(Received for publication, January 30, 1997, and in revised form, March 6, 1997)
From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523 bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage.
Proteolytic processing of sterol regulatory element-binding proteins (SREBPs)1 controls the metabolism of cholesterol and fatty acids in animal cells (1-3). SREBPs are transcription factors that are bound to membranes of the ER and nuclear envelope. Each SREBP is composed of three segments: 1) an NH2-terminal segment of ~485 amino acids that is a transcription factor of the basic helix-loop-helix-leucine zipper family, 2) a membrane attachment segment of ~75 amino acids composed of two membrane-spanning sequences separated by a short hydrophilic loop of 31 amino acids, and 3) a COOH-terminal segment of ~585 amino acids that plays a regulatory role. The proteins are oriented so that the NH2- and COOH-terminal segments project into the cytoplasm, and only the short hydrophilic loop projects into the lumen of the ER or nuclear envelope (4).
Before it can activate transcription, the NH2-terminal segment is released from the membrane in a complex two-step proteolytic sequence (2, 3). First, a protease cleaves the protein at Site-1, which is near an arginine in the lumenal loop, thereby breaking the attachment between the two transmembrane sequences. This allows a second protease to cleave the protein at Site-2, which is near the middle of the first transmembrane sequence (2, 3). The NH2-terminal fragment leaves the membrane with a portion of the first transmembrane sequence still attached. It enters the nucleus, where it activates transcription of genes encoding the LDL receptor (5, 6), several enzymes of cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A synthase (5, 6), 3-hydroxy-3-methylglutaryl coenzyme A reductase (7), farnesyl diphosphate synthase (8), and squalene synthase (9)), and enzymes of fatty acid biosynthesis (10, 11) and desaturation (12). The net result is to increase the cell's supply of cholesterol and fatty acids.
The Site-1 protease is the target of feedback regulation by cholesterol and other sterols (3). When these sterols accumulate within cells, the rate of proteolysis at Site-1 declines markedly. Cleavage at Site-2 also declines because this cleavage requires prior cleavage at Site-1 (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 cholesterol and fatty acids when intracellular sterol levels are already high.
Three isoforms of SREBP are known (5, 13, 14). SREBP-1a and 1c are derived from a single gene through use of alternate promoters that encode alternate first exons (5, 13, 14). SREBP-1a is much more active than SREBP-1c in stimulating transcription of all known target genes (15). The third protein, SREBP-2, is the product of a separate gene (6, 13), and it is also more active than SREBP-1c (15). In view of the regulatory role of the Site-1 protease, further knowledge of its structure and mode of regulation is desirable. A first step would be the identification of the precise site at which the Site-1 protease cuts SREBPs. In previous studies we have shown that this cleavage is abolished when arginine 519 in the lumenal loop of SREBP-2 (or the corresponding arginine of SREBP-1a) is changed to alanine by in vitro mutagenesis (2). The size of the cleavage product, as determined by SDS-PAGE, is consistent with cleavage at or near this arginine (3).
In the current studies we have used a combination of in vitro mutagenesis, epitope tagging, immunoprecipitation, and radiochemical sequencing to determine the precise location of Site-1 in SREBP-2. We found, surprisingly, that cleavage does not occur at arginine 519, but rather it occurs 3 residues further toward the COOH terminus, namely at leucine 522. Arginine 519 seems to be the NH2-terminal residue in a tetrapeptide sequence, RXXL, that serves as a recognition signal for the Site-1 protease.
We obtained HSV-TagTM and T7-TagTM monoclonal antibodies from Novagen; v-H-Ras(Ab-1)-agarose linked monoclonal antibody was obtained from Oncogene. Protein G-SepharoseR 4 Fast Flow beads were obtained from Pharmacia Biotech Inc., L-[35S]methionine (>1000 Ci/mmol) was obtained from DuPont NEN, and glycosidases were obtained from New England Biolabs.
Construction of pTK-HSV-BP2-Ras-T7pTK-HSV-BP2-Ras-T7 encodes an epitope-tagged SREBP-2/Ras fusion protein (1391 amino acids) consisting of an initiator methionine, two tandem copies of the HSV epitope (QPELAPEDPED), six novel amino acids (IDGTVP) encoded by a sequence that consists of restriction sites for BspDI and KpnI, human SREBP-2 (amino acids 14-1141), two novel amino acids (HM) encoded by the sequence for restriction site NdeI, human H-Ras (amino acids 2-189), and three tandem copies of the T7 epitope (HMASMTGGQQMGAAAMASMTGGQQMGGGPMASMTGGQQMGLINM).
pTK-HSV-BP2-Ras-T7 was constructed from a previously described plasmid,
pTK-HSV-BP2-T7 (16), by insertion of a cDNA segment encoding human
H-Ras between the sequences for SREBP-2 and the first copy of the T7
epitope. The nucleotide sequence encoding amino acids 2-189 of human
H-Ras was obtained by polymerase chain reaction on pRcCMV-H-Ras (17)
with a pair of primers containing an NdeI site at each 5
end using Pfu DNA polymerase. The amplified product was
digested with NdeI and cloned into the unique
NdeI site between human SREBP-2 and the three tandem copies
of the T7 epitope. Two independent clones were used in each of the
transfections.
pTK-HSV-BP2/LDLRTM
encodes an epitope-tagged SREBP-2 fusion protein in which a 26-amino
acid region that includes the second transmembrane domain of human
SREBP-2 (amino acids 535-560) is replaced with the transmembrane
domain of the human LDL receptor (amino acids 708-729) (18). To
construct pTK-HSV-BP2/LDLRTM, we used oligonucleotide site-directed
mutagenesis to produce an intermediate plasmid in which amino acids
535-560 of human SREBP-2 were replaced by two novel amino acids (TG)
corresponding to an AgeI restriction site. A pair of
complementary oligonucleotides (top strand,
5-CCGGTGCTCTGTCCATTGTCCTCCCCATCGTGCTCCTCGTCTTCCTTTGCCTGGGGGTCTTCCTTCTATGGT-3
; bottom strand,
5
-CCGGACCATACAAGGAAGACCCCCAGGCAAAGGAAGACGAGGAGCACGATGGGGAGGACAATGGACAGAGCA-3
) were annealed at 94-83 °C for 5 min and then at 83-23 °C for 60 min. These oligonucleotides correspond to amino acids 708-729 of the
human LDL receptor flanked by the single-stranded sequence 5
-CCGG-3
.
The annealed oligonucleotides were cloned into the unique
AgeI restriction site of the intermediate plasmid (described above). The resulting pTK-HSV-BP2/LDLRTM encodes an 1157-amino acid
chimeric protein consisting of an initiator methionine, two tandem
copies of the HSV epitope, six novel amino acids (IDGTVP), human
SREBP-2 (amino acids 14-534), two novel amino acids (TG), human LDL
receptor (amino acids 708-729), two novel amino acids (SG), and human
SREBP-2 (amino acids 561-1141).
Plasmid pTK-HSV-BP2(R519A)/LDLRTM is identical to pTK-HSV-BP2/LDLRTM except for the R519A point mutation in the lumenal loop of the SREBP-2 sequence. This plasmid was constructed by site-directed mutagenesis.
Construction of pTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/NGTpTK-HSV-BP2-NGT and pTK-HSV-BP2-NSS/NGT encode epitope-tagged SREBP-2 fusion proteins in which serine 515 in the loop region of SREBP-2 is replaced by a novel amino acid sequence containing one or two N-linked glycosylation sites, either SNGT or NSSGSSGNGT, respectively. Both plasmids were constructed by site-directed mutagenesis.
Site-directed MutagenesisOligonucleotide site-directed mutagenesis was carried out with single-stranded, uracil-containing DNA (19) using the Muta-gene Phagemid In Vitro Mutagenesis Version-2 kit (Bio-Rad) (2). The mutations were confirmed by sequencing the relevant region, and at least two independent clones of each mutant were independently transfected.
Cell Culture, Transfection, and Cell FractionationMonolayers of human embryonic kidney 293 cells were set up on day 0 (4 × 105 cells/60-mm dish) and cultured in 8-9% CO2 at 37 °C in medium A (Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 µg/ml streptomycin) supplemented with 10% (v/v) fetal calf serum (4). On day 2, cells were transfected with 4 µg of pTK empty vector (mock) or the indicated plasmid as described (2). 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) in the absence or presence of sterols as indicated in the legends. After incubation for 20 h, the cells received N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 µg/ml (2), and the cells were harvested 3 h later (2). The pooled cell suspension from 2 dishes was allowed to swell in hypotonic buffer (4) for 30 min at 0 °C, passed through a 22.5-gauge needle 30 times, and centrifuged at 1000 × g at 4 °C for 7 min. The 1000 × g pellet was resuspended in 0.1 ml of buffer C (10 mM Hepes-KOH (pH 7.4), 0.42 M NaCl, 2.5% (v/v) glycerol, 1.5 mM MgCl2, 0.5 mM sodium EDTA, 0.5 mM sodium EGTA, 1 mM dithiothreitol, and a mixture of protease inhibitors (4)). The suspension was rotated at 4 °C for 1 h and centrifuged at top speed in a microfuge for 15 min at 4 °C. The supernatant is designated nuclear extract. The supernatant from the original 1000 × g spin was centrifuged at 105 × g for 30 min at 4 °C in a Beckman TLA 100.2 rotor, and the pellet was dissolved in 0.1 ml of SDS lysis buffer (1) and designated membrane fraction.
Glycosidase Sensitivity of Membrane-bound SREBP-2Glycosidase sensitivity of SREBP-2 was carried out as
described (4). Monolayers of 293 cells were set up on day 0 (4 × 105 cells/60-mm dish) and cultured as described above. On
day 2, the cells were transfected with 7 µg of pTK empty vector
(mock), pTK-HSV-BP2-NGT, and pTK-HSV-BP2-NSS/NGT, respectively. Three h
after transfection, the cells were switched to medium B in the presence
of sterols as described in Fig. 1. After incubation for 20 h, the
cells were harvested, and the pooled cell suspension from four dishes
was fractionated. The 105 × g membrane pellet
was washed once with buffer A (4) and resuspended in 180 µl of buffer
A containing 1% (v/v) Triton X-100 without protease inhibitors.
Aliquots of the 105 × g membrane fraction (0.16 mg in 40 µl of buffer A) were boiled for 5 min in the presence
(peptide N-glycosidase F and endoglycosidase H reactions) or
in the absence (neuraminidase reactions) of 0.5% (w/v) SDS and 1%
(v/v) -mercaptoethanol for 5 min, after which the indicated amount
of glycosidase was added and incubated at 37 °C for 2 h as
described in Fig. 10.
Immunoblot Analysis
Samples of the nuclear extract and the 105 × g membrane fraction were mixed with 5× SDS loading buffer (20). Protein concentration was measured with a BCA kit (Pierce). After SDS-PAGE in 8% gels, proteins were transferred to Hybond-C extra nitrocellulose membranes (Amersham Corp.). Immunoblot analysis was carried out with a horseradish peroxidase detection kit using the SuperSignalTM CL-HRP Substrate System according to the manufacturer's instructions except that the nitrocellulose sheets were blocked in phosphate-buffered saline containing 0.05% (v/v) Tween 20, 5% (v/v) nonfat dry milk, and 5% (v/v) heat-inactivated newborn calf serum. The chimeric proteins were visualized with 0.5 µg/ml HSV-TagTM monoclonal antibody or with 10 µg/ml IgG-1C6, a mouse monoclonal antibody directed against amino acids 833-1141 of human SREBP-2 (4). Gels were calibrated with prestained molecular weight markers. Filters were exposed at room temperature to ReflectionTM NEF-496 film (DuPont NEN).
Metabolic Labeling and Immunoprecipitation of Epitope-tagged SREBP-2/Ras Fusion Protein and Its COOH-terminal FragmentMonolayers of 293 cells were set up on day 0 (7 × 105 cells/60-mm dish) and cultured as described above. On day 1, the cells were transfected with 4 µg of the wild-type or mutant version of pTK-HSV-BP2-Ras-T7, 1 µg of pCMV-SCAP(D443N) (16), and 2 µg of pVAI as described above. pVAI encodes the adenovirus virus-associated I RNA gene, which enhances translation of transfected cDNAs (21). The cells were incubated for 20 h with 50 µM compactin and 50 µM sodium mevalonate, at which time the medium was changed to 1.3 ml of methionine-free Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 10% newborn calf lipoprotein-deficient serum, 50 µM compactin, and 50 µM sodium mevalonate. After incubation for 1 h at 37 °C, 25 µg/ml N-acetyl-leucinal-leucinal-norleucinal was added, and the cells were pulse-labeled with 700 µCi/ml of [35S]methionine for 6 h at 37 °C. The cells from four dishes were harvested and pooled, and the membrane fraction was prepared as described above.
The pooled membrane fraction from the four dishes was resuspended in
0.1 ml of SDS lysis buffer at room temperature. All subsequent operations were carried out at 4 °C unless otherwise stated. The suspension was rotated for 30 min in 5 ml of buffer D (50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.5% (v/v)
SDS, 1.25% (v/v) Triton X-100, 1.25% (v/v) deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, 25 µg/ml
N-acetyl-leucinal-leucinal-norleucinal, and 1 mM
dithiothreitol), after which 20 µl of preimmune whole rabbit serum,
40 µg 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 × g for 7 min.
The resulting supernatant was mixed with an additional 20 µl of
preimmune whole rabbit serum, 40 µg of irrelevant monoclonal antibody
IgG-2001, and 0.2 ml of Protein G-Sepharose beads, rotated for 2 h, and centrifuged at 1000 × g for 3-7 min. To the
supernatant were added 1 µg of T7-Tag monoclonal antibody, 25 µg of
v-H-Ras(Ab-1)-agarose linked monoclonal antibody, and 20 µg of
anti-COOH-terminal SREBP-2 monoclonal antibody IgG-1C6. After rotation
for 2.5 h, 50 µl of Protein G-Sepharose beads were added,
followed by rotation for 2.5 h and centrifugation at 1000 × g for 3-7 min. The beads were washed once by rotation with
buffer D for 16 h, followed by four washes in buffer D for 1 h each. The washed beads were resuspended in 0.1 ml of 2× SDS loading
buffer (20) containing 10% (v/v) -mercaptoethanol and boiled for 5 min. After centrifugation at 1000 × g at room
temperature for 3 min, the supernatant was transferred to a fresh tube
(tube A), and the beads were re-eluted with 40 µl of 5× SDS loading buffer containing 25%
-mercaptoethanol and boiled for 5 min. After
centrifugation at 1000 × g at room temperature for 3 min, the supernatant was transferred to tube A, and the entire volume was boiled again for 5 min before SDS-PAGE.
Aliquots of the immunoprecipitated samples (from 1.3 dishes of cells) were subjected to SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride membranes (Immobilon-PSQ; Millipore). After drying, the membranes were exposed to an imaging plate and scanned in a Fuji X Bas 1000 phosphorimager. The band containing the COOH-terminal product of the cleavage reaction (Mr ~83,000) was excised and subjected directly to multiple cycles of Edman degradation on an Applied Biosystems model 477A sequencer. Fractions from each cycle (148 µl) were collected and counted in a scintillation counter.
To identify the exact position of Site-1, we prepared a cDNA (pTK-HSV-BP2-Ras-T7) encoding a triply tagged version of SREBP-2, which we designate SREBP-2/Ras (Fig. 1). The NH2 terminus contains two copies of an epitope tag from the HSV glycoprotein that we used previously (2). At the COOH terminus we inserted amino acids 2-189 of H-Ras followed by three copies of an 11-residue epitope derived from the gene 10 protein of bacteriophage T7 (23). The combination of epitopes at the COOH terminus allowed efficient precipitation of the COOH-terminal fragment with a mixture of antibodies directed against the Ras and T7 epitopes plus a monoclonal antibody directed against the COOH-terminal domain of SREBP-2 (see below).
To demonstrate that SREBP-2/Ras is cleaved at Site-1 in a physiologic
fashion, we transfected 293 cells with a vector encoding this construct
and another encoding a mutated version in which arginine 519 was
changed to alanine (Fig. 1). We used the relatively weak thymidine
kinase promoter, which produces near physiological levels of this
protein (2). Cells were incubated in inducing medium that contains the
3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor compactin to
block cholesterol synthesis plus a low concentration of mevalonate to
provide nonsterol end products. The medium was either devoid of sterols
( sterols) or it was supplemented with a mixture of
25-hydroxycholesterol and cholesterol (+ sterols), which suppresses
cleavage at Site-1 (3). Cell membranes were isolated, solubilized with
SDS, subjected to SDS-PAGE, and immunoblotted with IgG-1C6, a
monoclonal antibody directed against the COOH terminus of SREBP-2
(4).
When cells were transfected with the cDNA encoding the SREBP-2/Ras chimeric protein, the membranes contained the 83-kDa COOH-terminal fragment, which was the product of cleavage at Site-1 (Fig. 1, lane 3, transfected C). The amount of this fragment was reduced in the presence of sterols (lane 4). The IgG-1C6 antibody also visualized the 62-kDa COOH-terminal fragment of endogenous SREBP-2 (lane 3, endogenous C), and this was also decreased by sterols (lane 4). To test the physiologic relevance of the observed cleavage of SREBP-2/Ras, we cotransfected a cDNA encoding the D443N mutant version of SCAP, a protein that was previously shown to render the cleavage of SREBPs at Site-1 resistant to suppression by sterols (16). Indeed, in the presence of SCAP, sterols no longer prevented the appearance of the COOH-terminal fragment of SREBP-2/Ras (lane 6). Note that SCAP did not prevent suppression of the cleavage of endogenous SREBP-2. We believe that this is due to the small percentage of cells that received the SCAP cDNA. Unlike SREBP-2/Ras, which is expressed only in transfected cells, endogenous SREBP-2 is present in all cells in the dish, most of which did not receive the SCAP cDNA. To increase the expression of the transfected SREBP-2/Ras chimera, we cotransfected pVAI, which encodes a protein that stimulates translation of mRNAs produced by transfected cDNAs (21). In the presence of pVAI plus pSCAP, the COOH-terminal fragment of the chimeric protein was increased by at least 8-fold, and there was still no suppression by sterols (lanes 7 and 8). The R519A mutant of the chimeric protein did not give rise to detectable amounts of COOH-terminal cleavage product in either the absence or presence of SCAP (lanes 9-12). In the presence of pVAI plus pSCAP, a trace amount of the R519A mutant COOH-terminal fragment was detectable, but its amount was reduced by at least 12-fold when compared with the wild-type COOH-terminal fragment (compare lanes 13 and 14 with lanes 7 and 8). These data indicate that Site-1 cleavage of SREBP-2/Ras obeys all of the rules established for wild-type SREBP-2. Moreover, as discussed below, the use of pVAI and pSCAP enabled the production of sufficient amounts of SREBP-2/Ras to allow radiochemical sequencing.
In view of the high specific radioactivity of
[35S]methionine, we developed a strategy to use this
amino acid for radiochemical sequencing. Assuming that the cleavage
site is located near arginine 519, we searched for amino acids in the
region that could be replaced with methionine without abolishing
cleavage at Site-1. Fig. 2 shows two experiments in
which methionine residues were introduced individually into positions
525, 526, or 529 of the triply taggged SREBP-2/Ras protein (Fig.
2A). To follow the cleavage, we prepared nuclear extracts
and immunoblotted with an antibody against the NH2-terminal
HSV epitope tag. The E525M, S526M, and G529M mutants were each cleaved
efficiently as judged from the amount of NH2-terminal fragment in the nucleus, and all of the cleavages were suppressed by
sterols (Fig. 2B). In the experiment shown in lanes
1-6, both the cleaved wild-type and E525M proteins migrated as a
doublet. Such doublets are seen rarely and inconsistently in our
experiments, and their significance is unknown.
To determine whether the COOH-terminal fragment could be labeled with
[35S]methionine and immunoprecipitated, we transfected
293 cells with a cDNA encoding the SREBP-2/Ras chimera and labeled
the cells with [35S]methionine. A membrane fraction was
prepared, solubilized with detergents, and immunoprecipitated with a
mixture of three antibodies directed against H-Ras, the T7 epitope tag,
and the COOH-terminal segment of SREBP-2. The immunoprecipitate was
subjected to SDS-PAGE and phosphorimager analysis, which revealed a
band of ~83 kDa that was absent from mock-transfected cells (Fig.
3A). Immunoblot analysis of the precipitate
(Fig. 3B) and the supernatant (data not shown) confirmed
that all of the COOH-terminal fragment was precipitated. In future
experiments, the 83-kDa radiolabeled band was excised from the
nitrocellulose membrane and used for radiochemical sequencing.
Fig. 4 shows a series of experiments in which 293 cells
were transfected with cDNAs encoding the SREBP-2/Ras chimera with the wild-type sequence in the lumenal loop or with the three methionine substitutions at positions 525, 526, and 529. The cells were then incubated with [35S]methionine. After immunoprecipitation
and electrophoresis, the COOH-terminal fragments were eluted and
subjected to automated Edman degradation, and the radioactivity
released in each cycle was determined. The protein with the wild-type
SREBP-2 sequence did not show any peak of 35S radioactivity
in any of the early cycles (Fig. 4A). The E525M mutant
showed a clear peak of 35S radioactivity in cycle 3 from
the Edman degradation (Fig. 4B). The S526M and G529M mutants
showed peaks at cycles 4 and 7, respectively (Fig. 4, C and
D). These findings indicate strongly that the COOH-terminal fragment begins with serine 523 and that Site-1 cleavage occurs between
this residue and leucine 522 (indicated by the arrows in the
amino acid sequences of Fig. 4).
To determine the amino acid residues that are important for cleavage of SREBP-2 at Site-1, we made a systematic series of mutations in which alanine was individually substituted for most of the amino acids in the lumenal loop between the two membrane-spanning sequences. The expression vector was a cDNA encoding SREBP-2 with an NH2-terminal epitope tag derived from the HSV glycoprotein. Expression was driven by the relatively weak thymidine kinase promoter, which produces sufficient SREBP-2 to allow detection, yet not so much as to overwhelm the system for regulated proteolysis (2). Transfected 293 cells were incubated in inducing medium plus or minus sterols, after which nuclear extracts and membrane fractions were subjected to SDS-PAGE and immunoblotted with an antibody against the NH2-terminal epitope tag.
Fig. 5 shows illustrative results from one of the
alanine scanning experiments. When the cDNA encoded the wild-type
SREBP-2 sequence, we observed the mature NH2-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 loops of
four of the SREBPs that have been sequenced, two from the human (5, 6)
and two from the hamster (24, 25). Conserved residues are
highlighted. Only two single alanine substitutions reduced
cleavage dramatically: the R519A and the L522A mutations (Mutant
No. 11 and 14, respectively). Three of the multiple
alanine replacements reduced cleavage (Mutant No. 21, 24,
and 25). All of these mutations included the L522A
substitution. The other multiple alanine replacements did not reduce
cleavage. These no-effect mutations included the change of
Arg519-Ser-Val-Leu522 to
Arg519-Ala-Ala-Leu522 (Mutant No.
23). Cleavage was also not affected when the serine following the
cleavage site was changed to alanine (S523A, Mutant No. 15).
These data indicate that arginine 519 at the P4 position and leucine
522 at the P1 position are the only 2 residues in the region of Site-1
that cannot tolerate alanine substitutions.
To further dissect the requirement for arginine 519, we systematically
replaced this residue with 12 different residues (Fig. 7). When lysine was substituted for this arginine, only
a partial loss of cleavage activity was observed. Two negatively
charged residues (glutamic acid and aspartic acid) permitted a barely detectable level of cleavage. All other substitutions abolished cleavage. The location of arginine 519 was critical. Cleavage was
abolished when we replaced arginine 519 with alanine and then inserted
a new arginine at positions 517, 520, or 521. These mutations effectively moved the position of the arginine either toward the NH2 terminus or COOH terminus.
We also tested the specificity of the requirement for leucine 522 at
the cleavage site (Fig. 8). Cleavage was markedly
reduced but not totally abolished when this residue was changed to
arginine, alanine, or phenylalanine. It was abolished when leucine 522 was changed to glutamic acid or valine. The residue at the P1 position was not critical. This residue is serine in human and hamster SREBP-2,
but it is glutamic acid and glycine in hamster and human SREBP-1,
respectively. Replacement of serine 523 in human SREBP-2 with glutamic
acid or glycine preserved normal cleavage. The cleavage was also
unaffected when this residue was changed to arginine, alanine, leucine,
or phenylalanine. A moderate reduction was observed when it was changed
to cysteine.
To determine whether the second transmembrane domain contributes to
recognition by the Site-1 protease, we replaced this sequence with the
membrane-spanning region of another 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 NH2 terminus in the extracellular space and its COOH terminus in the cytosol (18). This orientation is the same
as the orientation of the second transmembrane domain of SREBP-2 (4).
The chimeric protein containing the LDL receptor transmembrane domain
was cleaved as efficiently as the wild-type protein, as judged by the
amount of NH2-terminal fragment found in the nucleus (Fig.
9, lane 5). Cleavage was abolished by sterols (lane
6). It was also abolished when arginine 519 of the chimera was
changed to alanine (lane 7). These data indicate that the precise sequence of the second transmembrane domain is not important for cleavage at Site-1 (or Site-2). A hydrophobic sequence at this
position is required, however. When the second transmembrane domain was
deleted without replacement by another transmembrane domain
(HSV-SREBP-2535-560), cleavage at Site-1 and -2 was abolished (data
not shown).
Previously we presented evidence that the lumenal loop sequence is indeed in the ER lumen (4). This evidence was based on studies of a cDNA encoding a chimeric protein that contained an epitope-tagged peptide segment (218 amino acids) inserted into the lumenal loop (4). We were forced to insert a long protein segment because shorter epitopes were not recognized by antibodies after insertion into the lumenal loop of SREBP-2. Sealed membrane vesicles were prepared from cells expressing this protein, and the segment containing the epitope was shown to be protected from digestion by trypsin in the absence, but not the presence, of detergents. The epitope-containing segment was also demonstrated to undergo N-linked glycosylation. The problem with these studies is that the chimeric protein was not cleaved by the Site-1 protease. Therefore, we could not be certain that the long epitope-containing peptide had not altered the orientation of the protein.
To circumvent this problem, we took advantage of the observation that
the precise sequence on the NH2-terminal side of arginine 519 is not essential for Site-1 cleavage. Therefore, we prepared a
plasmid encoding forms of SREBP-2 in which we inserted either 3 or 9 amino acids into this sequence in place of serine 515 (Fig. 10A). The inserted amino acids included
either one or two sites for N-linked glycosylation
(Asn-Xaa-Ser/Thr). After transfection, membrane pellets containing the
SREBP-2 precursor were digested with glycosidases, and the change in
mobility on SDS-PAGE was determined by immunoblotting. As shown in Fig.
10B, the mobility of the SREBP-2 precursors 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 (lanes 4 and 9). The changes were
greater for the construct with two glycosylation sites. This pattern
indicates that the lumenal loop sequence contained N-linked
carbohydrates of the high mannose type. The lack of processing to an
endoglycosidase H-resistant form indicates that the precursor is
located in the ER and not the Golgi complex. Fig. 10C shows
immunoblots of nuclear extracts and membrane pellets from cells
expressing SREBP-2 with the wild-type lumenal sequence, the R519A
mutation, or the insertion of one or two N-linked
glycosylation sites. The cells were incubated under conditions that
induce SREBP-2 cleavage ( sterols) or suppress cleavage (+ sterols).
The membrane-bound precursor form with one glycosylation site
(lanes 7 and 8) migrated slower than the
wild-type protein (lanes 3 and 4), and the form
with two glycosylation sites migrated even slower (lanes 9 and 10). Under inducing conditions the nuclear extracts
contained the NH2-terminal fragments of all proteins except
the R519A mutant. Cleavage of all proteins was suppressed by sterols.
This experiment demonstrates that the glycosylated precursor of SREBP-2
remained susceptible to sterol-regulated cleavage at Site-1 and
subsequent cleavage at Site-2. The presence of the carbohydrate chains
did, however, impede cleavage. The absolute amount of cleavage was
reduced by ~50% for the construct containing one glycosylation site
and by ~80-90% for the protein containing two glycosylation
sites.
Considered together, the data in this paper indicate that the Site-1 protease cleaves the peptide bond between leucine 522 and serine 523 in the lumenal loop of SREBP-2. The only residues that seem to be crucial for recognition are arginine 519 and leucine 522. The location of the arginine relative to the leucine also seems to be crucial. The recognition sequence seems to be RXXL where X can be serine, valine, or alanine, at least. The RXXL sequence is conserved in the four hamster and human SREBPs shown in Fig. 6 and also in SREBP-1c/ADD1 from the rat (RSMLE) (26) and SREBP/HLH106 from Drosophila (RRILS) (27). We believe that other features of the lumenal loop are also crucial for cleavage because moving the RXXL sequence to other sites in the lumenal loop substantially reduced cleavage (data not shown).
Although the current studies identify the site within SREBP-2 that is cleaved by the Site-1 protease, they do not reveal where in the cell this cleavage occurs. Immunofluorescence (24) and cell fractionation studies2 have shown that SREBPs are initially found on membranes of the nuclear envelope and ER. We do not yet know whether the Site-1 protease operates in these organelles or whether the SREBPs must be transported to some other site where cleavage takes place.
We thank Mark Daris, Gloria Brunschede, and Georgeanna Cantrell for excellent technical assistance; Constance Martinelli for invaluable help with tissue culture; Jeff Cormier and Michelle Laremore for synthesis of oligonucleotides and DNA sequencing; and Dr. Clive Slaughter and Carolyn Moomaw for excellent help with radiochemical amino acid sequence analysis.