Autocatalytic Processing of Site-1 Protease Removes Propeptide and Permits Cleavage of Sterol Regulatory Element-binding Proteins
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22795
ISSN1083-351X
AutoresPeter J. Espenshade, Dong Cheng, Joseph L. Goldstein, Michael S. Brown,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoSite-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH2-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH2-terminal signal sequence of S1P generates S1P-A (amino acids 23–1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138–1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187–1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805–22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P. Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH2-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH2-terminal signal sequence of S1P generates S1P-A (amino acids 23–1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138–1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187–1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805–22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P. Cholesterol metabolism in animal cells is controlled by the sterol-regulated proteolysis of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; CMV, cytomegalovirus; endo H, endoglycosidase H; ER, endoplasmic reticulum; HEK-293 cells, human embryonic kidney 293 cells; PAGE, polyacrylamide gel electrophoresis; S1P, Site-1 protease; S2P, Site-2 protease; SCAP, SREBP cleavage-activating protein; TK, thymidine kinase; kb, kilobase(s) The sterol-regulated reaction is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves SREBPs in a hydrophilic loop that projects into the lumen of the endoplasmic reticulum (ER) and other organelles (2Sakai 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 (328) Google Scholar). An understanding of the mechanism of regulation of S1P is essential if we are to understand how animals control the cholesterol content of cells and blood. SREBPs are a family of three proteins, each of ∼1150 amino acids in length. After synthesis, each SREBP is inserted into the membranes of the 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 (3029) Google Scholar). The NH2-terminal segment of ∼480 amino acids is a transcription factor of the basic helix-loop-helix-leucine zipper family that projects into the cytoplasm. The middle segment of ∼80 amino acids consists of two membrane-spanning helices separated by a luminal hydrophilic loop of ∼30 amino acids. The COOH-terminal segment of ∼590 amino acids extends into the cytoplasm, where it forms a complex with the COOH-terminal segment of a membrane-bound regulatory protein designated SREBP-cleavage activating protein (SCAP). The SREBP/SCAP complex is the true substrate for S1P; disruption of this complex in intact cells abrogates the proteolytic reaction (2Sakai 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 (328) 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). S1P initiates the processing of SREBPs by cleaving at a site in the middle of the luminal loop. This reaction has been studied most extensively for human SREBP-2. S1P cleaves this protein between the leucine and serine of the sequence RSVLS (4Duncan 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). The specificity of recognition has been studied by transfecting cDNAs encoding mutant forms of SREBP-2 into cultured cells. Cleavage of SREBP-2 absolutely requires arginine or lysine at the P4 position. Although the full range of residues at the P1 position was not studied, cleavage was markedly reduced when the leucine at P1 was replaced by alanine or by the closely related valine. The serine at the P1′position and the serine and valine at the P3 and P2 positions could be replaced by alanine with no effect on cleavage. Cleavage by S1P separates the SREBP into two fragments, each of which has a single membrane-spanning sequence. This separation allows a second protease, designated Site-2 protease (S2P), to cleave the NH2-terminal fragment at a position within its membrane-spanning sequence (5Rawson 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 (395) Google Scholar, 6Duncan E.A. Davé U.P. Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 17801-17809Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This cleavage releases the NH2-terminal segment of SREBP, allowing it to enter the nucleus, where it binds to enhancers and activates transcription of genes encoding the low density lipoprotein (LDL) receptor and multiple enzymes of cholesterol and fatty acid biosynthesis (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). When sterols accumulate in cells, the cleavage of SREBPs by S1P is abolished; SREBPs remain attached to membranes; and transcription of the target genes declines. This regulation is mediated by the sterol-sensing domain of SCAP (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar, 7Hua 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 mechanism by which SCAP stimulates the Site-1 cleavage reaction and the mechanism by which sterols block this stimulation is unknown. S1P was identified by expression cloning in SRD-12B cells, a mutant line of Chinese hamster ovary cells that is unable to synthesize cholesterol or to take up LDL. The defect in these cells was traced to a block in the Site-1 cleavage reaction (8Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and this defect was corrected when the cells were transfected with pools of cDNAs obtained from wild-type hamster cells (2Sakai 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 (328) Google Scholar). The complementing cDNA was isolated and shown to encode S1P. Subsequent analysis showed that the SRD-12B cells harbor mutations in the S1P gene that abrogate the production of any S1P mRNA. A DNA data base search revealed that the sequence of human S1P had been deduced previously as a part of a random cDNA sequencing project in Japan (9Nagase T. Miyajima N. Tanaka A. Sazuka T. Seki N. Sato S. Tabata S. Ishikawa K.-i. Kawarabayasi Y. Kotani H. Nomura N. DNA Res. 1995; 2: 37-43Crossref PubMed Scopus (114) Google Scholar). The hamster and human proteins are 97% identical (2Sakai 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 (328) Google Scholar). Hamster S1P is a large polypeptide of 1052 amino acids. The NH2-terminal 22 amino acids are hydrophobic and constitute a classic signal sequence. This is followed by a domain of ∼290 amino acids that distinguishes S1P as a subtilisin-related protease (2Sakai 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 (328) Google Scholar). By analogy with other family members, it was possible to identify the classic catalytic triad as aspartate 218, histidine 249, and serine 414 (2Sakai 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 (328) Google Scholar, 10Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (789) Google Scholar, 11Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; (in press)Google Scholar). Replacement of any one of these amino acids rendered S1P unable to complement the defect in SRD-12B cells, confirming that these residues are required for catalytic activity (2Sakai 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 (328) Google Scholar). Following the subtilisin homology domain, there is a stretch of ∼520 amino acids that has no resemblance to other proteins. This is followed by a hydrophobic sequence of 25 amino acids that appears to be a classic membrane-spanning anchor and a short COOH-terminal tail of 30 amino acids that is predicted to extend into the cytosol. This cytosolic sequence is unusual because it is extremely rich in proline and basic residues. Cell fractionation studies confirmed that S1P is membrane-bound (2Sakai 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 (328) Google Scholar). The superfamily of subtilisin-related proteases contains more than 200 members that are found in all living organisms (10Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (789) Google Scholar). The mammalian members of this family consist of furin and the prohormone convertases, which act in the Golgi apparatus or secretory vesicles to process peptides prior to secretion or movement to the cell surface (12Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (705) Google Scholar). All of these enzymes cleave after basic residues, often after dibasic sequences. They also require a basic residue at the P4 position. The classic recognition sequence is RX(R/K)R, where Xstands for any amino acid (12Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (705) Google Scholar). S1P differs from the mammalian members of the subtilisin family because it cleaves after a hydrophobic amino acid, leucine. In this respect, it resembles some of the bacterial subtilases, typified by Savinase from Bacillus lentus(13Sørensen S.B. Bech L.M. Meldal M. Breddam K. Biochemistry. 1993; 32: 8994-8999Crossref PubMed Scopus (32) Google Scholar). Seidah et al. (14Seidah N.G. Mowla S.J. Hamelin J. Mamarbachi A.M. Benjannet S. Touré B.B. Basak A. Munzer J.S. Marcinkiewicz J. Zhong M. Barale J.-C. Lazure C. Murphy R.A. Chrétien M. Marcinkiewicz M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1321-1326Crossref PubMed Scopus (249) Google Scholar) recently reported the cloning of a human subtilisin-related cDNA, called SKI-1, which is identical to S1P. These authors identified SKI-1 in a polymerase chain reaction-based screen for subtilisin-like proteins that cleave after nonbasic residues. In transfection experiments, they demonstrated that SKI-1 cleaved the precursor form of a secreted peptide, brain-derived neurotrophic factor, after the threonine of the sequence RGLTS. The physiologic significance of this cleavage event is not yet established because this cleavage site does not correspond to the major site at which native pro-brain-derived neurotrophic factor is processedin vivo (15Leibrock J. Lottspeich F. Hohn A. Hofer M. Hengerer B. Masiakowski P. Thoenen H. Barde Y.-A. Nature. 1989; 341: 149-152Crossref PubMed Scopus (1236) Google Scholar, 16Rosenfeld R.D. Zeni L. Haniu M. Talvenheimo J. Radka S.F. Bennett L. Miller J.A. Welcher A.A. Protein Expression Purif. 1995; 6: 465-471Crossref PubMed Scopus (136) Google Scholar). All of the known subtilisin family members are synthesized as inactive precursors that are activated by a cleavage reaction that releases an NH2-terminal propeptide (10Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (789) Google Scholar, 12Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (705) Google Scholar). Seidah et al.(14Seidah N.G. Mowla S.J. Hamelin J. Mamarbachi A.M. Benjannet S. Touré B.B. Basak A. Munzer J.S. Marcinkiewicz J. Zhong M. Barale J.-C. Lazure C. Murphy R.A. Chrétien M. Marcinkiewicz M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1321-1326Crossref PubMed Scopus (249) Google Scholar) provided evidence that SKI-1/S1P undergoes a processing reaction based on an increase in mobility on SDS-PAGE, but the site of cleavage was not identified. In the current studies, we show that S1P is indeed synthesized as an inactive membrane-bound precursor and that it undergoes several cleavages that produce active enzymes. The most important cleavage occurs after the sequence RSLK and appears to be autocatalytic. In an accompanying paper, we produce a truncated, secreted form of activated S1P and describe its catalytic properties (17Cheng D. Espenshade P.J. Slaughter C.A. Jaen J.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1999; 274: 22805-22812Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We obtained monoclonal antibodies IgG-HSV-Tag™ from Novagen, anti-c-Myc (clone 9E10) from Roche Molecular Biochemicals or Invitrogen, anti-c-Myc agarose-conjugated beads from Santa Cruz Biotechnology, Inc., anti-BiP from StressGene Biotechnologies Corp., horseradish peroxidase-conjugated donkey anti-rabbit whole antibody from Amersham Pharmacia Biotech, horseradish peroxidase-conjugated donkey anti-mouse IgG (affinity-purified) from Jackson Immunoresearch Laboratories, maleimide-activated keyhole limpet hemocyanin from Pierce, and glycosidases from New England Biolabs. Other reagents were obtained from sources as described previously (2Sakai 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 (328) Google Scholar, 17Cheng D. Espenshade P.J. Slaughter C.A. Jaen J.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1999; 274: 22805-22812Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 18Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar, 19Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). Newborn and fetal calf lipoprotein-deficient serum (d > 1.215 g/ml) were prepared as described (20Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1287) Google Scholar). The following plasmids contain the cytomegalovirus (CMV) promoter-enhancer driving expression of various cDNAs. pCMV-SCAP(D443N) is a previously described plasmid that encodes hamster SCAP with a D443N mutation that renders the protein resistant to sterols (7Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Other previously described plasmids (2Sakai 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 (328) Google Scholar) include pCMV-S1P, which encodes hamster S1P; pCMV-Myc-S1P, which encodes hamster S1P containing three tandem copies of the c-Myc epitope tag inserted between amino acids 23 and 24; and pCMV-Myc-S1P(S414A), which is identical to pCMV-Myc-S1P except that serine 414 has been changed to alanine. pCMV-Myc-S1P(Site-B mutant) is identical to pCMV-Myc-S1P except that amino acids 134–137 of S1P have been mutated from RSLK to AAAA. pCMV-Myc-S1P(Site-C mutant) is identical to pCMV-Myc-S1P except that amino acids 163–164 (RR) and 183–187 (RRLLR) have been mutated to VA and AAAAA, respectively. To generate pCMV-Myc-S1P(Site-B mutant) and pCMV-Myc-S1P(Site-C mutant), we used the QuikChange site-directed mutagenesis kit (Stratagene) to mutagenize a 2.6-kb HindIII fragment of pCMV-Myc-S1P subcloned into pBluescript KS− (Stratagene). Oligonucleotides corresponding to the following sequences were used to create pCMV-Myc-S1P(Site-B mutant) and pCMV-Myc-S1P(Site-C mutant): 5′-CCTCAACGCAAAGTCTTTGCCGCGGCTGCTTTTGCTGAATCTGACCC-3′ for the Site-B mutant, and 5′-GGCAGTCATCACGACCCCTGGTTGCCGCTAGCCTCTCCCTGGGC-3′ and 5′-AGGAAGACATTCAAGCGCCGCGGCTGCTGCTGCCATTCCTCGACAGG-3′ for the Site-C mutant. Following mutagenesis, the mutated 1.5-kbBstEII-HpaI fragment was ligated to an 8.3-kbBstEII-HpaI fragment of pCMV-Myc-S1P to yield the final plasmid. pCMV-Myc-S1P(Site-B + C mutant) contains both the Site-B and Site-C mutations and was constructed by sequential mutagenesis. pCMV-S1P-Myc encodes full-length hamster S1P (amino acids 1–1052) containing three tandem copies of the c-Myc epitope at the COOH terminus followed by six histidine residues. Construction of this expression vector is described in the accompanying paper, in which it is referred to as pCMV-S1P(1052)Myc-His (17Cheng D. Espenshade P.J. Slaughter C.A. Jaen J.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1999; 274: 22805-22812Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). pCMV-S1P-Myc(S414A) is identical to pCMV-S1P-Myc except that serine 414 has been changed to alanine. To generate pCMV-S1P-Myc(S414A), a 0.4-kb EcoRI fragment of pCMV-S1P was ligated to a 9.3-kb EcoRI fragment of pCMV-Myc-S1P(S414A) to create pCMV-S1P(S414A). Then, a 1.4-kbBstEII-HpaI fragment of pCMV-S1P(S414A) was ligated to a 8.3-kb BstEII-HpaI fragment of pCMV-S1P-Myc to create pCMV-S1P-Myc(S414A). The following plasmids contain the thymidine kinase (TK) promoter from herpes simplex virus, driving expression of the indicated cDNA. pTK-Myc-S1P encodes Myc-S1P and was constructed by ligating a 4.0-kbBamHI (blunted by Klenow)-XbaI fragment of pCMV-Myc-S1P to a 5.8-kb SpeI (blunted by Klenow)-XbaI fragment from pTK-HSV-BP2 (see below). pTK-Myc-S1P(Site-B mutant) is identical to pTK-Myc-S1P except that amino acids 134–137 have been mutated from RSLK to AAAA. pTK-Myc-S1P(Site-C mutant) is identical to pTK-Myc-S1P except that amino acids 163–164 (RR) and 183–187 (RRLLR) have been mutated to VA and AAAAA, respectively. pTK-Myc-S1P(Site-B + C mutant) contains both Site-B and Site-C mutations. A 3.1-kb SpeI-BspDI fragment from pCMV-Myc-S1P(Site-B mutant), pCMV-Myc-S1P(Site-C mutant), or pCMV-Myc-S1P(Site-B + C mutant) was ligated to a 6.7-kbSpeI-BspDI fragment from pTK-Myc-S1P to generate pTK-Myc-S1P(Site-B mutant), pTK-Myc-S1P(Site-C mutant), and pTK-Myc-S1P(Site-B + C mutant), respectively. pTK-HSV-BP2 encodes human SREBP-2 with two tandem copies of the HSV epitope at the NH2 terminus and has been 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). pTK-HSV-BP2(RSLK) and pTK-HSV-BP2(RRLL) encode mutant versions of pTK-HSV-BP2 in which the wild-type recognition sequence for S1P in human SREBP-2 (RSVL) has been changed to RSLK and RRLL, respectively. These two plasmids were generated by oligonucleotide site-directed mutagenesis using single-stranded, uracil-containing DNA as described (4Duncan 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). Mutagenesis was performed on a plasmid containing a 0.8-kb HindIII fragment of human SREBP-2 ligated to a 3.0-kb fragment of pBluescript KS− linearized by digestion withHindIII. Using this plasmid, we mutated amino acids 519–522 of SREBP-2 (RSVL) to either RSLK or RRLL with the oligonucleotide 5′-AGAACCTGACTCGAATGACTTCAGAGATCTGCCAGAGCCTGAGTGTGG-3′ or 5′-AGAACCTGACTCGAATGACAGCAGCCGGCGGCCAGAGCCTGAGTGTGG-3′, respectively. Subsequently, 0.8-kb HindIII fragments of SREBP-2 containing the mutated sequences were ligated into pTK-HSV-BP2 to generate pTK-HSV-BP2(RSLK) and pTK-HSV-BP2(RRLL). In all plasmid constructions, mutations were confirmed by sequencing the relevant regions. pVAI encodes the adenovirus-associated I RNA gene, which enhances translation of transfected cDNAs (22Akusjarvi G. Svensson C. Nygard O. Mol. Cell. Biol. 1987; 7: 549-551Crossref PubMed Scopus (54) Google Scholar). A polyclonal antibody (U1683) against amino acids 1023–1052 of hamster S1P (2Sakai 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 (328) Google Scholar) was generated by immunizing rabbits with a mixture of two synthetic peptides corresponding to amino acids 1023–1037 (KAKSRPKRRRPRAKR) and 1038–1052 (PQLTQQTHPPRTPSV), each with an NH2-terminal cysteine residue that was conjugated to keyhole limpet hemocyanin using a standard protocol (23Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Monoclonal antibodies were obtained commercially as described above. Monolayer cultures of human embryonic kidney 293 cells (HEK-293 cells) were set up for experiments 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 using an MBS kit (Stratagene) with the indicated plasmids as described previously (24Hua 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 refed with 2 ml of Medium A supplemented with 1% (v/v) newborn calf lipoprotein-deficient serum and cultured for 25 h prior to harvest. The medium from two dishes was pooled and centrifuged at 20,000 × g at 4 °C for 15 min. An aliquot (2 ml) of the resulting supernatant was mixed with 8 ml of cold acetone and incubated at −20 °C for 15 min. The precipitated proteins were collected at 3300 × g at 4 °C for 15 min and resuspended in 0.1 ml of SDS lysis buffer (10 mm Tris-HCl at pH 6.8, 0.1 m NaCl, 1% SDS, 1 mm EDTA, 1 mm EGTA). The 105 × g membrane fraction from cell lysates was prepared as described previously (25Sakai 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 (435) Google Scholar). Monolayers of SRD-12B cells (8Rawson R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 28261-28269Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) were set up on day 0 (4 × 105 cells/60-mm dish) and cultured in 8–9% CO2 at 37 °C in Medium B (1:1 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) supplemented with 5% fetal calf serum, 5 μg/ml cholesterol, 1 mm sodium mevalonate, and 20 μm sodium oleate. On day 1, the cells were transfected with the indicated plasmids using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions as described (2Sakai 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 (328) Google Scholar). Three h after transfection, the cells were refed with 5 ml of Medium B supplemented with 5% fetal calf serum. The cells were incubated for 17 h, after whichN-acetyl-leucinal-leucinal-norleucinal was added to each dish at a final concentration of 25 μg/ml, and the cells were harvested 1 h later. Pooled cells from four 60-mm dishes were used to prepare nuclear extract and 105 × g membrane fractions as described previously (25Sakai 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 (435) Google Scholar). For experiments in which only membrane fractions were prepared, cells were not incubated withN-acetyl-leucinal-leucinal-norleucinal. Protein samples were mixed at a ratio of 5:1 with 5× loading buffer (1× loading buffer contains 30 mm Tris-HCl at pH 7.4, 3% SDS (w/v), 5% (v/v) glycerol, 0.004% (v/v) bromphenol blue, and 2.5% (v/v) β-mercaptoethanol) and heated at 100 °C for 3 min prior to SDS-PAGE. Following SDS-PAGE, proteins were transferred to nitrocellulose filters, incubated with antibodies indicated in the figure legends, and detected by chemiluminescence, as described previously (2Sakai 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 (328) Google Scholar). Gels were calibrated using prestained molecular mass markers (New England Biolabs or Bio-Rad). Protein concentration was measured with a BCA kit (Pierce). Transfected HEK-293 cells were cultured for 18 h in 5 ml of Medium A supplemented with 5% fetal calf serum, after which cell membranes were isolated and treated with trypsin either in the absence or presence of Triton X-100 for 30 min at 30 °C as described previously (19Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). Treated samples were subjected to SDS-PAGE on 8% gels followed by immunoblot analysis. Membrane fractions from transfected SRD-12B cells were prepared as described above, after which 5 μg of membrane protein was treated with either peptideN-glycosidase F or endoglycosidase H (endo H) as described previously (2Sakai 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 (328) Google Scholar). SRD-12B cells and SRD-12B cells stably transfected with pCMV-S1P-Myc (designated S1P(1052) cells) (17Cheng D. Espenshade P.J. Slaughter C.A. Jaen J.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1999; 274: 22805-22812Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) were grown in roller bottles and set up at 4 × 107cells/850-cm2 bottle in Medium B supplemented with 5% fetal calf serum. The medium was changed every other day. On day 6, the medium was changed to Medium B supplemented with 5% newborn calf lipoprotein-deficient serum. On day 7, the cells were collected and washed with ice-cold phosphate-buffered saline. The washed cell pellet (∼10 ml of packed cells) was lysed in 40 ml of Buffer A (50 mm Tris-HCl at pH 8.0, 1.5% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholic acid, 150 mm NaCl, 2 mm MgCl2) supplemented with 1 mmPefabloc®, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μg/ml pepstatin A, and 5 μg/ml aprotinin. The cell lysate was rocked for 1 h at 4 °C followed by centrifugation at 2 × 105 × g for 1 h at 4 °C. The resulting supernatant was incubated with 500 μg of monoclonal 9E10 anti-Myc conjugated to agarose beads (Santa Cruz Biotechnology, Inc.) with continuous rocking for 6 h at 4 °C. The beads were then
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