insig-dependent ubiquitination and degradation of mammalian 3
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Insig-dependent Ubiquitination and Degradation of Mammalian
3-Hydroxy-3-methylglutaryl CoA Reductase Stimulated by Sterols
and Geranylgeraniol*
Navdar Sever‡, Bao-Liang Song‡, Daisuke Yabe‡, Joseph L. Goldstein§,
Michael S. Brown§, and Russell A. DeBose-Boyd¶
From the Department of Molecular Genetics, University of Texas Southwestern Medical Center,
Dallas, Texas 75390-9046
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on October 16, 2003 as Manuscript M310053200 by guest on M
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Running Title: Insig-dependent ubiquitination of HMG CoA Reductase
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SUMMARY
The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl CoA reductase
produces mevalonate, which is converted to sterols and to other products, including
geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and
rapidly degraded when sterols and nonsterol end-products of mevalonate metabolism
accumulate in cells. Here, we use RNA interference to show that sterol-accelerated
ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the
endoplasmic reticulum that were shown previously to accelerate degradation of reductase
when overexpressed by transfection. Alanine substitution experiments reveal that binding
of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence
YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also
found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a
sterol-stimulated fashion. When lysine-248 of reductase is substituted with arginine, Insig
binding persists, but the reductase is no longer ubiquitinated and degradation is markedly
slowed. Lysine-248 is predicted to lie immediately adjacent to a membrane-spanning helix,
suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show
that Insig-dependent, sterol-stimulated degradation of reductase is further accelerated
when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-
carbon farnesol, raising the possibility that the nonsterol potentiator of reductase
regulation is a geranylgeranylated protein.
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INTRODUCTION
The enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase)1 catalyzes
the two-step reduction of HMG CoA to mevalonate, a crucial intermediate in the synthesis of
cholesterol and nonsterol isoprenoids (1). Mevalonate-derived products include ubiquinone,
heme, and the farnesyl and geranylgeranyl groups that become attached to many proteins,
directing them to membranes. HMG CoA reductase is subject to tight regulation by a
multivalent feedback mechanism mediated by nonsterol and sterol end-products of mevalonate
metabolism (2). These end products decrease reductase activity by inhibiting transcription of the
reductase gene, blocking translation of the reductase mRNA, and accelerating degradation of the
reductase protein. The transcriptional effects are mediated through the action of sterol regulatory
element-binding proteins (SREBPs), membrane-bound transcription factors that enhance
transcription of genes encoding cholesterol biosynthetic enzymes and the LDL receptor (3). The
translational effects are mediated by nonsterol mevalonate-derived isoprenoids, which act by an
undefined mechanism (4). The accelerated degradation of reductase is mediated both by sterols
and by nonsterol end products of mevalonate metabolism (4, 5) and appears to be carried out by
the 26S proteasome (6, 7).
Hamster HMG CoA reductase is an endoplasmic reticulum (ER) resident glycoprotein of 887
amino acids and consists of two distinct domains (8). The NH2-terminal domain of 339 amino
acids contains eight membrane spanning regions separated by short loops (9); it anchors the
protein to ER membranes. The COOH-terminal domain of 548 amino acids projects into the
cytoplasm and exerts all of the catalytic activity (8). Two lines of evidence indicate that the
membrane domain is crucial for sterol-accelerated degradation of the enzyme: 1) the truncated
cytoplasmic COOH-terminal domain, expressed by transfection, retains full catalytic activity and
exhibits a long half-life that is not shortened by sterols (10); and 2) the transmembrane domain of
reductase fused to soluble β-galactosidase exhibits sterol-regulated degradation in a manner
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similar to wild-type reductase (11). Sterol-accelerated degradation of reductase occurs in the ER
and is blocked by inhibitors of the 26S proteasome, which lead to the accumulation of
ubiquitinated forms of the enzyme (6, 7).
A series of studies in the yeast Saccharomyces cerevisiae support a role for the ubiquitin-
proteasome pathway in the control of HMG CoA reductase degradation (12). HMG2p, one of
two reductase isozymes expressed in yeast, undergoes accelerated degradation when flux through
the mevalonate pathway is high (12, 13). Degradation of HMG2p is dependent upon a
membrane-bound ubiquitin ligase complex that interacts with HMG2p and mediates its
ubiquitination in response to degradative signals (14).
Although accelerated degradation of mammalian reductase and that of yeast HMG2p seem
similar, major differences exist. Although the membrane domain of yeast HMG2p includes
multiple membrane spanning segments (13), it bears no primary sequence resemblance to that of
mammalian reductase (8, 15). Moreover, accelerated degradation of HMG2p in yeast is
triggered maximally by a nonsterol isoprenoid (16), while maximal degradation of reductase in
mammalian cells requires sterols plus nonsterol isoprenoids (4, 5).
Recent clues to the mechanism of degradation of mammalian HMG CoA reductase have
emerged from the study of the transcriptional axis of sterol regulation as mediated by SREBPs.
The key discovery was SREBP cleavage-activating protein (SCAP), a polytopic membrane
protein that resembles the reductase by virtue of eight membrane spanning segments. SCAP is a
sterol-responsive escort protein that forms a complex with SREBPs and escorts them from the
ER to the Golgi where SREBPs are proteolytically released from membranes, thereby allowing
transcriptionally active fragments to enter the nucleus where they activate the reductase gene (3,
17, 18). Sterols block SREBP activation by preventing the exit of SCAP/SREBP complexes
from the ER, leading to decreased synthesis of cholesterol and other lipids. The block in SREBP
export is achieved through sterol-induced binding of SCAP to one of a pair of ER retention
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proteins called Insig-1 and Insig-2 (19, 20). The binding site for Insig in SCAP has been
localized to a ~170-amino acid segment that comprises transmembrane segments 2-6. These
segments show significant sequence similarities to helices 2-6 of the membrane domain of HMG
CoA reductase, and this region has been termed the sterol-sensing domain (21-23). Point
mutations within the sterol-sensing domain of SCAP prevent it from binding to Insigs (19, 20),
thus preventing the sterol-mediated ER retention of SCAP/SREBP (18, 24, 25).
The resemblances between the sterol-sensing domains of SCAP and HMG CoA reductase led
us to test the possible role of Insig-1 in the sterol-regulated degradation of the reductase (26).
When reductase is overexpressed in Chinese hamster ovary (CHO) cells, the enzyme is no longer
degraded rapidly in response to sterol treatment. Overexpression of Insig-1 restores regulated
degradation of overexpressed reductase, and this degradation can be inhibited by overexpressing
the sterol-sensing domain of SCAP, suggesting that reductase and SCAP compete for limiting
amounts of Insig when sterol levels are high. Although both proteins bind to Insig-1, the binding
leads to markedly different consequences: binding leads to ER retention of SCAP and to
accelerated degradation of reductase. How Insigs orchestrate seemingly discordant modes of
cellular regulation requires further knowledge of the mechanisms underlying the sterol-mediated
effects on reductase and SCAP. In this paper, we explore the role of Insigs, sterols, and
nonsterol isoprenoids in regulating degradation of reductase.
EXPERIMENTAL PROCEDURES
Materials - We obtained MG-132 and digitonin from Cal Biochem and horseradish
peroxidase-conjugated, donkey anti-mouse IgG (affinity-purified) from Jackson Immunoresearch
Laboratories; farnesol and geranylgeraniol from Sigma; NB-598 from Banyu Pharmaceutical
Co., Ltd; and hydroxypropyl-β-cyclodextrin from Cyclodextrin Technologies, Inc. (Gainesville,
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FL). Other reagents were obtained from described sources (17). Lipoprotein-deficient serum
(LPDS, d > 1.215 g/ml) was prepared from newborn calf serum by ultracentrifugation (27).
Expression Plasmids - The following plasmids were described in the indicated reference:
pCMV-Insig-1-Myc, which encodes amino acids 1-277 of human Insig-1 followed by six tandem
copies of a c-myc epitope tag under control of the cytomegalovirus promoter (19); pRc/CMV7S-
Mev, which encodes amino acids 1-494 of the hamster Mev protein under control of the CMV
promoter (28, 29); and pCMV-HMG-Red-T7(TM1-8) which encodes amino acids 1-346 of
hamster HMG CoA reductase followed by three tandem copies of the T7 epitope tag under
control of the CMV promoter (26). pEF1a-HA-Ubiquitin (provided by Dr. Zhijian Chen,
University of Texas Southwestern Medical Center) encodes amino acids 1-76 of human ubiquitin
preceded by an epitope tag derived from the influenza hemagglutinin (HA) protein
(YPYDVPDY) under the control of the EF1a promoter.
pCMV-HMG-Red-T7, which encodes full-length hamster reductase (amino acids 1-887)
followed by three tandem copies of the T7-epitope tag (MASMTGGQQMG), was constructed by
standard methods. Lysine mutations (K89R, K248R, and K89R/K248R) and sterol sensing-
domain mutations (Y75A, Y77A, Y75A/Y77A, I76A, F78A, I76A/F78A, and YIYF(75-78) to
AAAA) in pCMV-HMG-Red-T7 and pCMV-HMG-Red-T7(TM1-8) were generated with the
Quikchange XL site-directed mutagenesis kit (Stratagene).
The integrity of all plasmids was confirmed by DNA sequencing of their open reading frames
and ligation joints.
Cell Culture - Monolayers of Chinese hamster ovary cells (CHO-K1) were maintained in
tissue culture at 37°C in 8%-9% CO2. Stock cultures of CHO-K1 cells were maintained in
medium A (1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle medium
containing 100 U/ml penicillin and 100 µg/ml streptomycin sulfate) supplemented with 5% (v/v)
fetal calf serum (FCS).
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Monolayers of SV-589 cells, an immortalized line of human fibroblasts expressing the SV40
large T antigen (30), were grown at 37°C in 5% CO2. Stock cultures of SV-589 cells were
maintained in medium B (Dulbecco’s modified Eagle medium containing 100 U/ml penicillin
and 100 µg/ml streptomycin sulfate) supplemented with 10% FCS. SV589/pMev cells are
derivatives of SV-589 cells stably expressing pMev, a cDNA encoding a mutated
monocarboxylate transporter that confers efficient uptake of mevalonate from tissue culture
medium (28). SV-589/pMev cells were generated as follows. On day 0, SV-589 cells were set
up at 2 x 105 cells per 60-mm dish in medium B supplemented with 10% FCS. On day 1, the
cells were transfected with 2 µg of pRc/CMV7S-Mev using the FuGene 6 transfection reagent as
described below. Twenty-four hours after transfection, cells were switched to selection medium
that consisted of medium B supplemented with 10% LPDS, 50 µM sodium compactin, 200 µM
sodium mevalonate, and 700 µg/ml G418. Fresh medium was added every 2-3 days until
colonies formed after about 10 days. Individual colonies were isolated with cloning cylinders,
and pMev expression was assessed by the ability of exogenous mevalonate to induce reductase
degradation. Once stably transfected cell lines were established, they were each maintained at
37°C, 5% CO2 in medium B containing 10% LPDS, 50 µM sodium compactin, 200 µM sodium
mevalonate, and 500 µg/ml G418. Sterols were added to the culture medium at a final
concentration of 0.1% or 0.2% (v/v) ethanol.
Transient Transfection, Cell Fractionation, and Immunoblot Analysis - Transfections were
performed as described (31) with minor modifications. CHO-K1 cells were transfected with 3
µg DNA per 60-mm dish. For each transfection, FuGENE-6 DNA Transfection Reagent (Roche
Molecular Biochemicals) was added to 0.2 ml medium A at a ratio of 3 µl FuGENE-6 per 1 µg
DNA. Conditions of incubation are described in the figure legends. At the end of the
incubation, dishes of cells were harvested and pooled for analysis.
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Pooled cell pellets were used to isolate either nuclear extracts, 2 x 104g membrane fractions
(26), or whole cell lysate fractions (18). All fractions were subjected to 8% SDS-PAGE and
immunoblot analysis as described (26). Primary antibodies used for immunoblotting were as
follows: mouse monoclonal anti-T7-Tag (IgG2b) (Novagen); mouse monoclonal anti-Myc (IgG
fraction) from the culture medium of hybridoma clone 9E10 (American Type Culture
Collection); IgG-A9, a mouse monoclonal antibody against the catalytic domain of hamster
reductase (amino acids 450-887) (32); IgG-1D2, a mouse monoclonal antibody against the NH2-
terminus of human SREBP-2 (amino acids 48-403) (20); IgG-P4D1, a mouse monoclonal
antibody against bovine ubiquitin (Santa Cruz Biotechnology); IgG-CD71, a mouse monoclonal
antibody against the human transferrin receptor (Zymed Laboratories); and IgG-HA-7, a mouse
monoclonal antibody against the HA epitope tag (Sigma).
Blue Native-PAGE for Detection of HMG CoA Reductase/Insig-1 Complex - Monolayers of
SRD-13A cells, a line of mutant CHO cells that lacks SCAP (31), were set up on day 0 (8 x 105
cells/100-mm dish) and cultured in 8-9% CO2 at 37°C in medium A supplemented with 5% FCS,
5 µg/ml cholesterol, 1 mM sodium mevalonate, and 20 µM sodium oleate. On day 2, the cells
were transfected as described in Fig. 5, after which they were harvested for preparation of a 1.6 x
104g membrane pellet, which was solubilized with 1% (w/v) digitonin and centrifuged as
described (19). One aliquot of the resulting 20,000g supernatant was mixed with a 6-amino-n-
hexanoic acid-containing buffer and subjected to electrophoresis on a 4%-16% Blue Native gel
as described (19). A second aliquot of the 20,000g supernatant was mixed with SDS-containing
buffer and subjected to 10% SDS-PAGE.
RNA Interference - The protocol was adapted from Elbashir et al. (33). Duplexes of small-
interfering RNA (siRNA) were synthesized by Dharmacon Research (Lafayette, CO). siRNA
sequences targeting human Insig-1 and human Insig-2 (GenBank accession nos. AY112745 and
AF527632, respectively) corresponded to the following nucleotide positions relative to the first
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nucleotides of the start codons, respectively: Insig-1, 691-711; and Insig-2, 324-344. The siRNA
sequence targeting an irrelevant control gene, vesicular stomatitis virus glycoprotein (VSV-G),
was reported (34).
On day 0, SV-589 cells were set up at a density of 5 x 104 cells per 60-mm dish in medium B
supplemented with 10% FCS. On day 1, the cells were transfected with siRNA using a ratio of 6
µl of OligofectamineTM Reagent (Invitrogen) to 400 pmol of siRNA duplexes per dish as
described by the manufacturer. Cells were washed once with 2 ml of medium B (without
antibiotics) and refed with 1.6 ml of medium B (without antibiotics). The
siRNA/Oligofectamine mixture (in a total volume of 0.4 ml) was added to each dish and
incubated at 37oC. After 6 h, the cells received a direct addition of 1 ml of medium B containing
(final concentrations) 10% FCS, 5 µg/ml cholesterol, 1 mM sodium mevalonate, and 20 µM
sodium oleate, and were then incubated at 37oC. On day 3, the cells were transfected with
siRNA duplexes as described above. Six hours after the final transfection, the cells received a
direct addition of 1 ml of medium B containing (final concentration) 10% LPDS, 50 µM sodium
compactin, and 50 µM sodium mevalonate. On day 4, the cells were treated as described in the
figure legends, and harvested for analysis.
Real-time PCR - The protocol was identical to that described by Liang et al. (35). Triplicate
samples of first-strand cDNA were subjected to real-time PCR quantification using forward and
reverse primers for human Insig-1, human Insig-2, and human 36B4 (invariant control) (Table
1). Relative amounts of mRNAs were calculated using the comparative CT method.
Pulse-chase Analysis of HMG CoA Reductase - Cells were pulse-labeled in medium C
(methionine/cysteine-free Dulbecco’s modified Eagle’s medium containing 100 U/ml penicillin,
100 µg/ml streptomycin sulfate, 10% LPDS, and 50 µM sodium compactin), and pulse-chase
analysis was carried out as described (26). Immunoprecipitation of labeled reductase was carried
out with 30 µg of a polyclonal antibody directed against the 60-kDa COOH-terminal domain of
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human reductase (36). Immunoprecipitates were subjected to SDS-PAGE and transferred to
Hybond C-extra nitrocellulose filters. Dried filters were exposed to an imaging plate at room
temperature and scanned in a Fuji X Bas 1000 Phosphorimager.
Ubiquitination of HMG CoA Reductase - Conditions of incubations are described in the
figure legends. At the end of the incubation, cells were harvested, and immunoprecipitations
with either polyclonal antibody against endogenous human reductase (see above) or monoclonal
anti-T7 IgG-coupled agarose beads (Novagen) against transfected reductase were carried out as
described (26) with two modifications: the lysis buffer used to harvest the cells was
supplemented with 10 mM N-ethyl maleimide and the pelleted beads were washed three times
(30 min each). Immunoprecipitates were subjected to SDS-PAGE on 5-12% gradient gels and
transferred to nitrocellulose filters.
RESULTS
The experiment in Fig. 1 was designed to examine the relationship between ubiquitination
and sterol-accelerated degradation of HMG CoA reductase. SV589 cells, a line of SV-40
transformed human fibroblasts, were depleted of sterols by incubation for 16 h in LPDS
containing the reductase inhibitor, compactin, and a low level of mevalonate (50 µM), which is
the lowest level that assures viability. Cells were then treated for either 2 h or 5 h with a mixture
of sterols (1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) and a high concentration of
mevalonate (10 mM) in various combinations. Additionally, some of the cells received the
proteasome inhibitor MG-132 to block the degradation of any ubiquitinated reductase molecules.
Detergent-solubilized extracts were subjected to immunoprecipitation with polyclonal antibodies
against reductase, and the resulting immunoprecipitates were subjected to SDS-PAGE and
blotted with anti-ubiquitin (Fig. 1A, top panel) or anti-reductase (Fig. 1A, lower panel)
monoclonal antibodies. In cells treated for 2 h, sterols caused a slight decrease in the amount of
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reductase. The addition of mevalonate had no effect (lower panel, lanes 1-4). The sterol-
dependent decrease of reductase was blocked by MG-132, (lower panel, lanes 5-8), indicating
that this decrease reflected accelerated degradation mediated by proteasomes. After 2 h of sterol
treatment in the presence of MG-132, we observed the appearance of ubiquitinated reductase as
indicated by the high molecular weight smear in the anti-ubiquitin immunoblots of the reductase
immunoprecipitates (lanes 6, 8, upper panel), but not in cells treated with mevalonate alone
(lane 7). The addition of mevalonate did not enhance the sterol effect (compare lanes 6 and 8).
After 5 h in the presence of sterols alone, reductase was still detectable, though diminished
(lower panel, lane 10). In the presence of sterols plus mevalonate, the reductase was further
reduced (lower panel, lane 12). This result is consistent with other studies demonstrating that
sterols alone accelerate the degradation of reductase and that nonsterol mevalonate-derived
products further accelerate degradation (4, 5). Accelerated degradation of reductase was
completely blocked by MG-132 (lower panel, lanes 13-16). In the presence of MG-132, sterols
caused a marked increase in the amount of ubiquitinated reductase, and there was no further
effect with mevalonate addition (compare lanes 14 and 16 in upper panel of Fig. 1A).
Mevalonate alone had only a slight effect (upper panel, lane 15), which is likely attributable to
the incorporation of mevalonate into sterols after 5 h.
The sterol stimulation of reductase ubiquitination was remarkably rapid (Fig. 1B). In the
absence or presence of the proteasome inhibitor, sterol-dependent ubiquitination of reductase
was observed after as little as 5 min of sterol treatment (lanes 2, 3) and reached a maximum after
10 min (lanes 5, 6). Thereafter, in the absence of MG-132 ubiquitinated reductase declined,
presumably owing to degradation (lanes 8, 11). This decline was prevented by MG-132 (lanes 9,
12).
Although the data of Fig. 1 indicate that reductase is ubiquitinated and degraded in a sterol-
dependent manner, the proportion of reductase that was detectably polyubiquitinated at any one
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time was very small, even when degradation was blocked by MG-132. Thus, in the presence of
MG-132 most of the reductase that accumulated was not polyubiquitinated, as indicated by its
continued migration on SDS-PAGE as a 97-kDa band (lower panel in Fig. 1). We interpret these
findings to indicate a balance between ubiquitination and de-ubiquitination reactions. According
to this hypothesis, sterols stimulate the ubiquitination of reductase. When proteasomal function
is intact, most of the ubiquitinated reductase is degraded. When proteasomal degradation is
blocked, the amount of ubiquitinated reductase at any instant reflects an equilibrium between
ubiquitination and de-ubiquitination reactions. Similar conclusions have been reached by others
in studies of the ubiquitination of other proteins (37). The addition of mevalonate enhances
reductase degradation, but it is not clear whether mevalonate increases reductase ubiquitination
or whether it acts to enhance degradation of reductase that has become ubiquitinated under the
influence of sterols. It is clear, however, that nonsterol mevalonate products cannot act by
themselves. They only act in the presence of sterols.
In previous studies of reductase that was overexpressed by transfection, the sterol-mediated
acceleration of degradation was blunted, but it could be restored by simultaneous overexpression
of Insig-1 (26) or its close relative Insig-2 (unpublished observations). These findings raised the
possibility that accelerated reductase degradation requires Insig protein. To demonstrate this
requirement directly, we decreased the amount of the two Insigs through the use of RNA
interference (RNAi) in human SV589 fibroblasts (Fig. 2). Cells were transfected twice with
duplexes of siRNA targeting a control mRNA encoding vesicular stomatitis virus glycoprotein
(VSV-G, lanes 1-4), which is not present in the cells, Insig-1 (lanes 5-8), Insig-2 (lanes 9-12), or
the combination of Insig-1 and Insig-2 (lanes 13-16). Cells were then treated for 5 h with sterols
and/or mevalonate, and detergent extracts were analyzed by immunoblotting with reductase-
specific antibody. In control cells receiving VSV-G siRNA duplexes, sterols alone caused a
modest, but detectable decrease in reductase (compare lanes 1 and 2), and this reduction was
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complete upon addition of sterols plus mevalonate (lane 4). Similar responses were observed
when Insig-1 and Insig-2 siRNA duplexes were transfected alone (lanes 5-8 and 9-12,
respectively). However, when both Insig-1 and Insig-2 siRNA duplexes were introduced into
cells, no degradation of reductase was observed under any conditions (lanes 13-16). As a
control, we immunoblotted identical extracts for the transferrin receptor, which remained
unchanged regardless of siRNA transfection or treatment (lower panel, lanes 1-16). In the same
experiment, we used quantitative real-time PCR to show that Insig-1 and Insig-2 mRNAs were
reduced by 90% and 50%, respectively, by RNAi (Fig. 2B). It should be pointed out that in SV-
589 cells Insig-1 mRNA is normally expressed at levels 10-fold higher than Insig-2 mRNA.
To demonstrate directly that the RNAi knockdown of Insig-1 and Insig-2 abolishes sterol-
accelerated degradation of reductase, we performed a pulse-chase experiment. Cells transfected
with siRNA duplexes were pulse-labeled for 1 h with 35S-labeled methionine plus cysteine, after
which they were washed and switched to medium containing an excess of unlabeled methionine
and cysteine in the absence or presence of sterols plus mevalonate. Radiolabeled reductase was
monitored by immunoprecipitation with anti-reductase antibodies followed by SDS-PAGE and
visualization in a phosphorimager. Fig. 2C shows the time-course of disappearance of
35S-labeled reductase in cells treated with siRNA targeting VSV-G (upper panel) or Insig-1 plus
Insig-2 (lower panel). In control cells treated with VSV-G siRNA, sterols plus mevalonate
caused a rapid disappearance of 35S-labeled reductase that was nearly complete after 5 h (upper
panel, lanes 5-7). However, in cells transfected with Insig-1 plus Insig-2 siRNA, labeled
reductase continued to be present throughout the chase (lower panel, lanes 5-7). These results
were quantified by scanning of images in a phosphorimager, and the data are plotted in the lower
panel of Fig. 2C. The half-life of labeled reductase in control cells declined from 11.5 h to 0.9 h
upon treatment with sterols plus mevalonate. In cells treated with siRNAs targeting the two
Insigs, reductase half-life was 10.4 h in the absence or presence of sterols plus mevalonate.
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We next addressed the question of whether Insigs are necessary for the ubiquitination of
reductase or whether they are required for a post-ubiquitination step. To this end, we transfected
cells with siRNA duplexes targeting VSV-G (Fig. 2D, lanes 1-4) or Insig-1 plus Insig-2 (lanes 5-
8). Cells were treated for 1 h with sterols and/or mevalonate in the presence of MG-132.
Detergent extracts were immunoprecipitated with anti-reductase polyclonal antibodies and
subjected to immunoblotting with anti-ubiquitin (upper panel) or anti-reductase (lower panel).
In control cells treated with VSV-G RNAi, sterols caused the appearance of ubiquitinated
reductase (upper panel, lanes 2, 4), and mevalonate alone had only a slight effect (upper panel,
lane 3). In cells transfected with siRNAs targeting Insig-1 and Insig-2, sterol-mediated
ubiquitination of reductase was greatly diminished (upper panel, lanes 5-8). Together, these
results indicate that Insig-1 and Insig-2 are necessary for sterol-induced ubiquitination of
reductase.
Ubiquitination of proteins is a multistep process, culminating in the covalent attachment of
ubiquitin to the ε-amino group of a lysine residue in the target protein. The topology of the
ubiquitination machinery makes it likely that potential sites of ubiquitination are accessible to the
cytosol. The membrane domain of reductase is not only necessary, but sufficient for regulated
degradation (10, 11), and we have determined in transfection experiments that the membrane
domain (amino acids 1-346 corresponding to transmembrane segments 1-8; see Fig. 3A) of
reductase retains the ability to undergo Insig-dependent, sterol-regulated degradation (data not
shown). As shown in Fig. 3A, amino acids 1-346 of reductase contain only two lysines that are
exposed to the cytosol (positions 89 and 248). To ascertain their participation in regulated
ubiquitination of reductase, we changed the lysine codons at positions 89 and 248 to arginine in
pCMV-HMG-Red-T7, an expression plasmid encoding full-length reductase with three tandem
copies of the T7 epitope tag at the COOH-terminus of the protein. To enhance sensitivity of
ubiquitin detection, cells were also transfected with pEF1a-HA ubiquitin, an expression plasmid
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encoding human ubiquitin with an NH2-terminal tag consisting of two copies of the HA epitope.
In the experiment shown in Fig. 3B, CHO cells were transfected with pEF1a-HA-ubiquitin and
wild-type (lanes 1-4) or mutant versions (lanes 5-16) of pCMV-HMG-Red-T7 in the absence or
presence of pCMV-Insig-1-Myc. After treatment for 2 h with MG-132, cells were harvested,
transfected reductase was immunoisolated from detergent lysates with anti-T7 agarose beads,
and samples were subjected to SDS-PAGE. For simplicity, the cells were incubated with a
mixture of sterols plus mevalonate instead of adding each one separately. Immunoblot analysis
with anti-HA revealed that ubiquitination of wild-type reductase required Insig-1 plus sterols and
mevalonate (upper panel, lanes 1-4). Mutating lysine-89 to arginine had no effect on
ubiquitination (upper panel, lanes 5-8). However, mutating lysine-248 to arginine individually
or in combination with the K89R mutation eliminated the regulated ubiquitination of reductase
(upper panel, lanes 11-12 and 15-16). The introduced mutations did not affect the total amount
of reductase that was produced (lower panel, lanes 1-16).
To test the effects of the lysine mutations on regulated degradation, wild-type or mutant
versions of pCMV-HMG-Red-T7 were transfected into cells with or without pCMV-Insig-1-
Myc, and cells were incubated with or without sterols plus mevalonate for 5 h in the absence of
the proteasome inhibitor. As expected, in the presence of Insig-1 sterols plus mevalonate led to
the disappearance of reductase (Fig. 3C, lane 4), and the K89R mutation had little effect (lane 8).
Mutating lysine-248 to arginine partially blocked regulated degradation of reductase (lane 12),
and the block was more pronounced with the K89R/K248R double mutant (lanes 13-16).
Together, the results of Figs. 3B and 3C suggests that the cytosolically oriented lysine-248 is the
major site of Insig-dependent ubiquitination of reductase. Lysine-89 may play some role when
lysine-248 is absent.
The overexpressed sterol-sensing domain of SCAP blocks sterol-mediated degradation of
reductase, suggesting that the two proteins compete for the same site on Insig (26). Binding of
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SCAP to Insig is abolished upon the substitution of a cysteine for tyrosine-298 (Y298C mutant),
which lies within one of the transmembrane helices in the sterol-sensing domain of SCAP (18,
20, 25). The Y298C mutant of SCAP fails to competitively inhibit sterol-mediated degradation
of reductase presumably because it fails to bind to Insigs (19). Fig. 4A shows an amino acid
alignment between the second transmembrane helices of reductase and SCAP. Reductase and
SCAP share a tetrapeptide (YIYF) beginning at residue 75 of reductase, which corresponds to
the crucial Y298 residue in SCAP. To determine whether this tetrapeptide sequence is essential
for reductase interaction with Insigs, we generated a series of mutations in pCMV-HMG-Red-T7
and tested their effects on sterol-accelerated degradation (Fig. 4B). CHO cells were transfected
with pCMV-Insig-1-Myc and wild-type or mutant versions of pCMV-HMG-Red-T7 containing
various combinations of alanine substitutions in the YIYF motif. The level of Insig expression is
sufficient to permit nearly complete degradation of reductase in 5 h in the presence of 25-
hydroxycholesterol without added mevalonate. After incubation with various concentrations of
25-hydroxycholesterol for 5 h, cells were harvested, fractionated, and subjected to SDS-PAGE
followed by immunoblot analysis with anti-T7 antibody. Under the conditions of this
experiment, 0.1 µg/ml of 25-hydroxycholesterol was sufficient to accelerate the degradation of
wild-type reductase, and complete disappearance was observed at 0.3 µg/ml of the oxysterol
(Fig. 4B, lanes 1-4 and 17-20). The Y75A mutant (corresponding to the Y298C mutant in
SCAP) reduced only slightly the effect of sterols on degradation of reductase (Fig. 4B, lanes 5-
8). However, substitution of the other tyrosine in this tetrapeptide (Y77A) rendered reductase
significantly resistant to the sterol effect (lanes 9-12). The combined effect of the two tyrosine
mutations (Y75A/Y77A) was more potent than either of the two substitutions alone. The
isoleucine at the second position of the tetrapeptide was also important for sterol-accelerated
degradation (lanes 21-24), but the phenylalanine at the fourth position was not (F78A, lanes
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25-28). Substitution of all four residues of the tetrapeptide (YIYF to AAAA) prevented sterols
from exerting any effect on the degradation of reductase (lanes 33-36).
We next tested whether ubiquitination of the various mutants of reductase correlates with
their resistance to sterol-accelerated degradation (Fig. 4C). Cells were transfected with pCMV-
Insig-1-Myc, pEF1a-HA-ubiquitin, and wild-type (lanes 1, 2) or mutant versions of pCMV-
HMG-Red-T7 (lanes 3-12). The cells were treated for 2 h with MG-132 in the absence or
presence of sterols plus mevalonate and harvested, and detergent extracts prepared. Transfected
reductase was immunoprecipitated with anti-T7-coupled agarose beads. Immunoblotting the
precipitates with anti-HA monoclonal antibody revealed that the Y75A mutant of reductase was
ubiquitinated in a regulated manner, similar to that of the wild-type protein (lanes 1-4). In
contrast, ubiquitination of all of the degradation-resistant mutants was markedly reduced (lanes
5-12). These results strengthen the view that the conserved YIYF sequence in reductase is
essential for the ability of Insig-1 to mediate the regulated ubiquitination and the subsequent
proteasomal degradation of reductase.
The results in Figs. 3 and 4 show that lysine-248 and the YIYF sequence are necessary for
sterol-mediated reductase ubiquitination. We utilized blue native-polyacrylamide gel
electrophoresis (BN-PAGE) to test the hypothesis that mutations in the YIYF sequence of
reductase decrease the binding of protein to Insig-1 whereas substitution of lysine-248 preserves
binding but prevents ubiquitination. This technique was recently employed to identify sterol-
induced complexes between Insig-1 and SCAP (19). To eliminate the potential competition
between SCAP and reductase for Insig, we utilized SRD-13A cells, a CHO-derived mutant cell
line that lacks SCAP (31). In the experiment shown in Fig. 5, we transfected SRD-13A cells
with pCMV-Insig-1-Myc in the absence (lanes 1, 2) or presence of wild-type or mutant versions
of pCMV-HMG-Red-T7(TM1-8) (lanes 3-8). pCMV-HMG-Red-T7(TM1-8) is an expression
plasmid encoding the membrane domain of hamster reductase (amino acids 1-346) with a
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COOH-terminal T7 epitope tag (26). Following sterol depletion with hydroxypropyl-β-
cyclodextrin, the cells were incubated in medium with or without sterols. The medium also
contained MG-132 to prevent proteasomal degradation of the ubiquitinated reductase membrane
domain. After treatments, the cells were harvested, membranes were isolated and solubilized
with digitonin. Soluble material was mixed with Coomassie blue and 6-amino-n-hexanoic acid
(to impart a negative charge and prevent precipitation of proteins) and subjected to PAGE. The
fractionated proteins were transferred to membranes and blotted with various antibodies. In the
absence of the reductase membrane domain, Insig-1 appeared as a single band as revealed by
immunoblotting with anti-Myc. Its migration was not altered upon sterol treatment (compare
lanes 1, 2 in upper panel of Fig. 5). This band was designated as unbound Insig-1. When the
cells expressed the wild-type reductase membrane domain, some of the Insig-1 migrated more
slowly (lane 3). This slower migrating band contained Insig-1 bound to the membrane domain
of reductase, as demonstrated by the fact that it was supershifted when the digitonin-solubilized
material was incubated prior to PAGE with anti-T7 monoclonal antibody, but not with a control
antibody (data not shown). Thus, we designated the slower migrating band as the reductase/Insig
complex. In the presence of sterols, unbound Insig-1 disappeared almost completely (lane 4).
The reductase-Insig-1 complex was also diminished, but to a lesser extent (lane 4). The decrease
in the reductase-Insig-1 complex is likely caused by incomplete inhibition of proteasomal
activity, as revealed by immunoblotting of another aliquot of the same extract that was subjected
to SDS-PAGE. This blot revealed a decrease in the total amount of the reductase membrane
fragment in the presence of sterols (SDS-PAGE, lower panel, lanes 3, 4).
In cells that expressed the K248R mutant of reductase, about half of the Insig-1 was found in
the reductase/Insig-1 complex in the absence of sterols (lane 5). Addition of sterols caused all of
the Insig-1 to migrate as a complex with reductase (lane 6), indicating that this mutation does not
block the ability of sterols to enhance binding of the reductase membrane fragment to Insig-1.
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Sterol addition caused only a slight decline in the total amount of the K248R reductase
membrane fragment (SDS-PAGE, lane 6), presumably because this mutant is resistant to
ubiquitination and degradation. A very different result was obtained with the tetrapeptide mutant
(YIYF to AAAA). In cells that expressed this mutant protein, the majority of Insig-1 was in the
unbound state, and there was no increase in the complex when sterols were added (BN-PAGE,
lanes 7, 8), nor was there a detectable decrease in the total amount of the reductase membrane
domain as revealed by SDS-PAGE (lanes 7, 8).
We next conducted a series of studies designed to determine which product of mevalonate
metabolism, in addition to sterols, is required to accelerate the degradation of reductase. To
facilitate cellular uptake of mevalonate, we used a permanently transfected line of human SV-
589 cells that expresses a cell surface mevalonate transporter (SV589-pMev cells). This
transporter is a mutant version of a monocarboxylate transporter (MCT-1) with a substitution of
phenylalanine for cysteine at position 360, which lies in a putative transmembrane helix (28).
The mutant protein, but not the native protein, transports mevalonate into cells with high
efficiency and thus facilitates studies of the effects of mevalonate and its products on reductase.
Fig. 6A show three of the branches of the mevalonate pathway in animal cells. Mevalonate,
produced by reductase, is converted to the five-carbon intermediate isopentenyl pyrophosphate
(isopentyl-PP), which is polymerized to form geranyl-PP (10 carbons) and farnesyl-PP (15
carbons). Two farnesyl-PP molecules can undergo head-to-head condensation to produce
squalene (30 carbons), which is the first committed intermediate in the cholesterol synthetic
pathway. Conversion of squalene to cholesterol is blocked by NB-598, which inhibits squalene
epoxidase (38). Farnesyl-PP can donate a farnesyl group to certain proteins, and it can condense
with another isopentenyl-PP to form geranylgeranyl-PP (20 carbons), which is added to other
proteins by two distinct geranylgeranyl transferases (39).
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When SV589-pMev cells were incubated with a higher concentration of mevalonate (10 mM)
in the presence of the proteasome inhibitor MG-132, reductase was ubiquitinated as determined
by immunoprecipitation and blotting with the anti-ubiquitin antibody (Fig. 6B, lane 2).
Ubiquitination was blocked by NB-598 (lane 4), indicating that it required the incorporation of
mevalonate into cholesterol. Exogenous sterols also provoked ubiquitination of reductase (lane
5), but this effect was not blocked by NB-598 (lane 7). In the presence of maximal levels of
sterols, mevalonate had no additional effect on ubiquitination (lanes 6, 8).
To determine the relation between ubiquitination and degradation of reductase, we made the
same additions to SV589-pMev cells in the absence of MG-132 and measured the total amount
of reductase by SDS-PAGE (Fig. 7C). Mevalonate at 10 mM caused the complete disappearance
of reductase (Fig. 6C, lane 2) and this was blocked by NB-598 (lane 4). Exogenous sterols
reduced the amount of reductase only partially (lane 5). Complete disappearance required the
addition of mevalonate (lane 6), and this mevalonate effect was not blocked by NB-598 (lane 8),
indicating that it did not require conversion of mevalonate to sterols.
To determine which of the nonsterol mevalonate-derived products is required for accelerated
degradation of reductase, we tested the ability of externally added farnesol or geranylgeraniol
(GG-OH) to reproduce the effects of mevalonate (Fig. 7). For these experiments, we used
native SV-589 cells that lack the mevalonate transporter. Sterols added to these SV-589 cells led
to a partial decrease in reductase protein after 5 h (Fig. 7A, lane 2). Addition of 10 mM
mevalonate alone had no effect (lane 3), presumably because of the relatively slow uptake of
mevalonate in these cells. However, mevalonate synergized with sterols to give a complete
disappearance of reductase (lane 4). Farnesol at 10 µM or 100 µM had no effect on reductase,
either in the absence or presence of sterols (lanes 5-8). In contrast, GG-OH at 10 µM synergized
with sterols (lane 10), and the effect was complete at 100 µM (lane 12).
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As discussed above, Insig proteins are required for the regulated degradation of reductase and
for the ability of sterols to block SCAP-dependent processing of SREBPs. To determine whether
mevalonate and GG-OH contribute to SCAP regulation as they do to reductase regulation, we
added these compounds to cells in the absence or presence of sterols and then measured the
amount of membrane-bound and nuclear SREBP-2 as well as membrane-bound reductase (Fig.
7B). As described previously, both mevalonate and GG-OH synergized with sterols in
eliminating reductase protein from membranes (Fig. 7B, lanes 4, 6). On the other hand, sterols
alone blocked SREBP processing completely (lane 8). Mevalonate and GG-OH had no
additional effect (lanes 10, 12). To confirm that GG-OH was acting by enhancing reductase
degradation, we performed a pulse-chase experiment (Fig. 7C). Sterols alone accelerated the
degradation of reductase (compare lanes e-g with lanes b-d in Fig. 7C), but some 35S-labeled
reductase remained detectable at 5 h (lane g). GG-OH had little effect on degradation by itself
(lanes i-k), but it synergized with sterols so that the reductase was markedly reduced at 1.5 h
(lane l) and barely detectable at 3 h (lane m).
DISCUSSION
The data in this paper confirm and extend previous observations by others that eukaryotic
HMG CoA reductase is degraded by a ubiquitin-proteasome mechanism that is enhanced by
sterols and nonsterol mevalonate-derived products (5, 7, 13). The new findings in the current
paper are as follows: 1) Ubiquitination and degradation of mammalian reductase is dependent
upon Insigs, as revealed by the elimination of both processes when the two Insig mRNAs are
removed through RNA interference (Fig. 2). 2) Ubiquitination occurs primarily on lysine-248 of
reductase (Fig. 3). 3) Replacement of lysine-248 with arginine does not interfere with sterol-
stimulated binding of reductase to Insigs (Fig. 5), but it does block the subsequent ubiquitination
and degradation (Fig 3). 4) Sterol-stimulated binding of reductase to Insigs requires the
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tetrapeptide YIYF that is also present in the sterol-sensing domain of SCAP (Fig. 5). 5)
Mutations within the YIYF tetrapeptide prevent sterol-mediated ubiquitination and degradation
(Fig. 4). 6) GG-OH, but not farnesol, can synergize with sterols in accelerating degradation of
reductase, suggesting that a geranylgeranylated protein may be involved (Fig. 7).
The data of Fig. 1B show that the kinetics of sterol-stimulated reductase ubiquitination in
human fibroblasts are complex. The initial response is unexpectedly rapid, with ubiquitination
detectable within 5 min of sterol addition, suggesting that the added sterols quickly reach the ER
membrane. In the absence of a proteasome inhibitor, the amount of ubiquitinated reductase
reaches a maximum at 10-30 min, then declines dramatically by 60 min even though substantial
reductase remains within the cell. The fall in ubiquitinated reductase is blocked by MG-132,
implicating proteasomal degradation. But why isn’t the remaining reductase ubiquitinated? One
explanation is that the rate of ubiquitination slows after 30 min; another is that the rate of de-
ubiquitination increases. It is also possible that ubiquitination continues, but the partial decline
in total reductase lowers the absolute amount of ubiquitinated reductase below the threshold of
detection by the immunoblotting assay. Part of the difficulty in interpretation arises because
only a small amount of reductase appears to be ubiquitinated at any instant in time, perhaps
owing to de-ubiquitinating enzymes operating in the cell.
Nonsterol, mevalonate-derived products are not capable of stimulating reductase degradation
when cells are sterol-deficient, but they accelerate degradation when sterols are abundant (4, 5,
40). Two lines of evidence in the current study indicate that the major nonsterol product is
geranylgeranyl pyrophosphate. First, the nonsterol acceleration is not blocked by the squalene
epoxidase inhibitor NB-598 (Fig. 6), suggesting that it occurs at a branch prior to sterol
synthesis. Second, the effect can be reproduced by addition to the culture medium of GG-OH,
but not farnesol. Previous studies have shown that cells have a salvage pathway by which they
can activate GG-OH to the metabolically active pyrophosphate (41). Although there is some
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suggestion that GG-OH can be converted to cholesterol, there is no known enzymatic pathway
for this conversion. Moreover, the failure of NB-598 to inhibit the mevalonate effect suggests
that sterol synthesis is not required. It seems more likely that GG-OH, after conversion to the
pyrophosphate, is being incorporated into a geranylgeranylated protein that is required for
maximally rapid reductase degradation. Possible candidates include geranylgeranylated Rab
proteins, which play roles in vesicular transport (42). We cannot exclude the possibility that
GG-OH itself, or its pyrophosphate derivative, is the actual regulator.
Our finding that GG-OH, but not farnesol, synergizes with sterols in accelerating degradation
of mammalian reductase differs from previous studies. In one study farnesol, but not GG-OH,
promoted degradation of reductase in permeabilized cells (43). However, subsequent studies
showed that farnesol caused the reductase to become detergent-insoluble and thus resistant to
immunoprecipitation, which masqueraded as an apparent increase in degradation (44). In the
same study, farnesol synergized with sterols to accelerate reductase degradation in intact cells,
but GG-OH was not tested and kinetic data were not reported.
The data of Figs. 4 and 5 implicate the YIYF sequence in reductase as a binding site for
Insigs. We were attracted to the YIYF sequence because this sequence is conserved in the sterol-
sensing domain of SCAP (25) and because the first tyrosine of this sequence is required for
SCAP interaction with Insigs (19, 20). Surprisingly, the first tyrosine in the reductase YIYF
sequence is not required for sterol-stimulated ubiquitination and degradation, but the isoleucine
and the second tyrosine are required (IY sequence, Fig. 4). Replacement of the entire YIYF
sequence with AAAA blocks reductase binding to Insigs (Fig. 5), which explains the lack of
ubiquitination and degradation. Whether the IY sequence in SCAP is required for its interaction
with Insigs is currently being explored.
The conservative substitution of arginine for lysine-248 in reductase does not affect Insig
binding (Fig. 5), but it abolishes detectable reductase ubiquitination and delays degradation (Fig.
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3). A similar replacement of lysine-89, the only other lysine in the cytoplasmic loops of
reductase, has little effect on its own, but it further delays degradation when lysine-248 is also
replaced (Figs. 3 and 5). These findings indicate that lysine-248 is the primary site of reductase
ubiquitination and that lysine-89 can become ubiquitinated to a small degree when lysine-248 is
absent. It is striking that both of these lysines are predicted to lie at the COOH-terminal ends of
cytoplasmic loops where they join membrane-spanning helices (see diagram of Fig. 3). This
juxtamembrane location raises the possibility that ubiquitin is transferred to reductase by a
membrane-bound ubiquitin transferase. This would resemble the situation in Saccharomyces
cerevisiae where, Hrd1p, a membrane-bound ubiquitin transferase, is believed to attach
ubiquitins to reductase, earmarking it for degradation (12). In contrast to the mammalian system,
the yeast ubiquitination reaction appears to be stimulated primarily by a nonsterol mevalonate
metabolite, and not a sterol (12). Moreover, the yeast genome does not contain an easily
recognizable counterpart of Insigs.
A major remaining puzzle is how Insigs can interact with similar YIYF sequences in the
membrane domains of both SCAP and HMG CoA reductase while producing dramatically
different consequences. When SCAP binds to Insigs, it is not degraded, but rather it is retained
in the ER (19). Furthermore, we have been unable to find evidence that SCAP is ubiquitinated in
response to sterols.2 In contrast, the Insig-reductase interaction leads to ubiquitination and
degradation. Further progress in understanding these important differences will come through
the identification of other proteins that are recruited to the Insig-reductase and Insig-SCAP
complexes that form in response to sterols.
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FOOTNOTES
1Abbreviations used: BN-PAGE, blue native-polyacrylamide gel electrophoresis; CHO, Chinese
hamster ovary; ER, endoplasmic reticulum; GG-OH, geranylgeraniol; HMG CoA, 3-hydroxy-3-
methylglutaryl coenzyme A; LPDS, lipoprotein-deficient serum; PCR, polymerase chain
reaction; PP, pyrophosphate; RNAi, RNA interference; siRNA, small-interfering RNA; SREBP,
sterol regulatory element-binding proteins; SCAP, SREBP cleavage-activating protein; VSV-G,
vesicular stomatitis virus glycoprotein.
2Feramisco, J.D., Brown, M.S., and Goldstein, J.L. unpublished observations.
*This research was supported by Grant HL20948 from the National Institutes of Health and
grants from the Perot Family Foundation and W.M. Keck Foundation.
‡These authors contributed equally to this work.
§To whom correspondence may be addressed. E-mail: [email protected];
¶Recipient of an NIH Mentored Minority Faculty Development Award (HL70441).
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FIGURE LEGENDS
FIG. 1. Sterols promote ubiquitination of endogenous HMG CoA reductase in SV589
cells. A and B, SV-589 cells were set up for experiments on day 0 at 2 x 105 cells per 100-mm
dish in medium B with 10% FCS. On day 2, cells were washed with PBS and refed medium B
supplemented with 10% LPDS, 50 µM sodium compactin, and 50 µM sodium mevalonate.
After 16 h at 37°C, cells were switched to medium B containing 10% LPDS, 50 µM compactin,
and indicated additions. A, cells were treated in the absence (-) or presence (+) of sterols (1
µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol), 10 mM sodium mevalonate, and 10 µM
MG-132 as indicated. After 2 h (lanes 1-8) or 5 h (lanes 9-16) at 37°C, cells were harvested,
lysed, and subjected to immunoprecipitation with polyclonal anti-reductase as described in
“Experimental Procedures.” Aliquots of immunoprecipitates were subjected to SDS-PAGE,
transferred to nylon membranes, and immunoblotted with 5 µg/ml monoclonal IgG-A9 (against
reductase) or 0.2 µg/ml monoclonal IgG-P4D1 (against ubiquitin). Filters were exposed to film
for 2 s (top gel) and 30 s (bottom gel). B, cells were incubated at 37°C for the indicated time in
the absence (-) or presence (+) of sterols and 10 µM MG132 as indicated, harvested, and
subjected to immunoprecipitation and immunoblot analysis as in (A). Filters were exposed to
film for 30 s (top gel) and 10 s (bottom gel).
FIG. 2. Insig-1 and -2 are required for regulated degradation and ubiquitination of
HMG CoA reductase as determined by RNAi. SV-589 cells were set up on day 0 at 5 x 104
cells per 60-mm dish in medium B with 10% FCS. On days 1 and 3, cells were transfected with
the indicated siRNA as described in “Experimental Procedures.” The total amount of siRNA
was adjusted to 400 pmol/dish by addition of VSV-G siRNA. After the second transfection on
day 3, cells were incubated for 16 h at 37°C in medium B containing 10% LPDS, 50 µM
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compactin, and 50 µM mevalonate. A and B, on day 4, cells were switched to medium B
containing 10% LPDS and 50 µM compactin in the absence (-) or presence (+) of sterols (1
µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) and/or 10 mM mevalonate as indicated.
After 5 h, cells were harvested for preparation of whole cell extracts (A) and total RNA (B).
Aliquots of extracts (10 µg protein) were subjected to SDS-PAGE and immunoblot analysis with
5 µg/ml monoclonal IgG-A9 (against reductase) and 1 µg/ml monoclonal IgG-CD71 (against
transferrin receptor). Filters were exposed to film for 1 min (top) and 1 s (bottom). Total RNA
was prepared from cells treated with sterols and mevalonate (lanes 4, 8, 12, and 16). Aliquots of
reverse-transcribed total RNA (20 ng) were subjected to real-time PCR. Each value for cells
transfected with indicated siRNA represents the amount of Insig-1 mRNA or Insig-2 mRNA
relative to that in control cells transfected with siRNA targeting VSV-G. C, on day 4, cells were
preincubated for 1 h in medium C, after which they were pulse-labeled for 1 h with 100 µCi/ml
[35S]methionine in medium C. Cells were then chased in medium C supplemented with 0.5 mM
unlabeled methionine and 1 mM unlabeled cysteine in the absence (-) or presence (+) of sterols
(1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) plus 10 mM mevalonate as indicated.
After the indicated chase period, cells were harvested, lysed, and subjected to
immunoprecipitation with polyclonal anti-reductase. Aliquots of immunoprecipitates
(normalized for equal 35S-radioactivity incorporated into total cell protein) were subjected to
SDS-PAGE and transferred to nylon membranes. Filters were exposed for 48 h to an imaging
plate at room temperature, scanned, and quantified in a Fuji X Bas 1000 Phosphorimager, and
the image was photographed. The amount of 35S-labeled reductase during the pulse (lane 1 of
both gels) was arbitrarily set at 100%. D, on day 4, cells were switched to medium B containing
10% LPDS, 50 µM compactin, and 10 µM MG-132 in the absence (-) or presence (+) of sterols
(1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) and/or 10 mM mevalonate as
indicated. After 1 h cells were harvested, lysed, and subjected sequentially to
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immunoprecipitation, SDS-PAGE, and immunoblot analysis as described in Fig. 1. Filters were
exposed to film for 3 s (top gel) and 30 s (bottom gel).
FIG. 3. Lysines 89 and 248 are required for regulated ubiquitination and degradation
of HMG CoA reductase. A, amino acid sequence and topology of the membrane domain of
hamster reductase. Lysine residues are shown in black with those located in the cytosol denoted
by arrows. B and C, CHO-K1 cells were set up on day 0 at 5 x 105 cells per 60-mm dish in
medium A with 5% FCS. On day 1, cells were transfected in 2 ml of medium A containing 5%
LPDS and 1 µg of wild-type (lanes 1-4), K89R (lanes 5-8), K248R (lanes 9-12), or
K89R/K248R (lanes 13-16) versions of pCMV-HMG-Red-T7 in the absence or presence of 30
ng pCMV-Insig-1-Myc as indicated. In B, cells in each lane were also transfected with 0.1 µg of
pEF1a-HA-ubiquitin. The total DNA in each lane was adjusted to 3 µg/dish by the addition of
pcDNA3 mock vector. Six hours after transfection, cells received a direct addition of 2 ml of
medium A containing 5% LPDS, 10 µM compactin, and 50 µM mevalonate (final
concentrations). After 16 h at 37°C, cells were switched to medium A containing 5% LPDS and
50 µM compactin in the absence (-) or presence (+) of sterols (1 µg/ml 25-hydroxycholesterol
and 10 µg/ml cholesterol) plus 10 mM mevalonate as indicated. In B, the medium also contained
10 µM MG-132. B, following incubation at 37°C for 2 h, cells were harvested, lysed, and
subjected to immunoprecipitation with monoclonal anti-T7 coupled agarose beads. Aliquots of
immunoprecipitates were subjected to SDS-PAGE and transferred to nylon membranes.
Immunoblot analysis was carried out with 1:1000 dilution of monoclonal anti-HA-IgG (against
ubiquitin) or 1 µg/ml monoclonal anti-T7 IgG (against reductase). Filters were exposed to film
for 5 s. C, after incubation for 5 h, cells were harvested, membrane fractions prepared, and
aliquots (10 µg) were subjected to SDS-PAGE. Immunoblot analysis was carried out with 1
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µg/ml of monoclonal anti-T7 IgG (against reductase) and 1 µg/ml of monoclonal anti-Myc IgG
(against Insig-1). Filters were exposed to film for 7-10 s.
FIG. 4. Mutations in sterol-sensing domain of HMG CoA reductase confer resistance to
sterol-mediated degradation. A, comparison of the amino acid sequence of the second
transmembrane domain of reductase with that of the comparable sterol-sensing region in SCAP.
Sequences are from Chinese hamster reductase (amino acids 57-84) and Chinese hamster SCAP
(amino acids 280-307). GenBank accession numbers are L00165 and U67060, respectively.
Identical amino acids are highlighted in black. Asterisk denotes site of sterol-sensing mutation
(Y298C) in SCAP. B and C, on day 0, CHO-K1 cells were set up at 5 x 105 cells per 60-mm
dish in medium A with 5% FCS. On day 1, cells were transfected with 1 µg of the indicated
wild-type or mutant version of pCMV-HMG-Red-T7 together with 30 ng pCMV-Insig-1-Myc.
Cells in C were also transfected with 0.1 µg of pEF1a-HA-ubiquitin. Six hours after
transfection, cells received a direct addition of 2 ml of medium A to produce final concentrations
of 5% LPDS, 10 µM compactin, and 50 µM mevalonate. B, after 16 h, cells were switched to 3
ml of medium A containing 5% LPDS, 50 µM compactin, and the indicated concentration of 25-
hydroxycholesterol. After incubation for 5 h, cells were harvested, membrane fractions
prepared, and aliquots (15-20 µg) were subjected sequentially to SDS-PAGE and immunoblot
analysis as in Fig. 3. Filters were exposed to film for 5-20 s. C, following incubation for 16 h,
cells were switched to 3 ml of medium A containing 5% LPDS, 50 µM compactin, 10 µM MG-
132 in the absence (-) or presence (+) of sterols (1 µg/ml 25-hydroxycholesterol and 10 µg/ml
cholesterol) plus 10 mM mevalonate. After 2 h at 37°C, cells were harvested, lysed, and
subjected sequentially to immunoprecipitation, SDS-PAGE, and immunoblot analysis as
described in Fig. 3. Filters were exposed to film for 1-5 s.
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FIG. 5. Sterols promote interaction of Insig-1 with wild-type HMG-CoA reductase, but
not sterol-sensing domain mutant. On day 0, SRD-13A cells were set up for experiments as
described in “Experimental Procedures.” On day 2, the cells were transfected in medium A
containing 5% FCS and 60 ng of pCMV-Insig-1-Myc and/or 4 µg of the wild-type or mutant
version of pCMV-HMG-Red-T7(TM1-8) as indicated. The total amount of DNA in each
experiment was adjusted to 5 µg/dish by addition of pcDNA3 mock vector. On day 3, cells were
switched to medium A containing 5% LPDS, 1% hydroxypropyl-β-cyclodextrin, and 50 µM
compactin. After incubation for 1 h at 37°C, the cells were washed and switched to the same
medium (without hydroxypropyl-β-cyclodextrin) containing 10 µM MG-132 in the absence or
presence of 1 µg/ml of 25-hydroxycholesterol plus 10 µg/ml cholesterol (sterols) as indicated.
After incubation for 2 h at 37°C, cells from 4 dishes were pooled, and the resulting digitonin-
solubilized extracts were subjected to BN-PAGE and immunoblot analysis with 5 µg/ml
monoclonal anti-Myc IgG for measurement of reductase/Insig-1 complex. Filter was exposed to
film for 30 s at room temperature. Digitonin-solubilized extracts were also subjected to SDS-
PAGE and immunoblot analysis with either 5 µg/ml of monoclonal anti-Myc IgG (against Insig-
1) or 1:1000 dilution of monoclonal anti-T7 IgG (against reductase). Filters were exposed to
films for 30 s.
FIG. 6. Sterol and nonsterol requirements for degradation and ubiquitination of
endogenous HMG CoA reductase in SV-589 cells stably expressing mevalonate transporter,
pMev. A, schematic representation of the mevalonate pathway. B and C, SV-589/pMev cells
were set up on day 0 at 4 x 105 cells per 100-mm dish in medium B with 10% LDPS, 50 µM
compactin, 200 µM mevalonate, and 500 µg/ml G418. On day 2, the cells were switched to
medium B with 10% LPDS, 50 µM compactin, and 50 µM mevalonate. After 16 h at 37°C, the
cells were switched to medium B containing 10% LPDS and 50 µM compactin in the absence (-)
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or presence (+) of 10 mM mevalonate, 30 µM NB-598, and sterols (1 µg/ml 25-
hydroxycholesterol and 10 µg/ml cholesterol), as indicated. In B, the medium also contained 10
µM MG-132. B, after incubation for 1 h at 37°C, cells were harvested, lysed, and sequentially
subjected to immunoprecipitation, SDS-PAGE, and immunoblot analysis as described in Fig. 1.
Filters were exposed to film for 2 min (top gel) and 30 s (bottom gel). C, following incubation at
37°C for 5 h, the cells were harvested and subjected to cell fractionation as described in
“Experimental Procedures.” Aliquots of membranes (3 µg protein) were subjected to SDS-
PAGE, transferred to nylon membranes, and immunoblotted with 5 µg/ml monoclonal IgG-A9
(against reductase). The filter was exposed to film for 1 min.
FIG. 7. Geranylgeraniol accelerates sterol-induced degradation of endogenous HMG
CoA reductase in SV-589 cells. A and B, SV-589 cells were set up on day 0 at 2 x 105 cells per
100-mm dish in medium B with 10% FCS. On day 2, the cells were refed medium B with 10%
LPDS, 50 µM compactin, and 50 µM mevalonate. Following incubation for 16 h at 37°C, the
cells were switched to medium B containing 10% LPDS and 50 µM compactin in the absence (-)
or presence (+) of sterols (1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol), 10 mM
mevalonate, and the indicated concentration of farnesol or GG-OH. After incubation for 5 h at
37°C, the cells were harvested and subjected to cell fractionation. A, aliquots of membrane
fractions (5 µg) were subjected to SDS-PAGE and immunoblot analysis as in Fig. 6. Filters
were exposed to film for 30 s. B, aliquots of membrane and nuclear extract fractions (3-12 µg
and 20 µg, respectively) were subjected to SDS-PAGE and immunoblot analysis as above.
Filters were exposed to film for 30 s (top gel) and 1 min (bottom gel). P and N denote precursor
and nuclear cleaved forms of SREBP-2, respectively. C, SV-589 cells were set up on day 0 at 2
x 105 cells per 60-mm dish in medium B with 10% FCS. On day 1, the cells were refed medium
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B with 10% LPDS, 50 µM compactin, and 50 µM mevalonate. Following incubation for 16 h at
37°C, cells were preincubated and pulse-labeled with [35S]methionine as in Fig. 2. Cells were
then chased in medium C with 0.5 mM methionine and 1 mM unlabeled cysteine in the absence
(-) or presence (+) of sterols (1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) and 30
µM GG-OH as indicated. Following the indicated chase period, cells were harvested, lysed, and
subjected to immunoprecipitation with polyclonal anti-reductase and analyzed as in Fig. 2.
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TABLE 1 Nucleotide sequences of gene-specific primers used for quantitative real-time PCR mRNA Sequences of forward and GenBank reverse primers (5' to 3') accession no.
Insig-1 CCCAGATTTCCTCTATATTCGTTCTT AY112745 CACCCATAGCTAACTGTCGTCCTA
Insig-2 TGTCTCTCACACTGGCTGCACTA AF527632 CTCCAAGGCCAAAACCACTTC 36B4 TGCATCAGTACCCCATTCTATCA XM_065715 AAGGTGTAATCCGTCTCCACAGA
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Ubiquitin200
120
94
HMG-CoA
Reductase
120
94
kDa
1 2 3 4 5 6 7 8
- + - + - + - +- -+ +
Sterols Mevalonate
None 10 µMMG-1322 hours
9 10 11 12 13 14 15 16
- + - + - + - +- -+ +None 10 µM
5 hoursA
200
120
94
120
94
kDa1 2 3 4 5 6 7 8 9 10 11 12
- + + - + + - + + - + +- - + - - + - - + - - +
5 10 30 60
SterolsMG-132
Time (min)
Ubiquitin
HMG-CoA
Reductase
B
Fig. 1
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B Real-time PCR
Insig-1 Insig-2
VS
V-G
Insi
g-1
Insi
g-2
Insi
g-1
& -
2
siRNA
Rel
ati
ve
Am
ou
nt
of
mR
NA
s
0
0.2
0.4
0.6
0.8
1.0
VS
V-G
Insi
g-1
Insi
g-2
Insi
g-1
& -
2
siRNA
A Immunoblot
Mevalonate
Sterols
1 2 3 4 5 6 7 8 9 10
VSV-G
Insig-1
Insig-2
siRNA
(pmol)-
-
400
-
-
-200 200
200
200
200
200
11 12 13 14 15 16
HMG CoAReductase
TransferrinReceptor
- + - +
- - + +
- + - +
- - + +
- + - +
- - + +
- + - +
- - + +
0 1.5 3 5 1.5 3 5Time of Chase (hours)
Sterols & Mevalonate - - - - + + +
31 2 4 5 6 7
VSV-G
Insig-1 & -2siR
NA
C Radiolabeling and Immunoprecipitation D Immunoprecipitation
kDa
200
120
94
Ubiquitin
Sterols
Mevalonate
1 2 3 4 5 6 7 8
VSV-G
siRNA
Insig-1 & -2
siRNA
- + - + - + - +
- -+ +
120
94
HMG-CoA
Reductase
Time of Chase (hours)
35S
-Rad
ioac
tivi
ty(%
Co
ntr
ol)
10
50
100
0 1 2 3 4 5
VSV-G siRNA
0 1 2 3 4 5
NoneSterols & Mevalonate
Additions
Insig-1 & Insig-2 siRNA
Quantification
Fig. 2
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B
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
- + - + - + - + - + - + - + - +- - - -+ + + +
Sterols & Mevalonate
pCMV-Insig-1-Myc
pCMV-HMG-Red-T7 Wild-Type K89R K248R K89R/K248R
Ubiquitin
HMG-CoA
Reductase
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
- + - + - + - + - + - + - + - +
- - - -+ + + +
Wild-type K89R K248R K89R/K248R
Sterols & Mevalonate
pCMV-Insig-1-Myc
pCMV-HMG-Red-T7
HMG-CoA
Reductase
Insig-1
C
200
120
94
120
94
kDa
SW Q L
E D
Lumen
Cytosol
1
W CN
E
E
KF
P
D E
LE
TL
G
NE
LQR D
PS
ET
T
P
SV
S
W
L
L
L
C
TL
I
S
Y
RA
T
I
S
I
I
I
ID
L V
M
H
M
R
F
LR
SL
Q
K
R
S
L G
Q
N
LR
E
S
ER
S
LE
E
E
E
V
L
R
E
N
K
G
RP
I H
AF
Q
SE
TE
A
E
GLFV
A S H PWEVI
V G T V
TLTIC M M S
MNMFT G
N
N
I
Y E
D
CG
I
FY
FQ
G
I
SV
T
I
A
FF
F
S
V
L
T
S
FV
H F
I
LGYI
L
A
L
LA
I
F
F
AS
R
L
P
L
A
S
LD
F L
NS SS
LA
E
R
V
AE I
Q
D
NR
IAMG
T P G L
DLTF
E V L A
IVLC
T G V G
VGSM
LVLS
V C A PFFTM
F V F Y
NALVS M C G
FCCM
I
PNPV
T Q R V
MIM
S L G L
VLVH
A H S R
HS
GLS
V
DL
Q
N
S
R
QFY
SV
SP
EI
FFIY
V A L
LFAL
S L T V
VQEI
D M S I
M
K
K
K
K
KK
K
K
24889346 Catalytic
Domain
Sterol-Sensing Domain
WIA
S
Fig. 3
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A
B
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
0 .1 .3 1 0 .1 .3 1 0 .1 .3 1 0 .1 .3 1 0 .1 .3 1
I76A F78AWild-type
I76A/F78A
YIYF
to AAAA
HMG-CoA Reductase
(Full-length)
Insig-1
25-Hydroxychol. (µg/ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 .1 .3 1 0 .1 .3 1 0 .1 .3 1 0 .1 .3 1
Y75A Y77AWild-type
Y75A/Y77A
C
Sterols & Mevalonate
200
120
94
120
94
kDa
Ubiquitin
HMG-CoA
Reductase
1 2 3 4 5 6 7 8 9 10 11 12
- + - + - + - + - + - +
Y75A Y75A/Y77A
I76A/F78A
YIYF to
AAAA
K89R/K248R
Wild-
type
IGIAELIPLVTTYIILFAYIYFSTRKIDSCAP 307VLSSDIIILTITRCIAILYIYFQFQNLRHMG-CoA Reductase 8457
280*
Fig. 4
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1 2 3 4 5 6 7 8
- - - + -+ + +
+ + + + + + + +
None Wild-type K248RYIYF
to AAAApCMV-HMG-Red-T7(TM1-8)
pCMV-Insig-1-Myc
Sterols
Blu
e Nativ
e
PA
GE
SD
S
PA
GE
Unbound Insig-1
Insig-1
HMG-Red(TM1-8)
Complex
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Cholesterol
Squalene
Farnesylated
proteins
Geranyl-
geranyl-PP
Geranylgeranylated
proteins
Squalene
epoxide
Squalene
EpoxidaseNB-598
HMG-CoA Reductase
Mevalonate
Isopentyl-PP
Geranyl-PP
Farnesyl-PP
HMG-CoA
Acetyl CoA
A Mevalonate Pathway C Immunoblot
1 2 3 4 5 6 7 8
- + - + - + - +
- - + + - - + +
- - - - + + + +
Mevalonate (10mM)
NB-598
Sterols
HMG-CoA
Reductase
B Immunoprecipitation
kDa
12094
200
12094
Ubiquitin
HMG-CoA
Reductase
1 2 3 4 5 6 7 8
- + - + - + - +
- - + + - - + +
- - - - + + + +
Mevalonate (10mM)
NB-598
Sterols
Fig. 6
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1 2 3 4 5 6 7 8 9 10 11 12
- + - + - + - + - + - +
- + -10 10100 100
Farnesol
(µM)
GG-OH
(µM)
Sterols
Mevalonate (10 mM)
HMG-CoA
Reductase
A Immunoblot
B Immunoblot
Sterols
1 2 3 4 5 6
- + - + - +
- -+
- +-GG-OH (30µM)
7 8 9 10 11 12
- + - + - +
- -+
- +-
SREBP-2
HMG-CoA
Reductase
Membranes Nuclear Extracts
P
N
C Radiolabeling and Immunoprecipitation
Time of Chase (hr)
Sterols
HMG-CoA
Reductase
GG-OH (30µM)
a b c d e f g
0 1.5 3 5 1.5 3 5
- - -- - - -- - +
h i j k l m n
0 1.5 3 5 1.5 3 5
- + ++ + + +
- - +
Mevalonate (10 mM)
- - -
Fig. 7
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Russell A. DeBose-BoydNavdar Sever, Bao-Liang Song, Daisuke Yabe, Joseph L. Goldstein, Michael S. Brown andhydroxy-3-methylglutaryl CoA reductase stimulated by sterols and geranylgeraniol
Insig-dependent ubiquitination and degradation of mammalian 3
published online October 16, 2003J. Biol. Chem.
10.1074/jbc.M310053200Access the most updated version of this article at doi:
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