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• W2007334178 abstract "A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage. A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage. Proteolytic processing of sterol regulatory element-binding proteins (SREBPs) 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticulum; LDL, low density lipoprotein; SCAP, SREBP cleavage-activating protein; PAGE, polyacrylamide gel electrophoresis; VAI, virus-associated I. controls the metabolism of cholesterol and fatty acids in animal cells (1Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 2Hua 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, 3Sakai 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). SREBPs are transcription factors that are bound to membranes of the ER and nuclear envelope. Each SREBP is composed of three segments: 1) an NH2-terminal segment of ∼485 amino acids that is a transcription factor of the basic helix-loop-helix-leucine zipper family, 2) a membrane attachment segment of ∼75 amino acids composed of two membrane-spanning sequences separated by a short hydrophilic loop of 31 amino acids, and 3) a COOH-terminal segment of ∼585 amino acids that plays a regulatory role. The proteins are oriented so that the NH2- and COOH-terminal segments project into the cytoplasm, and only the short hydrophilic loop projects into the lumen of the ER or nuclear envelope (4Hua 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). Before it can activate transcription, the NH2-terminal segment is released from the membrane in a complex two-step proteolytic sequence (2Hua 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, 3Sakai 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). First, a protease cleaves the protein at Site-1, which is near an arginine in the lumenal loop, thereby breaking the attachment between the two transmembrane sequences. This allows a second protease to cleave the protein at Site-2, which is near the middle of the first transmembrane sequence (2Hua 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, 3Sakai 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 NH2-terminal fragment leaves the membrane with a portion of the first transmembrane sequence still attached. It enters the nucleus, where it activates transcription of genes encoding the LDL receptor (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar,6Hua 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), several enzymes of cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A synthase (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 6Hua 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), 3-hydroxy-3-methylglutaryl coenzyme A reductase (7Vallett S.M. Sanchez H.B. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1996; 271: 12247-12253Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), farnesyl diphosphate synthase (8Ericsson J. Jackson S.M. Edwards P.A. J. Biol. Chem. 1996; 271: 24359-24364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and squalene synthase (9Guan G. Jiang G. Koch R.L. Shechter I. J. Biol. Chem. 1995; 270: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)), and enzymes of fatty acid biosynthesis (10Kim J.B. Spiegelman B.M. Genes & Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (846) Google Scholar, 11Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and desaturation (12Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar). The net result is to increase the cell's supply of cholesterol and fatty acids. The Site-1 protease is the target of feedback regulation by cholesterol and other sterols (3Sakai 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). When these sterols accumulate within cells, the rate of proteolysis at Site-1 declines markedly. Cleavage at Site-2 also declines because this cleavage requires prior cleavage at Site-1 (3Sakai 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). As a result, the amounts of nuclear SREBPs decline, and transcription of the target genes falls. The net effect is to prevent overaccumulation of cholesterol and fatty acids when intracellular sterol levels are already high. Three isoforms of SREBP are known (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 13Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar, 14Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (641) Google Scholar). SREBP-1a and 1c are derived from a single gene through use of alternate promoters that encode alternate first exons (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 13Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar, 14Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (641) Google Scholar). SREBP-1a is much more active than SREBP-1c in stimulating transcription of all known target genes (15Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (684) Google Scholar). The third protein, SREBP-2, is the product of a separate gene (6Hua 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,13Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar), and it is also more active than SREBP-1c (15Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (684) Google Scholar). In view of the regulatory role of the Site-1 protease, further knowledge of its structure and mode of regulation is desirable. A first step would be the identification of the precise site at which the Site-1 protease cuts SREBPs. In previous studies we have shown that this cleavage is abolished when arginine 519 in the lumenal loop of SREBP-2 (or the corresponding arginine of SREBP-1a) is changed to alanine by in vitro mutagenesis (2Hua 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). The size of the cleavage product, as determined by SDS-PAGE, is consistent with cleavage at or near this arginine (3Sakai 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). In the current studies we have used a combination of in vitro mutagenesis, epitope tagging, immunoprecipitation, and radiochemical sequencing to determine the precise location of Site-1 in SREBP-2. We found, surprisingly, that cleavage does not occur at arginine 519, but rather it occurs 3 residues further toward the COOH terminus, namely at leucine 522. Arginine 519 seems to be the NH2-terminal residue in a tetrapeptide sequence, RXXL, that serves as a recognition signal for the Site-1 protease. We obtained HSV-TagTM and T7-TagTM monoclonal antibodies from Novagen; v-H-Ras(Ab-1)-agarose linked monoclonal antibody was obtained from Oncogene. Protein G-SepharoseR 4 Fast Flow beads were obtained from Pharmacia Biotech Inc.,l-[35S]methionine (>1000 Ci/mmol) was obtained from DuPont NEN, and glycosidases were obtained from New England Biolabs. pTK-HSV-BP2-Ras-T7 encodes an epitope-tagged SREBP-2/Ras fusion protein (1391 amino acids) consisting of an initiator methionine, two tandem copies of the HSV epitope (QPELAPEDPED), six novel amino acids (IDGTVP) encoded by a sequence that consists of restriction sites for BspDI andKpnI, human SREBP-2 (amino acids 14–1141), two novel amino acids (HM) encoded by the sequence for restriction siteNdeI, human H-Ras (amino acids 2–189), and three tandem copies of the T7 epitope (HMASMTGGQQMGAAAMASMTGGQQMGGGPMASMTGGQQMGLINM). pTK-HSV-BP2-Ras-T7 was constructed from a previously described plasmid, pTK-HSV-BP2-T7 (16Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), by insertion of a cDNA segment encoding human H-Ras between the sequences for SREBP-2 and the first copy of the T7 epitope. The nucleotide sequence encoding amino acids 2–189 of human H-Ras was obtained by polymerase chain reaction on pRcCMV-H-Ras (17James G.L. Brown M.S. Cobb M.H. Goldstein J.L. J. Biol. Chem. 1994; 269: 27705-27714Abstract Full Text PDF PubMed Google Scholar) with a pair of primers containing an NdeI site at each 5′ end using Pfu DNA polymerase. The amplified product was digested with NdeI and cloned into the uniqueNdeI site between human SREBP-2 and the three tandem copies of the T7 epitope. Two independent clones were used in each of the transfections. pTK-HSV-BP2/LDLRTM encodes an epitope-tagged SREBP-2 fusion protein in which a 26-amino acid region that includes the second transmembrane domain of human SREBP-2 (amino acids 535–560) is replaced with the transmembrane domain of the human LDL receptor (amino acids 708–729) (18Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (979) Google Scholar). To construct pTK-HSV-BP2/LDLRTM, we used oligonucleotide site-directed mutagenesis to produce an intermediate plasmid in which amino acids 535–560 of human SREBP-2 were replaced by two novel amino acids (TG) corresponding to an AgeI restriction site. A pair of complementary oligonucleotides (top strand, 5′-CCGGTGCTCTGTCCATTGTCCTCCCCATCGTGCTCCTCGTCTTCCTTTGCCTGGGGGTCTTCCTTCTATGGT-3′; bottom strand, 5′-CCGGACCATACAAGGAAGACCCCCAGGCAAAGGAAGACGAGGAGCACGATGGGGAGGACAATGGACAGAGCA-3′) were annealed at 94–83 °C for 5 min and then at 83–23 °C for 60 min. These oligonucleotides correspond to amino acids 708–729 of the human LDL receptor flanked by the single-stranded sequence 5′-CCGG-3′. The annealed oligonucleotides were cloned into the uniqueAgeI restriction site of the intermediate plasmid (described above). The resulting pTK-HSV-BP2/LDLRTM encodes an 1157-amino acid chimeric protein consisting of an initiator methionine, two tandem copies of the HSV epitope, six novel amino acids (IDGTVP), human SREBP-2 (amino acids 14–534), two novel amino acids (TG), human LDL receptor (amino acids 708–729), two novel amino acids (SG), and human SREBP-2 (amino acids 561–1141). Plasmid pTK-HSV-BP2(R519A)/LDLRTM is identical to pTK-HSV-BP2/LDLRTM except for the R519A point mutation in the lumenal loop of the SREBP-2 sequence. This plasmid was constructed by site-directed mutagenesis. pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/NGT encode epitope-tagged SREBP-2 fusion proteins in which serine 515 in the loop region of SREBP-2 is replaced by a novel amino acid sequence containing one or two N-linked glycosylation sites, either SNGT or NSSGSSGNGT, respectively. Both plasmids were constructed by site-directed mutagenesis. Oligonucleotide site-directed mutagenesis was carried out with single-stranded, uracil-containing DNA (19Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar) using the Muta-gene Phagemid In Vitro Mutagenesis Version-2 kit (Bio-Rad) (2Hua 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). The mutations were confirmed by sequencing the relevant region, and at least two independent clones of each mutant were independently transfected. 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) supplemented with 10% (v/v) fetal calf serum (4Hua 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). On day 2, cells were transfected with 4 μg of pTK empty vector (mock) or the indicated plasmid as described (2Hua 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). 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 as indicated in the legends. After incubation for 20 h, the cells receivedN-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 μg/ml (2Hua 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 the cells were harvested 3 h later (2Hua 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). The pooled cell suspension from 2 dishes was allowed to swell in hypotonic buffer (4Hua 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 7 min. The 1000 × g pellet was resuspended in 0.1 ml of buffer C (10 mm Hepes-KOH (pH 7.4), 0.42 m NaCl, 2.5% (v/v) glycerol, 1.5 mmMgCl2, 0.5 mm sodium EDTA, 0.5 mmsodium EGTA, 1 mm dithiothreitol, and a mixture of protease inhibitors (4Hua 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)). The suspension was rotated at 4 °C for 1 h and centrifuged at top speed in a microfuge 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 100.2 rotor, and the pellet was dissolved in 0.1 ml of SDS lysis buffer (1Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (854) Google Scholar) and designated membrane fraction. Glycosidase sensitivity of SREBP-2 was carried out as described (4Hua 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). 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 7 μg of pTK empty vector (mock), pTK-HSV-BP2-NGT, and pTK-HSV-BP2-NSS/NGT, respectively. Three h after transfection, the cells were switched to medium B in the presence of sterols as described in Fig. 1. After incubation for 20 h, the cells were harvested, and the pooled cell suspension from four dishes was fractionated. The 105 × g membrane pellet was washed once with buffer A (4Hua 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) and resuspended in 180 μl of buffer A containing 1% (v/v) Triton X-100 without protease inhibitors. Aliquots of the 105 × g membrane fraction (0.16 mg in 40 μl of buffer A) were boiled for 5 min in the presence (peptide N-glycosidase F and endoglycosidase H reactions) or in the absence (neuraminidase reactions) of 0.5% (w/v) SDS and 1% (v/v) β-mercaptoethanol for 5 min, after which the indicated amount of glycosidase was added and incubated at 37 °C for 2 h as described in Fig. 10.Figure 10Insertion of N-linked glycosylation sites into the lumenal loop of SREBP-2. A, sequences of the loop region of human SREBP-2, showing the sites of insertion of one (pTK-HSV-BP2-NGT) or two (pTK-HSV-BP2-NSS/NGT)N-linked glycosylation sites. B, glycosidase treatment of transfected SREBP-2. Aliquots of the 105 ×g membrane fraction from 293 cells transfected with the indicated cDNA were boiled for 5 min as described under “Experimental Procedures” and incubated for 2 h at 37 °C with one of the following glycosidases: lanes 1, 5, and6, none; lanes 2 and 7, 0.038 IU of peptide N-glycosidase F (PNGase F); lanes 3and 8, 0.25 IU of endoglycosidase H; and lanes 4and 9, 0.83 IU of neuraminidase. Aliquots of the membrane fraction (50 μg) were subjected to SDS-PAGE and immunoblot analysis with 0.5 μg/ml HSV-TagTM antibody. The filter was exposed to film for 10 s. Asterisk (∗) denotes an immunoreactive protein that is present in membranes from mock-transfected cells (lane 5). C, proteolytic processing of HSV-tagged SREBP-2 with inserted N-linked glycosylation sites in the lumenal loop. Aliquots of nuclear extracts (60 μg of protein) and membranes (80 μg) from 293 cells transfected with the indicated plasmid and incubated in the absence or presence of sterols as described in the legend to Fig. 1 were subjected to SDS-PAGE and immunoblot analysis with 0.5 μg/ml HSV-TagTMantibody. The filters for nuclear extracts and membranes were exposed to film for 5 min and 20 s, respectively. P andM denote the uncleaved precursor and cleaved NH2-terminal mature forms of SREBP-2, respectively. The other bands are present in mock-transfected cells and represent proteins that cross-react with the anti-HSV tag antibody.View Large Image Figure ViewerDownload (PPT) Samples of the nuclear extract and the 105 × g membrane fraction were mixed with 5× SDS loading buffer (20Bollag D.M. Edelstein S.J. Protein Methods. Wiley-Liss, Inc., New York1991: 100Google Scholar). Protein concentration was measured with a BCA kit (Pierce). After SDS-PAGE in 8% gels, proteins were transferred to Hybond-C extra nitrocellulose membranes (Amersham Corp.). Immunoblot analysis was carried out with a horseradish peroxidase detection kit using the SuperSignalTM CL-HRP Substrate System according to the manufacturer's instructions except that the nitrocellulose sheets were blocked in phosphate-buffered saline containing 0.05% (v/v) Tween 20, 5% (v/v) nonfat dry milk, and 5% (v/v) heat-inactivated newborn calf serum. The chimeric proteins were visualized with 0.5 μg/ml HSV-TagTM monoclonal antibody or with 10 μg/ml IgG-1C6, a mouse monoclonal antibody directed against amino acids 833–1141 of human SREBP-2 (4Hua 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). Gels were calibrated with prestained molecular weight markers. Filters were exposed at room temperature to Reflection™ NEF-496 film (DuPont NEN). Monolayers of 293 cells were set up on day 0 (7 × 105 cells/60-mm dish) and cultured as described above. On day 1, the cells were transfected with 4 μg of the wild-type or mutant version of pTK-HSV-BP2-Ras-T7, 1 μg of pCMV-SCAP(D443N) (16Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), and 2 μg of pVAI as described above. pVAI encodes the adenovirus virus-associated I RNA gene, which enhances translation of transfected cDNAs (21Akusjärvi G. Svensson C. Nygard O. Mol. Cell. Biol. 1987; 7: 549-551Crossref PubMed Scopus (54) Google Scholar). The cells were incubated for 20 h with 50 μm compactin and 50 μm sodium mevalonate, at which time the medium was changed to 1.3 ml of methionine-free Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 10% newborn calf lipoprotein-deficient serum, 50 μm compactin, and 50 μm sodium mevalonate. After incubation for 1 h at 37 °C, 25 μg/ml N-acetyl-leucinal-leucinal-norleucinal was added, and the cells were pulse-labeled with 700 μCi/ml of [35S]methionine for 6 h at 37 °C. The cells from four dishes were harvested and pooled, and the membrane fraction was prepared as described above. The pooled membrane fraction from the four dishes was resuspended in 0.1 ml of SDS lysis buffer at room temperature. All subsequent operations were carried out at 4 °C unless otherwise stated. The suspension was rotated for 30 min in 5 ml of buffer D (50 mm Tris-HCl (pH 7.5), 125 mm NaCl, 0.5% (v/v) SDS, 1.25% (v/v) Triton X-100, 1.25% (v/v) deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin A, 25 μg/mlN-acetyl-leucinal-leucinal-norleucinal, and 1 mmdithiothreitol), after which 20 μl of preimmune whole rabbit serum, 40 μg of irrelevant mouse monoclonal antibody IgG-2001 (22Tolleshaug H. Goldstein J.L. Schneider W.J. Brown M.S. Cell. 1982; 30: 715-724Abstract Full Text PDF PubMed Scopus (148) Google Scholar), and 0.2 ml of Protein G-Sepharose beads were added. After rotation for 16 h, the mixture was centrifuged at 1000 × g for 7 min. The resulting supernatant was mixed with an additional 20 μl of preimmune whole rabbit serum, 40 μg of irrelevant monoclonal antibody IgG-2001, and 0.2 ml of Protein G-Sepharose beads, rotated for 2 h, and centrifuged at 1000 × g for 3–7 min. To the supernatant were added 1 μg of T7-Tag monoclonal antibody, 25 μg of v-H-Ras(Ab-1)-agarose linked monoclonal antibody, and 20 μg of anti-COOH-terminal SREBP-2 monoclonal antibody IgG-1C6. After rotation for 2.5 h, 50 μl of Protein G-Sepharose beads were added, followed by rotation for 2.5 h and centrifugation at 1000 ×g for 3–7 min. The beads were washed once by rotation with buffer D for 16 h, followed by four washes in buffer D for 1 h each. The washed beads were resuspended in 0.1 ml of 2× SDS loading buffer (20Bollag D.M. Edelstein S.J. Protein Methods. Wiley-Liss, Inc., New York1991: 100Google Scholar) containing 10% (v/v) β-mercaptoethanol and boiled for 5 min. After centrifugation at 1000 × g at room temperature for 3 min, the supernatant was transferred to a fresh tube (tube A), and the beads were re-eluted with 40 μl of 5× SDS loading buffer containing 25% β-mercaptoethanol and boiled for 5 min. After centrifugation at 1000 × g at room temperature for 3 min, the supernatant was transferred to tube A, and the entire volume was boiled again for 5 min before SDS-PAGE. Aliquots of the immunoprecipitated samples (from 1.3 dishes of cells) were subjected to SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride membranes (Immobilon-PSQ; Millipore). After drying, the membranes were exposed to an imaging plate and scanned in a Fuji X Bas 1000 phosphorimager. The band containing the COOH-terminal product of the cleavage reaction (M r ∼83,000) was excised and subjected directly to multiple cycles of Edman degradation on an Applied Biosystems model 477A sequencer. Fractions from each cycle (148 μl) were collected and counted in a scintillation counter. To identify the exact position of Site-1, we prepared a cDNA (pTK-HSV-BP2-Ras-T7) encoding a triply tagged version of SREBP-2, which we designate SREBP-2/Ras (Fig. 1). The NH2terminus contains two copies of an epitope tag from the HSV glycoprotein that we used previously (2Hua 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). At the COOH terminus we inserted amino acids 2–189 of H-Ras followed by three copies of an 11-residue epitope derived from the gene 10 protein of bacteriophage T7 (23Tsai D.E. Kenan D.J. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8864-8868Crossref PubMed Scopus (70) Google Scholar). The combination of epitopes at the COOH terminus allowed efficient precipitation of the COOH-terminal fragment with a mixture of antibodies directed against the Ras and T7 epitopes plus a monoclonal antibody directed against the COOH-terminal domain of SREBP-2 (see below). To demonstrate that SREBP-2/Ras is cleaved at Site-1 in a physiologic fashion, we transfected 293 cells with a vector encoding this construct and another encoding a mutated version in which arginine 519 was changed to alanine (Fig. 1). We used the relatively weak thymidine kinase promoter, which produces near physiological levels of this protein (2Hua 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). Cells were incubated in inducing medium that contains the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor compactin to block cholesterol synthesis plus a low concentration of mevalonate to provide nonsterol end products. The medium was either devoid of sterols (−" @default.
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• W2007334178 title "Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2" @default.
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