(Received for publication, January 4, 1996; and in revised form, February 12, 1996)
From the
Sterol regulatory element binding proteins (SREBP-1 and SREBP-2)
are attached to the endoplasmic reticulum (ER) and nuclear envelope by
a hairpin domain consisting of two transmembrane regions connected by a
short lumenal loop of 30 hydrophilic amino acids. In
sterol-depleted cells, a protease cleaves the protein in the region of
the first transmembrane domain, releasing an NH
-terminal
fragment of
500 amino acids that activates transcription of genes
encoding the low density lipoprotein receptor and enzymes of
cholesterol synthesis. In sterol-overloaded cells, proteolysis does not
occur, and transcription is repressed. Through mutational analysis in
transfected cells, we identify two segments of SREBPs that are required
for proteolysis, one on either side of the ER membrane. An arginine in
the lumenal loop is essential. A tetrapeptide sequence (DRSR) on the
cytosolic face adjacent to the first transmembrane domain is also
required for maximal cleavage. Both of these elements are conserved in
the human and hamster versions of SREBP-1 and SREBP-2. Sterol-mediated
suppression of cleavage of SREBP-1 was found to be dependent on the
extreme COOH-terminal region (residue 1034 to the COOH terminus), which
exists in two forms as a result of alternative splicing. The form
encoded by the ``a'' class exons (exons 18a and 19a)
undergoes sterol-regulated cleavage. The form encoded by the
``c'' class exons (18c and 19c) is cleaved less efficiently
and is not suppressed by sterols. These studies were made possible
through use of a vector that achieves low level expression of
epitope-tagged SREBPs under control of the relatively weak thymidine
kinase promoter from herpes simplex virus. In contrast to SREBPs
overproduced by high level expression vectors, the SREBPs produced at
low levels were subject to the same regulated cleavage pattern as the
endogenous SREBPs. These results indicate that sterol-regulated
proteolysis of SREBPs is a complex process, requiring sequences on both
sides of the ER membrane.
Cholesterol homeostasis is maintained by sterol-regulated
cleavage of two membrane-bound transcription factors designated sterol
regulatory element binding protein-1 and -2 (SREBP-1 and -2). ()These proteins, each nearly 1150 amino acids in length,
are attached to the endoplasmic reticulum (ER) and nuclear
envelope(1, 2, 3) . When cells require
cholesterol, a protease cleaves each of the SREBPs, releasing an
NH
-terminal fragment of
500 amino acids that contains
basic helix-loop-helix-leucine zipper and transcription activation
domains. These fragments enter the nucleus and bind to sterol
regulatory elements in the promoters of genes encoding the low density
lipoprotein receptor, 3-hydroxy-3-methylglutaryl CoA synthase and
possibly other enzymes of cholesterol and fatty acid
biosynthesis(1, 2, 3, 4, 5) .
These actions lead to enhanced cholesterol uptake from plasma
lipoproteins as well as enhanced cholesterol synthesis. When sterols
overaccumulate in cells, proteolysis of the SREBPs is suppressed, the
proteins remain bound to the ER, and transcription of the
sterol-regulated genes declines.
In order to unravel the mechanism
for sterol-regulated proteolysis, it is necessary first to understand
the mechanism by which the SREBPs are attached to membranes. We
recently used protease protection and N-linked glycosylation
methods to demonstrate that SREBP-2 has a hairpin
orientation(6) . The NH-terminal and COOH-terminal
segments of
500 amino acids each face the cytosol. They are
separated by a hairpin membrane attachment domain that consists of one
transmembrane segment, a short 30-amino acid hydrophilic loop on the
lumenal side of the ER membrane, and a second transmembrane segment.
The precise cleavage site and the domains within the SREBP that are
required for proteolysis and for sterol-mediated suppression of
proteolysis are unknown.
One way to answer these questions is through in vitro mutagenesis of expressible cDNAs. We have not been able to use this method in the past because we were unable to observe sterol-mediated regulation when SREBPs were overexpressed in human embryonic kidney 293 cells as a result of transient transfection(2, 3, 4) . Overexpression seemed to overwhelm the regulatory machinery, leading to aberrant and unregulated proteolysis. In the current studies, we have overcome this problem through the use of an expression vector containing a relatively weak promoter from the herpes simplex virus thymidine kinase (TK) gene (7) . The promoter drives low level expression of SREBPs containing an epitope tag derived from a herpes simplex virus glycoprotein. The tag allows the products of the transfected cDNAs to be differentiated from the endogenous SREBPs. Under these conditions cleavage of the epitope-tagged SREBPs is tightly regulated by sterols. Through mutagenesis studies, we identify two crucial segments in the SREBPs, one on either side of the membrane, that are required for proteolysis and a region in the COOH-terminal segment that is necessary in order for sterols to suppress this proteolysis.
Figure 1:
Map of pTK-HSV-BP2 (A) and
model for membrane orientation of SREBPs (B). A,
expression vector pTK-HSV-BP2 was constructed as described under
``Experimental Procedures.'' The plasmid is driven by the HSV
TK promoter (pTK), and it encodes an 1157-amino acid fusion protein
consisting of the following coding sequences from NH terminus to COOH terminus: an initiator methionine, two tandem
copies of the 11-amino acid HSV epitope tag, six novel amino acids
(IDGTVP corresponding to BspDI and KpnI restriction
sites), and amino acids 14-1141 of human SREBP-2. BGH pA denotes bovine growth hormone polyadenylation sequence; f1 ori denotes the origin of replication of filamentous phage for
generation of single-stranded DNA. neo denotes
neomycin-resistance gene; amp denotes ampicillin-resistance
gene; thin lines denote unspecified vector sequences. B, this model shows the hairpin orientation of SREBP-1 and
SREBP-2 in cell membranes. The two transmembrane regions are connected
by a short lumenal loop of
30 hydrophilic amino acids. The arrows denote the positions of the two sequences that are
important for sterol-regulated proteolysis.
The above three fragments, namely, the 7.9-kb NruI-KpnI fragment of the pcDNA3-SREBP-2
backbone containing amino acids 315-1141 of SREBP-2, the 1.1-kb SalI (blunted)-NotI fragment of the TK
promoter, and the 124-base pair annealed oligonucelotides (5` NotI and 3` KpnI), were ligated together to create an
intermediate plasmid, designated ``triple ligation
construct.'' To clone the NH terminus of SREBP-2 into
the triple ligation construct, the nucleotide sequence encoding amino
acids 14-315 of human SREBP-2 was amplified by polymerase chain
reaction in a 100-µl reaction containing 200 ng of
pXH4(3) , 2.5 units of pfu DNA polymerase, 5 nmol of
dNTPs, and 20 pmol of each primer, which had a KpnI site at
its 5` end. The polymerase chain reaction was carried out at an initial
temperature of 80 °C followed by 94 °C for 3 min and 20 cycles
of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min
for each cycle. The amplified product was digested with KpnI
and cloned into the KpnI site of the triple ligation construct
to create pTK-HSV-BP2 (see Fig. 1A). This plasmid
encodes an 1157-amino acid fusion protein of SREBP-2 (amino acids
14-1141) preceded by an initiator methionine, the two tandem
copies of the HSV epitope, and six novel amino acids (see above).
Because methionine at residue 13 of SREBP-2 (3) is surrounded
by a favorable Kozak consensus sequence(8) , the
NH
-terminal 13 amino acids were not included in the vector
in order to prevent multiple initiations of translation.
The
following steps were used to construct an expression vector for human
SREBP-1a modeled after pTK-HSV-BP2: 1) pTK-HSV-BP2 was digested with BspDI and XbaI to remove the 4.2-kb SREBP-2 sequence
but retain the HSV epitope sequence. 2) The sequence corresponding to
amino acids 2-474 of SREBP-1a was amplified by polymerase chain
reaction of pSREBP-1a (2) with an NH-terminal
primer flanked by a BspDI site and a COOH-terminal primer
flanked by an EagI site using conditions described above. 3)
The 2.6-kb EagI-XbaI fragment corresponding to
amino acids 475-1147 of SREBP-1a was isolated from
pSREBP-1a(2) . The above three fragments were ligated to
generate pTK-HSV-BP1a. This plasmid encodes an 1171-amino acid fusion
protein of SREBP-1a (amino acids 2-1147) preceded by an initiator
methionine, the two tandem copies of the HSV epitope, and two novel
amino acids (ID) corresponding to the BspDI restriction site.
To construct an expression vector for human SREBP-1c, the following
steps were used: 1) pSREBP-1c (2) was digested with Eco47III and XbaI to isolate a 2.5-kb Eco47III-XbaI fragment encoding amino acids
311-1047 of human SREBP-1c. 2) The sequence corresponding to amino
acids 2-310 of SREBP-1c was amplified by polymerase chain
reaction of pSREBP-1c (2) with an NH-terminal
primer flanked by BspDI and a COOH-terminal primer flanked by Eco47III using conditions described above. The amplified
product was then digested with BspDI and Eco47III. 3)
pTK-HSV-BP2 was digested with BspDI and XbaI to
remove the 4.2-kb SREBP-2 sequence but retain the HSV epitope sequence.
The three fragments were ligated to generate pTK-HSV-BP1c. This plasmid
encodes an 1071-amino acid fusion protein of SREBP-1c (amino acids
2-1047) preceded by an initiator methionine, the two tandem
copies of the HSV epitope, and two novel amino acids (ID) corresponding
to the BspDI restriction site.
To construct an expression vector for human SREBP-1b, pTK-HSV-BP1a was digested with SrfI and XbaI to remove the 1.2-kb COOH-terminal sequences of SREBP-1a (amino acids 927-1147), after which this sequence was replaced with the 0.8-kb SrfI-XbaI fragment from pSREBP-1c (2) encoding amino acids 903-1047. The resulting plasmid is designated pTK-HSV-BP1b.
A control vector without any insert, designated pTK, was constructed by digesting pTK-HSV-BP2 with SpeI and XbaI to remove the 4.2-kb SREBP-2 sequence, followed by ligation. To construct a CMV promoter-driven expression vector for SREBP-1a, a 4.1-kb SpeI (blunted)-XbaI-digested fragment encoding the HSV-epitope tagged SREBP-1a fusion protein of 1171 amino acids (see above) was inserted into the EcoRV-XbaI sites of pcDNA3. This plasmid is designated pCMV-HSV-BP1a. All of the above plasmids were sequenced in the region of the construction junctions to confirm the DNA sequence.
Fig. 1A shows the TK-driven vector that was
used to express SREBP-2 in these studies. The NH terminus
of the encoded protein contains a 22-amino acid epitope tag derived
from the HSV envelope glycoprotein D (HSV tag). Expression is driven by
the HSV TK promoter, and the polyadenylation signal is derived from the
bovine growth hormone gene. A similar expression vector was constructed
for SREBP-1a as described under ``Experimental Procedures.'' Fig. 1B shows the topology of the SREBPs and indicates
the location of the two segments that are required for proteolysis: the
DRSR immediately external to the first transmembrane sequence and the
Arg in the lumenal loop.
Fig. 2compares immunoblots of nuclear extracts and crude membrane pellets from human embryonic kidney 293 cells that were transfected with plasmids encoding HSV-tagged SREBP-1a under control of the strong CMV promoter or the weak TK promoter. The CMV vector gave extremely high level overexpression of the membrane-bound form of SREBP-1a, even when only 0.5 µg of plasmid was used (lanes 13-16 in Fig. 2B). The immunoblots showed multiple overlapping bands, indicating extensive proteolysis of the overexpressed protein. The amounts of the precursor and the proteolytic fragments were similar when the cells were sterol-deprived by incubation with the cholesterol synthesis inhibitor compactin (-sterols) or when they were sterol-overloaded by incubation with 25-hydroxycholesterol and cholesterol in the presence of compactin (+sterols). Nuclear extracts of cells transfected with the CMV-driven vector contained relatively large amounts of the mature nuclear form of tagged SREBP-1a, but the amount was not affected by sterols (lanes 5-8 in Fig. 2A).
Figure 2:
Immunoblot analysis of HSV-tagged
SREBP-1a in 293 cells transfected with expression vectors driven by the
CMV and TK promoters. On day 0, 293 cells were set up at 4
10
cells/60-mm dish 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.
On day 2, the cells were transfected with the indicated amount of the
indicated plasmid as described under ``Experimental
Procedures.'' The total DNA in each transfection was adjusted to 5
µg/dish with an empty vector pTK(6) . 3 h after
transfection, the cells were switched to medium B (medium A containing
10% lipoprotein-deficient serum, 50 µM compactin, and 50
µM sodium mevalonate) in the absence (-) or presence
(+) of 1 µg/ml 25-hydroxycholesterol plus 10 µg/ml
cholesterol (sterols) as indicated. On day 3, the cells were harvested
and fractionated into a nuclear extract fraction (A) and a
membrane fraction (B) as described under ``Experimental
Procedures.'' Aliquots of the nuclear extracts (60 µg of
protein) and membranes (80 µg) were subjected to SDS-PAGE and
immunoblot analysis with 0.5 µg/ml HSV-Tag(TM) antibody. The
filters were exposed to film for 20 s. N and P denote
the NH
-terminal mature and precursor forms of SREBP-1a,
respectively.
When the TK promoter was used, the amount of membrane-bound precursor was much lower than observed with the CMV promoter, and the smear of membrane-bound proteolytic fragments was not observed (Fig. 2, compare lanes 11-12 with lanes 13-16). The nuclei of cells expressing the TK-driven protein showed abundant mature form of SREBP-1a in the absence of sterols and a disappearance of this protein in the presence of sterols (Fig. 2, lanes 3 and 4). This behavior reflects that of endogenous SREBP-1a in untransfected cells(1, 13) .
Fig. 3A shows the effect of different concentrations of 25-hydroxycholesterol on the amount of the mature nuclear form of tagged SREBP-2 produced by the TK vector. The protein disappeared at a sterol concentration between 0.3 and 1 µg/ml (Fig. 3A), which is similar to that previously observed for the endogenous protein(1, 13) . The sterol had no effect on the amount of the membrane-bound precursor (Fig. 3B).
Figure 3:
Immunoblot analysis of HSV-tagged SREBP-2
in 293 cells transfected with pTK-HSV-BP2: regulation by
25-hydroxycholesterol. On day 0, 293 cells were set up as described in
the legend to Fig. 2. On day 2, the cells were transfected with
5 µg of pTK-HSV-BP2 as described under ``Experimental
Procedures.'' 3 h after transfection, the cells were switched to
medium B supplemented with 0.2% ethanol containing the indicated final
concentrations of 25-hydroxycholesterol. On day 3, the cells were
harvested and fractionated as described under ``Experimental
Procedures.'' Aliquots of nuclear extract (60 µg of protein) (A) and membranes (80 µg) (B) were subjected to
SDS-PAGE and immunoblot analysis with 0.5 µg/ml HSV-Tag(TM)
antibody. The filters A and B were exposed to film
for 1 and 2 min, respectively. N and P denote the
NH-terminal mature and precursor forms of SREBP-2,
respectively.
In
an initial attempt to localize sequences in SREBP-1a that are required
for sterol-regulated proteolysis, we created a series of point
mutations in the linker region between the basic helix-loop-helix
leucine zipper region and the first transmembrane domain, which is the
approximate region at which cleavage takes
place(4, 13, 14, 15) . We previously
identified an apoptosis-related cysteine protease that cleaves SREBP-1a
at Asp and SREBP-2 at Asp
(15) .
This protease, designated CPP32, becomes activated only during
apoptosis, and it is not regulated by sterols. Changing the Asp at the
cleavage site to Ala abrogates cleavage by CPP32 in
vitro(15) . However, changing these residues in SREBP-1a (Fig. 4B, lanes 5 and 6) or SREBP-2 (Fig. 4C, lanes 15 and 16) did not
affect cleavage by the sterol-regulated protease in transfected 293
cells. These results provide further evidence that CPP32 is not
responsible for sterol-regulated proteolysis.
Figure 4:
Immunoblot analysis of HSV-tagged SREBP-1a (B) and SREBP-2 (C) in 293 cells transfected with
wild-type and linker region mutants. A, schematic diagram of
the domain structures of SREBP-1a and SREBP-2, showing the sites of
mutations in the linker region between amino acids 456/450 and 487/481.
The 4-amino acid sequence DRSR (residues 484-487 in SREBP-1a and
residues 478-481 in SREBP-2) were each replaced by AS. For
reference, the first amino acid in the first transmembrane domain is
residue 488 and 482 for SREBP-1 and -2, respectively. B and C, immunoblot analysis of wild-type and linker region mutants
of HSV-tagged SREBPs in nuclear extracts and membranes from 293 cells
transfected with pTK-HSV-BP1a and its mutants (B) and
pTK-HSV-BP2 and its mutants (C). On day 0, 293 cells were set
up as described in the legend to Fig. 2. On day 2, the cells
were transfected with 5 µg of the indicated plasmid. 3 h after
transfection, the cells were switched to medium B in the
absence(-) or the presence (+) of 1 µg/ml
25-hydroxycholesterol plus 10 µg/ml cholesterol (sterols) as
indicated. On day 3, the cells were harvested and fractionated as
described under ``Experimental Procedures.'' Aliquots of 40
µg of protein (B) or 60 µg (C) from nuclear
extracts (upper panels) and aliquots of 60 (B) or 80
µg (C) from membranes (lower panels) were
subjected to SDS-PAGE and immunoblot analysis with 0.5 µg/ml
HSV-Tag(TM) antibody. The filters B and C were
exposed to film for 30 s and 1 min, respectively. N and P denote the NH-terminal mature and precursor forms of
SREBPs, respectively.
SREBP-1a and -2
contain a conserved sequence DRSR that marks the boundary of the first
transmembrane domain (Fig. 1B and 4A). The
following residue, leucine (SREBP-1) or isoleucine (SREBP-2), initiates
the hydrophobic sequence of the first transmembrane domain(6) .
We eliminated the DRSR by changing it to AS in both SREBP-1a and -2. We
used AS because its coding sequence is a site for the restriction
enzyme NheI, which facilitated the identification of the
desired mutant. In both SREBP-1a and SREBP-2 the DRSR AS
substitution severely reduced cleavage by the sterol-regulated protease (Fig. 4, B, lanes 7 and 8, and C, lanes 17 and 18). A single substitution
within this sequence (D484A) abolished cleavage of SREBP-1a (lanes
9 and 10), whereas the analogous substitution in SREBP-2 (D478A)
did not affect cleavage (lanes 19 and 20). None of
these changes affected the amount of the precursor form in the
membranes (lower panels of Fig. 4, B and C).
In addition to the above experiments, we produced other mutations in the linker region that had no effects on the cleavage or sterol-mediated regulation of SREBP-1a or SREBP-2 (Fig. 5). These mutations included nine point mutations (alanine substitutions) in SREBP-1a and three deletion mutations in SREBP-2.
Figure 5:
Linker region mutations in SREBP-1a (A) and SREBP-2 (B) that do not affect cleavage or
regulation. A, the bold letters denote amino acid
residues that were individually changed to alanine and had no effect on
cleavage or regulation in SREBP-1a. Asterisks denote point
mutations that abolished or markedly decreased cleavage in SREBP-1a. B, the dashed line denotes residues in the linker
region of SREBP-2 that were deleted and replaced by two amino acids
(Ala-Ser for 433-477 and
402-469 or Thr-Gly for
433-449).
We next turned our attention to the short hydrophilic loop that lies on the lumenal side of the ER membrane between the two transmembrane regions(6) . This loop contains a conserved Arg (residue 527 of SREBP-1a and 519 of SREBP-2) (Fig. 1B and 6A). Replacement of this Arg with an Ala abolished cleavage of both proteins (Fig. 6, B, lanes 5 and 6, and C, lanes 11 and 12). Changing the Arg to a Lys in SREBP-2 preserved the cleavage (lanes 13 and 14). This latter mutation was not made in SREBP-1a.
Figure 6:
Immunoblot analysis of HSV-tagged SREBP-1a (B) and SREBP-2 (C) from 293 cells transfected with
wild-type and loop region mutants. A, schematic diagram of the
loop regions of human SREBP-1a and SREBP-2, showing the site of the
mutated arginine (R) in both SREBPs. B and C, immunoblot analysis of wild-type and loop region mutants of
HSV-tagged SREBPs in nuclear extracts and membranes from 293 cells
transfected with pTK-HSV-BP1a and its mutants (B) and
pTK-HSV-BP2 and its mutants (C). 293 cells were set up,
transfected with the indicated plasmid, and fractionated as described
in the legend to Fig. 2. Aliquots of 40 µg of protein (B) or 60 µg (C) from nuclear extracts (upper
panels) and aliquots of 60 (B) and 80 µg (C)
from membranes (lower panels) were subjected to SDS-PAGE and
immunoblot analysis with 0.5 µg/ml HSV-Tag(TM) antibody. The
filters in B and C were exposed to film for 30 s and
1 min, respectively. N and P denote the
NH-terminal mature and precursor forms of SREBPs,
respectively.
We
previously showed that SREBP-1 can exist in several forms, owing to the
use of alternative NH-terminal exons (designated 1a and 1c)
and alternative pairs of COOH-terminal exons that are designated
18a/19a and 18c/19c(2, 16) . This alternative splicing
can result in at least three versions of the protein, designated
SREBP-1a, 1b, and 1c (Fig. 7A). All of the experiments
up to this point were conducted with the 1a version. Surprisingly, when
we transfected 293 cells with a cDNA encoding the 1c version, which
contains the c class exons at both ends, we observed two differences in
comparison with SREBP-1a (Fig. 7B). First, the amount
of mature form in the nucleus was reduced. Second, there was no longer
any suppression by sterols (Fig. 7B, compare lanes
5 and 6 with lanes 3 and 4). The sizes of the precursor
form and the nuclear form were also reduced because the c class exons
encode a shorter protein sequence than the a class exons. To determine
whether the differences in cleavage and regulation were attributable to
the c class exons at the NH
terminus or COOH terminus, we
prepared SREBP-1b, which contains exon 1a at the NH
terminus and exons 18c and 19c at the COOH terminus (Fig. 7A). Cleavage of this version also failed to be
suppressed by sterols, indicating that the resistance to sterol
suppression is dictated by the exons at the COOH terminus (Fig. 7B, lanes 7 and 8).
Figure 7:
Immunoblot analysis of alternatively
spliced forms of human SREBP-1: differential regulation by sterols. A, schematic diagram of alternatively spliced forms of human
SREBP-1(16) . B, immunoblot analysis of alternatively
spliced forms of human HSV-tagged SREBP-1a, -1b, and -1c. 293 cells
were set up, transfected with the indicated plasmid, and fractionated
as described in the legend to Fig. 2. Aliquots of 60 µg of
protein from nuclear extracts (upper panel) and aliquots of 80
µg from membranes (lower panel) were subjected to SDS-PAGE
and immunoblot analysis with 0.5 µg/ml HSV-Tag(TM) antibody. The
filters were exposed to film for 1 min. N and P denote the NH-terminal mature and precursor forms of
SREBPs, respectively.
In the current studies, we have developed an expression
vector that produces physiologic amounts of SREBP-1 and -2, thereby
allowing sterol-regulated cleavage to occur in transfected 293 cells.
The amount of SREBP produced by this vector is about the same as the
amount of endogenous SREBP in the cells as judged from comparative
immunoblots (6) . ()Using this vector as a basis for
a mutational analysis, we have identified two conserved regions of
SREBP-1a and -2 that are required for high level cleavage by the
sterol-regulated protease. Surprisingly, these two sequences lie on
opposite sides of the ER membrane.
The most essential residue of
either SREBP-1a or -2 is the Arg in the lumenal loop. Changing this Arg
to an Ala abolished cleavage. In numerous experiments with this mutant
protein, we have never observed any cleavage band, even when the gels
were exposed for prolonged periods. Changing the Arg to a Lys in
SREBP-2 preserved cleavage, indicating that the positive charge is the
important feature. The precise location of this Arg is important
because its removal abolished cleavage of SREBP-1a even though the
lumenal loop of this protein contains two other arginines (Arg and Arg
). (The lumenal loop of SREBP-2 contains
only one Arg.) We have not yet conducted an extensive mutational
analysis of the lumenal loop to determine whether other residues are
required. We note, however, that this sequence is not well conserved
between SREBP-1a and -2 (Fig. 6A). One conspicuous
conserved residue is a leucine that is three residues to the
COOH-terminal side of the crucial Arg (Leu
in SREBP-1a
and Leu
in SREBP-2). In experiments not shown, we changed
this leucine to alanine in SREBP-2 without any effect on
sterol-regulated cleavage.
The exact role played by the lumenal Arg is not clear as yet. It may be a site of initial cleavage that is followed by a second cleavage on the cytoplasmic side in the region of the DRSR sequence. It might also be part of a recognition sequence that binds a regulatory protein that facilitates cleavage on the cytoplasmic face. Several other possibilities exist, and they should be susceptible to analysis with the expression vector that is now in hand.
The requirement for the DRSR sequence on the
cytoplasmic side of the membrane is not as absolute as the requirement
for the lumenal Arg, at least for SREBP-2. The DRSR AS mutant
form of SREBP-1a never yielded a mature nuclear protein. However, in
some experiments with the DRSR
AS version of SREBP-2, we
observed a small amount of the nuclear form that was up to 10-20%
of the amount generated by the wild-type protein in the same
experiments. These data indicate that the DRSR sequence is important
but not absolutely essential for sterol-regulated proteolysis. It may
not be a cleavage site itself, but rather it may be part of the
recognition site for a protease that cleaves elsewhere.
Aside from
the DRSR sequence, the spacer region between the leucine zipper and the
first transmembrane region does not appear to contain any residues that
are crucial for cleavage of SREBP-1a or -2. As shown in Fig. 5B, we were able to delete all of the amino acids
between residues 402 and 477 of SREBP-2 without affecting
sterol-regulated cleavage. This eliminates all of the spacer region
except for the DRSR sequence immediately adjacent to the membrane. A
similar conclusion was drawn with regard to SREBP-1a. Here we observed
normal sterol-regulated cleavage of mutant forms of SREBP-1a containing
any of nine point mutations in the juxtamembrane portion (Fig. 5A). These substitutions included the GM sequence
at positions 481 and 482, which is two residues to the
NH-terminal side of the DRSR sequence (Fig. 5A). Like the DRSR sequence, the GM sequence is
conserved in human and hamster SREBP-1 and -2, but replacement of Gly
or Met with Ala did not affect sterol-regulated cleavage in the
transfected 293 cells.
The finding that cleavage of SREBP-1c is not regulated by sterols was a surprise, and it may have physiologic implications. Immunity to regulation was traced to the COOH-terminal two exons. We do not yet know whether the a class exons are required for regulation or whether the c class exons dominantly interfere with regulation. The COOH-terminal c class exons cannot be a major contributor to the total amount of SREBP-1 in tissue culture cells, because cleavage of endogenous SREBP-1 is totally suppressed by sterols(1, 13, 14) . A different situation may pertain in liver. We previously observed that livers of chow-fed hamsters contain the nuclear form of SREBP-1 but not SREBP-2(17) . It is possible that this nuclear form results from the protein with the COOH-terminal c class exons. Inasmuch as we have no evidence for an alternatively spliced form of SREBP-2, cleavage of this protein may always be regulated by sterols. Experiments are currently underway to determine whether hamster liver contains mRNAs encoding the c class of SREBP-1.