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Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2

Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 19, Issue of May 9, pp. 12778 –12785, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2* (Received for publication, January 30, 1997, and in revised form March 6, 1997) Elizabeth A. Duncan‡, Michael S. Brown, Joseph L. Goldstein§, and Juro Sakai From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235 A sterol-regulated protease initiates release of the envelope. Each SREBP is composed of three segments: 1) an NH -terminal segments of sterol regulatory element- NH -terminal segment of ;485 amino acids that is a transcrip- binding proteins (SREBPs) from cell membranes, tion factor of the basic helix-loop-helix-leucine zipper family, 2) thereby allowing them to enter the nucleus and to stim- a membrane attachment segment of ;75 amino acids composed ulate transcription of genes involved in the uptake and of two membrane-spanning sequences separated by a short synthesis of cholesterol and fatty acids. Using SREBP-2 hydrophilic loop of 31 amino acids, and 3) a COOH-terminal as a prototype, we here identify the site of sterol-regu- segment of ;585 amino acids that plays a regulatory role. The 522 523 lated cleavage as the Leu -Ser bond in the middle of proteins are oriented so that the NH - and COOH-terminal the 31-residue hydrophilic loop that projects into the segments project into the cytoplasm, and only the short hydro- lumen of the endoplasmic reticulum and nuclear enve- philic loop projects into the lumen of the ER or nuclear lope. This site was identified through use of a vector envelope (4). encoding an SREBP-2/Ras fusion protein with a triple Before it can activate transcription, the NH -terminal seg- epitope tag that allowed immunoprecipitation of the ment is released from the membrane in a complex two-step cleaved COOH-terminal fragment. The NH terminus of proteolytic sequence (2, 3). First, a protease cleaves the protein this fragment was pinpointed by radiochemical se- at Site-1, which is near an arginine in the lumenal loop, quencing after replacement of selected codons with me- thereby breaking the attachment between the two transmem- thionine codons and labeling the cells with [ S]methi- brane sequences. This allows a second protease to cleave the onine. Alanine scanning mutagenesis revealed that only protein at Site-2, which is near the middle of the first trans- two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage membrane sequence (2, 3). The NH -terminal fragment leaves site (leucine 522), and 2) the arginine at the P4 position the membrane with a portion of the first transmembrane se- (arginine 519). These define a tetrapeptide sequence, quence still attached. It enters the nucleus, where it activates RXXL, that is necessary for cleavage. Cleavage was not transcription of genes encoding the LDL receptor (5, 6), several affected when the second transmembrane helix of enzymes of cholesterol biosynthesis (3-hydroxy-3-methylglu- SREBP-2 was replaced with the membrane-spanning re- taryl coenzyme A synthase (5, 6), 3-hydroxy-3-methylglutaryl gion of the low density lipoprotein receptor, indicating coenzyme A reductase (7), farnesyl diphosphate synthase (8), that this sequence is not required for regulation. Glyco- and squalene synthase (9)), and enzymes of fatty acid biosyn- sylation-site insertion experiments confirmed that thesis (10, 11) and desaturation (12). The net result is to in- leucine 522 is located in the lumen of the endoplasmic crease the cell’s supply of cholesterol and fatty acids. reticulum. We conclude that the sterol-regulated prote- The Site-1 protease is the target of feedback regulation by ase is a novel enzyme whose active site faces the lumen cholesterol and other sterols (3). When these sterols accumu- of the nuclear envelope, endoplasmic reticulum, or an- late within cells, the rate of proteolysis at Site-1 declines mark- other membrane organelle to which the SREBPs may be edly. Cleavage at Site-2 also declines because this cleavage transported before cleavage. requires prior cleavage at Site-1 (3). As a result, the amounts of nuclear SREBPs decline, and transcription of the target genes falls. The net effect is to prevent overaccumulation of choles- Proteolytic processing of sterol regulatory element-binding 1 terol and fatty acids when intracellular sterol levels are al- proteins (SREBPs) controls the metabolism of cholesterol and ready high. fatty acids in animal cells (1–3). SREBPs are transcription Three isoforms of SREBP are known (5, 13, 14). SREBP-1a factors that are bound to membranes of the ER and nuclear and 1c are derived from a single gene through use of alternate promoters that encode alternate first exons (5, 13, 14). * This work was supported by National Institutes of Health Grant SREBP-1a is much more active than SREBP-1c in stimulating HL20948 and by a research grant from the Perot Family Foundation. transcription of all known target genes (15). The third protein, The costs of publication of this article were defrayed in part by the SREBP-2, is the product of a separate gene (6, 13), and it is also payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to more active than SREBP-1c (15). In view of the regulatory role indicate this fact. of the Site-1 protease, further knowledge of its structure and ‡ Supported by Medical Scientists Training Grant GM08014. mode of regulation is desirable. A first step would be the iden- § To whom correspondence should be addressed: Dept. of Molecular tification of the precise site at which the Site-1 protease cuts Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2141; Fax: 214-648- SREBPs. In previous studies we have shown that this cleavage is abolished when arginine 519 in the lumenal loop of SREBP-2 The abbreviations used are: SREBP, sterol regulatory element- (or the corresponding arginine of SREBP-1a) is changed to binding protein; ER, endoplasmic reticulum; LDL, low density lipopro- alanine by in vitro mutagenesis (2). The size of the cleavage tein; SCAP, SREBP cleavage-activating protein; PAGE, polyacrylamide gel electrophoresis; VAI, virus-associated I. product, as determined by SDS-PAGE, is consistent with cleav- 12778 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Identification of a Sterol-regulated Cleavage Site in SREBP-2 12779 region, and at least two independent clones of each mutant were inde- age at or near this arginine (3). pendently transfected. In the current studies we have used a combination of in vitro Cell Culture, Transfection, and Cell Fractionation—Monolayers of mutagenesis, epitope tagging, immunoprecipitation, and radio- human embryonic kidney 293 cells were set up on day 0 (4 3 10 chemical sequencing to determine the precise location of Site-1 cells/60-mm dish) and cultured in 8 –9% CO at 37 °C in medium A in SREBP-2. We found, surprisingly, that cleavage does not (Dulbecco’s modified Eagle’s medium containing 100 units/ml penicillin occur at arginine 519, but rather it occurs 3 residues further and 100 mg/ml streptomycin) supplemented with 10% (v/v) fetal calf serum (4). On day 2, cells were transfected with 4 mg of pTK empty toward the COOH terminus, namely at leucine 522. Arginine vector (mock) or the indicated plasmid as described (2). Three h after 519 seems to be the NH -terminal residue in a tetrapeptide transfection, the cells were switched to medium B (medium A contain- sequence, RXXL, that serves as a recognition signal for the ing 10% newborn calf lipoprotein-deficient serum, 50 mM compactin, and Site-1 protease. 50 mM sodium mevalonate) in the absence or presence of sterols as indicated in the legends. After incubation for 20 h, the cells received EXPERIMENTAL PROCEDURES N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 TM TM Materials—We obtained HSV-Tag and T7-Tag monoclonal an- mg/ml (2), and the cells were harvested 3 h later (2). The pooled cell tibodies from Novagen; v-H-Ras(Ab-1)-agarose linked monoclonal anti- suspension from 2 dishes was allowed to swell in hypotonic buffer (4) for body was obtained from Oncogene. Protein G-Sepharose 4 Fast Flow 30 min at 0 °C, passed through a 22.5-gauge needle 30 times, and beads were obtained from Pharmacia Biotech Inc., L-[ S]methionine centrifuged at 1000 3 g at 4 °C for 7 min. The 1000 3 g pellet was (.1000 Ci/mmol) was obtained from DuPont NEN, and glycosidases resuspended in 0.1 ml of buffer C (10 mM Hepes-KOH (pH 7.4), 0.42 M were obtained from New England Biolabs. NaCl, 2.5% (v/v) glycerol, 1.5 mM MgCl , 0.5 mM sodium EDTA, 0.5 mM Construction of pTK-HSV-BP2-Ras-T7—pTK-HSV-BP2-Ras-T7 en- sodium EGTA, 1 mM dithiothreitol, and a mixture of protease inhibitors codes an epitope-tagged SREBP-2/Ras fusion protein (1391 amino ac- (4)). The suspension was rotated at 4 °C for 1 h and centrifuged at top ids) consisting of an initiator methionine, two tandem copies of the HSV speed in a microfuge for 15 min at 4 °C. The supernatant is designated epitope (QPELAPEDPED), six novel amino acids (IDGTVP) encoded by nuclear extract. The supernatant from the original 1000 3 g spin was a sequence that consists of restriction sites for BspDI and KpnI, human centrifuged at 10 3 g for 30 min at 4 °C in a Beckman TLA 100.2 rotor, SREBP-2 (amino acids 14 –1141), two novel amino acids (HM) encoded and the pellet was dissolved in 0.1 ml of SDS lysis buffer (1) and by the sequence for restriction site NdeI, human H-Ras (amino acids designated membrane fraction. 2–189), and three tandem copies of the T7 epitope (HMASMTGGQQM- Glycosidase Sensitivity of Membrane-bound SREBP-2—Glycosidase GAAAMASMTGGQQMGGGPMASMTGGQQMGLINM). sensitivity of SREBP-2 was carried out as described (4). Monolayers of pTK-HSV-BP2-Ras-T7 was constructed from a previously described 293 cells were set up on day 0 (4 3 10 cells/60-mm dish) and cultured plasmid, pTK-HSV-BP2-T7 (16), by insertion of a cDNA segment en- as described above. On day 2, the cells were transfected with 7 mgof coding human H-Ras between the sequences for SREBP-2 and the first pTK empty vector (mock), pTK-HSV-BP2-NGT, and pTK-HSV-BP2- copy of the T7 epitope. The nucleotide sequence encoding amino acids NSS/NGT, respectively. Three h after transfection, the cells were 2–189 of human H-Ras was obtained by polymerase chain reaction on switched to medium B in the presence of sterols as described in Fig. 1. pRcCMV-H-Ras (17) with a pair of primers containing an NdeI site at After incubation for 20 h, the cells were harvested, and the pooled cell each 59 end using Pfu DNA polymerase. The amplified product was suspension from four dishes was fractionated. The 10 3 g membrane digested with NdeI and cloned into the unique NdeI site between pellet was washed once with buffer A (4) and resuspended in 180 mlof human SREBP-2 and the three tandem copies of the T7 epitope. Two buffer A containing 1% (v/v) Triton X-100 without protease inhibitors. independent clones were used in each of the transfections. Aliquots of the 10 3 g membrane fraction (0.16 mg in 40 ml of buffer A) Construction of pTK-HSV-BP2/LDLRTM—pTK-HSV-BP2/LDL- were boiled for 5 min in the presence (peptide N-glycosidase F and RTM encodes an epitope-tagged SREBP-2 fusion protein in which a endoglycosidase H reactions) or in the absence (neuraminidase reac- 26-amino acid region that includes the second transmembrane domain tions) of 0.5% (w/v) SDS and 1% (v/v) b-mercaptoethanol for 5 min, after of human SREBP-2 (amino acids 535–560) is replaced with the trans- which the indicated amount of glycosidase was added and incubated at membrane domain of the human LDL receptor (amino acids 708 –729) 37 °Cfor2has described in Fig. 10. (18). To construct pTK-HSV-BP2/LDLRTM, we used oligonucleotide Immunoblot Analysis—Samples of the nuclear extract and the 10 3 site-directed mutagenesis to produce an intermediate plasmid in which g membrane fraction were mixed with 53 SDS loading buffer (20). amino acids 535–560 of human SREBP-2 were replaced by two novel Protein concentration was measured with a BCA kit (Pierce). After amino acids (TG) corresponding to an AgeI restriction site. A pair of SDS-PAGE in 8% gels, proteins were transferred to Hybond-C extra complementary oligonucleotides (top strand, 59-CCGGTGCTCTGTCC- nitrocellulose membranes (Amersham Corp.). Immunoblot analysis was ATTGTCCTCCCCATCGTGCTCCTCGTCTTCCTTTGCCTGGGGGTC- carried out with a horseradish peroxidase detection kit using the Su- TM TTCCTTCTATGGT-39; bottom strand, 59-CCGGACCATACAAGGAAG- perSignal CL-HRP Substrate System according to the manufactur- ACCCCCAGGCAAAGGAAGACGAGGAGCACGATGGGGAGGACAA- er’s instructions except that the nitrocellulose sheets were blocked in TGGACAGAGCA-39) were annealed at 94 – 83 °C for 5 min and then at phosphate-buffered saline containing 0.05% (v/v) Tween 20, 5% (v/v) 83–23 °C for 60 min. These oligonucleotides correspond to amino acids nonfat dry milk, and 5% (v/v) heat-inactivated newborn calf serum. The TM 708 –729 of the human LDL receptor flanked by the single-stranded chimeric proteins were visualized with 0.5 mg/ml HSV-Tag mono- sequence 59-CCGG-39. The annealed oligonucleotides were cloned into clonal antibody or with 10 mg/ml IgG-1C6, a mouse monoclonal antibody the unique AgeI restriction site of the intermediate plasmid (described directed against amino acids 833–1141 of human SREBP-2 (4). Gels above). The resulting pTK-HSV-BP2/LDLRTM encodes an 1157-amino were calibrated with prestained molecular weight markers. Filters acid chimeric protein consisting of an initiator methionine, two tandem were exposed at room temperature to Reflectiony NEF-496 film copies of the HSV epitope, six novel amino acids (IDGTVP), human (DuPont NEN). SREBP-2 (amino acids 14 –534), two novel amino acids (TG), human Metabolic Labeling and Immunoprecipitation of Epitope-tagged LDL receptor (amino acids 708 –729), two novel amino acids (SG), and SREBP-2/Ras Fusion Protein and Its COOH-terminal Fragment— human SREBP-2 (amino acids 561–1141). Monolayers of 293 cells were set up on day 0 (7 3 10 cells/60-mm dish) Plasmid pTK-HSV-BP2(R519A)/LDLRTM is identical to pTK-HSV- and cultured as described above. On day 1, the cells were transfected BP2/LDLRTM except for the R519A point mutation in the lumenal loop with 4 mg of the wild-type or mutant version of pTK-HSV-BP2-Ras-T7, of the SREBP-2 sequence. This plasmid was constructed by site-di- 1 mg of pCMV-SCAP(D443N) (16), and 2 mg of pVAI as described above. rected mutagenesis. pVAI encodes the adenovirus virus-associated I RNA gene, which en- Construction of pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/ hances translation of transfected cDNAs (21). The cells were incubated NGT—pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/NGT encode for 20 h with 50 mM compactin and 50 mM sodium mevalonate, at which epitope-tagged SREBP-2 fusion proteins in which serine 515 in the loop time the medium was changed to 1.3 ml of methionine-free Dulbecco’s region of SREBP-2 is replaced by a novel amino acid sequence contain- modified Eagle’s medium supplemented with 100 units/ml penicillin, ing one or two N-linked glycosylation sites, either SNGT or NSSGSS- 100 mg/ml streptomycin, 10% newborn calf lipoprotein-deficient serum, GNGT, respectively. Both plasmids were constructed by site-directed 50 mM compactin, and 50 mM sodium mevalonate. After incubation for mutagenesis. 1hat37 °C,25 mg/ml N-acetyl-leucinal-leucinal-norleucinal was added, Site-directed Mutagenesis—Oligonucleotide site-directed mutagene- and the cells were pulse-labeled with 700 mCi/ml of [ S]methionine for sis was carried out with single-stranded, uracil-containing DNA (19) 6 h at 37 °C. The cells from four dishes were harvested and pooled, and using the Muta-gene Phagemid In Vitro Mutagenesis Version-2 kit the membrane fraction was prepared as described above. (Bio-Rad) (2). The mutations were confirmed by sequencing the relevant The pooled membrane fraction from the four dishes was resuspended 12780 Identification of a Sterol-regulated Cleavage Site in SREBP-2 in 0.1 ml of SDS lysis buffer at room temperature. All subsequent operations were carried out at 4 °C unless otherwise stated. The sus- pension 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 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mg/ml pepstatin A, 25 mg/ml N-acetyl-leucinal- leucinal-norleucinal, and 1 mM dithiothreitol), after which 20 mlof preimmune whole rabbit serum, 40 mg of irrelevant mouse monoclonal antibody IgG-2001 (22), and 0.2 ml of Protein G-Sepharose beads were added. After rotation for 16 h, the mixture was centrifuged at 1000 3 g for 7 min. The resulting supernatant was mixed with an additional 20 ml of preimmune whole rabbit serum, 40 mg of irrelevant monoclonal antibody IgG-2001, and 0.2 ml of Protein G-Sepharose beads, rotated for 2 h, and centrifuged at 1000 3 g for 3–7 min. To the supernatant were added 1 mg of T7-Tag monoclonal antibody, 25 mg of v-H-Ras(Ab- 1)-agarose linked monoclonal antibody, and 20 mg of anti-COOH-termi- nal SREBP-2 monoclonal antibody IgG-1C6. After rotation for 2.5 h, 50 ml of Protein G-Sepharose beads were added, followed by rotation for 2.5 h and centrifugation at 1000 3 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 23 SDS loading buffer (20) containing 10% (v/v) b-mercaptoethanol and boiled for 5 min. After centrifugation at 1000 3 g at room temper- ature for 3 min, the supernatant was transferred to a fresh tube (tube A), and the beads were re-eluted with 40 mlof53 SDS loading buffer containing 25% b-mercaptoethanol and boiled for 5 min. After centrif- ugation at 1000 3 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. NH -terminal Sequence Analysis of S-Labeled COOH-terminal FIG.1. Sterol-regulated cleavage of epitope-tagged SREBP-2/ Fragment of Epitope-tagged SREBP-2 Fusion Protein—Aliquots of the Ras fusion protein in transfected 293 cells. A, schematic diagram immunoprecipitated samples (from 1.3 dishes of cells) were subjected to of the fusion protein encoded by pTK-HSV-BP2-Ras-T7, showing addi- tional COOH-terminal sequences encoding H-Ras followed by three T7 SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride mem- SQ epitope tags. Encircled numbers 1 and 2 denote the two sequential sites branes (Immobilon-P ; Millipore). After drying, the membranes were of proteolytic cleavage. B, immunoblot analysis of the membrane frac- exposed to an imaging plate and scanned in a Fuji X Bas 1000 phos- tion of 293 cells transfected with the wild-type and R519A mutant phorimager. The band containing the COOH-terminal product of the version of pTK-HSV-BP2-Ras-T7. On day 0, 293 cells were set up for cleavage reaction (M ;83,000) was excised and subjected directly to experiments as described under “Experimental Procedures.” On day 2, multiple cycles of Edman degradation on an Applied Biosystems model the cells were transfected with the indicated plasmids as follows: 4 mg 477A sequencer. Fractions from each cycle (148 ml) were collected and of pTK mock vector (lanes 1 and 2), 4 mg of wild-type pTK-HSV-BP2- counted in a scintillation counter. Ras-T7 (lanes 3– 8), or 4 mg of R519A mutant version of pTK-HSV-BP2- Ras-T7 (lanes 9 –14); 2 mg of pVAI (21) (lanes 7, 8, 13, and 14); and 1 mg RESULTS AND DISCUSSION of pCMV-SCAP(D443N) (16) (lanes 5– 8 and 11–14). After transfection, To identify the exact position of Site-1, we prepared a cDNA the cells were incubated in the absence (2) or presence (1)of1 mg/ml (pTK-HSV-BP2-Ras-T7) encoding a triply tagged version of 25-hydroxycholesterol plus 10 mg/ml cholesterol (sterols) as indicated. On day 3, the cells were harvested and fractionated as described under SREBP-2, which we designate SREBP-2/Ras (Fig. 1). The NH “Experimental Procedures.” Aliquots of the membranes (80 mg of pro- terminus contains two copies of an epitope tag from the HSV tein) were subjected to SDS-PAGE and immunoblot analysis with 10 glycoprotein that we used previously (2). At the COOH termi- mg/ml IgG-1C6, an antibody directed to the COOH-terminal domain of nus we inserted amino acids 2–189 of H-Ras followed by three SREBP-2. The filters were exposed to film for 30 s. P and C denote the uncleaved precursor form and the cleaved COOH-terminal fragment of copies of an 11-residue epitope derived from the gene 10 protein SREBP-2, respectively. of bacteriophage T7 (23). The combination of epitopes at the COOH terminus allowed efficient precipitation of the COOH- terminal fragment with a mixture of antibodies directed 83-kDa COOH-terminal fragment, which was the product of against the Ras and T7 epitopes plus a monoclonal antibody cleavage at Site-1 (Fig. 1, lane 3, transfected C). The amount of directed against the COOH-terminal domain of SREBP-2 (see this fragment was reduced in the presence of sterols (lane 4). below). The IgG-1C6 antibody also visualized the 62-kDa COOH-ter- To demonstrate that SREBP-2/Ras is cleaved at Site-1 in a minal fragment of endogenous SREBP-2 (lane 3, endogenous physiologic fashion, we transfected 293 cells with a vector C), and this was also decreased by sterols (lane 4). To test the encoding this construct and another encoding a mutated ver- physiologic relevance of the observed cleavage of SREBP-2/Ras, sion in which arginine 519 was changed to alanine (Fig. 1). We we cotransfected a cDNA encoding the D443N mutant version used the relatively weak thymidine kinase promoter, which of SCAP, a protein that was previously shown to render the produces near physiological levels of this protein (2). Cells were cleavage of SREBPs at Site-1 resistant to suppression by ste- incubated in inducing medium that contains the 3-hydroxy-3- rols (16). Indeed, in the presence of SCAP, sterols no longer methylglutaryl coenzyme A reductase inhibitor compactin to prevented the appearance of the COOH-terminal fragment of block cholesterol synthesis plus a low concentration of meval- SREBP-2/Ras (lane 6). Note that SCAP did not prevent sup- onate to provide nonsterol end products. The medium was pression of the cleavage of endogenous SREBP-2. We believe either devoid of sterols (2 sterols) or it was supplemented with that this is due to the small percentage of cells that received the a mixture of 25-hydroxycholesterol and cholesterol (1 sterols), SCAP cDNA. Unlike SREBP-2/Ras, which is expressed only in which suppresses cleavage at Site-1 (3). Cell membranes were transfected cells, endogenous SREBP-2 is present in all cells in isolated, solubilized with SDS, subjected to SDS-PAGE, and the dish, most of which did not receive the SCAP cDNA. To immunoblotted with IgG-1C6, a monoclonal antibody directed increase the expression of the transfected SREBP-2/Ras chi- against the COOH terminus of SREBP-2 (4). mera, we cotransfected pVAI, which encodes a protein that When cells were transfected with the cDNA encoding the stimulates translation of mRNAs produced by transfected SREBP-2/Ras chimeric protein, the membranes contained the cDNAs (21). In the presence of pVAI plus pSCAP, the COOH- Identification of a Sterol-regulated Cleavage Site in SREBP-2 12781 FIG.2. Immunoblot analysis of epitope-tagged SREBP-2/Ras fusion protein in 293 cells transfected with wild-type and me- thionine loop mutants. A, amino acid sequence of the lumenal loop region of human SREBP-2. The first amino acid in the loop region follows the first transmembrane domain (TM1) and is designated resi- due 503. Residues individually mutated to methionine in the epitope- 35 FIG.3. Analysis of the [ S]methionine-labeled COOH-terminal tagged SREBP-2/Ras fusion protein are indicated below the sequence. fragment of human SREBP-2 after immunoprecipitation of B, immunoblot analysis of SREBP-2/Ras fusion protein. 293 cells were 5 transfected 293 cells. On day 0, 293 cells were set up at 7 3 10 set up for experiments, transfected with the indicated wild-type or cells/60-mm dish in medium A. On day 1, cells were transfected with mutant version of pTK-HSV-BP2-Ras-T7, incubated in the absence or either 4 mg of pTK mock vector (lanes 1 and 2)or4 mg of pTK-HSV- presence of sterols, and fractionated as described in the legend of Fig. 1. BP2-Ras-T7, 2 mg of pVAI, and 1 mg of pCMV-SCAP(D443N) (lanes 3 Aliquots of the nuclear extracts (60 mg of protein) were subjected to 35 and 4). On day 2, cells were radiolabeled with [ S]methionine, har- TM SDS-PAGE and immunoblot analysis with 0.5 mg/ml HSV-Tag anti- vested, and subjected to immunoprecipitation. The immunoprecipitated body. The filters were exposed to film for 7 s. M denotes the cleaved samples (from 0.5 dish of cells in lanes 1 and 3 and 2.5 dishes in lanes NH -terminal mature form of the SREBP-2/Ras fusion protein. The 2 and 4) were subjected to SDS-PAGE, after which the nitrocellulose other bands are present in mock-transfected cells and represent pro- filter was exposed to an imaging plate for 15 min at room temperature teins that cross-react with the anti-HSV tag antibody. 35 to detect S radioactivity (A). The same filter was subjected to immu- noblot analysis with 10 mg/ml IgG-1C6, an antibody directed against the COOH-terminal domain of SREBP-2 (B). Chemiluminescence from terminal fragment of the chimeric protein was increased by at the bound secondary antibody was detected by exposure to x-ray film for least 8-fold, and there was still no suppression by sterols (lanes 1s. Asterisk (p) denotes the 83-kDa COOH-terminal fragment of the 7 and 8). The R519A mutant of the chimeric protein did not give SREBP-2/Ras fusion protein. rise to detectable amounts of COOH-terminal cleavage product in either the absence or presence of SCAP (lanes 9 –12). In the transfected 293 cells with a cDNA encoding the SREBP-2/Ras presence of pVAI plus pSCAP, a trace amount of the R519A chimera and labeled the cells with [ S]methionine. A mem- mutant COOH-terminal fragment was detectable, but its brane fraction was prepared, solubilized with detergents, and amount was reduced by at least 12-fold when compared with immunoprecipitated with a mixture of three antibodies di- the wild-type COOH-terminal fragment (compare lanes 13 and rected against H-Ras, the T7 epitope tag, and the COOH- 14 with lanes 7 and 8). These data indicate that Site-1 cleavage terminal segment of SREBP-2. The immunoprecipitate was of SREBP-2/Ras obeys all of the rules established for wild-type subjected to SDS-PAGE and phosphorimager analysis, which SREBP-2. Moreover, as discussed below, the use of pVAI and revealed a band of ;83 kDa that was absent from mock-trans- pSCAP enabled the production of sufficient amounts of SREBP- fected cells (Fig. 3A). Immunoblot analysis of the precipitate 2/Ras to allow radiochemical sequencing. (Fig. 3B) and the supernatant (data not shown) confirmed that In view of the high specific radioactivity of [ S]methionine, all of the COOH-terminal fragment was precipitated. In future we developed a strategy to use this amino acid for radiochem- experiments, the 83-kDa radiolabeled band was excised from ical sequencing. Assuming that the cleavage site is located near the nitrocellulose membrane and used for radiochemical arginine 519, we searched for amino acids in the region that sequencing. could be replaced with methionine without abolishing cleavage Fig. 4 shows a series of experiments in which 293 cells were at Site-1. Fig. 2 shows two experiments in which methionine transfected with cDNAs encoding the SREBP-2/Ras chimera residues were introduced individually into positions 525, 526, with the wild-type sequence in the lumenal loop or with the or 529 of the triply taggged SREBP-2/Ras protein (Fig. 2A). To three methionine substitutions at positions 525, 526, and 529. follow the cleavage, we prepared nuclear extracts and immu- The cells were then incubated with [ S]methionine. After im- noblotted with an antibody against the NH -terminal HSV munoprecipitation and electrophoresis, the COOH-terminal epitope tag. The E525M, S526M, and G529M mutants were fragments were eluted and subjected to automated Edman each cleaved efficiently as judged from the amount of NH - degradation, and the radioactivity released in each cycle was terminal fragment in the nucleus, and all of the cleavages were determined. The protein with the wild-type SREBP-2 sequence suppressed by sterols (Fig. 2B). In the experiment shown in did not show any peak of S radioactivity in any of the early lanes 1– 6, both the cleaved wild-type and E525M proteins cycles (Fig. 4A). The E525M mutant showed a clear peak of S migrated as a doublet. Such doublets are seen rarely and in- radioactivity in cycle 3 from the Edman degradation (Fig. 4B). consistently in our experiments, and their significance is The S526M and G529M mutants showed peaks at cycles 4 and unknown. 7, respectively (Fig. 4, C and D). These findings indicate To determine whether the COOH-terminal fragment could strongly that the COOH-terminal fragment begins with serine be labeled with [ S]methionine and immunoprecipitated, we 523 and that Site-1 cleavage occurs between this residue and 12782 Identification of a Sterol-regulated Cleavage Site in SREBP-2 FIG.5. Immunoblot analysis of HSV-tagged SREBP-2 in 293 cells transfected with wild-type and alanine loop mutants. 293 cells were set up for experiments, transfected with 4 mg of either wild-type pTK-HSV-BP2 or the indicated mutant version of the same plasmid, incubated in the absence or presence of sterols, and fraction- ated as described in the Fig. 1 legend. Aliquots of the nuclear extracts (60 mg of protein) and membranes (80 mg) were subjected to SDS-PAGE TM and immunoblot analysis with 0.5 mg/ml HSV-Tag antibody. The filters were exposed to film for 15 s. M and P denote the cleaved NH -terminal mature and uncleaved precursor forms of SREBP-2, re- spectively. The other bands are present in mock-transfected cells and represent proteins that cross-react with the anti-HSV tag antibody. SREBP-2 sequence, we observed the mature NH -terminal fragment of SREBP-2 in nuclear extracts (Fig. 5, lane 3). This fragment was abolished in the presence of sterols (lane 4). The R519A mutant was not cleaved (lane 5), but the D510A and Q511A mutants were cleaved as efficiently as the wild-type protein (lanes 7 and 9, respectively). Fig. 6 summarizes the results of all 27 alanine scanning experiments. At the top we show the sequences of the lumenal FIG.4. NH -terminal sequence of COOH-terminal fragment of SREBP-2 after cleavage of transfected epitope-tagged SREBP- loops of four of the SREBPs that have been sequenced, two from 2/Ras fusion protein. 293 cells were set up for experiments and the human (5, 6) and two from the hamster (24, 25). Conserved transfected with the wild-type (A) or the indicated methionine mutant residues are highlighted. Only two single alanine substitutions version of pTK-HSV-BP2-Ras-T7 (B–D) plus 2 mg of pVAI and 1 mgof reduced cleavage dramatically: the R519A and the L522A mu- pCMV-SCAP(D443N). On day 2, the cells were radiolabeled with [ S]methionine, harvested, and fractionated, after which the cleaved tations (Mutant No. 11 and 14, respectively). Three of the COOH-terminal fragment of human SREBP-2 was immunoprecipi- multiple alanine replacements reduced cleavage (Mutant No. tated. Aliquots of the immunoprecipitated samples (from 1.3 dishes of 21, 24, and 25). All of these mutations included the L522A cells) were subjected to SDS-PAGE and transferred to polyvinylidene substitution. The other multiple alanine replacements did not fluoride membranes. The membranes were exposed to an imaging plate reduce cleavage. These no-effect mutations included the change and scanned in a Fuji X Bas 1000 phosphorimager. The band containing 519 522 519 522 the COOH-terminal product of the cleavage reaction was excised and of Arg -Ser-Val-Leu to Arg -Ala-Ala-Leu (Mutant No. subjected to multiple cycles of Edman degradation. The radioactivity 23). Cleavage was also not affected when the serine following recovered at each cycle of Edman degradation is plotted. Amino acid the cleavage site was changed to alanine (S523A, Mutant No. sequences shown at the top of each panel correspond to the SREBP-2 15). These data indicate that arginine 519 at the P4 position sequence in the region of the postulated site of sterol-regulated cleavage. and leucine 522 at the P1 position are the only 2 residues in the region of Site-1 that cannot tolerate alanine substitutions. leucine 522 (indicated by the arrows in the amino acid se- To further dissect the requirement for arginine 519, we sys- quences of Fig. 4). tematically replaced this residue with 12 different residues To determine the amino acid residues that are important for (Fig. 7). When lysine was substituted for this arginine, only a cleavage of SREBP-2 at Site-1, we made a systematic series of partial loss of cleavage activity was observed. Two negatively mutations in which alanine was individually substituted for charged residues (glutamic acid and aspartic acid) permitted a most of the amino acids in the lumenal loop between the two barely detectable level of cleavage. All other substitutions abol- membrane-spanning sequences. The expression vector was a ished cleavage. The location of arginine 519 was critical. Cleav- cDNA encoding SREBP-2 with an NH -terminal epitope tag age was abolished when we replaced arginine 519 with alanine derived from the HSV glycoprotein. Expression was driven by and then inserted a new arginine at positions 517, 520, or 521. the relatively weak thymidine kinase promoter, which pro- These mutations effectively moved the position of the arginine duces sufficient SREBP-2 to allow detection, yet not so much as either toward the NH terminus or COOH terminus. to overwhelm the system for regulated proteolysis (2). Trans- We also tested the specificity of the requirement for leucine fected 293 cells were incubated in inducing medium plus or 522 at the cleavage site (Fig. 8). Cleavage was markedly re- minus sterols, after which nuclear extracts and membrane duced but not totally abolished when this residue was changed fractions were subjected to SDS-PAGE and immunoblotted to arginine, alanine, or phenylalanine. It was abolished when with an antibody against the NH -terminal epitope tag. leucine 522 was changed to glutamic acid or valine. The residue Fig. 5 shows illustrative results from one of the alanine at the P91 position was not critical. This residue is serine in scanning experiments. When the cDNA encoded the wild-type human and hamster SREBP-2, but it is glutamic acid and Identification of a Sterol-regulated Cleavage Site in SREBP-2 12783 FIG.7. Schematic diagram of the loop region of SREBP and summary of mutational analysis at arginine 519. Amino acid alignment of the loop region between transmembrane (TM) regions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown as in Fig. 6. The arginine at position 519 in human SREBP-2 was mutated to the indicated amino acid. The cleavage of each mutant relative to that of wild-type human SREBP-2 is shown at the right. FIG.6. Schematic diagram of the loop region of SREBP and summary of the results of alanine point mutations. Amino acid alignment of the loop region between transmembrane (TM) regions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown at the top. Highlighted letters denote amino acid residues that are identical in all four SREBPs. Residues individually mutated to alanine in human SREBP-2 are indicated below the alignment. The cleavage of each mutant relative to that of wild-type human SREBP-2, as determined by immunoblot analysis of transfected cells (see Fig. 5), is shown at the right. A value of 0 denotes undetectable cleavage; a value of 41 denotes cleavage of the mutant protein equivalent to that of wild-type human SREBP-2. glycine in hamster and human SREBP-1, respectively. Re- placement of serine 523 in human SREBP-2 with glutamic acid or glycine preserved normal cleavage. The cleavage was also FIG.8. Schematic diagram of the loop region of SREBP and unaffected when this residue was changed to arginine, alanine, summary of mutational analysis at leucine 522 and serine 523. leucine, or phenylalanine. A moderate reduction was observed Amino acid alignment of the loop region between transmembrane re- when it was changed to cysteine. gions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown To determine whether the second transmembrane domain as in Fig. 6. The leucine at position 522 and the serine at position 523 in human SREBP-2 were individually mutated to the indicated amino contributes to recognition by the Site-1 protease, we replaced acids. The cleavage of each mutant relative to that of wild-type human this sequence with the membrane-spanning region of another SREBP-2 is shown at the right. protein, namely, the LDL receptor (Fig. 9). The LDL receptor is a type 1 transmembrane protein of the plasma membrane with a single membrane-spanning segment oriented with its NH inserted into the lumenal loop (4). We were forced to insert a terminus in the extracellular space and its COOH terminus in long protein segment because shorter epitopes were not recog- the cytosol (18). This orientation is the same as the orientation nized by antibodies after insertion into the lumenal loop of of the second transmembrane domain of SREBP-2 (4). The SREBP-2. Sealed membrane vesicles were prepared from cells chimeric protein containing the LDL receptor transmembrane expressing this protein, and the segment containing the domain was cleaved as efficiently as the wild-type protein, as epitope was shown to be protected from digestion by trypsin in judged by the amount of NH -terminal fragment found in the the absence, but not the presence, of detergents. The epitope- nucleus (Fig. 9, lane 5). Cleavage was abolished by sterols (lane containing segment was also demonstrated to undergo 6). It was also abolished when arginine 519 of the chimera was N-linked glycosylation. The problem with these studies is that changed to alanine (lane 7). These data indicate that the pre- the chimeric protein was not cleaved by the Site-1 protease. cise sequence of the second transmembrane domain is not Therefore, we could not be certain that the long epitope-con- important for cleavage at Site-1 (or Site-2). A hydrophobic taining peptide had not altered the orientation of the protein. sequence at this position is required, however. When the sec- To circumvent this problem, we took advantage of the obser- ond transmembrane domain was deleted without replacement vation that the precise sequence on the NH -terminal side of by another transmembrane domain (HSV-SREBP-2D535–560), arginine 519 is not essential for Site-1 cleavage. Therefore, we cleavage at Site-1 and -2 was abolished (data not shown). prepared a plasmid encoding forms of SREBP-2 in which we Previously we presented evidence that the lumenal loop se- inserted either 3 or 9 amino acids into this sequence in place of quence is indeed in the ER lumen (4). This evidence was based serine 515 (Fig. 10A). The inserted amino acids included either on studies of a cDNA encoding a chimeric protein that con- one or two sites for N-linked glycosylation (Asn-Xaa-Ser/Thr). tained an epitope-tagged peptide segment (218 amino acids) After transfection, membrane pellets containing the SREBP-2 12784 Identification of a Sterol-regulated Cleavage Site in SREBP-2 FIG. 10. Insertion of N-linked glycosylation sites into the lu- menal 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, glycosi- dase treatment of transfected SREBP-2. Aliquots of the 10 3 g mem- FIG.9. Sterol-regulated cleavage of epitope-tagged SREBP-2/ brane fraction from 293 cells transfected with the indicated cDNA were LDLRTM chimeric protein in transfected 293 cells. A, schematic boiled for 5 min as described under “Experimental Procedures” and diagram of the chimeric protein encoded by pTK-HSV-BP2/LDLRTM, incubated for2hat37 °C with one of the following glycosidases: lanes showing the replacement of the second transmembrane domain of hu- 1, 5, and 6, none; lanes 2 and 7, 0.038 IU of peptide N-glycosidase F man SREBP-2 with the transmembrane domain of the LDL receptor. B, (PNGase F); lanes 3 and 8, 0.25 IU of endoglycosidase H; and lanes 4 293 cells were set up for experiments, transfected with 4 mg of either and 9, 0.83 IU of neuraminidase. Aliquots of the membrane fraction (50 wild-type pTK-HSV-BP2 or the indicated mutant plasmid, incubated in mg) were subjected to SDS-PAGE and immunoblot analysis with 0.5 the absence or presence of sterols, and fractionated as described in the TM mg/ml HSV-Tag antibody. The filter was exposed to film for 10 s. legend of Fig. 1. Aliquots of the nuclear extracts (60 mg of protein) and Asterisk (p) denotes an immunoreactive protein that is present in mem- membranes (80 mg) were subjected to SDS-PAGE and immunoblot TM branes from mock-transfected cells (lane 5). C, proteolytic processing of analysis with 0.5 mg/ml HSV-Tag antibody. The filters were exposed HSV-tagged SREBP-2 with inserted N-linked glycosylation sites in the to film for 7 s. M and P denote the cleaved NH -terminal mature and lumenal loop. Aliquots of nuclear extracts (60 mg of protein) and mem- uncleaved precursor forms of SREBP-2, respectively. The other bands branes (80 mg) from 293 cells transfected with the indicated plasmid are present in mock-transfected cells and represent proteins that cross- and incubated in the absence or presence of sterols as described in the react with the anti-HSV tag antibody. legend to Fig. 1 were subjected to SDS-PAGE and immunoblot analysis TM with 0.5 mg/ml HSV-Tag antibody. The filters for nuclear extracts and membranes were exposed to film for 5 min and 20 s, respectively. P precursor were digested with glycosidases, and the change in and M denote the uncleaved precursor and cleaved NH -terminal ma- mobility on SDS-PAGE was determined by immunoblotting. As ture forms of SREBP-2, respectively. The other bands are present in shown in Fig. 10B, the mobility of the SREBP-2 precursors mock-transfected cells and represent proteins that cross-react with the anti-HSV tag antibody. with either one or two glycosylation sites was increased by treatment with peptide N-glycosidase F (lanes 2 and 7) and by endoglycosidase H (lanes 3 and 8), but not by neuraminidase migrated even slower (lanes 9 and 10). Under inducing condi- (lanes 4 and 9). The changes were greater for the construct with tions the nuclear extracts contained the NH -terminal frag- two glycosylation sites. This pattern indicates that the lumenal ments of all proteins except the R519A mutant. Cleavage of all loop sequence contained N-linked carbohydrates of the high proteins was suppressed by sterols. This experiment demon- mannose type. The lack of processing to an endoglycosidase strates that the glycosylated precursor of SREBP-2 remained H-resistant form indicates that the precursor is located in the susceptible to sterol-regulated cleavage at Site-1 and subse- ER and not the Golgi complex. Fig. 10C shows immunoblots of quent cleavage at Site-2. The presence of the carbohydrate nuclear extracts and membrane pellets from cells expressing chains did, however, impede cleavage. The absolute amount of SREBP-2 with the wild-type lumenal sequence, the R519A cleavage was reduced by ;50% for the construct containing one mutation, or the insertion of one or two N-linked glycosylation glycosylation site and by ;80 –90% for the protein containing sites. The cells were incubated under conditions that induce two glycosylation sites. SREBP-2 cleavage (2 sterols) or suppress cleavage (1 sterols). Considered together, the data in this paper indicate that the The membrane-bound precursor form with one glycosylation Site-1 protease cleaves the peptide bond between leucine 522 site (lanes 7 and 8) migrated slower than the wild-type protein and serine 523 in the lumenal loop of SREBP-2. The only (lanes 3 and 4), and the form with two glycosylation sites residues that seem to be crucial for recognition are arginine 519 Identification of a Sterol-regulated Cleavage Site in SREBP-2 12785 Chem. 270, 29422–29427 and leucine 522. The location of the arginine relative to the 5. Yokoyama, C., Wang, X., Briggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein, leucine also seems to be crucial. The recognition sequence J. L., and Brown, M. S. (1993) Cell 75, 187–197 seems to be RXXL where X can be serine, valine, or alanine, at 6. Hua, X., Yokoyama, C., Wu, J., Briggs, M. R., Brown, M. S., Goldstein, J. L., and Wang, X. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11603–11607 least. The RXXL sequence is conserved in the four hamster and 7. Vallett, S. M., Sanchez, H. B., Rosenfeld, J. M., and Osborne, T. F. (1996) human SREBPs shown in Fig. 6 and also in SREBP-1c/ADD1 J. Biol. Chem. 271, 12247–12253 from the rat (RSMLE) (26) and SREBP/HLH106 from Drosoph- 8. Ericsson, J., Jackson, S. M., and Edwards, P. A. (1996) J. Biol. Chem. 271, 24359 –24364 ila (RRILS) (27). We believe that other features of the lumenal 9. Guan, G., Jiang, G., Koch, R. L., and Shechter, I. (1995) J. Biol. Chem. 270, loop are also crucial for cleavage because moving the RXXL 21958 –21965 10. Kim, J. B., and Spiegelman, B. M. (1996) Genes & Dev. 10, 1096 –1107 sequence to other sites in the lumenal loop substantially re- 11. Bennett, M. K., Lopez, J. M., Sanchez, H. B., and Osborne, T. F. (1995) J. Biol. duced cleavage (data not shown). Chem. 270, 25578 –25583 Although the current studies identify the site within 12. Shimano, H., Horton, J. D., Hammer, R. E., Shimomura, I., Brown, M. S., and Goldstein, J. L. (1996) J. Clin. Invest. 98, 1575–1584 SREBP-2 that is cleaved by the Site-1 protease, they do not 13. Hua, X., Wu, J., Goldstein, J. L., Brown, M. S., and Hobbs, H. H. (1995) reveal where in the cell this cleavage occurs. Immunofluores- Genomics 25, 667– 673 cence (24) and cell fractionation studies have shown that 14. Shimomura, I., Shimano, H., Horton, J. D., Goldstein, J. L., and Brown, M. S. (1997) J. Clin. Invest., 99, 838 – 845 SREBPs are initially found on membranes of the nuclear en- 15. Shimano, H., Horton, J. D., Shimomura, I., Hammer, R. E., Brown, M. S., and velope and ER. We do not yet know whether the Site-1 protease Goldstein, J. L. (1997) J. Clin. Invest., 99, 846 – 854 operates in these organelles or whether the SREBPs must be 16. Hua, X., Nohturfft, A., Goldstein, J. L., and Brown, M. S. (1996) Cell 87, 415– 426 transported to some other site where cleavage takes place. 17. James, G. L., Brown, M. S., Cobb, M. H., and Goldstein, J. L. (1994) J. Biol. Chem. 269, 27705–27714 Acknowledgments—We thank Mark Daris, Gloria Brunschede, and 18. Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Georgeanna Cantrell for excellent technical assistance; Constance Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27–38 Martinelli for invaluable help with tissue culture; Jeff Cormier and 19. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, Michelle Laremore for synthesis of oligonucleotides and DNA sequenc- 367–382 ing; and Dr. Clive Slaughter and Carolyn Moomaw for excellent help 20. Bollag, D. M., and Edelstein, S. J. (1991) Protein Methods, p. 100, Wiley-Liss, with radiochemical amino acid sequence analysis. Inc., New York 21. Akusja ¨ rvi, G., Svensson, C., and Nygard, O. (1987) Mol. Cell. Biol. 7, 549 –551 REFERENCES 22. Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715–724 1. Wang, X., Sato, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994) Cell 77, 23. Tsai, D. E., Kenan, D. J., and Keene, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 53– 62 89, 8864 – 8868 2. Hua, X., Sakai, J., Brown, M. S., and Goldstein, J. L. (1996) J. Biol. Chem. 271, 10379 –10384 24. Sato, R., Yang, J., Wang, X., Evans, M. J., Ho, Y. K., Goldstein, J. L., and Brown, M. S. (1994) J. Biol. Chem. 269, 17267–17273 3. Sakai, J., Duncan, E. A., Rawson, R. B., Hua, X., Brown, M. S., and Goldstein, J. L. (1996) Cell 85, 1037–1046 25. Yang, J., Brown, M. S., Ho, Y. K., and Goldstein, J. L. (1995) J. Biol. Chem. 4. Hua, X., Sakai, J., Ho, Y. K., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. 270, 12152–12161 26. Tontonoz, P., Kim, J. B., Graves, R. A., and Spiegelman, B. M. (1993) Mol. Cell. Biol. 13, 4753– 4759 J. Sakai, E. A. Duncan, M. S. Brown, and J. L. Goldstein, unpub- 27. Theopold, U., Ekengren, S., and Hultmark, D. (1996) Proc. Natl. Acad. Sci. lished observations. U. S. A. 93, 1195–1199 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2

Duncan, Elizabeth A.; Brown, Michael S.; Goldstein, Joseph L.; Sakai, Juro
Journal of Biological Chemistry – May 1, 1997
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0021-9258
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10.1074/jbc.272.19.12778
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 19, Issue of May 9, pp. 12778 –12785, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2* (Received for publication, January 30, 1997, and in revised form March 6, 1997) Elizabeth A. Duncan‡, Michael S. Brown, Joseph L. Goldstein§, and Juro Sakai From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235 A sterol-regulated protease initiates release of the envelope. Each SREBP is composed of three segments: 1) an NH -terminal segments of sterol regulatory element- NH -terminal segment of ;485 amino acids that is a transcrip- binding proteins (SREBPs) from cell membranes, tion factor of the basic helix-loop-helix-leucine zipper family, 2) thereby allowing them to enter the nucleus and to stim- a membrane attachment segment of ;75 amino acids composed ulate transcription of genes involved in the uptake and of two membrane-spanning sequences separated by a short synthesis of cholesterol and fatty acids. Using SREBP-2 hydrophilic loop of 31 amino acids, and 3) a COOH-terminal as a prototype, we here identify the site of sterol-regu- segment of ;585 amino acids that plays a regulatory role. The 522 523 lated cleavage as the Leu -Ser bond in the middle of proteins are oriented so that the NH - and COOH-terminal the 31-residue hydrophilic loop that projects into the segments project into the cytoplasm, and only the short hydro- lumen of the endoplasmic reticulum and nuclear enve- philic loop projects into the lumen of the ER or nuclear lope. This site was identified through use of a vector envelope (4). encoding an SREBP-2/Ras fusion protein with a triple Before it can activate transcription, the NH -terminal seg- epitope tag that allowed immunoprecipitation of the ment is released from the membrane in a complex two-step cleaved COOH-terminal fragment. The NH terminus of proteolytic sequence (2, 3). First, a protease cleaves the protein this fragment was pinpointed by radiochemical se- at Site-1, which is near an arginine in the lumenal loop, quencing after replacement of selected codons with me- thereby breaking the attachment between the two transmem- thionine codons and labeling the cells with [ S]methi- brane sequences. This allows a second protease to cleave the onine. Alanine scanning mutagenesis revealed that only protein at Site-2, which is near the middle of the first trans- two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage membrane sequence (2, 3). The NH -terminal fragment leaves site (leucine 522), and 2) the arginine at the P4 position the membrane with a portion of the first transmembrane se- (arginine 519). These define a tetrapeptide sequence, quence still attached. It enters the nucleus, where it activates RXXL, that is necessary for cleavage. Cleavage was not transcription of genes encoding the LDL receptor (5, 6), several affected when the second transmembrane helix of enzymes of cholesterol biosynthesis (3-hydroxy-3-methylglu- SREBP-2 was replaced with the membrane-spanning re- taryl coenzyme A synthase (5, 6), 3-hydroxy-3-methylglutaryl gion of the low density lipoprotein receptor, indicating coenzyme A reductase (7), farnesyl diphosphate synthase (8), that this sequence is not required for regulation. Glyco- and squalene synthase (9)), and enzymes of fatty acid biosyn- sylation-site insertion experiments confirmed that thesis (10, 11) and desaturation (12). The net result is to in- leucine 522 is located in the lumen of the endoplasmic crease the cell’s supply of cholesterol and fatty acids. reticulum. We conclude that the sterol-regulated prote- The Site-1 protease is the target of feedback regulation by ase is a novel enzyme whose active site faces the lumen cholesterol and other sterols (3). When these sterols accumu- of the nuclear envelope, endoplasmic reticulum, or an- late within cells, the rate of proteolysis at Site-1 declines mark- other membrane organelle to which the SREBPs may be edly. Cleavage at Site-2 also declines because this cleavage transported before cleavage. requires prior cleavage at Site-1 (3). As a result, the amounts of nuclear SREBPs decline, and transcription of the target genes falls. The net effect is to prevent overaccumulation of choles- Proteolytic processing of sterol regulatory element-binding 1 terol and fatty acids when intracellular sterol levels are al- proteins (SREBPs) controls the metabolism of cholesterol and ready high. fatty acids in animal cells (1–3). SREBPs are transcription Three isoforms of SREBP are known (5, 13, 14). SREBP-1a factors that are bound to membranes of the ER and nuclear and 1c are derived from a single gene through use of alternate promoters that encode alternate first exons (5, 13, 14). * This work was supported by National Institutes of Health Grant SREBP-1a is much more active than SREBP-1c in stimulating HL20948 and by a research grant from the Perot Family Foundation. transcription of all known target genes (15). The third protein, The costs of publication of this article were defrayed in part by the SREBP-2, is the product of a separate gene (6, 13), and it is also payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to more active than SREBP-1c (15). In view of the regulatory role indicate this fact. of the Site-1 protease, further knowledge of its structure and ‡ Supported by Medical Scientists Training Grant GM08014. mode of regulation is desirable. A first step would be the iden- § To whom correspondence should be addressed: Dept. of Molecular tification of the precise site at which the Site-1 protease cuts Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2141; Fax: 214-648- SREBPs. In previous studies we have shown that this cleavage is abolished when arginine 519 in the lumenal loop of SREBP-2 The abbreviations used are: SREBP, sterol regulatory element- (or the corresponding arginine of SREBP-1a) is changed to binding protein; ER, endoplasmic reticulum; LDL, low density lipopro- alanine by in vitro mutagenesis (2). The size of the cleavage tein; SCAP, SREBP cleavage-activating protein; PAGE, polyacrylamide gel electrophoresis; VAI, virus-associated I. product, as determined by SDS-PAGE, is consistent with cleav- 12778 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Identification of a Sterol-regulated Cleavage Site in SREBP-2 12779 region, and at least two independent clones of each mutant were inde- age at or near this arginine (3). pendently transfected. In the current studies we have used a combination of in vitro Cell Culture, Transfection, and Cell Fractionation—Monolayers of mutagenesis, epitope tagging, immunoprecipitation, and radio- human embryonic kidney 293 cells were set up on day 0 (4 3 10 chemical sequencing to determine the precise location of Site-1 cells/60-mm dish) and cultured in 8 –9% CO at 37 °C in medium A in SREBP-2. We found, surprisingly, that cleavage does not (Dulbecco’s modified Eagle’s medium containing 100 units/ml penicillin occur at arginine 519, but rather it occurs 3 residues further and 100 mg/ml streptomycin) supplemented with 10% (v/v) fetal calf serum (4). On day 2, cells were transfected with 4 mg of pTK empty toward the COOH terminus, namely at leucine 522. Arginine vector (mock) or the indicated plasmid as described (2). Three h after 519 seems to be the NH -terminal residue in a tetrapeptide transfection, the cells were switched to medium B (medium A contain- sequence, RXXL, that serves as a recognition signal for the ing 10% newborn calf lipoprotein-deficient serum, 50 mM compactin, and Site-1 protease. 50 mM sodium mevalonate) in the absence or presence of sterols as indicated in the legends. After incubation for 20 h, the cells received EXPERIMENTAL PROCEDURES N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 TM TM Materials—We obtained HSV-Tag and T7-Tag monoclonal an- mg/ml (2), and the cells were harvested 3 h later (2). The pooled cell tibodies from Novagen; v-H-Ras(Ab-1)-agarose linked monoclonal anti- suspension from 2 dishes was allowed to swell in hypotonic buffer (4) for body was obtained from Oncogene. Protein G-Sepharose 4 Fast Flow 30 min at 0 °C, passed through a 22.5-gauge needle 30 times, and beads were obtained from Pharmacia Biotech Inc., L-[ S]methionine centrifuged at 1000 3 g at 4 °C for 7 min. The 1000 3 g pellet was (.1000 Ci/mmol) was obtained from DuPont NEN, and glycosidases resuspended in 0.1 ml of buffer C (10 mM Hepes-KOH (pH 7.4), 0.42 M were obtained from New England Biolabs. NaCl, 2.5% (v/v) glycerol, 1.5 mM MgCl , 0.5 mM sodium EDTA, 0.5 mM Construction of pTK-HSV-BP2-Ras-T7—pTK-HSV-BP2-Ras-T7 en- sodium EGTA, 1 mM dithiothreitol, and a mixture of protease inhibitors codes an epitope-tagged SREBP-2/Ras fusion protein (1391 amino ac- (4)). The suspension was rotated at 4 °C for 1 h and centrifuged at top ids) consisting of an initiator methionine, two tandem copies of the HSV speed in a microfuge for 15 min at 4 °C. The supernatant is designated epitope (QPELAPEDPED), six novel amino acids (IDGTVP) encoded by nuclear extract. The supernatant from the original 1000 3 g spin was a sequence that consists of restriction sites for BspDI and KpnI, human centrifuged at 10 3 g for 30 min at 4 °C in a Beckman TLA 100.2 rotor, SREBP-2 (amino acids 14 –1141), two novel amino acids (HM) encoded and the pellet was dissolved in 0.1 ml of SDS lysis buffer (1) and by the sequence for restriction site NdeI, human H-Ras (amino acids designated membrane fraction. 2–189), and three tandem copies of the T7 epitope (HMASMTGGQQM- Glycosidase Sensitivity of Membrane-bound SREBP-2—Glycosidase GAAAMASMTGGQQMGGGPMASMTGGQQMGLINM). sensitivity of SREBP-2 was carried out as described (4). Monolayers of pTK-HSV-BP2-Ras-T7 was constructed from a previously described 293 cells were set up on day 0 (4 3 10 cells/60-mm dish) and cultured plasmid, pTK-HSV-BP2-T7 (16), by insertion of a cDNA segment en- as described above. On day 2, the cells were transfected with 7 mgof coding human H-Ras between the sequences for SREBP-2 and the first pTK empty vector (mock), pTK-HSV-BP2-NGT, and pTK-HSV-BP2- copy of the T7 epitope. The nucleotide sequence encoding amino acids NSS/NGT, respectively. Three h after transfection, the cells were 2–189 of human H-Ras was obtained by polymerase chain reaction on switched to medium B in the presence of sterols as described in Fig. 1. pRcCMV-H-Ras (17) with a pair of primers containing an NdeI site at After incubation for 20 h, the cells were harvested, and the pooled cell each 59 end using Pfu DNA polymerase. The amplified product was suspension from four dishes was fractionated. The 10 3 g membrane digested with NdeI and cloned into the unique NdeI site between pellet was washed once with buffer A (4) and resuspended in 180 mlof human SREBP-2 and the three tandem copies of the T7 epitope. Two buffer A containing 1% (v/v) Triton X-100 without protease inhibitors. independent clones were used in each of the transfections. Aliquots of the 10 3 g membrane fraction (0.16 mg in 40 ml of buffer A) Construction of pTK-HSV-BP2/LDLRTM—pTK-HSV-BP2/LDL- were boiled for 5 min in the presence (peptide N-glycosidase F and RTM encodes an epitope-tagged SREBP-2 fusion protein in which a endoglycosidase H reactions) or in the absence (neuraminidase reac- 26-amino acid region that includes the second transmembrane domain tions) of 0.5% (w/v) SDS and 1% (v/v) b-mercaptoethanol for 5 min, after of human SREBP-2 (amino acids 535–560) is replaced with the trans- which the indicated amount of glycosidase was added and incubated at membrane domain of the human LDL receptor (amino acids 708 –729) 37 °Cfor2has described in Fig. 10. (18). To construct pTK-HSV-BP2/LDLRTM, we used oligonucleotide Immunoblot Analysis—Samples of the nuclear extract and the 10 3 site-directed mutagenesis to produce an intermediate plasmid in which g membrane fraction were mixed with 53 SDS loading buffer (20). amino acids 535–560 of human SREBP-2 were replaced by two novel Protein concentration was measured with a BCA kit (Pierce). After amino acids (TG) corresponding to an AgeI restriction site. A pair of SDS-PAGE in 8% gels, proteins were transferred to Hybond-C extra complementary oligonucleotides (top strand, 59-CCGGTGCTCTGTCC- nitrocellulose membranes (Amersham Corp.). Immunoblot analysis was ATTGTCCTCCCCATCGTGCTCCTCGTCTTCCTTTGCCTGGGGGTC- carried out with a horseradish peroxidase detection kit using the Su- TM TTCCTTCTATGGT-39; bottom strand, 59-CCGGACCATACAAGGAAG- perSignal CL-HRP Substrate System according to the manufactur- ACCCCCAGGCAAAGGAAGACGAGGAGCACGATGGGGAGGACAA- er’s instructions except that the nitrocellulose sheets were blocked in TGGACAGAGCA-39) were annealed at 94 – 83 °C for 5 min and then at phosphate-buffered saline containing 0.05% (v/v) Tween 20, 5% (v/v) 83–23 °C for 60 min. These oligonucleotides correspond to amino acids nonfat dry milk, and 5% (v/v) heat-inactivated newborn calf serum. The TM 708 –729 of the human LDL receptor flanked by the single-stranded chimeric proteins were visualized with 0.5 mg/ml HSV-Tag mono- sequence 59-CCGG-39. The annealed oligonucleotides were cloned into clonal antibody or with 10 mg/ml IgG-1C6, a mouse monoclonal antibody the unique AgeI restriction site of the intermediate plasmid (described directed against amino acids 833–1141 of human SREBP-2 (4). Gels above). The resulting pTK-HSV-BP2/LDLRTM encodes an 1157-amino were calibrated with prestained molecular weight markers. Filters acid chimeric protein consisting of an initiator methionine, two tandem were exposed at room temperature to Reflectiony NEF-496 film copies of the HSV epitope, six novel amino acids (IDGTVP), human (DuPont NEN). SREBP-2 (amino acids 14 –534), two novel amino acids (TG), human Metabolic Labeling and Immunoprecipitation of Epitope-tagged LDL receptor (amino acids 708 –729), two novel amino acids (SG), and SREBP-2/Ras Fusion Protein and Its COOH-terminal Fragment— human SREBP-2 (amino acids 561–1141). Monolayers of 293 cells were set up on day 0 (7 3 10 cells/60-mm dish) Plasmid pTK-HSV-BP2(R519A)/LDLRTM is identical to pTK-HSV- and cultured as described above. On day 1, the cells were transfected BP2/LDLRTM except for the R519A point mutation in the lumenal loop with 4 mg of the wild-type or mutant version of pTK-HSV-BP2-Ras-T7, of the SREBP-2 sequence. This plasmid was constructed by site-di- 1 mg of pCMV-SCAP(D443N) (16), and 2 mg of pVAI as described above. rected mutagenesis. pVAI encodes the adenovirus virus-associated I RNA gene, which en- Construction of pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/ hances translation of transfected cDNAs (21). The cells were incubated NGT—pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/NGT encode for 20 h with 50 mM compactin and 50 mM sodium mevalonate, at which epitope-tagged SREBP-2 fusion proteins in which serine 515 in the loop time the medium was changed to 1.3 ml of methionine-free Dulbecco’s region of SREBP-2 is replaced by a novel amino acid sequence contain- modified Eagle’s medium supplemented with 100 units/ml penicillin, ing one or two N-linked glycosylation sites, either SNGT or NSSGSS- 100 mg/ml streptomycin, 10% newborn calf lipoprotein-deficient serum, GNGT, respectively. Both plasmids were constructed by site-directed 50 mM compactin, and 50 mM sodium mevalonate. After incubation for mutagenesis. 1hat37 °C,25 mg/ml N-acetyl-leucinal-leucinal-norleucinal was added, Site-directed Mutagenesis—Oligonucleotide site-directed mutagene- and the cells were pulse-labeled with 700 mCi/ml of [ S]methionine for sis was carried out with single-stranded, uracil-containing DNA (19) 6 h at 37 °C. The cells from four dishes were harvested and pooled, and using the Muta-gene Phagemid In Vitro Mutagenesis Version-2 kit the membrane fraction was prepared as described above. (Bio-Rad) (2). The mutations were confirmed by sequencing the relevant The pooled membrane fraction from the four dishes was resuspended 12780 Identification of a Sterol-regulated Cleavage Site in SREBP-2 in 0.1 ml of SDS lysis buffer at room temperature. All subsequent operations were carried out at 4 °C unless otherwise stated. The sus- pension 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 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mg/ml pepstatin A, 25 mg/ml N-acetyl-leucinal- leucinal-norleucinal, and 1 mM dithiothreitol), after which 20 mlof preimmune whole rabbit serum, 40 mg of irrelevant mouse monoclonal antibody IgG-2001 (22), and 0.2 ml of Protein G-Sepharose beads were added. After rotation for 16 h, the mixture was centrifuged at 1000 3 g for 7 min. The resulting supernatant was mixed with an additional 20 ml of preimmune whole rabbit serum, 40 mg of irrelevant monoclonal antibody IgG-2001, and 0.2 ml of Protein G-Sepharose beads, rotated for 2 h, and centrifuged at 1000 3 g for 3–7 min. To the supernatant were added 1 mg of T7-Tag monoclonal antibody, 25 mg of v-H-Ras(Ab- 1)-agarose linked monoclonal antibody, and 20 mg of anti-COOH-termi- nal SREBP-2 monoclonal antibody IgG-1C6. After rotation for 2.5 h, 50 ml of Protein G-Sepharose beads were added, followed by rotation for 2.5 h and centrifugation at 1000 3 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 23 SDS loading buffer (20) containing 10% (v/v) b-mercaptoethanol and boiled for 5 min. After centrifugation at 1000 3 g at room temper- ature for 3 min, the supernatant was transferred to a fresh tube (tube A), and the beads were re-eluted with 40 mlof53 SDS loading buffer containing 25% b-mercaptoethanol and boiled for 5 min. After centrif- ugation at 1000 3 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. NH -terminal Sequence Analysis of S-Labeled COOH-terminal FIG.1. Sterol-regulated cleavage of epitope-tagged SREBP-2/ Fragment of Epitope-tagged SREBP-2 Fusion Protein—Aliquots of the Ras fusion protein in transfected 293 cells. A, schematic diagram immunoprecipitated samples (from 1.3 dishes of cells) were subjected to of the fusion protein encoded by pTK-HSV-BP2-Ras-T7, showing addi- tional COOH-terminal sequences encoding H-Ras followed by three T7 SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride mem- SQ epitope tags. Encircled numbers 1 and 2 denote the two sequential sites branes (Immobilon-P ; Millipore). After drying, the membranes were of proteolytic cleavage. B, immunoblot analysis of the membrane frac- exposed to an imaging plate and scanned in a Fuji X Bas 1000 phos- tion of 293 cells transfected with the wild-type and R519A mutant phorimager. The band containing the COOH-terminal product of the version of pTK-HSV-BP2-Ras-T7. On day 0, 293 cells were set up for cleavage reaction (M ;83,000) was excised and subjected directly to experiments as described under “Experimental Procedures.” On day 2, multiple cycles of Edman degradation on an Applied Biosystems model the cells were transfected with the indicated plasmids as follows: 4 mg 477A sequencer. Fractions from each cycle (148 ml) were collected and of pTK mock vector (lanes 1 and 2), 4 mg of wild-type pTK-HSV-BP2- counted in a scintillation counter. Ras-T7 (lanes 3– 8), or 4 mg of R519A mutant version of pTK-HSV-BP2- Ras-T7 (lanes 9 –14); 2 mg of pVAI (21) (lanes 7, 8, 13, and 14); and 1 mg RESULTS AND DISCUSSION of pCMV-SCAP(D443N) (16) (lanes 5– 8 and 11–14). After transfection, To identify the exact position of Site-1, we prepared a cDNA the cells were incubated in the absence (2) or presence (1)of1 mg/ml (pTK-HSV-BP2-Ras-T7) encoding a triply tagged version of 25-hydroxycholesterol plus 10 mg/ml cholesterol (sterols) as indicated. On day 3, the cells were harvested and fractionated as described under SREBP-2, which we designate SREBP-2/Ras (Fig. 1). The NH “Experimental Procedures.” Aliquots of the membranes (80 mg of pro- terminus contains two copies of an epitope tag from the HSV tein) were subjected to SDS-PAGE and immunoblot analysis with 10 glycoprotein that we used previously (2). At the COOH termi- mg/ml IgG-1C6, an antibody directed to the COOH-terminal domain of nus we inserted amino acids 2–189 of H-Ras followed by three SREBP-2. The filters were exposed to film for 30 s. P and C denote the uncleaved precursor form and the cleaved COOH-terminal fragment of copies of an 11-residue epitope derived from the gene 10 protein SREBP-2, respectively. of bacteriophage T7 (23). The combination of epitopes at the COOH terminus allowed efficient precipitation of the COOH- terminal fragment with a mixture of antibodies directed 83-kDa COOH-terminal fragment, which was the product of against the Ras and T7 epitopes plus a monoclonal antibody cleavage at Site-1 (Fig. 1, lane 3, transfected C). The amount of directed against the COOH-terminal domain of SREBP-2 (see this fragment was reduced in the presence of sterols (lane 4). below). The IgG-1C6 antibody also visualized the 62-kDa COOH-ter- To demonstrate that SREBP-2/Ras is cleaved at Site-1 in a minal fragment of endogenous SREBP-2 (lane 3, endogenous physiologic fashion, we transfected 293 cells with a vector C), and this was also decreased by sterols (lane 4). To test the encoding this construct and another encoding a mutated ver- physiologic relevance of the observed cleavage of SREBP-2/Ras, sion in which arginine 519 was changed to alanine (Fig. 1). We we cotransfected a cDNA encoding the D443N mutant version used the relatively weak thymidine kinase promoter, which of SCAP, a protein that was previously shown to render the produces near physiological levels of this protein (2). Cells were cleavage of SREBPs at Site-1 resistant to suppression by ste- incubated in inducing medium that contains the 3-hydroxy-3- rols (16). Indeed, in the presence of SCAP, sterols no longer methylglutaryl coenzyme A reductase inhibitor compactin to prevented the appearance of the COOH-terminal fragment of block cholesterol synthesis plus a low concentration of meval- SREBP-2/Ras (lane 6). Note that SCAP did not prevent sup- onate to provide nonsterol end products. The medium was pression of the cleavage of endogenous SREBP-2. We believe either devoid of sterols (2 sterols) or it was supplemented with that this is due to the small percentage of cells that received the a mixture of 25-hydroxycholesterol and cholesterol (1 sterols), SCAP cDNA. Unlike SREBP-2/Ras, which is expressed only in which suppresses cleavage at Site-1 (3). Cell membranes were transfected cells, endogenous SREBP-2 is present in all cells in isolated, solubilized with SDS, subjected to SDS-PAGE, and the dish, most of which did not receive the SCAP cDNA. To immunoblotted with IgG-1C6, a monoclonal antibody directed increase the expression of the transfected SREBP-2/Ras chi- against the COOH terminus of SREBP-2 (4). mera, we cotransfected pVAI, which encodes a protein that When cells were transfected with the cDNA encoding the stimulates translation of mRNAs produced by transfected SREBP-2/Ras chimeric protein, the membranes contained the cDNAs (21). In the presence of pVAI plus pSCAP, the COOH- Identification of a Sterol-regulated Cleavage Site in SREBP-2 12781 FIG.2. Immunoblot analysis of epitope-tagged SREBP-2/Ras fusion protein in 293 cells transfected with wild-type and me- thionine loop mutants. A, amino acid sequence of the lumenal loop region of human SREBP-2. The first amino acid in the loop region follows the first transmembrane domain (TM1) and is designated resi- due 503. Residues individually mutated to methionine in the epitope- 35 FIG.3. Analysis of the [ S]methionine-labeled COOH-terminal tagged SREBP-2/Ras fusion protein are indicated below the sequence. fragment of human SREBP-2 after immunoprecipitation of B, immunoblot analysis of SREBP-2/Ras fusion protein. 293 cells were 5 transfected 293 cells. On day 0, 293 cells were set up at 7 3 10 set up for experiments, transfected with the indicated wild-type or cells/60-mm dish in medium A. On day 1, cells were transfected with mutant version of pTK-HSV-BP2-Ras-T7, incubated in the absence or either 4 mg of pTK mock vector (lanes 1 and 2)or4 mg of pTK-HSV- presence of sterols, and fractionated as described in the legend of Fig. 1. BP2-Ras-T7, 2 mg of pVAI, and 1 mg of pCMV-SCAP(D443N) (lanes 3 Aliquots of the nuclear extracts (60 mg of protein) were subjected to 35 and 4). On day 2, cells were radiolabeled with [ S]methionine, har- TM SDS-PAGE and immunoblot analysis with 0.5 mg/ml HSV-Tag anti- vested, and subjected to immunoprecipitation. The immunoprecipitated body. The filters were exposed to film for 7 s. M denotes the cleaved samples (from 0.5 dish of cells in lanes 1 and 3 and 2.5 dishes in lanes NH -terminal mature form of the SREBP-2/Ras fusion protein. The 2 and 4) were subjected to SDS-PAGE, after which the nitrocellulose other bands are present in mock-transfected cells and represent pro- filter was exposed to an imaging plate for 15 min at room temperature teins that cross-react with the anti-HSV tag antibody. 35 to detect S radioactivity (A). The same filter was subjected to immu- noblot analysis with 10 mg/ml IgG-1C6, an antibody directed against the COOH-terminal domain of SREBP-2 (B). Chemiluminescence from terminal fragment of the chimeric protein was increased by at the bound secondary antibody was detected by exposure to x-ray film for least 8-fold, and there was still no suppression by sterols (lanes 1s. Asterisk (p) denotes the 83-kDa COOH-terminal fragment of the 7 and 8). The R519A mutant of the chimeric protein did not give SREBP-2/Ras fusion protein. rise to detectable amounts of COOH-terminal cleavage product in either the absence or presence of SCAP (lanes 9 –12). In the transfected 293 cells with a cDNA encoding the SREBP-2/Ras presence of pVAI plus pSCAP, a trace amount of the R519A chimera and labeled the cells with [ S]methionine. A mem- mutant COOH-terminal fragment was detectable, but its brane fraction was prepared, solubilized with detergents, and amount was reduced by at least 12-fold when compared with immunoprecipitated with a mixture of three antibodies di- the wild-type COOH-terminal fragment (compare lanes 13 and rected against H-Ras, the T7 epitope tag, and the COOH- 14 with lanes 7 and 8). These data indicate that Site-1 cleavage terminal segment of SREBP-2. The immunoprecipitate was of SREBP-2/Ras obeys all of the rules established for wild-type subjected to SDS-PAGE and phosphorimager analysis, which SREBP-2. Moreover, as discussed below, the use of pVAI and revealed a band of ;83 kDa that was absent from mock-trans- pSCAP enabled the production of sufficient amounts of SREBP- fected cells (Fig. 3A). Immunoblot analysis of the precipitate 2/Ras to allow radiochemical sequencing. (Fig. 3B) and the supernatant (data not shown) confirmed that In view of the high specific radioactivity of [ S]methionine, all of the COOH-terminal fragment was precipitated. In future we developed a strategy to use this amino acid for radiochem- experiments, the 83-kDa radiolabeled band was excised from ical sequencing. Assuming that the cleavage site is located near the nitrocellulose membrane and used for radiochemical arginine 519, we searched for amino acids in the region that sequencing. could be replaced with methionine without abolishing cleavage Fig. 4 shows a series of experiments in which 293 cells were at Site-1. Fig. 2 shows two experiments in which methionine transfected with cDNAs encoding the SREBP-2/Ras chimera residues were introduced individually into positions 525, 526, with the wild-type sequence in the lumenal loop or with the or 529 of the triply taggged SREBP-2/Ras protein (Fig. 2A). To three methionine substitutions at positions 525, 526, and 529. follow the cleavage, we prepared nuclear extracts and immu- The cells were then incubated with [ S]methionine. After im- noblotted with an antibody against the NH -terminal HSV munoprecipitation and electrophoresis, the COOH-terminal epitope tag. The E525M, S526M, and G529M mutants were fragments were eluted and subjected to automated Edman each cleaved efficiently as judged from the amount of NH - degradation, and the radioactivity released in each cycle was terminal fragment in the nucleus, and all of the cleavages were determined. The protein with the wild-type SREBP-2 sequence suppressed by sterols (Fig. 2B). In the experiment shown in did not show any peak of S radioactivity in any of the early lanes 1– 6, both the cleaved wild-type and E525M proteins cycles (Fig. 4A). The E525M mutant showed a clear peak of S migrated as a doublet. Such doublets are seen rarely and in- radioactivity in cycle 3 from the Edman degradation (Fig. 4B). consistently in our experiments, and their significance is The S526M and G529M mutants showed peaks at cycles 4 and unknown. 7, respectively (Fig. 4, C and D). These findings indicate To determine whether the COOH-terminal fragment could strongly that the COOH-terminal fragment begins with serine be labeled with [ S]methionine and immunoprecipitated, we 523 and that Site-1 cleavage occurs between this residue and 12782 Identification of a Sterol-regulated Cleavage Site in SREBP-2 FIG.5. Immunoblot analysis of HSV-tagged SREBP-2 in 293 cells transfected with wild-type and alanine loop mutants. 293 cells were set up for experiments, transfected with 4 mg of either wild-type pTK-HSV-BP2 or the indicated mutant version of the same plasmid, incubated in the absence or presence of sterols, and fraction- ated as described in the Fig. 1 legend. Aliquots of the nuclear extracts (60 mg of protein) and membranes (80 mg) were subjected to SDS-PAGE TM and immunoblot analysis with 0.5 mg/ml HSV-Tag antibody. The filters were exposed to film for 15 s. M and P denote the cleaved NH -terminal mature and uncleaved precursor forms of SREBP-2, re- spectively. The other bands are present in mock-transfected cells and represent proteins that cross-react with the anti-HSV tag antibody. SREBP-2 sequence, we observed the mature NH -terminal fragment of SREBP-2 in nuclear extracts (Fig. 5, lane 3). This fragment was abolished in the presence of sterols (lane 4). The R519A mutant was not cleaved (lane 5), but the D510A and Q511A mutants were cleaved as efficiently as the wild-type protein (lanes 7 and 9, respectively). Fig. 6 summarizes the results of all 27 alanine scanning experiments. At the top we show the sequences of the lumenal FIG.4. NH -terminal sequence of COOH-terminal fragment of SREBP-2 after cleavage of transfected epitope-tagged SREBP- loops of four of the SREBPs that have been sequenced, two from 2/Ras fusion protein. 293 cells were set up for experiments and the human (5, 6) and two from the hamster (24, 25). Conserved transfected with the wild-type (A) or the indicated methionine mutant residues are highlighted. Only two single alanine substitutions version of pTK-HSV-BP2-Ras-T7 (B–D) plus 2 mg of pVAI and 1 mgof reduced cleavage dramatically: the R519A and the L522A mu- pCMV-SCAP(D443N). On day 2, the cells were radiolabeled with [ S]methionine, harvested, and fractionated, after which the cleaved tations (Mutant No. 11 and 14, respectively). Three of the COOH-terminal fragment of human SREBP-2 was immunoprecipi- multiple alanine replacements reduced cleavage (Mutant No. tated. Aliquots of the immunoprecipitated samples (from 1.3 dishes of 21, 24, and 25). All of these mutations included the L522A cells) were subjected to SDS-PAGE and transferred to polyvinylidene substitution. The other multiple alanine replacements did not fluoride membranes. The membranes were exposed to an imaging plate reduce cleavage. These no-effect mutations included the change and scanned in a Fuji X Bas 1000 phosphorimager. The band containing 519 522 519 522 the COOH-terminal product of the cleavage reaction was excised and of Arg -Ser-Val-Leu to Arg -Ala-Ala-Leu (Mutant No. subjected to multiple cycles of Edman degradation. The radioactivity 23). Cleavage was also not affected when the serine following recovered at each cycle of Edman degradation is plotted. Amino acid the cleavage site was changed to alanine (S523A, Mutant No. sequences shown at the top of each panel correspond to the SREBP-2 15). These data indicate that arginine 519 at the P4 position sequence in the region of the postulated site of sterol-regulated cleavage. and leucine 522 at the P1 position are the only 2 residues in the region of Site-1 that cannot tolerate alanine substitutions. leucine 522 (indicated by the arrows in the amino acid se- To further dissect the requirement for arginine 519, we sys- quences of Fig. 4). tematically replaced this residue with 12 different residues To determine the amino acid residues that are important for (Fig. 7). When lysine was substituted for this arginine, only a cleavage of SREBP-2 at Site-1, we made a systematic series of partial loss of cleavage activity was observed. Two negatively mutations in which alanine was individually substituted for charged residues (glutamic acid and aspartic acid) permitted a most of the amino acids in the lumenal loop between the two barely detectable level of cleavage. All other substitutions abol- membrane-spanning sequences. The expression vector was a ished cleavage. The location of arginine 519 was critical. Cleav- cDNA encoding SREBP-2 with an NH -terminal epitope tag age was abolished when we replaced arginine 519 with alanine derived from the HSV glycoprotein. Expression was driven by and then inserted a new arginine at positions 517, 520, or 521. the relatively weak thymidine kinase promoter, which pro- These mutations effectively moved the position of the arginine duces sufficient SREBP-2 to allow detection, yet not so much as either toward the NH terminus or COOH terminus. to overwhelm the system for regulated proteolysis (2). Trans- We also tested the specificity of the requirement for leucine fected 293 cells were incubated in inducing medium plus or 522 at the cleavage site (Fig. 8). Cleavage was markedly re- minus sterols, after which nuclear extracts and membrane duced but not totally abolished when this residue was changed fractions were subjected to SDS-PAGE and immunoblotted to arginine, alanine, or phenylalanine. It was abolished when with an antibody against the NH -terminal epitope tag. leucine 522 was changed to glutamic acid or valine. The residue Fig. 5 shows illustrative results from one of the alanine at the P91 position was not critical. This residue is serine in scanning experiments. When the cDNA encoded the wild-type human and hamster SREBP-2, but it is glutamic acid and Identification of a Sterol-regulated Cleavage Site in SREBP-2 12783 FIG.7. Schematic diagram of the loop region of SREBP and summary of mutational analysis at arginine 519. Amino acid alignment of the loop region between transmembrane (TM) regions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown as in Fig. 6. The arginine at position 519 in human SREBP-2 was mutated to the indicated amino acid. The cleavage of each mutant relative to that of wild-type human SREBP-2 is shown at the right. FIG.6. Schematic diagram of the loop region of SREBP and summary of the results of alanine point mutations. Amino acid alignment of the loop region between transmembrane (TM) regions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown at the top. Highlighted letters denote amino acid residues that are identical in all four SREBPs. Residues individually mutated to alanine in human SREBP-2 are indicated below the alignment. The cleavage of each mutant relative to that of wild-type human SREBP-2, as determined by immunoblot analysis of transfected cells (see Fig. 5), is shown at the right. A value of 0 denotes undetectable cleavage; a value of 41 denotes cleavage of the mutant protein equivalent to that of wild-type human SREBP-2. glycine in hamster and human SREBP-1, respectively. Re- placement of serine 523 in human SREBP-2 with glutamic acid or glycine preserved normal cleavage. The cleavage was also FIG.8. Schematic diagram of the loop region of SREBP and unaffected when this residue was changed to arginine, alanine, summary of mutational analysis at leucine 522 and serine 523. leucine, or phenylalanine. A moderate reduction was observed Amino acid alignment of the loop region between transmembrane re- when it was changed to cysteine. gions 1 and 2 of hamster and human SREBP-1 and SREBP-2 is shown To determine whether the second transmembrane domain as in Fig. 6. The leucine at position 522 and the serine at position 523 in human SREBP-2 were individually mutated to the indicated amino contributes to recognition by the Site-1 protease, we replaced acids. The cleavage of each mutant relative to that of wild-type human this sequence with the membrane-spanning region of another SREBP-2 is shown at the right. protein, namely, the LDL receptor (Fig. 9). The LDL receptor is a type 1 transmembrane protein of the plasma membrane with a single membrane-spanning segment oriented with its NH inserted into the lumenal loop (4). We were forced to insert a terminus in the extracellular space and its COOH terminus in long protein segment because shorter epitopes were not recog- the cytosol (18). This orientation is the same as the orientation nized by antibodies after insertion into the lumenal loop of of the second transmembrane domain of SREBP-2 (4). The SREBP-2. Sealed membrane vesicles were prepared from cells chimeric protein containing the LDL receptor transmembrane expressing this protein, and the segment containing the domain was cleaved as efficiently as the wild-type protein, as epitope was shown to be protected from digestion by trypsin in judged by the amount of NH -terminal fragment found in the the absence, but not the presence, of detergents. The epitope- nucleus (Fig. 9, lane 5). Cleavage was abolished by sterols (lane containing segment was also demonstrated to undergo 6). It was also abolished when arginine 519 of the chimera was N-linked glycosylation. The problem with these studies is that changed to alanine (lane 7). These data indicate that the pre- the chimeric protein was not cleaved by the Site-1 protease. cise sequence of the second transmembrane domain is not Therefore, we could not be certain that the long epitope-con- important for cleavage at Site-1 (or Site-2). A hydrophobic taining peptide had not altered the orientation of the protein. sequence at this position is required, however. When the sec- To circumvent this problem, we took advantage of the obser- ond transmembrane domain was deleted without replacement vation that the precise sequence on the NH -terminal side of by another transmembrane domain (HSV-SREBP-2D535–560), arginine 519 is not essential for Site-1 cleavage. Therefore, we cleavage at Site-1 and -2 was abolished (data not shown). prepared a plasmid encoding forms of SREBP-2 in which we Previously we presented evidence that the lumenal loop se- inserted either 3 or 9 amino acids into this sequence in place of quence is indeed in the ER lumen (4). This evidence was based serine 515 (Fig. 10A). The inserted amino acids included either on studies of a cDNA encoding a chimeric protein that con- one or two sites for N-linked glycosylation (Asn-Xaa-Ser/Thr). tained an epitope-tagged peptide segment (218 amino acids) After transfection, membrane pellets containing the SREBP-2 12784 Identification of a Sterol-regulated Cleavage Site in SREBP-2 FIG. 10. Insertion of N-linked glycosylation sites into the lu- menal 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, glycosi- dase treatment of transfected SREBP-2. Aliquots of the 10 3 g mem- FIG.9. Sterol-regulated cleavage of epitope-tagged SREBP-2/ brane fraction from 293 cells transfected with the indicated cDNA were LDLRTM chimeric protein in transfected 293 cells. A, schematic boiled for 5 min as described under “Experimental Procedures” and diagram of the chimeric protein encoded by pTK-HSV-BP2/LDLRTM, incubated for2hat37 °C with one of the following glycosidases: lanes showing the replacement of the second transmembrane domain of hu- 1, 5, and 6, none; lanes 2 and 7, 0.038 IU of peptide N-glycosidase F man SREBP-2 with the transmembrane domain of the LDL receptor. B, (PNGase F); lanes 3 and 8, 0.25 IU of endoglycosidase H; and lanes 4 293 cells were set up for experiments, transfected with 4 mg of either and 9, 0.83 IU of neuraminidase. Aliquots of the membrane fraction (50 wild-type pTK-HSV-BP2 or the indicated mutant plasmid, incubated in mg) were subjected to SDS-PAGE and immunoblot analysis with 0.5 the absence or presence of sterols, and fractionated as described in the TM mg/ml HSV-Tag antibody. The filter was exposed to film for 10 s. legend of Fig. 1. Aliquots of the nuclear extracts (60 mg of protein) and Asterisk (p) denotes an immunoreactive protein that is present in mem- membranes (80 mg) were subjected to SDS-PAGE and immunoblot TM branes from mock-transfected cells (lane 5). C, proteolytic processing of analysis with 0.5 mg/ml HSV-Tag antibody. The filters were exposed HSV-tagged SREBP-2 with inserted N-linked glycosylation sites in the to film for 7 s. M and P denote the cleaved NH -terminal mature and lumenal loop. Aliquots of nuclear extracts (60 mg of protein) and mem- uncleaved precursor forms of SREBP-2, respectively. The other bands branes (80 mg) from 293 cells transfected with the indicated plasmid are present in mock-transfected cells and represent proteins that cross- and incubated in the absence or presence of sterols as described in the react with the anti-HSV tag antibody. legend to Fig. 1 were subjected to SDS-PAGE and immunoblot analysis TM with 0.5 mg/ml HSV-Tag antibody. The filters for nuclear extracts and membranes were exposed to film for 5 min and 20 s, respectively. P precursor were digested with glycosidases, and the change in and M denote the uncleaved precursor and cleaved NH -terminal ma- mobility on SDS-PAGE was determined by immunoblotting. As ture forms of SREBP-2, respectively. The other bands are present in shown in Fig. 10B, the mobility of the SREBP-2 precursors mock-transfected cells and represent proteins that cross-react with the anti-HSV tag antibody. with either one or two glycosylation sites was increased by treatment with peptide N-glycosidase F (lanes 2 and 7) and by endoglycosidase H (lanes 3 and 8), but not by neuraminidase migrated even slower (lanes 9 and 10). Under inducing condi- (lanes 4 and 9). The changes were greater for the construct with tions the nuclear extracts contained the NH -terminal frag- two glycosylation sites. This pattern indicates that the lumenal ments of all proteins except the R519A mutant. Cleavage of all loop sequence contained N-linked carbohydrates of the high proteins was suppressed by sterols. This experiment demon- mannose type. The lack of processing to an endoglycosidase strates that the glycosylated precursor of SREBP-2 remained H-resistant form indicates that the precursor is located in the susceptible to sterol-regulated cleavage at Site-1 and subse- ER and not the Golgi complex. Fig. 10C shows immunoblots of quent cleavage at Site-2. The presence of the carbohydrate nuclear extracts and membrane pellets from cells expressing chains did, however, impede cleavage. The absolute amount of SREBP-2 with the wild-type lumenal sequence, the R519A cleavage was reduced by ;50% for the construct containing one mutation, or the insertion of one or two N-linked glycosylation glycosylation site and by ;80 –90% for the protein containing sites. The cells were incubated under conditions that induce two glycosylation sites. SREBP-2 cleavage (2 sterols) or suppress cleavage (1 sterols). Considered together, the data in this paper indicate that the The membrane-bound precursor form with one glycosylation Site-1 protease cleaves the peptide bond between leucine 522 site (lanes 7 and 8) migrated slower than the wild-type protein and serine 523 in the lumenal loop of SREBP-2. The only (lanes 3 and 4), and the form with two glycosylation sites residues that seem to be crucial for recognition are arginine 519 Identification of a Sterol-regulated Cleavage Site in SREBP-2 12785 Chem. 270, 29422–29427 and leucine 522. The location of the arginine relative to the 5. Yokoyama, C., Wang, X., Briggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein, leucine also seems to be crucial. The recognition sequence J. L., and Brown, M. S. (1993) Cell 75, 187–197 seems to be RXXL where X can be serine, valine, or alanine, at 6. Hua, X., Yokoyama, C., Wu, J., Briggs, M. R., Brown, M. S., Goldstein, J. L., and Wang, X. (1993) Proc. 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A. 93, 1195–1199

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Published: May 1, 1997

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