insig-dependent ubiquitination and degradation of mammalian 3

1

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];

[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


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


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

Fig. 5 by guest on March 26, 2018

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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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