From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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In response to sterol deprivation, two sequential
proteolytic cleavages release the NH2-terminal
fragments of sterol regulatory element-binding proteins (SREBPs) from
cell membranes. The fragments translocate to the nucleus where they
activate genes involved in cholesterol and fatty acid metabolism. The
SREBPs are bound to membranes in a hairpin fashion. The
NH2-terminal and COOH-terminal domains face the cytoplasm,
separated by two membrane spanning segments and a short lumenal loop.
The first cleavage occurs at Site-1 in the lumenal loop. The
NH2-terminal fragment is then released by cleavage at
Site-2, which is believed to lie within the first transmembrane
segment. Here, we use a novel cysteine panning method to identify the
second cleavage site (Site-2) in human SREBP-2 as the
Leu484-Cys485 bond that lies at the junction
between the cytoplasmic NH2-terminal fragment and the first
transmembrane segment. We transfected cells with cDNAs encoding
fusion proteins with single cysteine residues at positions to the
NH2-terminal and COOH-terminal sides of cysteine 485. The
NH2-terminal fragments were tested for susceptibility to
modification with
N-(3-maleimidylpropionyl)biocytin,
which attaches a biotin group to cysteine sulfhydryls. Cysteines to the
NH2-terminal side of cysteine 485 were retained on the
NH2-terminal fragment, but cysteines to the COOH-terminal
side of leucine 484 were lost. Leucine 484 is three residues to the
COOH-terminal side of the tetrapeptide Asp-Arg-Ser-Arg, which
immediately precedes the first transmembrane segment and is required
for Site-2 cleavage.
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INTRODUCTION |
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Cells maintain cholesterol homeostasis through a feedback pathway that depends upon the regulated proteolytic processing of a pair of membrane-bound transcription factors, sterol regulatory element-binding proteins-1 and -2 (SREBP-1 and SREBP-2)1 (reviewed in Ref. 1). Cells deprived of cholesterol process the SREBPs proteolytically to release the NH2-terminal domains which travel to the nucleus to activate transcription of genes that regulate several metabolic pathways: uptake of cholesterol and fatty acids (low density lipoprotein receptor and lipoprotein lipase) (1, 2), synthesis of cholesterol (3-hydroxy-3-methylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, farnesyl diphosphate synthase, squalene synthase) (1, 3-5), synthesis of fatty acids (acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase) (1, 2, 6, 7), and synthesis of triglycerides (glycerol-3-phosphate acyltransferase) (8). When cells accumulate sterols, proteolysis of the SREBPs is suppressed, nuclear SREBPs decline, and the transcription of the target genes is reduced. This regulation assures a steady supply of cholesterol and fatty acids while preventing their overaccumulation.
SREBPs are unique among transcription factors because they are synthesized as membrane-bound precursors with three domains. The precursors are bound to the endoplasmic reticulum and nuclear envelope in a hairpin orientation (9). The cytoplasmic NH2-terminal domain (~480 amino acids) contains an acidic transcriptional activation sequence and a basic helix-loop-helix leucine zipper element that is responsible for DNA binding and dimerization. The middle segment (~80 amino acids) consists of two-membrane spanning helices separated by a short hydrophilic loop of 31 amino acids that projects into the lumen of the endoplasmic reticulum and nuclear envelope. The COOH-terminal segment (~590 amino acids) projects into the cytoplasm. It is designated the regulatory domain because it is necessary for the sterol-regulated proteolysis of the SREBPs (10, 11).
In response to sterol deprivation, the NH2-terminal domains of the SREBPs are released from membranes by a two-step proteolytic cascade (1, 12). The first proteolytic cleavage is regulated by the putative sterol sensor, SCAP (SREBP cleavage-activating protein) (13), which forms a complex with the COOH-terminal regulatory domain of the SREBPs (10, 11). The complex triggers the first cleavage, which occurs at Site-1 in the middle of the lumenal loop (14). This separates the NH2-terminal half of the SREBP from the COOH-terminal half, but both halves remain membrane bound. The membrane-bound NH2-terminal fragment of SREBP is termed the intermediate (12). Next, a second protease, designated the Site-2 protease (S2P), cleaves the NH2-terminal intermediate, apparently within the first transmembrane segment (12, 15). The cleaved NH2-terminal domain now travels to the nucleus where it activates transcription by binding to sterol regulatory elements in the promoters of the genes described above. The activity of S2P is not itself regulated by sterols, but it cleaves the NH2-terminal domain at Site-2 only after the protein has been cleaved at Site-1 (12, 15).
The cDNA encoding S2P, a multiple membrane-spanning protein that is required for Site-2 proteolysis, was recently identified by complementation cloning in a mutant line of Chinese hamster ovary cells (M19 cells) (16) that lack this activity (12, 15). Lacking the ability to cleave the SREBPs at Site-2, M19 cells are unable to transcribe cholesterol biosynthetic genes and are thus cholesterol auxotrophs (12, 15, 16). When transfected into M19 cells, the S2P cDNA restores cleavage at Site-2 and relieves the need for exogenous cholesterol. S2P contains the consensus metalloprotease zinc-binding site, HEXXH (15). Mutation of this site abolishes S2P activity, and it was concluded that S2P is indeed the Site-2 protease (15).
We recently mapped Site-1 of human SREBP-2 to the Leu522-Ser523 bond in the lumenal loop by a combination of mutagenesis, [35S]methionine labeling, and radiochemical sequencing of the cleaved COOH-terminal fragment (14). To estimate the location of Site-2, we previously replaced the NH2 terminus of SREBP-2 with H-Ras and used truncated forms of this fusion protein as size standards for comparison with the fully processed fusion protein (12). These studies suggested that Site-2 is located somewhere within the first transmembrane segment, but the precise peptide bond was not identified. Proteolysis of the SREBPs at Site-2 is abolished when the sequence D478RSR, which is immediately proximal to the first transmembrane segment, is replaced with AS (12).
In designing a strategy to identify the precise location of Site-2, we faced several obstacles. Radiochemical sequencing was not possible because this technique relies upon the ability to isolate the COOH-terminal product of the cleavage reaction (14). Following Site-2 proteolysis, this small fragment (~40 amino acids) is likely to be rapidly degraded. Mass spectrometry could be used to determine the precise mass of the nuclear NH2-terminal fragment. However, this would require the isolation of relatively large amounts of this fragment from cell nuclei. This is not feasible in cultured cells because overexpression of SREBPs by transfection leads to artifactual proteolytic processing (17). Faithful processing is observed only when the cells express low levels of SREBPs through the use of weak promoters, such as the herpes simplex virus (HSV) thymidine kinase promoter (17).
We herein report a novel technique that permits the identification of the precise peptide bond cleaved during proteolysis at Site-2. We produced a fusion protein whose NH2-terminal fragment lacks cysteine. Each residue near Site-2 was singly replaced with cysteine, the released NH2-terminal fragment was isolated, and its susceptibility to a cysteine modification reagent was studied. The results demonstrate that Site-2 cleavage occurs at a bond that appears to lie just inside the first transmembrane segment of human SREBP-2, i.e. the Leu484-Cys485 bond. This cleavage site is located three residues to the COOH-terminal side of the D478RSR sequence that was shown previously to be required for the Site-2 cleavage reaction (12).
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EXPERIMENTAL PROCEDURES |
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Materials--
We obtained IgG-HSV-TagTM monoclonal antibody
from Novagen; streptavidin-agarose from ProZyme, Inc.; Ni-NTA-agarose
from Qiagen; cytochrome c and bovine serum albumin from
Sigma-Aldrich; Rat Brain QUICK-Clone cDNA from
CLONTECH; N-ethylmaleimide (NEM) from U. S. Biochemical Corp.; and
N-(3-maleimidylpropionyl)biocytin
(MPB) from Molecular Probes, Inc. Other reagents were obtained from
previously reported sources (9, 14).
Plasmid Constructions-- The expression vector pTK-HSV-ACBP/BP2 encodes an epitope-tagged ACBP (acyl-CoA-binding protein)/SREBP-2 fusion protein (782 amino acids) consisting of an initiator methionine, two tandem copies of the HSV epitope (QPELAPEDPED) (17), one novel amino acid (I) encoded by the last three bases of an EcoRV restriction site, rat ACBP (amino acids 2-87) (Ref. 18), four novel amino acids (TGAS) that correspond to tandem AgeI and NheI restriction sites, and human SREBP-2 (amino acids 473-1141) (Ref. 19). pTK-HSV-ACBP/BP2 was constructed from a previously described plasmid, pTK-HSV-Ras-BP2 (473-1141) (Ref. 12), by replacing the cDNA for human H-Ras with the cDNA for rat ACBP followed by four novel amino acids (TGAS). To construct pTK-HSV-ACBP/BP2, we first used oligonucleotide site-directed mutagenesis to produce an intermediate plasmid with a unique AgeI site immediately following the H-Ras sequence in pTK-HSV-Ras-BP2(473-1141). The cDNA encoding amino acids 2-87 of rat ACBP was obtained by polymerase chain reaction using rat brain cDNA as a template (CLONTECH), an NH2-terminal primer flanked by an EcoRV site, and a COOH-terminal primer flanked by an AgeI site. The amplified product was digested with EcoRV and AgeI and ligated into the unique EcoRV and AgeI sites of the intermediate plasmid (described above).
The expression vector pTK-HSV-8His-ACBP/BP2 encodes the sequence described above with an insertion of 11 amino acids between the HSV tag and ACBP/BP2. To construct pTK-HSV-8His-ACBP/BP2, a pair of complementary oligonucleotides (top strand, 5'-ATCCATCACCATCACCATCACCATCACACCGGT-3'; bottom strand, 5'-ACCGGTGTGATGGTGATGGTGATGGTGATGGAT-3') were phosphorylated, annealed, and ligated into the unique EcoRV restriction site of pTK-HSV-BP2-ACBP/BP2. The inserted oligonucleotides encode 11 amino acids (isoleucine, 8 histidines, threonine, and glycine). The expression vector pTK-HSV-8His-BP2 encodes the same sequence as pTK-HSV-BP2 (Ref. 17) except for the insertion of 10 amino acids (isoleucine, alanine, and 8 histidines) between the HSV tag and SREBP-2. To construct pTK-HSV-8His-BP2, complementary oligonucleotides (top strand, 5'-CGCACATCACCATCACCATCACCATCACAT-3'; bottom strand, 5'-CGATGTGATGGTGATGGTGATGGTGATGTG-3') were phosphorylated, annealed, and ligated into the unique BspDI restriction site of pTK-HSV-BP2. The structures of the above expression vectors were confirmed by sequencing all ligation junctions and the entire ACBP coding region.Site-directed Mutagenesis-- Oligonucleotide site-directed mutagenesis was carried out with single-stranded, uracil-containing DNA using the Muta-gene Phagemid In Vitro Mutagenesis Version-2 kit (Bio-Rad). The mutations were confirmed by sequencing of the relevant region; at least two independent clones of each mutant were independently transfected.
Transfection of 293 Cells and Cell Fractionation-- Monolayers of human embryonic kidney 293 cells were set up for experiments on day 0 (4 × 105 cells/60-mm dish) and cultured in 8-9% CO2 at 37 °C in medium A (Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 µg/ml streptomycin sulfate) supplemented with 10% (v/v) fetal calf serum. On day 2, the cells were transfected with 4 µg of pTK empty vector (mock) or the indicated plasmid using an MBS Transfection Kit (Stratagene) as described previously (9). Three h after transfection, the cells were switched to medium B (medium A containing 10% newborn calf lipoprotein-deficient serum, 50 µM compactin, and 50 µM sodium mevalonate) (9) in the absence or presence of sterols as indicated in the legends. For transfections involving pTK-HSV-8His-BP2 and its mutant versions, the cells received ALLN (N-acetyl-leucinal-leucinal-norleucinal) at a final concentration of 25 µg/ml 3 h prior to harvest. Cells transfected with constructs encoding ACBP/SREBP-2 fusion proteins did not receive ALLN prior to harvest. Homogenates of cells were fractionated as described previously to yield three fractions: nuclear extract, membranes (105 × g pellet), and cytosol (105 × g supernatant) (12).
Transfection of CHO-7 and M19 Cells and Cell Fractionation-- Monolayers of CHO-7 and M19 cells (15) were set up for experiments on day 0 (7 × 105 cells/60-mm dish) in medium C (a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 µg/ml streptomycin sulfate, 5% (v/v) fetal calf serum, 5 µg/ml cholesterol, 1 mM sodium mevalonate, and 20 µM sodium oleate). On day 1, cells were transfected with 6.5 µg of DNA/dish using LipofectAMINETM Reagent (Life Technologies) as described previously (15), except that all cells were grown in the absence of sterols and the cells did not receive ALLN. Cell fractionation was carried out as described previously (12) with the following modifications. The pooled cell suspension from two dishes was allowed to swell for 45 min at 0 °C in hypotonic buffer containing 50 mM Hepes-KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, and 1 mM sodium EGTA, and protease inhibitors as described previously (9). Samples were passed through a 22.5-gauge needle 30 times, and centrifuged at 890 × g at 4 °C for 4 min. The supernatant was centrifuged at 105 × g for 30 min at 4 °C in a Beckman TLA 100.2 rotor, and the resulting pellet was dissolved in 0.1 ml of SDS lysis buffer (20) and designated the membrane fraction. The supernatant was centrifuged again at 105 × g for 30 min at 4 °C in a Beckman TLA 100.2 rotor. The cytosol fraction was prepared by precipitating the final 105 × g supernatant with 5 volumes of acetone for 10 min at 0 °C, followed by centrifugation at top speed in a microcentrifuge at 4 °C for 15 min and resuspension of the pellet in 0.1 ml of SDS lysis buffer.
SDS-PAGE Immunoblot Analysis-- Protein concentration was measured with a BCA kit (Pierce). Samples of nuclear extract, 105 × g membrane fraction, and cytosol were mixed with 5 × SDS loading buffer (21) and boiled for 5 min. SDS-PAGE was carried out using gels of 7, 12, or 15% acrylamide. Proteins were transferred to Hybond-C extra nitrocellulose membranes (Amersham Corp.), and immunoblot analysis was carried out with IgG-HSV-TagTM monoclonal antibody as described previously (14). Bound antibody was visualized with peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratory) using the Super SignalTM CL-HRP Substrate System (Pierce) as described previously (14).
Tricine SDS-PAGE Immunoblot Analysis-- Separating gels (16.5% T, 3% C, where T denotes the total % concentration of acrylamide and bisacrylamide and C denotes the % concentration of the cross-linker relative to the total concentration of T) and stacking gels (4% T, 3% C) were cast as described by Schägger and von Jagow (22) except that a 18 × 24-cm Vertical Slab Sturdier Electrophoresis Unit (Hoefer) was used. Cytosol fractions from transfected 293 cells were mixed with 3 × Tricine Sample Buffer (Bio-Rad) and boiled for 5 min. Electrophoresis was performed at 50 mA for 23 h at room temperature, and the gels were calibrated with Polypeptide SDS-PAGE Standards (Bio-Rad). Gels were transferred to Hybond-C extra nitrocellulose membranes, and immunoblot analysis was carried out as described above.
Identification of Site-2 Cleavage Site by Streptavidin Precipitation of MPB-derivatized SREBP-2-- Monolayers of 293 cells were set up as described above. On day 2, the cells were transfected with 4 µg/dish of the wild-type or mutant version of either pTK-HSV-8His-ACBP/BP2 or pTK-HSV-8His-BP2, 0.25 µg of pCMV-Myc-S2P (Ref. 15), 0.5 µg of pCMV-SCAP(D443N) (Ref. 13), and 0.5 µg of pVAI (23) using an MBS Transfection Kit as described above (9). The cells were incubated for 20 h in medium B in the absence of sterols and harvested as described above. The cells were then fractionated into nuclear extracts (for pTK-HSV-8His-BP2 transfections) or cytosol (for pTK-HSV-8His-ACBP/BP2 transfections) as described previously (12), except that EDTA, EGTA, and dithiothreitol were omitted from all buffers. All buffers contained the following protease inhibitors: 25 µg/ml ALLN, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin A. The cytosol fraction was not precipitated with acetone, but used directly in hypotonic buffer containing 50 mM Hepes-KOH (pH 7.4), 10 mM KCl, and 1.5 mM MgCl2.
The procedure used to identify the Site-2 cleavage site in SREBP-2 is outlined in Fig. 4. All buffers were freshly prepared before each experiment. Aliquots of nuclear extracts (500 µg of protein; 0.2 ml from four pooled 60-mm dishes of transfected 293 cells) were mixed with 2 ml of buffer A (50 mM Tris-HCl at pH 8.0, 3 M urea, and 0.05% Tween 20) supplemented with 5 mM imidazole-HCl (pH 8.0) and incubated at room temperature for 15 min followed by 37 °C for 3 min. All subsequent operations were carried out at room temperature unless otherwise stated. Each sample was then mixed with 75 µl of Ni-NTA-agarose (washed in buffer A), rotated for 30 min, and centrifuged at top speed in a microcentrifuge for 3 min. The agarose beads were washed 3 times (2 min each) in 1 ml of buffer A supplemented with 5 mM imidazole-HCl (pH 8.0). The His-tagged SREBP-2 was eluted from the Ni-NTA-agarose by mixing the beads for 10 min in 0.4 ml of buffer B (50 mM Tris-HCl at pH 7.0, 8 M urea, and 0.05% Tween 20) supplemented with 100 mM imidazole-HCl (pH 7.0). The mixture was centrifuged at top speed in a microcentrifuge for 3 min, and the supernatant was adjusted to 90 µM dithiothreitol and incubated for 20 min. The volume was then brought to 1.3 ml with PBS-Tween (10 mM phosphate-buffered saline (pH 7.4) and 0.05% Tween 20), and each sample then received a 0.2-ml addition of either NEM or MPB at a final concentration of 0.35 mM. (Stock solutions of 1.6 mg/ml NEM and MPB were made up in PBS-Tween and stored at ![]() |
RESULTS |
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We previously replaced the 472-amino acid NH2-terminal segment of human SREBP-2 with the 188-amino acid H-Ras protein and used prematurely terminated forms of this fusion protein as size standards for comparison with the NH2-terminal fragment generated from the fusion protein within cells (12). Immunoblot analysis after SDS-PAGE suggested that the Site-2 protease cleaves SREBP-2 somewhere within the ~20-amino acid sequence that comprises the first transmembrane segment (12). To increase the resolution of this technique, we sought a smaller protein that could replace the NH2-terminal segment of SREBP-2 while maintaining proper two-step processing. The acyl-CoA binding protein (ACBP) fits this criterion (Fig. 1A). ACBP is an 87-amino acid cytosolic protein (18). When fused to amino acids 473-1141 of human SREBP-2, the ACBP is predicted to produce a mature NH2-terminal segment of ~123 to 143 amino acids, which is one-fourth the size of the mature NH2-terminal segment of native SREBP-2.
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To demonstrate that ACBP/SREBP-2 is cleaved through the normal two-step process, we performed a co-transfection experiment in wild-type CHO-7 cells and in M19 cells, a mutant line of CHO cells that lack S2P (12, 15). The cells were transiently transfected with a cDNA encoding ACBP/SREBP-2 tagged at its NH2 terminus with an epitope derived from the HSV envelope glycoprotein D (24) and driven by the HSV-thymidine kinase promoter. We also transfected cDNAs encoding pVAI, pSCAP(D443N), and either S2P or empty vector. pVAI encodes an RNA that stimulates translation of mRNAs produced from transfected cDNAs (23). It enhances expression of the ACBP/SREBP-2 fusion protein by severalfold in our experiments. pSCAP(D443N) encodes a mutant version of SCAP that was previously shown to increase the efficiency of cleavage of SREBPs at Site-1 (13). Membranes and cytosol were isolated from transfected cells and subjected to SDS-PAGE and immunoblot analysis with an antibody directed against the HSV epitope tag. The chimeric ACBP/SREBP-2 plasmid produced a full-length membrane-bound precursor with an apparent molecular mass of ~80 kDa (designated P in Fig. 1B, upper panel). In the absence of co-transfected S2P, the CHO-7 cells cleaved ACBP/SREBP-2, generating a mature form that was found in the cytosol and migrated at ~20 kDa on SDS-PAGE (designated M in lane 3). The M19 cells cleaved ACBP/SREBP-2 only at Site-1, generating an intermediate form of ~24 kDa that remained bound to membranes (designated I in lane 7). The M19 cells produced the cytosolic mature form when we co-transfected a plasmid encoding Myc-tagged S2P (designated M in lane 8). This was associated with a disappearance of the membrane-bound intermediate form (designated I in lane 8). As expected, the expression of exogenous S2P did not alter the cleavage of ACBP/SREBP-2 in the CHO-7 cells, which possess endogenous S2P (designated M in Fig. 1B, lane 4). These data demonstrate that the proteolysis of ACBP/SREBP-2, like that of native SREBP-2, occurs sequentially at Site-1 followed by Site-2.
We used three truncated forms of ACBP/SREBP-2 as size standards to refine our estimate of the location of Site-2. The locations of the three premature stop codons are shown in Fig. 2A. The amino acids are numbered according to the amino acid sequence of human SREBP-2. Residue 473 is the first residue of SREBP-2 in the fusion protein. cDNAs encoding the full-length fusion protein and the truncated proteins were transfected separately into human embryonic kidney 293 cells, and the cytosol fractions were obtained. The sizes of the fragments were compared after separation on a long Tricine polyacrylamide gel and visualized by immunoblotting (Fig. 2B). The top panel shows that the migration of an irrelevant 56-kDa protein detected by the anti-HSV tag antibody on the same Tricine gel did not vary from lane to lane. As shown in the bottom panel of Fig. 2B, the mature 126-amino acid product that was generated from cleavage of full-length ACBP/SREBP-2 (lanes 2, 3, 5, 7, 9, and 10) migrated more slowly than the Stop-482 protein, which terminates immediately after the final R of the D478RSR sequence, and the Stop-484 protein, which terminates two residues into the first transmembrane segment (lanes 4 and 6). The mature product migrated slightly faster than the Stop-488 standard, which terminates immediately after leucine 487, six amino acids into the transmembrane segment (lane 8). These studies indicate that Site-2 lies within the 3-amino acid segment between residues 483 and 487.
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To determine the amino acid residues that are required for cleavage of SREBP-2 at Site-2, we made a systematic series of mutations in which an alanine or phenylalanine residue was substituted individually for most of the amino acids in the first transmembrane segment that are conserved in the seven mammalian SREBPs (Fig. 3). 293 cells were transfected with a cDNA encoding wild-type or mutant human SREBP-2 that was tagged at its NH2 terminus with the HSV tag, and the cells were incubated in the absence or presence of sterols. Nuclear extracts and membrane fractions from the transfected cells were subjected to SDS-PAGE and immunoblotted with an antibody against the NH2-terminal HSV epitope tag. The results from analysis of all 15 mutations are summarized in Fig. 3. The data reveal that all mutants were cleaved in a fashion similar to that of wild-type human SREBP-2.
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During the course of these studies, we noticed that ACBP lacks cysteine residues and that the first cysteine in the fusion protein occurs at amino acid 485 of SREBP-2, which is within the range previously defined as the site of cleavage (see Fig. 2). Moreover, the data of Fig. 3 indicated that this cysteine and the other residues near the suggested cleavage site could be replaced with alanine or phenylalanine without affecting cleavage. We hypothesized that replacement with cysteine would be equally well tolerated. To take advantage of this situation, we first prepared a series of plasmids encoding ACBP/SREBP-2 fusion proteins with an NH2-terminal HSV-epitope tag followed by 8 histidines and then performed the procedure illustrated in Fig. 4. Each fusion protein had a cysteine residue that was located either before or after the suspected site of cleavage. After partial purification by Ni-NTA-agarose chromatography, the cytosolic NH2-terminal fragment was reacted with MPB, which attaches biotin to cysteine sulfhydryls. Excess MPB was removed by a second round of Ni-NTA chromatography, and the MPB-derivatized proteins were trapped by binding to streptavidin-agarose. The supernatant and pellet fractions from the streptavidin-agarose precipitation were subjected to SDS-PAGE and immunoblotted with an antibody directed against the HSV epitope tag. If the cysteine is on the NH2-terminal side of Site-2, the NH2-terminal fragment should be found in the streptavidin pellet. If the cysteine is distal to Site-2, the NH2-terminal fragment should not be derivatized with biotin and should be found in the streptavidin supernatant.
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As shown in Fig. 5, residues 483-486 of the ACBP/SREBP-2 fusion protein were individually replaced with cysteine. To remove any ambiguity, we replaced cysteine 485 with alanine in all constructs except the cysteine 485 construct (Fig. 5A). The position of the first cysteine in each fusion protein is indicated. The cysteine 485 construct represents the wild-type version of ACPB/SREBP-2. To confirm that all of these proteins would be cleaved by S2P, we transfected these cDNAs into M19 cells in the absence or presence of S2P. We prepared membrane and cytosol fractions from the transfected cells and immunoblotted with an antibody directed against the NH2-terminal HSV epitope. In the absence of S2P, each of the mutants was cleaved at Site-1 to generate the membrane-bound intermediate form (Fig. 5B, upper panel; compare lanes 3, 5, and 7, and 11 with lane 9). When the M19 cells were co-transfected with S2P, the intermediate form of each mutant protein was lost, and the mature cytosolic form appeared (Fig. 5B, lower panel; compare lanes 4, 6, 8, and 12 with lane 10).
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To confirm the efficiency of biotinylation and streptavidin precipitation, we transfected 293 cells with the plasmid encoding ACBP/SREBP-2 with the first cysteine at position 483 and reacted the cytosolic NH2-terminal fragment with MPB or with a control reagent NEM. To enhance production of the NH2-terminal fragment, the cells were co-transfected with pVAI, pCMV-SCAP(D443N), and pCMV-Myc-S2P. Based on the previous data (Fig. 2), we hypothesized that residue 483 would be proximal to Site-2 (Fig. 6A), allowing precipitation of the NH2-terminal fragment by streptavidin-agarose after derivatization with MPB. After enrichment of the cytosolic NH2-terminal fragment by Ni-NTA chromatography, the samples were treated with NEM or MPB and the procedure of Fig. 4 was followed. After treatment with NEM, the treated NH2-terminal fragment was found primarily in the streptavidin-agarose supernatant (Fig. 6B, compare lane 2 with lane 4). After treatment with MPB, the protein was found in the streptavidin-agarose pellet (Fig. 6B, compare lane 6 with lane 8).
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To determine the cleavage site precisely, 293 cells were co-transfected with plasmids encoding ACBP/SREBP-2 fusion proteins with cysteines at each of the positions between residues 483 and 486 (Fig. 7A), and the cytosolic NH2-terminal fragments were subjected to the procedure of Fig. 4. As shown in Fig. 7B, when the first cysteine was at position 491, the fusion protein appeared in the supernatant after streptavidin precipitation (lanes 3 and 4), confirming that cysteine 491 is distal to Site-2. When the first cysteine was at position 483 or 484, the protein was found in the pellet after streptavidin precipitation (Fig. 7B, lanes 5-8). When the first cysteine was at position 485 or 486, the protein was in the supernatant (Fig. 7B, lanes 9-12). These data indicate that Site-2 proteolysis occurs at the peptide bond between residues 484 and 485 (Fig. 7A).
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We considered the possibility that cysteine at the extreme COOH-terminus of the NH2-terminal fragment might be resistant to MPB derivatization. To rule out this possibility, we produced a truncated ACBP/SREBP-2 that terminates immediately following cysteine 485 (Fig. 7A, Stop-486 cDNA). Following derivatization with MPB and streptavidin precipitation, this protein was found in the pellet (Fig. 7B, lanes 13 and 14), indicating that a cysteine at the COOH-terminal position of ACBP/SREBP-2 can be modified with MPB.
We quantified the chemiluminescence signal of ACBP/SREBP-2 protein in the supernatant or pellet fraction for each of the mutations (Fig. 7C). The results yielded a clear discrimination. In each case over 90% of the protein was found in either the pellet or the supernatant. These data indicate that S2P cuts the intermediate SREBP-2 clearly at a single peptide bond and that there is no significant further shortening of the protein.
The next experiment was designed to determine whether Site-2 proteolysis of full-length human SREBP-2 also occurs at the Leu484-Cys485 peptide bond. For this purpose, we took advantage of the observation that there is only a single cysteine in the NH2-terminal domain of SREBP-2 (Ref. 19). We mutated this cysteine, at residue 51, to alanine. We then made cDNAs encoding SREBP-2 (C51A) with the first cysteine at position 484 or 485. Immunoblotting of nuclear extracts confirmed that these proteins are proteolytically processed and regulated by sterols in the same manner as wild-type SREBP-2, i.e. the Cys51 cDNA (Fig. 8A). We then used the procedure of Fig. 4 to determine the site of cleavage. After biotinylation, the mature NH2 terminus of wild-type SREBP-2 was primarily found in the streptavidin pellet. This is the expected result since this protein contains cysteine 51, which is proximal to Site-2 (Fig. 8B, lanes 3-6). When we transfected a cDNA encoding the triple mutant (C51A,L484C,C485A) indicated as Cys484, the derivatized mature NH2 terminus was also found primarily in the pellet, indicating that cleavage occurs distal to residue 484 (Fig. 8B, lanes 7 and 8). Conversely, the mature NH2 terminus from the C51A mutant, designated Cys485, was found primarily in the supernatant (Fig. 8B, lanes 9 and 10). Inasmuch as cysteine 485 is intact in this protein, cleavage must occur proximal to residue 485. When quantified, these data show a clear distinction between MPB-reactive and nonreactive NH2-terminal fragments, demonstrating the homogeneity of Site-2 processing (Fig. 8C). Taken together, these data show that Site-2 proteolysis in native SREBP-2 occurs at the peptide bond between leucine 484 and cysteine 485.
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To begin to determine the sequence requirements for cleavage at Site-2,
we focused first on the D478RSR region, the sequence
immediately preceding the first transmembrane segment. Previously, we
found that replacement of these four residues with AS abolished
cleavage at Site-2 (Ref. 12). As a starting point for the further
definition of the sequence responsible for recognition of Site-2, we
mutated D478RSR to AAAA, preserving the length of the
segment but eliminating the four polar residues. In the experiment of
Fig. 9, wild-type human SREBP-2 was
processed to produce the nuclear NH2-terminal fragment in a
sterol-regulated manner (Fig. 9B, lanes 3 and 4, designated M), but the D478RSR AS and the
D478RSR
AAAA mutations failed to undergo Site-2
proteolysis, and therefore they generated the membrane-bound
intermediate forms (Fig. 9B, lanes 5-8, designated
I). When only the initial aspartic acid was preserved
(DAAA), Site-2 cleavage was also abolished (Fig. 9B,
lanes 9 and 10). These observations suggest that
one or both of the arginines in the DRSR sequence is essential for Site-2 cleavage.
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To study each of the arginines in the DRSR sequence separately, we prepared plasmids encoding mutant forms of SREBP-2 in which the first or second arginine was replaced with alanine (DASR and DRSA, respectively). Both of these proteins were cleaved by the Site-2 protease to generate mature nuclear forms (Fig. 9B, lanes 11 and 13), indicating that either arginine is sufficient. To determine whether the position of the arginine affects the precise site of cleavage, we prepared versions of the DASR and DRSA plasmids in which cysteine 51 was replaced with alanine in the encoded protein such that the first cysteine was located either at position 484 or 485. The results were the same for the DASR and the DRSA mutants (Fig. 9C, lanes 5-12). When the first cysteine was located at position 484, the NH2-terminal fragments were derivatized with MPB, and they were therefore found in the streptavidin pellet. When the first cysteine was at residue 485, the NH2-terminal fragments were found in the supernatant fractions, indicating that cleavage occurred at the 484/485 bond irrespective of whether the arginine was located at the second or fourth position of the DRSR sequence. The quantitative scanning data of Fig. 9C, shown in Fig. 9D, confirms that essentially all of the cleavage occurred at the 484/485 bond regardless of whether the first transmembrane sequence is preceded by DRSR, DASR, or DRSA.
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DISCUSSION |
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The cysteine panning technique that we developed for these studies has revealed that Site-2 cleavage of human SREBP-2 occurs at the Leu484-Cys485 bond, which is located three residues to the COOH-terminal side of the DRSR sequence that is required for cleavage. This Leu-Cys sequence is conserved not only in all seven mammalian SREBPs, but also in SREBP from Drosophila melanogaster (Fig. 3; Refs. 19 and 25-28).
The Leu-Cys cleavage site is within the range that was defined
previously on the basis of the size of the mature
NH2-terminal fragment (12). This site is also just within
the sequence that was previously defined as the first membrane-spanning
segment on the basis of hydrophobicity plots (9). These plots predict that the DRSR sequence marks the end of the NH2-terminal
cytoplasmic domain (see Fig. 3). In the mammalian SREBP-2s, the DRSR is
followed by the hydrophobic sequence ILLC (LALC in SREBP-1s), which
initiates a long hydrophobic stretch that is predicted to constitute
the first transmembrane domain. We believe that the transmembrane domain terminates at the conserved tryptophan that is 22 residues downstream from the DRSR sequence in all the SREBPs except human SREBP-1 and Drosophila SREBP where this position is occupied
by arginine (Fig. 3). The tryptophan or arginine is invariably followed by glycine. An additional glycine or proline is frequently found in the
immediate downstream sequence. Both of these residues would be expected
to disrupt the transmembrane -helix. In the first transmembrane
protein to be studied by crystallography, the photosynthetic reaction
center of Rhodopseudomonas viridis, two-thirds of the tryptophan residues of the L and M subunits were located at the junction between the hydrophobic surface and the polar transition zone
or in the polar regions near the hydrophobic surface (29).
We emphasize that there is no direct evidence that the Leu484-Cys485 bond actually lies within the phospholipid bilayer. We cannot exclude the possibility that this bond lies on the surface of the membrane and not within it.
If the Leu484-Cys485 peptide bond does lie within the membrane, it would be just beneath the polar head groups of the phospholipids. The bond might be close enough to the bilayer surface so that the Site-2 protease could surround it, sequestering it from the phospholipids and permitting attack by a molecule of water. Such an intramembrane location would be consistent with the predicted structure of the protein that has been postulated to be the Site-2 protease. This is a highly unusual polytopic membrane protein with a consensus metalloprotease zinc-binding site (HEIGH) that has a unique location within an otherwise hydrophobic region of the sequence (15). This zinc atom might be in an ideal position to cleave a peptide bond that was located just beneath the surface of the lipid bilayer.
A major unresolved question relates to the specificity of the
recognition site that directs the Site-2 protease to the SREBP. The
current data suggest that only a single juxtamembranous arginine is
required. It is possible that even this residue is not directly recognized by the Site-2 protease, but that its role is to position the
transmembrane -helix. Consistent with this notion are the observations of Fig. 9, which demonstrate that the cleavage site remains at the Leu484-Cys485 bond whether the
required arginine is at position 479 or 481. Thus, cleavage does not
occur at a fixed position following the arginine, suggesting that the
arginine is not part of a peptide recognition site.
The postulated transmembrane sequences of all seven mammalian SREBPs shown in Fig. 3 contain 10 identically conserved residues, six of which are leucines that are spaced an average of three residues apart (LXXLXXLXLXXXXLXXL). Remarkably, most of these conserved leucines could be replaced with alanine without affecting Site-2 cleavage of human SREBP-2 (Fig. 3). The same was true for the other four conserved residues in the transmembrane segment, including the phenylalanine and the proline that are conserved in the Drosophila sequence as well as the mammalian sequences (Fig. 3). Even though the Leu484-Cys485 is conserved as far back as Drosophila, these residues are not required for cleavage. Both residues could be replaced with phenylalanine without altering the cleavage (mutant 15 in Fig. 3). Additional studies will be required to determine how the Site-2 protease discriminates SREBP-2 from the many other transmembrane proteins that have arginine residues at the water-lipid interface.
We note that the relative position of Site-2 in SREBP differs from the
relative position of the cleavage site in the amyloid precursor
protein, the only protein in which intramembrane cleavage previously
has been documented (30). Amyloid precursor protein is cleaved
predominantly at two sites that are two residues apart, giving rise to
peptides of 40 and 42 amino acids in length, respectively. Both sites
are near the middle of the postulated transmembrane sequence, which
contrasts with the junctional location of the SREBP Site-2. The enzyme
that cleaves amyloid precursor protein within the membrane, termed the
-secretase, has not yet been isolated. At present it is unclear as
to whether a single
-secretase makes both cleavages, or whether two
-secretases exist. The double site of cleavage of amyloid precursor
protein differs from the single intramembrane site of cleavage that is
documented by the cysteine panning technique used in these studies.
Indeed, one advantage of this technique is that it gives a quantitative
readout of the percentage of observed cleavage that occurs at the
Leu484-Cys485 bond. These data indicate that
this site of cleavage represents more than 90% of the cleavage that
occurs within SREBP-2 and that there is no trimming back of the cleaved
peptide beyond residue 484. Inasmuch as this technique measures only
the final cleavage site, we cannot exclude the unlikely possibility
that intramembrane cleavage of SREBP-2 is initiated at a site that is
distal to the 484/485 bond and that there is further precise trimming
of all cleaved molecules back to residue 484.
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ACKNOWLEDGEMENTS |
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We thank our colleagues Rodney K. Guy and Steven Jon Russell for helpful discussions, Lisa Beatty for invaluable assistance with cultured cells, Jeff Cormier and Michelle Laremore for efficient DNA sequencing and oligonucleotide synthesis, and Tanisha Brown for superb help with gel electrophoresis.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Research Grant HL20948 and a grant from the Perot Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Medical Scientist Training Grant GM08014.
§ Recipient of a Postdoctoral Fellowship for Physicians from the Howard Hughes Medical Institute.
1
The abbreviations used are: SREBP, sterol
regulatory element-binding protein; ACBP, acyl-CoA-binding protein;
ALLN, N-acetyl-leucinal-leucinal-norleucinal; HSV, herpes
simplex virus; MPB,
N-(3-maleimidylpropionyl) biocytin;
NEM, N-ethylmaleimide; SCAP, SREBP cleavage-activating
protein; S2P, Site-2 protease; TK, thymidine kinase; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE,
polyacrylamide gel electrophoresis.
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REFERENCES |
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