-KOH at pH 7.4, 0.42 mNaCl, 2.5% (v/v) glycerol, 1.5 mm MgCl2, 1 mm sodium EDTA, 1 mm sodium EGTA, 1 mm dithiothreitol, and a mixture of protease inhibitors). The suspension was rotated at 4 °C for 1 h and centrifuged at top speed in a microcentrifuge for 15 min at 4 °C. The supernatant is designated nuclear extract. The supernatant from the original 1000 × g spin was centrifuged at 105 ×g for 30 min at 4 °C in a Beckman TLA 120.2 rotor, and the pellet was dissolved in 0.1 ml of SDS lysis buffer (10 mm Tris-HCl at pH 6.8, 100 mm NaCl, 1% (v/v) SDS, 1 mm sodium EDTA, and 1 mm sodium EGTA) and designated membrane fraction. M19 cells are a mutant line of CHO-K1 cells (19Hasan M.T. Chang T.Y. Somat. Cell Mol. Genet. 1994; 20: 481-491Crossref PubMed Scopus (13) Google Scholar) that have a deletion that eliminates the gene encoding the SREBP Site-2 protease (2Sakai 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, 20Rawson 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 cells were grown in monolayer culture as described previously (19Hasan M.T. Chang T.Y. Somat. Cell Mol. Genet. 1994; 20: 481-491Crossref PubMed Scopus (13) Google Scholar, 20Rawson 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 method is described in Ref.3Duncan 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. Monolayers of 293 cells were set up on day 0 (4 × 105 cells/60-mm dish) and cultured as described above. On day 2, the cells were transfected with 3 μg/dish pCMV-PSS/BP2-(504–1141)NSS/NGT and pCMV-PSS/BP2-(504–587)NSS/NGT, respectively. Three h after transfection, the cells were switched to medium B in the presence of sterols. After incubation for 20 h, the cells were harvested, and the pooled cells from four dishes were fractionat" @default.
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