(Received for publication, September 6, 1995)
From the
Sterol regulatory element-binding proteins (SREBP-1 and SREBP-2)
are proteins of 1150 amino acids each that are attached to
membranes of the endoplasmic reticulum (ER). In sterol-depleted cells,
a protease releases an NH
-terminal fragment of
500
amino acids that contains a basic helix-loop-helix leucine zipper
motif. This fragment enters the nucleus and stimulates transcription of
genes encoding the low density lipoprotein receptor and enzymes of
cholesterol biosynthesis. Prior evidence indicates that the SREBPs are
attached to membranes by virtue of an 80-residue segment located
80 amino acids to the COOH-terminal side of the leucine zipper.
This segment contains two long hydrophobic sequences separated by a
short hydrophilic sequence of
30 amino acids. We have proposed a
hairpin model in which the two hydrophobic sequences span the membrane,
separated by the short hydrophilic sequence which projects into the
lumen of the ER (the ``lumenal loop''). The model predicts
that the NH
- and COOH-terminal segments face the cytosol.
To test this model, we constructed a cDNA encoding human SREBP-2 with
epitope tags at the NH
terminus and in the lumenal loop.
The COOH-terminal region was visualized with a newly developed
monoclonal antibody against this region. Sealed membrane vesicles were
isolated from cells expressing the epitope-tagged version of SREBP-2.
Trypsin treatment of these vesicles destroyed the NH
- and
COOH-terminal segments and reduced the lumenal epitope to a size
consistent with protection of the lumenal sequence plus the two
membrane-spanning segments. The lumenal epitope tag contained two
potential sites for N-linked glycosylation. The size of the
trypsin-protected fragment was reduced by treatment with N-Glycanase® and endoglycosidase H, indicating that this
segment was located in the lumen of the ER where it was glycosylated.
These data provide strong support for the hairpin model.
Sterol regulatory element-binding proteins (SREBPs) ()are membrane-bound transcription factors that regulate
genes involved in cholesterol
homeostasis(1, 2, 3) . Each full-length SREBP
contains between 1133 and 1147 amino acids. The NH
-terminal
portion, comprised of
500 amino acids, includes an acidic domain
that activates transcription and a basic helix-loop-helix leucine
zipper (bHLH-Zip) domain that mediates homodimerization and DNA
binding. This is followed by a hydrophobic segment of
80 amino
acids that attaches the protein to membranes of the endoplasmic
reticulum (ER) and a COOH-terminal domain of
550 amino acids whose
function has not been assigned.
When cultured cells are depleted of
cholesterol, the SREBPs are cleaved by a protease that releases the
NH-terminal regions(4) . These fragments of
500 amino acids enter the nucleus and bind to a 10-base pair
sterol regulatory element (SRE-1) in the promoter of the genes encoding
3-hydroxy-3-methylglutaryl-coenzyme A synthase and perhaps other
cholesterol biosynthetic enzymes, thereby increasing cholesterol
synthesis. They also bind to SRE-1 in the promoter of the gene encoding
the low density lipoprotein receptor, thereby increasing cholesterol
uptake from plasma lipoproteins. When sterols accumulate in the cell,
proteolysis of the SREBPs is suppressed, and the residual nuclear
fragments are rapidly degraded by a protease that is sensitive to
inhibition by acetyl-leucinal-leucinal-norleucinal (ALLN)(4) .
As a result, transcription of the SRE-containing genes declines. The
fate of the COOH-terminal segments of the SREBPs has not been studied
because of the lack of antibodies that react with this fragment.
Cultured cells such as human HeLa cells and hamster fibroblasts
produce two SREBPs, designated 1 and 2. The two human proteins are
50% identical to each other, and they share all of the landmark
features outlined
above(1, 2, 3, 5) . They bind to the
same 10-base pair SRE-1, and they activate transcription of the same
genes. The two SREBPs act independently in cultured cells, and there is
no evidence that heterodimer formation is required(2) .
Proteolysis of both proteins is activated in parallel by sterol
depletion and inhibited in parallel by overloading with sterols such as
25-hydroxycholesterol(4, 5) .
Both SREBPs behave
biochemically as integral membrane proteins. They are removed from
membranes only by detergents and not by treatment with high salt or
alkali(3, 4) . The membrane attachment domain consists
of two long hydrophobic segments of at least 20 residues each that are
separated by a short hydrophilic sequence of 30 residues. Deletion
of this domain markedly reduces the proportion of SREBP-1 that is bound
to membranes(3) . Based on these observations, we have proposed
a hairpin model for the orientation of SREBP in the membrane. The model
postulates that the two hydrophobic segments span the membrane bilayer
in opposite directions separated by the short hydrophilic sequence of
30 amino acids, which projects into the lumen of the ER or the
nuclear envelope. The NH
- and COOH-terminal segments both
face the cytosol(3, 4) .
In the current experiments we use the classic method of protease protection (6) to test the hairpin model for the orientation of human SREBP-2 in the membrane. The ability to perform this test is based on two advances: 1) the development of a monoclonal antibody against the COOH-terminal domain of human SREBP-2 and 2) the development of an epitope tag containing sites for N-linked glycosylation that can be inserted into the lumenal loop. The results are consistent with the hairpin model.
Figure 5:
Map of pTK-HSV-BP2-7D4. This expression
vector was constructed as described under ``Experimental
Procedures.'' Expression is driven by the HSV thymidine kinase
promoter (pTK). The plasmid encodes a 1378-amino acid fusion protein
consisting, from NH terminus to COOH terminus, of the
following: an initiator methionine; two tandem copies of the 11-amino
acid HSV epitope tag (QPELAPEDPED); six amino acids (IDGTVP) encoded by BspDI and KpnI sites; amino acids 14-504 from
human SREBP-2; four amino acids (YPYD) from the influenza hemagglutinin
(HA) epitope; amino acids 33-250 from hamster SREBP-2 including
the 7D4 epitope; eight amino acids (PYDVPDYA) from the HA epitope; and
amino acids 514-1141 from human SREBP-2. BGH pA denotes the
bovine growth hormone polyadenylation sequence. Two putative N-linked glycosylation signals in the 7D4 epitope are underlined.
Monolayers of human embryonic kidney 293 cells (3) were set up on day 0 (4 10
cells/60-mm
dish) and cultured in 8-9% CO
at 37 °C in
Dulbecco's modified Eagle's medium supplemented with 100
units/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) fetal
calf serum. On day 2, the cells were transfected with 5 µg of
pTK-HSV-BP2-7D4 using a modified bovine serum transfection kit
(Stratagene) according to the manufacturer's instructions, except
that the monolayers were not washed prior to addition of 6% (v/v)
modified bovine serum in Dulbecco's modified Eagle's
medium. The transfection was carried out at 35 °C for 3 h in 3%
CO
, after which each dish of cells was washed once with 5
ml of phosphate-buffered saline and switched to 5 ml of
Dulbecco's modified Eagle's medium supplemented with
penicillin, streptomycin, 10% (v/v) calf lipoprotein-deficient serum, 1
µg/ml 25-hydroxycholesterol, 10 µg/ml cholesterol, 50
µM compactin, and 50 µM sodium mevalonate. On
day 3 (20 h later), 5 µl of solution containing 25 mg/ml ALLN was
added to each dish, and the cells were harvested 3 h later. To prepare
the membrane fraction, the cells from 24-32 dishes were pooled
and allowed to swell at 4 °C for 10 min in buffer B (buffer A
without NaCl and supplemented with 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 25 µg/ml
ALLN, and 1 mM dithiothreitol) and then homogenized with 12
strokes of a tight pestle in a Dounce homogenizer. The suspension was
pelleted at 1000
g for 10 min at 4 °C, and the
supernatant was centrifuged at 55,000 rpm for 30 min at 4 °C in a
TLA100.2 rotor to obtain the membrane fraction (10
g pellet). The pellets were washed once with buffer A and
resuspended in buffer A for use in trypsin proteolysis experiments.
For proteolysis of 293 cell
membranes, aliquots of the 10
g membrane
fraction (0.1 mg of protein in 40 µl of buffer A) were incubated at
room temperature in a model REAX 2000 mixer with mild shaking for 30
min in the absence or presence of 1% Triton X-100. Trypsin and soybean
trypsin inhibitor were added as described above, followed by SDS-PAGE
and immunoblotting.
To determine the glycosidase sensitivity of the
trypsin-resistant fragment, aliquots of the 10
g membrane fraction of transfected 293 cells (0.1 mg of
protein in 40 µl of buffer A) were treated sequentially as follows:
1) incubation with 0.3 unit of trypsin (added in 3 µl) at room
temperature for 30 min; 2) addition of 150 units of soybean trypsin
inhibitor (added in 3 µl); 3) addition of Triton X-100 (final
concentration, 1%); 4) boiling for 5 min in the presence (N-Glycanase® and endo H reactions) or absence
(neuraminidase reaction) of SDS (final concentration, 0.5%) and
-mercaptoethanol (final concentration, 0.1%); and 5) addition of
the indicated glycosidase (added in 4 µl) and incubation at 37
°C for 2 h as described in the legend to Fig. 8. Each sample
(final volume, 59 µl) was mixed with 10 µl of 5
SDS
loading buffer (15) and used for SDS-PAGE and immunoblot
analysis.
Figure 8:
Sensitivity of the protease-resistant,
epitope-tagged loop region of SREBP-2 to treatment with glycosidases.
Aliquots of the 10
g membrane fraction
from pTK-HSV-BP2-7D4 transfected 293 cells (0.1 mg of protein) were
digested with trypsin at room temperature for 30 min as described under
``Experimental Procedures.'' After addition of soybean
trypsin inhibitor, the membrane fractions were treated with detergent,
boiled for 5 min, and then incubated for 2 h at 37 °C with one of
the following glycosidases: lane 1, none; lane 2,
1.25 IU of N-Glycanase®; lane 3, 2.5 IU of endo
H; and lane 4, 0.75 IU of neuraminidase. The samples were
processed for SDS-PAGE and immunoblot analysis with 8 µg/ml
IgG-7D4. The filter was exposed to film for 1 min. The arrow denotes trypsin-resistant fragment, and the asterisk denotes the deglycosylated form of the trypsin-resistant
fragment.
Fig. 1shows hydrophobicity plots for human SREBP-1
and -2 according to the method of Kyte and Doolittle (16) with
a window of 17 residues. The two hydrophobic segments, designated 1 and
2, are clearly visible, as is the short hydrophilic segment between
them. These segments are located 80 residues to the COOH-terminal
side of the bHLH-Zip region, which is underlined in Fig. 1.
Figure 1: Hydropathy plots of the amino acid sequence of human SREBP-1 and -2. Positive values represent hydrophobicity. The residue-specific hydropathy index was calculated over a window of 17 residues by the method of Kyte and Doolittle (16) using the Genetics Computer Group sequence analysis software package, version 7.1(21) . Horizontal black bars denote bHLH-Zip domains. Numbers 1 and 2 denote the two putative transmembrane segments.
To compare the fates of the NH- and
COOH-terminal segments of SREBP-2, we took advantage of a newly
developed monoclonal antibody, designated IgG-1C6, that was raised
against a fragment of human SREBP-2 extending from residue 833 to the
COOH terminus. In the experiment of Fig. 2, HeLa cells were
partially deprived of sterols by growth in the presence of a low
concentration of newborn calf serum (2.5%). We then overloaded the
cells with sterols by adding a mixture of 25-hydroxycholesterol and
cholesterol to the culture medium. At varying times, cells were
harvested, and high salt nuclear extracts and 10
g membrane pellets were subjected to SDS-PAGE and blotted with
a monoclonal antibody directed against the NH
terminus and
with the COOH-terminal monoclonal IgG-1C6. At zero time, when the cells
were in a sterol-depleted state, both antibodies stained the
full-length precursor form of SREBP-2, which was found in the membrane
fraction (designated P in Fig. 2, lane F). As
expected, the NH
-terminal antibody also stained a fragment
whose migration corresponded to a molecular mass of 68 kDa, which was
found in the nuclear extracts (lane A). The
NH
-terminal fragment was shown previously to migrate with
anomalous slowness, owing to the acidic NH
-terminal
activation domain(3) . The COOH-terminal antibody stained a
fragment of
65 kDa that was bound to the membranes (lane
F). Two h after sterol addition, the NH
-terminal
fragment had almost disappeared from the nucleus (lane D). The
COOH-terminal fragment was also rapidly degraded. By 2 h, it had been
reduced by more than 80% (lane I), but a small amount
continued to be detectable at 6 h (lane J). No COOH-terminal
fragment was detected in immunoblots of the 10
g supernatant fraction (data not shown).
Figure 2:
Disappearance of NH- and
COOH-terminal portions of SREBP-2 in HeLa cells following addition of
sterols. On day 0, HeLa cells were divided into five replicate spinner
cultures, each containing 1.2
10
cells in 500 ml.
On day 1, each culture received a mixture of sterols (1 µg/ml
25-hydroxycholesterol plus 10 µg/ml cholesterol) for the indicated
time at 37 °C. Staggered additions were made so that the cells were
harvested at the same time. A high salt nuclear extract and a membrane
fraction were prepared as described under ``Experimental
Procedures,'' and an aliquot of each fraction (50 µg of
protein) was subjected to SDS-PAGE and immunoblot analysis. Duplicate
filters were blotted with 5 µg/ml anti-NH
-terminal
IgG-3H8 (upper panel) or 5 µg/ml anti-COOH-terminal
IgG-1C6 (lower panel). The gels were calibrated with
prestained protein standards (New England Biolabs). The filters were
exposed to film for 60 s (upper panel) or 80 s (lower
panel). P, N, and C denote the
precursor form and the NH
-terminal and COOH-terminal
fragments of SREBP-2, respectively.
The COOH-terminal
fragment of SREBP-2 behaved as an integral membrane protein, just like
the full-length precursor form(4) . In the experiment of Fig. 3, membranes isolated from sterol-deprived cells were
treated with various protein-solubilizing agents and subjected to
centrifugation at 10
g. The pellets and
supernatants were subjected to SDS-PAGE and immunoblotted with IgG-1C6,
which visualized both the precursor form and the COOH-terminal fragment
of SREBP-2. The precursor and the COOH-terminal fragment were both
solubilized partially with 1% SDS and 1% Triton X-100. Neither was
solubilized with buffer alone, nor with 0.1 M
Na
CO
or 1 M hydroxylamine.
Figure 3:
Membrane association of the cleaved
COOH-terminal portion of SREBP-2 in HeLa cells. A 10
g membrane fraction was prepared from 500 ml of
HeLa cells as described under ``Experimental Procedures.''
Aliquots of the membrane fraction (0.4 mg of protein in 80 µl of
buffer A) were centrifuged at 10
g for 30
min at 4 °C. Each pellet was resuspended in 160 µl of one of
the following solutions: lane 1, 1% (w/v) SDS in buffer A; lane 2, buffer A; lane 3, 0.1 M
Na
CO
in water; lane 4, 1 M hydroxylamine (pH 10) in water; lane 5, 1% (v/v) Triton
X-100 in buffer A. After shaking for 15 min at room temperature, the
mixtures were centrifuged at 10
g for 30
min at 15 °C. An aliquot of the supernatant (32 µl) was mixed
with 5
SDS loading buffer (lanes 1-5). Each
pellet was mixed with 160 µl of lysis buffer (22) containing 1% SDS, and an aliquot (32 µl) was mixed
with 5
SDS loading buffer (lanes 6-10). After
boiling for 5 min, the samples were subjected to SDS-PAGE, and
immunoblot analysis was carried out with 5 µg/ml anti-COOH-terminal
IgG-1C6. The filter was exposed to film for 30 s. P and C denote the precursor form and the COOH-terminal fragment of
SREBP-2, respectively.
To determine the orientation of the COOH-terminal fragment with respect to the membrane, we homogenized HeLa cells gently with a Dounce homogenizer and isolated membrane vesicles by centrifugation (Fig. 4). The vesicles were incubated with varying concentrations of trypsin in the absence or presence of Triton X-100 followed by SDS-PAGE and immunoblotting with IgG-1C6. Trypsin at a concentration of 1.7 units/ml obliterated the IgG-1C6 epitope, whether it was present on the precursor or on the COOH-terminal fragment (Fig. 4A, upper panel, lane 7). Disruption of the membranes with Triton X-100 increased trypsin sensitivity only slightly (lower panel). These data indicate that the COOH-terminal fragment of SREBP-2 is exposed to the cytoplasmic face of the membrane. As a control for the intactness of the membrane vesicles, we immunoblotted the digested membranes with a monoclonal antibody against chaperone proteins, called BiPs, that are known to reside in the lumen of the ER (17) . The antibody visualizes two BiPs, designated BiP94 and BiP78. BiP94 was completely resistant to trypsin in the absence of Triton X-100 (Fig. 4B, upper panel), and it was readily destroyed when Triton X-100 was present (lower panel). This observation confirms that the membrane vesicles were sealed. BiP78 was not informative because the protein was resistant to trypsin either in the presence or absence of Triton X-100, apparently because of an intrinsic trypsin resistance.
Figure 4:
Topology of the COOH-terminal domain of
SREBP-2 as determined by trypsin proteolysis. Aliquots of the membrane
fractions from HeLa cells (0.3 mg of protein) were resuspended in 110
µl of buffer A in the absence (upper panels) or presence (lower panels) of 1% Triton X-100 as described under
``Experimental Procedures.'' After incubation at room
temperature for 30 min, each sample received trypsin as indicated. The
final trypsin concentrations (units/ml) were 0, 5.6
10
, 1.7
10
, 5.6
10
, 0.16, 0.56, 1.7, 5.6, and 16.7 in lanes
1-9 and lanes 10-18, respectively. After
incubation for 30 min at room temperature, each sample received 300
units of soybean trypsin inhibitor (added in 6 µl), after which a
40 µl-aliquot from each sample was subjected to SDS-PAGE and
immunoblot analysis with 5 µg/ml anti-COOH-terminal IgG-1C6 (A). A duplicate filter was blotted with 5 µg/ml anti-BiP
antibody (B). The filters were exposed to film for 10 min (A) or 10 s (B). P and C in A denote the precursor and COOH-terminal forms of SREBP-2,
respectively. B94 and B78 in B denote BiP-94
and BiP-78, respectively.
We next sought to determine the membrane orientation of the hydrophilic loop between the two putative transmembrane segments. Multiple attempts to raise monoclonal or polyclonal antibodies against this short 30-residue sequence failed. We then used recombinant DNA techniques to insert DNA segments encoding short epitope ``tags'' into this segment followed by expression in transfected cells. We tried DNA segments encoding epitopes for which monoclonal antibodies are commercially available. These included Myc(18) , influenza hemagglutinin(11) , T7 gene 10 leader peptide(19) , and the Flag(TM) epitope(14) . All of these attempts failed. Although the monoclonal antibodies recognized these epitopes when inserted into other proteins or at other locations in SREBP-2, they did not recognize the epitope when inserted into the 30-residue hydrophilic loop, even after the protein had been denatured by SDS-PAGE.
In a final attempt to circumvent this problem, we
decided to insert a DNA sequence encoding a much longer segment of
protein into the hydrophilic loop. We chose a 218-amino acid segment to
which we already had a potent monoclonal antibody. This segment
consisted of amino acids 33-250 of hamster SREBP-2 ((5) ), and the monoclonal antibody that recognizes it is
IgG-7D4 ((12) ). The antibody is specific for the hamster
sequence, and it does not recognize human SREBP-2. When this hamster
sequence is inserted into human SREBP-2, it constitutes a novel
epitope. It should also be noted that this segment is part of the
NH-terminal region of hamster SREBP-2 that is normally
located on the cytoplasmic side of the membrane.
Fig. 5shows
the plasmid that we constructed, which is designated pTK-HSV-BP2-7D4. Fig. 6A diagrams the resultant protein. The protein consists
of human SREBP-2 with two epitope tags. The first is a short epitope
tag from the HSV glycoprotein that is inserted at the NH terminus of the protein. The second is the 33-250-amino
acid segment of hamster SREBP-2 that is inserted into the loop between
the two transmembrane regions. By chance, this sequence contains two
potential N-linked glycosylation sites (Asn-X-Ser or
-Thr) that are underlined in Fig. 5. Expression is
driven by a promoter from the HSV thymidine kinase.
Figure 6:
Specificity of monoclonal antibody IgG-7D4
for hamster 7D4 epitope inserted into the loop region of human SREBP-2. A, schematic diagram of protein encoded by pTK-HSV-BP2-7D4,
showing site in loop between two transmembrane segments where hamster
7D4 epitope is inserted. B, immunoblot analysis of cells
expressing protein encoded by pTK-HSV-BP2-7D4. 293 cells were
transfected with 5 µg of one of the following plasmids: lanes 1 and 4, empty vector pTK; lanes 2 and 5, pTK-HSV-BP2 encoding HSV-tagged human SREBP-2; lanes 3 and 6, pTK-HSV-BP2-7D4 encoding HSV-tagged and 7D4-tagged
human SREBP-2. Aliquots of membrane fractions (0.1 mg of protein in
buffer A) were subjected to SDS-PAGE and immunoblot analysis with 0.5
µg/ml anti-HSV-Tag(TM) antibody (lanes 1-3) or 5
µg/ml IgG-7D4 (lanes 4-6). The filters were exposed
to film for 2 min (lanes 1-3) or 3 min (lanes
4-6).
To confirm the
specificity of IgG-7D4, we transfected human 293 cells with
pTK-HSV-BP2-7D4 or with a control plasmid (pTK-HSV-BP2) that contains
the NH-terminal HSV tag but lacks the 7D4 epitope (Fig. 6). Membranes from the transfected cells were subjected to
SDS-PAGE and immunoblotted with an antibody against the HSV tag or with
IgG-7D4. Neither antibody visualized any protein in mock-transfected
cells (lanes 1 and 4). In cells transfected with
pTK-HSV-BP2, the protein was visualized with the anti-HSV antibody (lane 2), but not IgG-7D4 (lane 5). This result
confirms the inability of IgG-7D4 to recognize the human protein. In
cells transfected with pTK-HSV-BP2-7D4, both antibodies visualized a
protein that migrated with an apparent molecular mass of 175 kDa (lanes 3 and 6), which is much larger than native
SREBP-2, owing to the presence of the long 7D4 epitope.
Fig. 7shows a trypsin protection experiment similar to that
of Fig. 4, but performed with membrane vesicles from 293 cells
that were transfected with pTK-HSV-BP2-7D4. In the absence of trypsin,
the membranes contained a protein of 175 kDa that was visualized by the
anti-HSV antibody which reacts with the NH-terminal epitope
tag (upper panel), IgG-1C6 which reacts with the COOH-terminal
segment (middle panel), and IgG-7D4 which reacts with the
epitope inserted into the putative lumenal loop (lower panel).
Treatment with trypsin abolished both the NH
- and
COOH-terminal segments (lanes 2-4). The 7D4 epitope was
preserved on a fragment of
47 kDa that resisted trypsin at a
concentration that was 10-fold above the concentration that destroyed
the other two epitopes (lane 4). When the digestion was
conducted in the presence of Triton X-100, the NH
- and
COOH-terminal segments were destroyed as before, and the 7D4 epitope
became trypsin-sensitive. These results are consistent with the hairpin
model for the orientation of SREBP-2, in which the NH
terminus and COOH terminus are exposed to the cytosol, and the
hydrophilic loop between the transmembrane segments projects into the
ER lumen.
Figure 7:
Resistance of epitope-tagged loop region
of SREBP-2 to trypsin proteolysis. 293 cells were transfected with
pTK-HSV-BP2-7D4, the 10
g membrane
fractions were prepared, and aliquots of these membranes (0.1 mg of
protein) were resuspended in 40 µl of buffer A in the absence (lanes 1-4) or presence (lanes 5-8) of 1%
Triton X-100 as described under ``Experimental Procedures.''
After incubation at room temperature for 30 min, each sample received
increasing amounts of trypsin. The final trypsin concentrations
(units/ml) were as follows: lanes 1 and 5, 0; lanes 2 and 6, 2.5; lanes 3 and 7,
7.5; and lanes 4 and 8, 25. Triplicate samples for
each reaction were incubated and processed for SDS-PAGE as described in
the legend to Fig. 4. Immunoblot analysis was carried out with
one of the following antibodies: upper panel, 0.5 µg/ml
anti-NH
-terminal antibody (HSV-Tag(TM) antibody); middle panel, 10 µg/ml anti-COOH-terminal antibody
(IgG-1C6); and lower panel, 8 µg/ml of IgG-7D4. The
filters were exposed to film for 10 s (upper and lower
panels) or 3 min (middle panel). Arrows denote
the position of migration of the endogenous and transfected precursor (P) forms of SREBP-2 and of the trypsin-resistant
fragment.
The apparent size of the trypsin-resistant fragment on
SDS-PAGE, as visualized with IgG-7D4 (47 kDa) was greater than would be
predicted if the protected fragment consisted of the 218 amino acid
peptide containing the epitope plus the remaining lumenal amino acids
and the two transmembrane regions (35 kDa). We suspected that this
might be attributable to glycosylation of the epitope tag at one or
both of the N-linked sites shown in Fig. 5. To test
this possibility, we digested intact membrane vesicles with trypsin as
before, then solubilized the membranes and denatured the proteins by
treatment with detergent and boiling. The trypsin-resistant fragment
was then digested with one of three glycosidases, and its size was
estimated by SDS-PAGE and immunoblotting with IgG-7D4. As shown in Fig. 8, in the absence of glycosidases the trypsin-resistant
fragment migrated as a 47-kDa protein. The apparent molecular mass was
reduced to about 35 kDa by treatment with N-Glycanase® and
endo H, but not neuraminidase. This pattern of glycosidase sensitivity
indicates that the N-linked sugars remained in their high
mannose unprocessed forms and that the protein had not been transported
to the Golgi complex(20) .
The current manuscript provides evidence to support the
hairpin model for the orientation of SREBP-2 in the membrane. According
to this model, the NH- and COOH-terminal regions of SREBP-2
are oriented toward the cytosol. They are separated by a membrane
attachment domain that consists of two membrane-spanning segments
separated by a short loop that projects into the lumen of the ER.
The evidence in support of this model comes from trypsin sensitivity
experiments performed with membrane vesicles. The NH- and
COOH-terminal regions were readily destroyed by trypsin, and the
lumenal loop epitope was reduced to a size consistent with protection
of the glycosylated loop plus the two membrane-spanning segments. The
addition of N-linked sugars to the loop was confirmed by the
observation that the protected fragment was reduced in size by
treatment with N-Glycanase® and endo H.
In order to visualize the lumenal loop in immunoblots, we had to resort to the unorthodox procedure of inserting a long 218-residue segment of protein containing an epitope. This was necessary because short epitopes inserted into this region failed to react with their cognate monoclonal antibodies even after the protein was denatured by SDS-PAGE. It seems likely that this region of the protein must refold during transfer to nitrocellulose, perhaps through hydrophobic interactions between the two membrane-spanning segments, thereby occluding the lumenal epitope. When a long protein segment was inserted, the epitope was no longer occluded.
It is possible that the insertion of such a long epitope
changed the orientation of the SREBP in the membrane. We believe that
this is unlikely for two reasons: 1) the NH-terminal
fragment and COOH-terminal fragment were trypsin-sensitive in the
native protein as well as in the protein bearing the epitope tag ( Fig. 4and Fig. 7); and 2) the sequence that we inserted
does not have lumenal targeting properties, since it is normally on the
cytoplasmic side of the membrane.
The current studies also provide
the first glimpse of the fate of the COOH-terminal segment of SREBP-2
after the NH-terminal domain has been released by the
sterol-regulated protease. The data suggest that this fragment remains
attached to membranes as an integral protein. When sterols are added,
most of the COOH-terminal fragment is degraded within 2 h, but a small
amount remains detectable for as long as 6 h. We do not know whether
this represents a slowly degraded pool or whether it represents a small
amount of COOH-terminal fragment that is continuously produced, even in
the presence of sterols.
Although the current study defines the orientation of the two transmembrane segments and the lumenal loop, the data do not make any statement about the mechanism by which more distal elements of the COOH-terminal segment interact with the membrane. This region has some affinity for membranes since a fraction of SREBP-2 remains associated with membranes even when the transmembrane segments are deleted(3) . Inspection of the hydrophobicity plots in Fig. 1reveals several moderately hydrophobic sequences in the COOH-terminal region. However, none of these is long enough nor of sufficient hydrophobicity to meet the criteria (16) of true transmembrane segments. It is possible that some of these segments dip into the lipid bilayer, but we do not believe that any of them span the membrane.
Although all of the current experiments were performed with SREBP-2, we believe that the results apply to SREBP-1, since the hydrophobicity profiles of the two proteins are nearly identical (Fig. 1) and because proteolysis of the two proteins is regulated in parallel(4, 5, 12) . Knowledge of the membrane orientation of these proteins is essential if we are to understand the site at which they are cleaved by the sterol-regulated protease and the mechanism of its regulation.