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J Biol Chem, Vol. 274, Issue 44, 31671-31678, October 29, 1999
From the Department of Biology, University of California, San
Diego, La Jolla, California 92093
Sterol synthesis by the mevalonate pathway is
modulated, in part, through feedback-regulated degradation of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals
and yeast, a non-sterol isoprenoid signal positively regulates the rate
of HMGR degradation. To define more precisely the molecule that serves
as the source of this signal, we have conducted both pharmacological
and genetic manipulations of the mevalonate pathway in yeast. We now
demonstrate that farnesyl diphosphate (FPP) is the source of the
positive signal for Hmg2p degradation in yeast. This FPP-derived signal
does not act by altering the endoplasmic reticulum degradation
machinery in general. Rather, the FPP-derived signal specifically
modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule
also serves as a positive signal for HMGR degradation. Thus, both yeast
and mammalian cells employ the same strategy for regulation of HMGR
degradation, perhaps by conserved molecular processes.
The mevalonate pathway is responsible for the biosynthesis of
numerous essential molecules including prenyl groups, coenzyme Q,
dolichol, and sterols such as cholesterol (1).
3-Hydroxy-3-methylglutaryl-coenzyme A reductase
(HMGR)1 is a key enzyme of
the pathway and is rate-determining for cholesterol synthesis in
mammals (2, 3). The mevalonate pathway is modulated in large part by
feedback control of the amount of HMGR protein (1), and a significant
portion of HMGR feedback control occurs through regulation of HMGR
degradation (4-6). HMGR is an integral endoplasmic reticulum (ER)
membrane protein, and its degradation occurs without exit from the ER
(6-8). The non-catalytic, N-terminal transmembrane anchor of HMGR is
both necessary and sufficient for regulated degradation (6, 8-10).
When there is abundant synthesis of pathway products, HMGR degradation
is fast, and steady-state levels of the protein tend to be low.
Conversely, when synthesis of pathway products is reduced, for instance
when a patient is given the HMGR inhibitor lovastatin, HMGR degradation
is slowed, and steady-state levels of the protein tend to increase.
However, neither the identity of the mevalonate-derived signal nor the mechanism by which this signal is coupled to HMGR degradation is known.
In order to discover and understand the mechanisms of HMGR-regulated
degradation, we have been studying the process in the yeast
Saccharomyces cerevisiae (6, 11, 12). Our earlier work has
shown that the yeast HMGR isozyme Hmg2p is degraded in a regulated
manner with many similarities to the analogous process in mammals.
Through the use of genetic selections and screens, we have been able to
identify genes required for the degradation of Hmg2p, called
HRD genes (11), and genes required for normal regulation of Hmg2p
degradation.2
Concurrently with these screens, we have been studying the nature of
the mevalonate-derived signals that control Hmg2p stability. Hmg2p
degradation is regulated by an unknown signal from the mevalonate pathway (Fig. 1a). Inhibiting early pathway enzymes, such as
HMGR itself or HMG-CoA synthase, decreases the rate of Hmg2p
degradation (6). These early pathway blocks decrease the availability
of a downstream signal for degradation. Conversely, inhibiting the enzyme squalene synthase, which is downstream of HMGR, stimulates degradation and ubiquitination of Hmg2p (12). Furthermore, the degradation-enhancing effect of squalene synthase inhibition is abolished by simultaneous inhibition of HMG-CoA synthase or HMGR.
These pharmacological studies imply that the signal for Hmg2p
degradation is a pathway molecule between mevalonate and squalene (Fig.
1a). The most reasonable candidate for this signal is
farnesyl diphosphate (FPP) or an off-pathway FPP derivative. The idea
that FPP, or a derivative, is a positive signal for Hmg2p degradation is particularly interesting since there is accumulating evidence from
in vitro and in vivo studies that farnesol, an
FPP-derivative, is a signal for regulation of mammalian HMGR stability
(13-17).
We have now tested the hypothesis that FPP provides a molecular signal
for control of Hmg2p stability using unique genetic opportunities
available in yeast. In conjunction with pharmacological and biochemical
approaches, we have constructed yeast strains that allowed either
overexpression or down-regulation of specific mevalonate pathway genes
(Fig. 1b). Our results indicated that FPP was indeed a
source of a signal for Hmg2p degradation in yeast, demonstrating that
there is striking conservation for this mode of HMGR regulation among eukaryotes.
Materials and Reagents--
All enzymes were obtained from New
England Biolabs (Beverly, MA). Chemical reagents were obtained from
Sigma. Lovastatin and zaragozic acid were generously donated by Merck.
Terbinafine was commercially obtained as a 1% Lamisil® solution from
Novartis (East Hanover, NJ). ECLTM chemiluminescence
immunodetection reagents were from Amersham Pharmacia Biotech. The
anti-Myc 9E10 antibody was used as a cell culture supernatant obtained
by growing the 9E10 hybridoma (ATCC CRL 1729) in RPMI 1640 culture
medium (Life Technologies, Inc.) with 10% fetal calf serum. The
anti-HA antibody was an ascites fluid obtained from Babco (Berkeley,
CA). The anti-HSV-Tag antibody was obtained from Novagen (Madison, WI).
Affinity-purified goat anti-mouse horseradish peroxidase-conjugated
antiserum was obtained from Sigma.
Recombinant DNA and Molecular Cloning--
PCR was performed as
described previously (18). The genes encoding squalene synthase
(ERG9), farnesyl-diphosphate synthase (ERG20),
and squalene epoxidase (ERG1) were PCR-amplified from yeast
strain RHY623 genomic DNA (18), isolated by the Winston method (19),
using separate primers that contained PstI and BamHI sites in the upstream primers and NheI and
SalI sites in the downstream primers. The amplified
ERG9 and ERG20 genes were cloned between the
PstI and SalI sites in pRH423 (12), thereby placed under control of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoter (PGAPDH) (20). The squalene
synthase-containing plasmid was named pRH440, and the
farnesyl-diphosphate synthase-containing plasmid was named pRH830.
Squalene synthase (ERG9) was tagged at the C terminus with
the HSV epitope sequence (21). The plasmid containing an HSV-tagged squalene synthase expressed from the GAPDH promoter was made by annealing primers that encoded the HSV epitope sequence (QPELAPEDPED) and cloning the resulting DNA fragment between the NheI and
SalI sites in pRH440 to yield pRH442 (ERG9-HSV).
The plasmid to tag ERG9 at its genomic locus with HSV was
made by digesting pRH442 with MunI, and the 5.4-kb vector
fragment was reclosed to yield pRH885. The remaining portion of
erg9 included codons 208-446 and the 3' HSV sequence.
Plasmids that allowed expression of the genomic copy of either
ERG9, ERG20, or ERG1 from the
MET3 promoter (PMET3) (22, 23) were constructed as
follows: pRH442 was digested with EcoRI, and pRH448 was
digested with KpnI. Each vector was reclosed with the insert
removed. The erg9 vector was named pRH948, and the
erg20 vector was named pRH950. The MET3 promoter
was cloned into pRH948 and pRH950 by digesting each plasmid with
SspI and PstI and replacing the insert with the
MET3 promoter containing SspI-PstI
fragment from pHAM8 obtained from Dr. Harry Mountain (Staffordshire,
UK). The PMET3-erg9 plasmid was named pRH973 and the
PMET3-erg20 plasmid was named pRH975. A PCR product
containing the ERG1 coding region was digested with BamHI and PvuII. The 840-base pair fragment was
then cloned between the BamHI and HpaI sites in
pRH973, and the resulting plasmid was named pRH1204.
The plasmid to delete HRD1 was constructed as follows. A
1.45-kb BglII-XhoI fragment from pUG6 (24), which
contained the kanMX expression module (25), replaced the
1.43-kb BglII-SalI fragment in pRH507, which
contained the HRD1 gene (11). The resulting plasmid was
named pRH1122.
Strains and Media--
Escherichia coli DH5
Yeast strain RHY871 (a his3
Yeast strain RHY1326 (a his3 Biochemical Assays--
Cycloheximide chase assays and log phase
steady-state assays were performed as described previously (18).
Methionine chase assays were performed as follows. Cells were grown to
early log phase with an absorbance (A600) of
0.01. Methionine was added to a final concentration of 2 mM, and the cells grown at 30 °C for 15 h. Cells
were then either used for the cycloheximide chase assay as described
above or for the FACS analysis described below.
Hmg2p ubiquitination assays were performed as described
previously.3 Strains were
transformed with pRH1100,3 which expressed a triple HA
epitope-tagged ubiquitin from the constitutive GAPDH promoter.
Transformants were selected for Ade+ prototrophy. Hmg2p
ubiquitination was assayed by immunoprecipitation of 1Myc-Hmg2p and
then immunoblotting the precipitate for covalently linked
HA-ubiquitin.
Flow Cytometry (FACS Analysis)--
FACS analysis was performed
as described previously (18). Living cells were analyzed by flow
microfluorimetry using a FACScaliburTM (Becton Dickinson,
Palo Alto, CA) flow microfluorimeter with settings for
fluorescein-labeled antibody analysis. Histograms were produced from
10,000 individual cells and were plotted with log fluorescence
(arbitrary units) on the horizontal axis and cell number on the
vertical axis.
Zaragozic Acid Increased Hmg2p Degradation by Squalene Synthase
Inhibition--
We previously discovered that addition of zaragozic
acid (ZA), a potent inhibitor of squalene synthase (30) (Fig.
1a), to yeast cells stimulated
degradation of Hmg2p (12). Squalene synthase inhibition would be
expected to cause a build-up of farnesyl diphosphate (FPP), the
substrate of the enzyme, implicating FPP as a source of the positive
signal for Hmg2p degradation. However, it was possible that the effect
of ZA was the result of some action of the drug. We addressed this by
testing the effect of ZA on Hmg2p degradation in a strain that
overexpressed squalene synthase. If ZA was inducing Hmg2p degradation
through squalene synthase inhibition, then cells expressing increased
levels of squalene synthase should require increased amounts of ZA to
cause the same degree of degradation.
We placed the squalene synthase gene (ERG9) under control of
the constitutive GAPDH promoter, PGAPDH (20), and transformed
the PGAPDH-ERG9 construct into yeast cells. PGAPDH-ERG9 was present as a single integrated copy
through targeted insertion of an integrating vector. The strain
expressing a single, integrated copy of PGAPDH-ERG9
was 8-fold more resistant to the growth-slowing effect of ZA than a
wild-type strain, consistent with approximately 8-fold higher squalene
synthase levels in the PGAPDH-ERG9 strain than the
wild-type strain, as measured by Western blot of HSV-tagged versions of
squalene synthase.4
We then tested if the Hmg2p degradation-enhancing effect of ZA required
higher doses upon squalene synthase overexpression. The yeast strains
used above also co-expressed two versions of Hmg2p, 1Myc-Hmg2p and the
fluorescence reporter protein Hmg2p-GFP which have identical
degradation behaviors as normal Hmg2p (12, 18). Cells containing a
single, integrated copy of PGAPDH-ERG9 required
8-fold more ZA to decrease 1Myc-Hmg2p and Hmg2p-GFP steady-state levels
as was required for wild-type cells (Fig.
2, a and b, 40 µg/ml for PGAPDH-ERG9 versus 5 µg/ml
for wt), consistent with the 8-fold overexpression of squalene
synthase. These results suggested that the mechanism for ZA-induced
degradation of Hmg2p was through squalene synthase inhibition.
Squalene Synthase Levels Determined the Degree of Hmg2p
Degradation--
The above results indicated that ZA altered Hmg2p
degradation by decreasing squalene synthase activity. In that case,
genetic down-regulation of the squalene synthase gene (ERG9)
should also increase Hmg2p degradation. Because mevalonate pathway
enzymes are essential for yeast viability, a null allele of
ERG9 in yeast results in cell death (32, 33). Therefore, we
made a conditional allele of squalene synthase by placing the wild-type
ERG9 gene under control of the MET3 promoter
(22), which is repressed by the presence of high extracellular
concentrations (>0.5 mM) of methionine (23). We
constructed a "promoter-switch" plasmid that contained a truncated
version of erg9 placed behind the MET3 promoter.
Targeted integration of this plasmid into the ERG9 locus resulted in the creation of a single, functional copy of
ERG9 under control of the regulated MET3 promoter
(PMET3) (Fig. 1b). This plasmid was used to
transform a methionine prototroph yeast strain to allow growth in any
concentration of methionine. The strain also co-expressed 1Myc-Hmg2p
and Hmg2p-GFP allowing a complete characterization of Hmg2p
degradation. When grown in the absence of methionine, normal regulated
Hmg2p degradation was observed in the yeast strain that expressed
squalene synthase from the MET3 promoter
(PMET3-ERG9).4
Genetic down-regulation of squalene synthase enhanced Hmg2p degradation
in a manner identical to inhibition with ZA. After 15 h growth in
2 mM methionine, Hmg2p degradation in the
PMET3-ERG9 strain was increased. This was indicated
by a lower steady-state level of Hmg2p-GFP and 1Myc-Hmg2p in the
PMET3-ERG9 strain compared with the wild-type strain
(Fig. 3, a and b,
PMET3-ERG9 versus wt). The
effect of down-regulation was similar to that in the wild-type strain
after 15 h incubation in the presence of ZA
(PMET3-ERG9 compared with wt,+ZA).
Hmg2p-GFP in the PMET3-ERG9 was stabilized by the
addition of lovastatin to a similar degree as the stabilization of
Hmg2p-GFP by lovastatin addition in the wild-type strain preincubated
with ZA (PMET3-ERG9, +Lov compared with
wt,+ZA+Lov), indicating that enhanced Hmg2p degradation
caused by squalene synthase down-regulation or ZA addition was still
regulated by the mevalonate pathway.
It was possible that enhanced Hmg2p degradation was the result of cell
death inadvertently caused by squalene synthase down-regulation rather
than the build-up of a positive signal for degradation. Cells
containing the PMET3-ERG9 allele ceased to grow after 15 h incubation in 2 mM methionine (6 doublings)
(see below, Fig. 8b). However, when the
PMET3-ERG9 cells from this 15-h time point were
transferred to media without methionine, they retained the same plating
efficiency4 and growth curve as identically treated
wild-type ERG9 cells (see below, Fig. 8b),
indicating that these cells were still viable.
The enhanced Hmg2p degradation by ZA addition or squalene synthase
down-regulation required the HRD gene-encoded proteins. Enhanced degradation by squalene synthase or ZA addition was completely eliminated by the presence of the hrd1
Additionally, we observed that overexpression of squalene synthase
stabilized Hmg2p. In the strains previously described in Fig. 2, which
overexpressed squalene synthase by the presence of the
PGAPDH-ERG9 allele, both versions of Hmg2p were significantly stabilized. This was observed as both an increase in the
steady-state level of 1Myc-Hmg2p (Fig. 4,
PGAPDH-ERG9 versus wt) and
Hmg2p-GFP,4 and as a decrease in 1Myc-Hmg2p degradation
when squalene synthase was overexpressed (Fig. 4,
PGAPDH-ERG9 versus wt). Thus, squalene synthase overexpression had the opposite effect on Hmg2p degradation as squalene synthase down-regulation.
Down-regulation of Farnesyl-diphosphate Synthase Stabilized
Hmg2p--
The above studies implicated the substrate of squalene
synthase, FPP, as a central molecule in the regulation of Hmg2p
stability. Manipulation of squalene synthase predicted to increase FPP
levels hastened Hmg2p degradation, whereas manipulation of squalene
synthase predicted to decrease FPP levels slowed Hmg2p degradation. We wanted to test further the hypothesis that FPP was the source of the
positive signal for Hmg2p degradation, by eliminating FPP production.
This could be accomplished by inhibition of farnesyl-diphosphate synthase (FPP synthase), which generates FPP as a product.
Unfortunately, no drugs are currently available that inhibit yeast FPP
synthase in vivo. Therefore, we again used a genetic
approach to lower FPP synthase production. As with the other enzymes of
the mevalonate pathway, yeast cells that contain a null allele of FPP
synthase are not viable (34), so we generated a conditional allele of the FPP synthase coding region (ERG20) that resulted in the
wild-type ERG20 gene placed under control of the
MET3 promoter, similar to ERG9 described above.
When grown in the absence of methionine, normal regulated Hmg2p
degradation was observed in the yeast strain that expressed FPP
synthase from the MET3 promoter
(PMET3-ERG20).4
In contrast to enhanced Hmg2p degradation caused by squalene synthase
down-regulation, FPP synthase down-regulation resulted in stabilization
of Hmg2p. When the PMET3-ERG20 strain was grown
15 h in 2 mM methionine, Hmg2p was exceedingly stable, as indicated by both a higher steady-state level and decreased degradation of 1Myc-Hmg2p in the PMET3-ERG20 strain
compared with the wild-type strain (Fig.
5a,
PMET3-ERG20 versus wt).
Similarly, Hmg2p-GFP steady-state levels were increased dramatically in
the PMET3-ERG20 strain (Fig. 5b,
PMET3-ERG20 versus wt), and
this effect was mimicked by growth of the wild-type strain in the
presence of lovastatin (PMET3-ERG20 compared with
wt,+Lov), which acts to slow Hmg2p degradation (6). The effect of FPP synthase down-regulation was reversed by the presence of
ZA in the degradation assay (Fig. 5a,
PMET3-ERG20, 4ZA lane), and in Hmg2p-GFP
steady-state fluorescence (Fig. 5b, PMET3-ERG20,+ZA compared with
wt,+ZA), indicating that regulated degradation of
Hmg2p was still operative. Although the methionine treatment halted the
growth of the PMET3-ERG20 strain after 15 h
(see below, Fig. 8b), if these cells were then transferred
to media that did not contain methionine, they retained a similar
plating efficiency4 and growth curve as the wild-type
ERG20 control cells (see below, Fig. 8b),
indicating that Hmg2p stabilization was not a result of inadvertent
cell death.
Thus, Hmg2p stabilization was caused by FPP synthase down-regulation,
further strengthening the model that FPP was the source of a positive
signal for Hmg2p degradation.
FPP Synthase Down-regulation Also Blocked Hmg2p
Ubiquitination--
The covalent attachment of ubiquitin is a critical
and regulated step in Hmg2p degradation (12). Because FPP synthase
down-regulation stabilized Hmg2p, we also determined its effect on
Hmg2p ubiquitination. To assay Hmg2p ubiquitination, strains containing
the appropriate wild-type or regulated alleles of the FPP synthase gene
were transformed with plasmids that expressed HA epitope-tagged
ubiquitin.3 Hmg2p ubiquitination was assayed by
immunoprecipitation of 1Myc-Hmg2p, followed by anti-HA immunoblotting
to detect covalently attached HA-ubiquitin.3 Regulation of
Hmg2p ubiquitination was assessed by performing each assay in the
presence of lovastatin (Lov), which decreases ubiquitination, or
zaragozic acid (ZA), which increases ubiquitination (12) (Fig.
6, wt).
Down-regulation of FPP synthase caused a drastic decrease in the level
of Hmg2p ubiquitination (Fig. 6, PMET3-ERG20 versus wt, no drug lanes). This effect was similar to the
addition of lovastatin to the wild-type strain during the
ubiquitination assay (wt, Lov lane). Furthermore, the
addition of ZA for 10 min during the ubiquitination assay, which
normally increases Hmg2p ubiquitination, had no effect on Hmg2p
ubiquitination in the FPP synthase down-regulated strain (wt ZA
lane versus PMET3-ERG20, ZA10
lane). This effect of FPP synthase down-regulation on ZA action
was identical to addition of lovastatin to the wild-type cells
(PMET3-ERG20, ZA10 lane compared with
wt, ZA+Lov lane). However, addition of ZA for 1 h did
increase Hmg2p ubiquitination in the FPP synthase down-regulated strain
similar to that in wild-type strain (PMET3-ERG20,
ZA60 lane compared with wt ZA lane). Thus,
pharmacological and genetic manipulations that altered the cellular
levels of FPP correspondingly altered Hmg2p ubiquitination in a manner
consistent with its role as a positive degradation signal.
Genetic Manipulations Did Not Alter General ER
Degradation--
The above genetic experiments were consistent with
FPP being the source of a positive signal for Hmg2p degradation.
However, the results could also be explained by effects on the
machinery for ER degradation, rather than on the signal controlling
Hmg2p degradation. To test this, we examined if these genetic
manipulations altered the degradation of 6Myc-Hmg2p, a previously
described unregulated mutant of Hmg2p (11). Degradation of 6Myc-Hmg2p requires the same degradation pathway as normal Hmg2p (11), but
6Myc-Hmg2p degradation is not regulated by signals from the mevalonate
pathway. Thus, if down-regulation of mevalonate pathway enzymes altered
only the regulatory signal for Hmg2p degradation, then 6Myc-Hmg2p
degradation should be unaffected.
The effect of squalene synthase or FPP synthase down-regulation on
6Myc-Hmg2p degradation was determined in a strain that was isogenic to
the wild-type strain described above, except that unregulated
6Myc-Hmg2p and 6Myc-Hmg2p-GFP were co-expressed rather than 1Myc-Hmg2p
and Hmg2p-GFP. As shown in the top left panel of Fig.
7, 6Myc-Hmg2p-GFP steady-state levels
were unaffected by addition of drugs, such as lovastatin or ZA, which
normally altered the steady-state levels of Hmg2p-GFP (top right
panel). Similarly, down-regulation of squalene synthase or FPP
synthase had no effect on the steady-state levels of the unregulated
6Myc-Hmg2p-GFP (bottom left panel), whereas these
perturbations appropriately altered the steady-state levels of the
regulated Hmg2p-GFP (bottom right panel). The co-expressed
6Myc-Hmg2p was similarly unresponsive to the same pharmacological or
genetic manipulations that affected 1Myc-Hmg2p or the optical Hmg2p-GFP
reporter.4 These results indicated that squalene synthase
and FPP synthase down-regulation affected the regulation of Hmg2p
stability and not the process of ER degradation.
Squalene Epoxidase Down-regulation Did Not Alter Hmg2p
Degradation--
The previous experiments provided strong evidence
that FPP levels within the cell controlled the rate of Hmg2p
degradation. However, it was not clear if this feature was unique to
FPP or if other downstream products could also affect Hmg2p
degradation. Squalene epoxidase is the next enzyme after squalene
synthase in the pathway and is responsible for the formation of
squalene epoxide from squalene (Fig. 1a). We wondered if
altering squalene epoxidase levels would have any effect on Hmg2p
degradation. Therefore, we generated a MET3-regulated allele
of the squalene epoxidase gene (ERG1), similar to the
ERG9 and ERG20 alleles described above. When
grown in the absence of methionine, normal regulated Hmg2p degradation
was observed in the yeast strain that expressed squalene epoxidase from
the MET3 promoter
(PMET3-ERG1).4
Unlike the other manipulations above, squalene epoxidase
down-regulation had no effect on Hmg2p degradation. When grown in methionine, the PMET3-ERG1 strain and the wild-type
strain had identical Hmg2p-GFP steady-state levels (Fig.
8a,
PMET3-ERG1 compared with wt), and
1Myc-Hmg2p degradation.4 This was in contrast to squalene
synthase down-regulation (see above and Fig. 8a,
PMET3-ERG1 versus
PMET3-ERG9), which had the expected effects in the
same experiment.
It was possible that squalene epoxidase down-regulation had no effect
on Hmg2p degradation because the conditional allele was not correctly
functioning. However, cells carrying the ineffective PMET3-ERG1 allele showed identical
methionine-dependent growth retardation as the other
alleles that affected degradation (Fig. 8b). Thus, the
conditional allele of squalene epoxidase was correctly integrated and
down-regulated. To confirm further that a block at squalene epoxidase
did not affect Hmg2p degradation, we treated the wild-type strain with
terbinafine, a specific inhibitor of fungal squalene epoxidase
(35-38). No concentration of terbinafine had any observable effect on
Hmg2p-GFP steady-state levels (Fig. 8c, +Tb
compared with no drug) or 1Myc-Hmg2p
degradation,4 despite examining the effect with lethal
doses of the drug. Together these results indicated that FPP was the
primary, if not the only, source of the positive signal for Hmg2p degradation.
HMGR degradation is regulated by signals generated downstream in
the mevalonate pathway. The identity of these signals and the mechanism
by which they control HMGR degradation is currently not known. To
understand better the nature of this regulatory mechanism, we have
examined the regulated degradation of the yeast HMGR isozyme Hmg2p
through genetic and pharmacological manipulation of the mevalonate pathway.
The results detailed in this work strongly implicated the mevalonate
pathway product FPP as the source of the positive signal for Hmg2p
degradation. ZA addition or squalene synthase down-regulation, both
predicted to increase FPP levels in the cell, increased Hmg2p degradation. Conversely, addition of lovastatin, FPP synthase down-regulation, or squalene synthase overexpression, all predicted to
decrease FPP levels in the cell, stabilized Hmg2p. Identical Hmg2p
steady-state levels in degradation-deficient strains containing either
normal or down-regulated squalene synthase levels indicated that FPP
levels affected only Hmg2p degradation.
Utility of the MET3 Promoter--
In order to examine the
molecular signals for Hmg2p degradation, we required a way to
manipulate the mevalonate pathway at any enzymatic step. Drugs that
inhibit some of the pathway enzymes are available, such as lovastatin
and ZA. However, there are no currently available drugs that inhibit
enzymes between HMGR and squalene synthase. Furthermore, because the
products of the mevalonate pathway are essential for yeast viability,
null alleles of mevalonate pathway genes result in cell death.
Therefore, we required a way to alter the level or activity of
mevalonate pathway enzymes in a controllable, yet viable, manner.
To accomplish this, we manipulated the levels of key mevalonate pathway
enzymes by placing them under the control of a regulated promoter. We
chose the MET3 promoter (22), which can be repressed by
incubation of cells in high concentrations of methionine (>0.5 mM) (23). In all cases, the conditional alleles were
similar to null alleles in that cells carrying the conditional alleles, when continuously incubated in methionine, were unable to grow after a
few doublings. However, unlike cells with null alleles, cells carrying
the conditional alleles could be induced to grow by transferring them
to media containing no methionine. This type of genetic manipulation
provided a facile way to alter the expression of target genes and could
be used, in theory, to create regulated alleles of any yeast gene.
FPP as the Source of the Signal for Hmg2p
Degradation--
Manipulation of FPP levels by both pharmacological
and genetic means resulted in the expected changes to Hmg2p degradation consistent with FPP, or a derivative, as a positive signal for Hmg2p
degradation. The idea that intracellular levels of FPP, or a
derivative, serve to modulate Hmg2p degradation in yeast paralleled
similar observations in mammalian cells. It has been proposed that
degradation of mammalian HMGR is regulated by the intracellular levels
of farnesol, a derivative of FPP (13-17). It may also be the case that
farnesol is the positive signal for Hmg2p degradation in yeast, and we
are currently in the process of examining whether FPP or farnesol
regulates Hmg2p degradation.
One way to distinguish between FPP and farnesol as the regulatory
signal would be by elimination of the pyrophosphatase activity required
to convert FPP to farnesol (17). Recently, two pyrophosphatases, LPP1 and DPP1, that appear to convert FPP to
farnesol in vitro have been described (39). Null alleles of
the genes encoding these enzymes have been made in yeast, and the cells
are viable. We examined Hmg2p degradation in these strains and found
that the absence of Lpp1p and Dpp1p had no effect on Hmg2p regulated degradation.4 This could mean that either FPP, not
farnesol, is the signal for HMGR degradation in yeast or that these
enzymes are not solely responsible for the conversion of FPP to
farnesol in vivo. In either case, it is apparent that FPP
serves as the source of a signal for HMGR degradation in both mammals
and yeast.
In mammalian cells, there is an additional sterol signal that acts to
modulate HMGR degradation. The addition of sterols to mammalian cells
results in increased degradation of HMGR (4, 5). However, this
downstream signal from sterols does not accelerate HMGR degradation in
the absence of the upstream FPP-derived signal (4, 17), indicating that
sterols provide an additional positive signal for HMGR degradation that
works only in conjunction with the FPP-derived signal. In this study,
our results demonstrated that no other pathway product downstream of
FPP was required for Hmg2p degradation in yeast. That is inhibition of
squalene epoxidase activity or down-regulation of squalene epoxidase
had no stabilizing effect on Hmg2p degradation. However, yeast may
contain abundant stores of sterols, and so the existence of other
signals cannot be ruled out by these negative results. We are further
exploring, by both genetic and pharmacological means, whether an
additional signal downstream of squalene can act in conjunction with
FPP to hasten Hmg2p degradation.
Mechanisms by Which FPP Regulates Hmg2p Degradation--
The
identification of FPP, or a derivative, as the positive signal for HMGR
degradation in yeast leads to several models for regulation of Hmg2p
degradation. The simplest model for the regulatory mechanism is that
the FPP-derived signal acts as an allosteric regulator and physically
binds to the transmembrane domain of Hmg2p, altering its susceptibility
to degradation. Alternatively, the FPP-derived molecule could modify or
interact with a separate effector protein that alters Hmg2p stability.
The effector could act to promote or prevent Hmg2p degradation, as
either of these possibilities have precedent in cellular degradation
(40, 41). It is also possible that the FPP-derived signal directly
affects the structure of the ER membrane. By this model, the Hmg2p
transmembrane domain would respond to the altered membrane by becoming
increasingly susceptible to degradation. In this regard, it is
interesting that the distance from the ER surface of key residues in
the Hmg2p transmembrane is critical in regulation of Hmg2p
stability.3
We are currently exploring the mechanism by which FPP acts to regulate
Hmg2p degradation. It is clear that both yeast and mammalian cells
measure FPP production as a means to control HMGR degradation. This
conservation in signaling strategy, together with the similar location
and machinery required for HMGR degradation, indicates that the
mechanism for regulation of HMGR degradation may also be conserved
among eukaryotes.
A final and important note should be made regarding a recent study on a
fascinating side effect of the HMGR inhibitor lovastatin. It has been
demonstrated that the closed We gratefully thank Merck for the generous
gifts of zaragozic acid and lovastatin, Dr. Paul Sternweis for the
lpp1 *
This work was supported by National Institutes of Health
Grant DK5199601 (to R. Y. H.) and a Searle scholarship (to
R. Y. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
S. Cronin and R. Hampton, manuscript in preparation.
3
Gardner, R. G., and Hampton, R. Y. (1999)
EMBO J., in press.
4
R. Gardner and R. Hampton, unpublished observations.
The abbreviations used are:
HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase;
FPP, farnesyl
diphosphate;
ER, endoplasmic reticulum;
ZA, zaragozic acid;
Lov, lovastatin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PCR, polymerase chain reaction;
kb, kilobase pair;
HSV, herpes simplex
virus;
FACS, fluorescence-activated cell sorter;
wt, wild type;
HA, hemagglutinin;
GFP, green fluorescent protein.
A Highly Conserved Signal Controls Degradation of
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) Reductase in
Eukaryotes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strains were grown at 37 °C in LB + amp (100 µg/ml). Yeast strains
were grown at 30 °C in minimal medium supplemented with glucose and
the appropriate amino acids, as described (6). The lithium acetate
method was used to transform yeast with plasmid DNA (26).
200 lys2-801 ade2-101 leu2
ura3-52::LEU2::hmg2-GFP met2 hmg1::LYS2
hmg2::HIS3::1MYC- HMG2) was the parent
strain for transformation of plasmids containing GAPDH-expressed mevalonate pathway genes. RHY871 co-expressed 1Myc-Hmg2p (12) and the
autofluorescent Hmg2p-GFP (27). Each integrating plasmid was introduced
by targeted integration at the StuI site of the ura3-52 genomic locus. Yeast transformants were selected for
Ura+ prototrophy.
200 lys2-801 ade2-101 leu2
ura3-52::LEU2::hmg2-GFP MET2 hmg1::LYS2
hmg2::HIS3::1MYC- HMG2) was made by
transforming RHY871 with a functional MET2 gene fragment from pGMET (28), followed by selection for Met+
prototrophy. RHY1326 and RHY1462 (a his3200 lys2-801 ade2-101 leu2::6myc-hmg2-GFP::LEU2
ura3-52::6MYC-HMG2 MET2 hmg1::LYS2
hmg2::HIS3) were the parent strains for
transformation of all plasmids containing PMET3-expressed
mevalonate pathway genes. Plasmid pRH973 (PMET3-erg9) was introduced by targeted integration
at the HpaI site of ERG9. Plasmid pRH975
(PMET3-erg20) was introduced by targeted integration
at the HindIII site of ERG20. Plasmid pRH1204
(PMET3-erg1) was introduced by targeted integration
at the AgeI site of ERG1. Yeast transformants
were selected for Ura+ prototrophy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Manipulation of the mevalonate pathway.
A, representation of the mevalonate (Mev) pathway
indicating key enzymes and their inhibitors. B, construction
of a MET3 promoter-regulated allele by "promoter
switching." A mevalonate pathway gene, ERGX, was placed
under control of the MET3 promoter through targeted
integration of a promoter-switch plasmid at the ERGX genomic
locus. The plasmid consisted of a non-functional, 5' portion of the
ERGX gene placed behind the MET3 promoter
(PMET3). Integration resulted in a functional copy of the
ERGX gene placed under control of the MET3
promoter and a non-functional, deleted copy behind the native
promoter.
View larger version (43K):
[in a new window]
Fig. 2.
Squalene synthase overexpression increased
the concentration of ZA required for Hmg2p degradation.
a, effect of ZA, which hastens Hmg2p degradation, on
1Myc-Hmg2p steady-state levels in a wild-type strain (wt) or
in an otherwise isogenic strain carrying a single integrant of the
PGAPDH-ERG9 allele (PGAPDH-ERG9).
Strains were treated for 4 h with the indicated concentrations of
ZA and then subjected to immunoblotting to determine 1Myc-Hmg2p
steady-state levels. In all figures, 1Myc-Hmg2p immunoreactivity was
detected with the anti-Myc 9E10 antibody. b, effect of ZA on
Hmg2p-GFP steady-state levels in a wild-type strain (wt) or
in the strain carrying a single integrant of the
PGAPDH-ERG9 allele (PGAPDH-ERG9).
Cells were grown 4 h in the presence of the indicated
concentrations of ZA and then analyzed by flow cytometry.
View larger version (40K):
[in a new window]
Fig. 3.
Squalene synthase down-regulation decreased
Hmg2p levels. a, effect of squalene synthase down-regulation
on Hmg2p-GFP steady-state levels. Cells expressing squalene synthase
from either the wild-type promoter (wt) or the
MET3 promoter (PMET3-ERG9) were grown for
15 h at 30 °C in the presence of 2 mM methionine.
ZA (+ZA, 10 µg/ml final concentration) or lovastatin
(+Lov, 25 µg/ml final concentration) was added to the
appropriate cultures, and all cultures were grown an additional 4 h at 30 °C. Hmg2p-GFP fluorescence was analyzed by flow cytometry.
b, increased 1Myc-Hmg2p degradation as a result of squalene
synthase down-regulation was HRD1-dependent.
Cycloheximide chase assay of strains expressing squalene synthase from
the wild-type promoter (wt) or from the MET3
promoter (PMET3-ERG9) in the presence of a normal
HRD1 gene or the hrd1 allele
(PMET3-ERG9, hrd1, and wt,
hrd1). Cells were grown for 15 h at 30 °C in the
presence of 2 mM methionine, and then cycloheximide was
added to 50 µg/ml final concentration. After addition of
cycloheximide, lysates were prepared at the indicated times and
immunoblotted to determine Hmg2p levels.
allele (Fig.
3b, PMET3-ERG9, hrd1, and wt
+ZA, hrd1), which normally stabilizes Hmg2p (11). This indicated that the degradation-enhancing effect of squalene synthase down-regulation or ZA addition required a functional HRD pathway and was not due to aberrant degradation by an
alternate pathway. Furthermore, identical steady-state levels of
1Myc-Hmg2p in the wild-type, hrd1 strain and the
PMET3-ERG9, hrd1 strain indicated that the lower
steady-state levels of Hmg2p in the PMET3-ERG9
strain were due only to enhanced degradation and not reduced
translation efficiency.
View larger version (20K):
[in a new window]
Fig. 4.
Squalene synthase overexpression decreased
Hmg2p degradation. Effect of squalene synthase overexpression on
1Myc-Hmg2p degradation. Otherwise identical strains expressing squalene
synthase from the wild-type promoter (wt) or from a single,
integrated allele with the GAPDH promoter
(PGAPDH-ERG9) were subjected to a cycloheximide
chase assay. Lysates for each indicated time point after addition of
cycloheximide were made and immunoblotted to determine Hmg2p
levels.
View larger version (42K):
[in a new window]
Fig. 5.
FPP synthase down-regulation decreased Hmg2p
degradation. a, effect of FPP synthase down-regulation on
1Myc-Hmg2p degradation. Otherwise identical strains expressing FPP
synthase from the wild-type promoter (wt) or from the
MET3 promoter (PMET3-ERG20) were grown
for 15 h at 30 °C in the presence of 2 mM
methionine and then subjected to a cycloheximide chase assay. Lysates
for each indicated time point were made and immunoblotted to determine
Hmg2p levels. ZA (10 µg/ml final concentration) was added to the
indicated sample (4ZA) at the same time as addition of
cycloheximide. Lovastatin (50 µg/ml final concentration) was added to
the indicated wild-type strain (wt,+Lov) at the same time as
addition of methionine. b, effect of FPP synthase
down-regulation on Hmg2p-GFP steady-state levels. Cells expressing FPP
synthase from either the wild-type promoter (wt) or the
MET3 promoter (PMET3-ERG20) were grown
for 15 h at 30 °C in the presence of 2 mM
methionine. ZA (+ZA, 10 µg/ml final concentration) or
lovastatin (+Lov, 25 µg/ml final concentration) was added
to the appropriate cultures, and all cultures were grown an additional
4 h at 30 °C. Hmg2p-GFP fluorescence was analyzed by flow
cytometry.
View larger version (73K):
[in a new window]
Fig. 6.
FPP synthase down-regulation decreased Hmg2p
ubiquitination. Regulated ubiquitination of 1Myc-Hmg2p in cells
expressing FPP synthase from the wild-type promoter (wt) or
the MET3 promoter (PMET3-ERG20). Cells
were grown 15 h at 30 °C in the presence of 2 mM
methionine. Ubiquitination assays were performed in the presence of no
drug ( 
), 25 µg/ml lovastatin (Lov), or 10 µg/ml ZA (ZA). For the wild-type strain, lovastatin was
added 30 min prior to cell lysis, and ZA was added 10 min prior to cell
lysis. For the PMET3-ERG20 strain, ZA was added
either 10 min (ZA10) or 60 min (ZA60) prior to
cell lysis. Upper panels are the result of anti-HA
(-HA) immunoblotting for covalently linked HA-tagged
ubiquitin. Lower panels are the result of parallel
immunoblotting an aliquot (1/8 total volume) of the same
immunoprecipitates with the 9E10 anti-Myc antibody (-myc)
to assess total immunoprecipitated Hmg2p.
View larger version (38K):
[in a new window]
Fig. 7.
Conditional alleles of squalene synthase and
FPP synthase altered regulation of Hmg2p degradation. Upper
panels, cells expressing either unregulated 6Myc-Hmg2p-GFP or
normally regulated Hmg2p-GFP were treated with either no drug, 10 µg/ml ZA (ZA), or 25 µg/ml lovastatin (Lov)
and grown for 4 h at 30 °C. Bottom panels, cells
from the upper panel expressing either squalene synthase
from the MET3 promoter (PMET3-ERG9), FPP
synthase from the MET3 promoter
(PMET3-ERG20), or both from their respective
wild-type promoters (wt) were grown for 15 h at
30 °C in the presence of 2 mM methionine. 6Myc-Hmg2p-GFP
or Hmg2p-GFP steady-state fluorescence was analyzed by flow
cytometry.
View larger version (33K):
[in a new window]
Fig. 8.
Squalene epoxidase down-regulation or
inhibition had no effect on Hmg2p-GFP steady-state levels.
a, effect of squalene epoxidase down-regulation on Hmg2p
steady-state levels. Cells expressing squalene epoxidase from either
the wild-type promoter (wt) or the MET3 promoter
(PMET3-ERG1) or squalene synthase from the
MET3 promoter (PMET3-ERG9) were grown for
15 h at 30 °C in the presence of 2 mM methionine.
Hmg2p-GFP fluorescence was analyzed by flow cytometry. b,
left panel, cells containing either the conditional alleles
of squalene synthase (PMET3-ERG9), FPP synthase
(PMET3-ERG20), or squalene epoxidase
(PMET3-ERG1) were grown to mid-log phase
(A600 approximately 0.5) in media that did not
contain methionine. New cultures containing 2 mM methionine
were inoculated from these previously grown cultures to an initial
A600 of 0.01 and then incubated for 32 h at
30 °C. The new A600 for these cultures was
measured at the indicated time points and plotted versus
time. Growth of each strain was compared with the wild-type strain
(wt). Right panel, new cultures, which contained
no methionine, were inoculated from the previous 15-h methionine-grown
cultures to an initial A600 of 0.01 and then
incubated for 32 h at 30 °C. The new
A600 for these cultures was measured at the
indicated time points and plotted versus time. c,
effect of terbinafine (Tb), a squalene epoxidase inhibitor,
on Hmg2p-GFP steady-state levels. Cells were treated with either no
drug, 40 µg/ml terbinafine (Tb), or 10 µg/ml ZA
(ZA) and grown for 4 h at 30 °C. Hmg2p-GFP
steady-state fluorescence was analyzed by flow cytometry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactone ring form of this particular
statin can directly inhibit the proteasome (29). At first thought, it
might be reasonable to conclude that this action is responsible for the
stabilizing effects of lovastatin on Hmg2p, which is degraded by the
proteasome (11). However, several observations show quite clearly that
all actions of lovastatin we have observed were due to altered signal
production and not proteasome inhibition. Degradation of 6Myc-Hmg2p,
which is proteasome-dependent, is unaffected by doses of
lovastatin that slow Hmg2p degradation (Ref. 11 and Fig. 7).
Furthermore, the pathway inhibitor L659,699, which inhibits HMG-CoA
synthase but contains no lactone moiety, is equally effective at
stabilizing Hmg2p as lovastatin (12). Third, genetic manipulations of
HMG-CoA synthase (6), or FPP synthase (this work), which slow
production of the degradation signals but do not involve drugs, have
identical actions to lovastatin on Hmg2p degradation but not 6Myc-Hmg2p
degradation. Finally, lovastatin decreases ubiquitination of Hmg2p
(Ref. 12 and this study), an effect opposite of that observed when the
proteasome is compromised or inhibited (11). Although proteasome
inhibition by the closed ring form of lovastatin is interesting and
perhaps important clinically, it plays no obvious or important role in the actions of this drug on regulating Hmg2p stability in yeast and is
most likely not involved in mammalian HMGR degradation, for similar reasons.
ACKNOWLEDGEMENTS
/dpp1 strains, Dr. Harry Mountain for the
MET2 and MET3 plasmids, and Dr. Robert Rickert
for the use of the FACScaliburTM flow microfluorimeter and software.
FOOTNOTES
To whom correspondence should be addressed.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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N. Sever, B.-L. Song, D. Yabe, J. L. Goldstein, M. S. Brown, and R. A. DeBose-Boyd Insig-dependent Ubiquitination and Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-CoA Reductase Stimulated by Sterols and Geranylgeraniol J. Biol. Chem., December 26, 2003; 278(52): 52479 - 52490. [Abstract] [Full Text] [PDF]
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 T. Granshaw, M. Tsukamoto, and S. Brody Circadian Rhythms in Neurospora Crassa: Farnesol or Geraniol Allow Expression of Rhythmicity in the Otherwise Arrhythmic Strains frq 10, wc-1, and wc-2 J Biol Rhythms, August 1, 2003; 18(4): 287 - 296. [Abstract] [PDF]
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 H. Hiyoshi, M. Yanagimachi, M. Ito, N. Yasuda, T. Okada, H. Ikuta, D. Shinmyo, K. Tanaka, N. Kurusu, I. Yoshida, et al. Squalene synthase inhibitors suppress triglyceride biosynthesis through the farnesol pathway in rat hepatocytes J. Lipid Res., January 1, 2003; 44(1): 128 - 135. [Abstract] [Full Text] [PDF]
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S. R. Cronin, R. Rao, and R. Y. Hampton Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis J. Cell Biol., June 10, 2002; 157(6): 1017 - 1028. [Abstract] [Full Text] [PDF]
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 N. W. Bays, S. K. Wilhovsky, A. Goradia, K. Hodgkiss-Harlow, and R. Y. Hampton HRD4/NPL4 Is Required for the Proteasomal Processing of Ubiquitinated ER Proteins Mol. Biol. Cell, December 1, 2001; 12(12): 4114 - 4128. [Abstract] [Full Text] [PDF]
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 R. G. Gardner, A. G. Shearer, and R. Y. Hampton In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation Mol. Cell. Biol., July 1, 2001; 21(13): 4276 - 4291. [Abstract] [Full Text] [PDF]
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H. Hiyoshi, M. Yanagimachi, M. Ito, I. Ohtsuka, I. Yoshida, T. Saeki, and H. Tanaka Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors J. Lipid Res., July 1, 2000; 41(7): 1136 - 1144. [Abstract] [Full Text]
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S. R. Cronin, A. Khoury, D. K. Ferry, and R. Y. Hampton Regulation of HMG-CoA Reductase Degradation Requires the P-Type ATPase Cod1p/Spf1p J. Cell Biol., March 6, 2000; 148(5): 915 - 924. [Abstract] [Full Text] [PDF]
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T. Ravid, R. Doolman, R. Avner, D. Harats, and J. Roitelman The Ubiquitin-Proteasome Pathway Mediates the Regulated Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase J. Biol. Chem., November 10, 2000; 275(46): 35840 - 35847. [Abstract] [Full Text] [PDF]
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R. G. Gardner, H. Shan, S. P. T. Matsuda, and R. Y. Hampton An Oxysterol-derived Positive Signal for 3-Hydroxy- 3-methylglutaryl-CoA Reductase Degradation in Yeast J. Biol. Chem., March 16, 2001; 276(12): 8681 - 8694. [Abstract] [Full Text] [PDF]
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