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(Received for publication, December 22, 1995) From the
We have previously reported that degradation of
3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting
enzyme in the isoprenoid pathway leading to cholesterol production, can
be accelerated in cultured cells by the addition of farnesyl compounds,
which are thought to mimic a natural, nonsterol mevalonate
metabolite(s). In this paper we report accelerated reductase
degradation by the addition of farnesol, a natural product of
mevalonate metabolism, to intact cells. We demonstrate that this
regulation is physiologically meaningful, shown by its blockage by
several inhibitory conditions that are known to block the degradation
induced by mevalonate addition. We further show that intracellular
farnesol levels increase significantly after mevalonate addition. Based
on these results, we conclude that farnesol is a nonsterol,
mevalonate-derived product that plays a role in accelerated reductase
degradation. Our conclusion is in agreement with a previous report
(Correll, C. C., Ng, L., and Edwards, P. A.(1994) J. Biol. Chem. 269, 17390-17393), in which an in vitro system was
used to study the effect of farnesol on reductase degradation. However,
the apparent stimulation of degradation in vitro appears to be
due to nonphysiological processes. Our findings demonstrate that in
vitro, farnesol causes reductase to become detergent insoluble and
thus lost from immunoprecipitation experiments, yielding apparent
degradation. We further show that another resident endoplasmic
reticulum protein, calnexin, similarly gives the appearance of protein
degradation after farnesol addition in vitro. However, after
the addition of farnesol to cells in vivo, calnexin remains
stable, whereas reductase is degraded, providing further evidence that
the in vivo effects of farnesol are physiologically meaningful
and specific for reductase, whereas the in vitro effects are
not. The isoprenoid metabolic pathway, which leads to the production
of cholesterol among other essential cellular products, is tightly
regulated at the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) ( We
have studied the regulation of the degradation rate of reductase.
Although production of sterols has a role in triggering accelerated
reductase degradation, it has become clear that an unidentified
nonsterol product is necessary for this acceleration to occur. This has
been shown by treating cells with a potent competitive inhibitor of
reductase, compactin, or by using a cell line, UT2, that lacks HMG-CoA
reductase. Under these conditions exogenous cholesterol does not
trigger accelerated degradation of reductase, unless the metabolic
block is bypassed with exogenous mevalonate(14, 15) .
The reciprocal relationship also appears to exist that the nonsterol
component requires the presence of the sterol regulatory component to
cause accelerated degradation. This has been demonstrated using
inhibitors of enzymes in the squalene branch of the isoprenoid
pathway(16) , and in our current study using a cell line
deficient in squalene synthase. In both of these instances endogenous
production of sterols, but not nonsterol products, was effectively
blocked, and in both instances accelerated reductase degradation did
not occur. The identity of the nonsterol mevalonate-derived regulatory
component, however, has remained unknown. Recently Bradfute and Simoni (17) and others (18) reported that reductase
degradation can be accelerated in intact cultured cells by the addition
of farnesyl derivatives, which appear to act by mimicking the elusive
mevalonate-derived metabolite. Here we report acceleration of
reductase turnover in intact cells by the addition of farnesol. We
demonstrate that this regulation is physiologically meaningful by
showing that the effect of farnesol is sensitive to inhibitory agents
and a mutational condition that are known to stunt the regulatory
effect of exogenously added mevalonate. Furthermore we report that
intracellular levels of farnesol increase after mevalonate addition, a
treatment known to accelerate reductase degradation, and decrease after
compactin addition, a treatment that blocks mevalonate production and
is known to increase reductase stability. The possible role of
farnesol in regulation of reductase degradation has been recently
suggested (19) based on findings in an in vitro system. However, several findings of ours suggest that reductase
protein loss in this system is largely due to nonphysiological causes.
We show that a significant fraction of reductase protein becomes
detergent-insoluble during incubation with farnesol in vitro,
causing a depletion of immunoprecipitable reductase and thus the
appearance of degradation. Also, our studies of another endoplasmic
reticulum resident protein, calnexin, reveal that this protein likewise
appears to be rapidly lost in permeabilized cells in response to
farnesol but actually is largely rendered detergent-insoluble. Neither
of these apparently nonphysiological effects occur in intact cells
treated with farnesol. Based on our findings, we conclude that farnesol
is a likely physiological, nonsterol regulatory molecule with a
critical role in accelerated reductase degradation.
For studies of calnexin degradation, the above procedure was
followed except for the following changes. After the pulse and chase
periods, cells were solubilized in 1.0 ml of HBS buffer (50 mM HEPES, pH 7.5, 200 mM NaCl) supplemented with 1% SDS and
boiled for 5 min. Lysates were centrifuged 30 min at 16,000
Figure 1:
Farnesol causes accelerated degradation
of HMG-CoA reductase in vivo. As described under
``Experimental Procedures,'' CHO cells were pulse-labeled,
then chase was done in the presence (closed symbols) or the
absence (open symbols) of 30 µM farnesol for 0,
4, 6, 8, or 10 h, and then cells were solubilized. Reductase was
immunoprecipitated and subjected to SDS-PAGE, and then bands were
quantified by Bio-Rad Molecular Imager. The image presented is from one
representative experiment of six, and the graph is a compilation of
these experiments.
Additionally, we found that 30
µM farnesol causes a decrease in the rate of reductase
synthesis (data not shown). This also has been demonstrated to occur
due to the addition of mevalonate (14) or the addition of
farnesyl acetate (17) in vivo.
Figure 2:
Farnesol-induced accelerated degradation
of HMG-CoA reductase is blocked by ALLN and by thapsigargin. CHO cells
were pulse-labeled, and then chase was done in the presence of 30
µM farnesol, with or without either 20 µg/ml ALLN (upper gel) or 1 µM thapsigargin (THAP, lower gel) for 2, 4, 6, or 8 h. Solubilization,
immunoprecipitation, and electrophoresis were performed as described
under ``Experimental Procedures.'' Bands were quantified by
densitometer, relative to a 100% value at time zero (not shown). A
graph of reductase degradation is presented, showing farnesol alone (open circles), farnesol + ALLN (closed
circles), or farnesol + thapsigargin (closed
triangles).
Figure 3:
SSD cells exhibit a stunted regulatory
effect of farnesol. CHO (upper gel) and SSD (lower
gel) cells were pulse-labeled, and then chase was done in the
presence of either 20 mM mevalonate (MVA) or 30
µM farnesol (FARN) for 0, 2, 4, or 8 h. For basal
degradation, chase was done with no addition for 0, 4, or 8 h.
Solubilization, immunoprecipitation, and electrophoresis were done as
described under ``Experimental Procedures.'' Band intensities
were quantified by densitometer. Graphs showing reductase degradation
in CHO cells and SSD cells are presented, with no addition (open
circles), mevalonate (closed circles), or farnesol (closed triangles) shown.
Figure 4:
Farnesol regulation requires exogenous
sterols in SSD cells. A pulse-chase study of reductase degradation was
done as described under ``Experimental Procedures.'' The
chase period was for 8 h with a range of 25-hydroxycholesterol
concentrations from 2.5 to 0.025 µM, each with no farnesol (open columns) or with 30 µM farnesol (shaded
columns) present. Band intensities were quantified by Molecular
Imager and are presented relative to a 100% value at time zero of the
chase period.
Figure 5:
Intracellular farnesol levels rise
following mevalonate addition. CHO and SSD cells were grown in ten
150-mm plates per sample and then were left untreated (open
columns) or treated with 20 mM mevalonate (shaded
columns) for 3 h. Cells were then subjected to lipid
saponification and extraction as described under ``Experimental
Procedures.'' Farnesol levels were determined by HPLC as described
under ``Experimental Procedures'' using a set of known
quantities of farnesol as standards.
Of further interest is a
comparison between intracellular farnesol levels in SSD cells and wild
type cells. The initial studies of SSD cells (26) revealed that
these cells secrete a considerable amount of farnesol into the culture
medium. Consistent with this observation, we found intracellular
farnesol levels to be approximately twice as high in SSD cells compared
with wild type cells, with or without mevalonate addition (Fig. 5).
To further examine this matter, we tested another protein as an
internal standard to determine whether the effect caused by farnesol in vitro is specific for the loss of reductase. Calnexin, a
chaperone protein implicated in protein folding and retention in the
endoplasmic reticulum, is a good control based on the fact that, like
reductase, it is an integral membrane-spanning resident protein of the
endoplasmic reticulum(31) . The effect of farnesol on the loss
of calnexin was measured both in permeabilized cells and in intact
cells. As shown in Fig. 6A, the addition of farnesol to
permeabilized cells results in very rapid loss of calnexin protein in a
dose-dependent manner. However, following farnesol addition to intact
cells, calnexin remains stable, whereas the degradation of reductase is
accelerated (Fig. 6B). These results suggest that in vitro, the loss of reductase caused by farnesol is
nonspecific and nonphysiological, whereas in vivo the effect
is specific for reductase and physiologically meaningful.
Figure 6:
Farnesol causes calnexin loss in
permeabilized cells but not in intact cells. A, protein loss in vitro is shown. Pulse-labeled CHO cells were permeabilized
as described under ``Experimental Procedures'' and then
incubated 4.5 h with 0, 25, 50, or 100 µM farnesol.
Calnexin was immunoprecipitated, and samples were subjected to
SDS-PAGE. Bands were quantified by Molecular Imager, and values are
shown relative to a 100% value at time zero of the chase period. B, protein loss in vivo is shown. Pulse-labeled CHO
cells were chased in the absence (open symbols) or the
presence (closed symbols) of 30 µM farnesol and
then solubilized. Samples were immunoprecipated with antibodies either
to reductase (HMGR, circles) or calnexin (CNX, triangles) and then subjected to SDS-PAGE, and
the bands were quantified by Molecular Imager. Reductase and calnexin
bands shown are from two different exposure times of the same gel. The
antibodies used to immunoprecipitate calnexin were successfully
characterized in an immunoinhibition experiment using a peptide
corresponding to the calnexin epitope (data not
shown).
Figure 7:
Farnesol causes HMGal protein to become
detergent insoluble in permeabilized cells. A, results of a
pulse-chase study in permeabilized cells are shown. CHO-HMGal cells
were metabolically labeled and permeabilized (see ``Experimental
Procedures''), then incubated ± 50 µM farnesol
for the indicated times, and then subjected to immunoprecipitation and
electrophoresis. Intensities of HMGal protein bands were quantified by
densitometry. B, results of HMGal activity determination for
samples at times and conditions in A are shown. Solubilized
cells were assayed for
Figure 8:
Immunoblot analysis shows that both
reductase (HMGR) and calnexin (CNX) are rendered
detergent insoluble by farnesol in permeabilized cells. CHO cells were
permeabilized and treated for 6 h with or without 50 µM farnesol and then solubilized and centrifuged at 16,000
Studies aimed toward determining the identity of the
regulatory nonsterol, mevalonate-derived metabolite involved in HMG-CoA
reductase degradation have been subject to certain limitations when
performed in living cells. This is because many of these candidate
pathway metabolites, such as isopentenyl pyrophosphate, geranyl
pyrophosphate, and farnesyl pyrophosphate, are polar molecules and are
thus not permeant to cells. This has led to the employment of other
strategies, one of which has involved the use of nonpolar artificial
isoprenoid analogues, which were added to intact cells to determine
whether reductase degradation could be accelerated, presumably by these
artificial products mimicking some natural product. This approach
showed some success, as farnesylated tocopherol analogs (18) and farnesyl acetate and farnesyl ethyl ether (17) were shown to accelerate reductase degradation in
vivo. These results suggested that some farnesyl metabolite(s) in
the isoprenoid pathway is the regulatory molecule. Another strategy
has involved the use of permeabilized cells, which do not present a
permeability barrier. We showed previously (13) that
mevalonate-induced accelerated reductase degradation persisted in cells
after permeabilization with digitonin, but only if cells were
pretreated with mevalonate before permeabilization. Presumably this
period was necessary for production/accumulation of adequate levels of
the mevalonate-derived regulator(s) so that regulated degradation could
be underway at the time of permeabilization. Using a modification of
our permeabilized cell system, Correll et al.(19) were able to demonstrate apparent accelerated
degradation of reductase by addition of farnesol, without the
pretreatment period required for mevalonate. Farnesol is produced
from farnesyl pyrophosphate in cells, in a reaction catalyzed by an
allyl pyrophosphatase(28, 29, 30) , which
diverts some farnesyl pyrophosphate from its primary metabolic route
toward cholesterol. We had not tested farnesol in our permeabilized
cell system(13) , and in the studies of farnesyl acetate in
vivo(17) , farnesol had been tested but at a very limited
set of concentrations, which in hindsight were either too low to elicit
a regulatory response or so high that toxicity resulted. In the current
study we have more rigorously tested farnesol in vivo, using
concentrations low enough to be nontoxic to cells but higher than those
previously found to be ineffectual and have found that farnesol indeed
causes accelerated degradation of reductase. This is an important
finding, because it is the first evidence of a natural nonsterol
mevalonate-derived metabolite causing this effect when added to cells in vivo. Also of importance are our findings that this effect
of exogenous farnesol in accelerating reductase degradation can be
blocked by ALLN, by thapsigargin, and in a CHO cell line missing the
enzyme squalene synthase (see Fig. 2and Fig. 3).
Accelerated degradation by exogenous mevalonate has been shown to be
sensitive to all three of these conditions, so our hypothesis regarding
the role of farnesol is supported by these findings. Although the in vitro results of Correll et al.(19) were
controlled for degradation of total cell protein in their study, for
the following reasons we feel that their reported loss of reductase in vitro is not due to physiologically meaningful causes. One,
the farnesol-induced degradation of reductase in permeabilized cells is
extremely rapid, considerably more so than observed in
mevalonate-pretreated permeabilized cells (13) or even intact
cells treated with mevalonate or farnesol. Two, in order to achieve
significant acceleration of reductase loss in vitro, farnesol
must be used at concentrations of 50 µM and higher, which
are levels we have found to be highly toxic when added to intact cells.
Three, our studies show that farnesol causes a second resident
endoplasmic reticulum membrane protein, calnexin, to be rapidly lost in
permeabilized cells. This loss of calnexin is not observed in
farnesol-treated intact cells. Four, our studies show that farnesol,
when added to permeabilized cells, causes a significant fraction of
reductase (and calnexin) to become detergent insoluble, and therefore
not recoverable in the usual immunoprecipitation from cell lysates.
Although the amount of reductase and calnexin protein that is rendered
detergent insoluble by farnesol is significant, it does not account for
the total amount of each protein that is lost (compare Fig. 8with Fig. 7A and Fig. 6A).
We suggest that a certain degree of nonspecific proteolysis occurs in vitro, perhaps brought about by farnesol disrupting the
endoplasmic reticulum. We believe that our findings, which
demonstrate physiologically meaningful reductase degradation in
vivo by a natural mevalonate product, serve as an important
extension of previous in vivo studies utilizing farnesyl
analogs and show that farnesol is a nonsterol mevalonate product with a
key role in the regulated degradation of reductase.
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 7916-7922
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)to mevalonate, catalyzed by the enzyme HMG-CoA
reductase, a 97-kDa glycoprotein of the endoplasmic reticulum
membrane(1, 2) . The levels of this enzyme are
governed by regulation of
transcription(3, 4, 5) , mRNA
translation(6, 7, 8, 9) , and enzyme
degradation(10, 11, 12, 13) .
Materials
Minimum essential medium (MEM), fetal
calf serum, and trypsin were from Life Technologies, Inc.
[
H]trans,trans-Farnesol (20 Ci/mmol) and
[1-H]farnesyl pyrophosphate (15 Ci/mmol) were
obtained from American Radiolabeled Chemicals (St. Louis, MO), and
trans,trans-farnesol was obtained from Aldrich.
Tran[
S]label metabolic labeling reagent
(>1000 Ci/mmol) was obtained from ICN (Costa Mesa, CA). Compactin
was the generous gift of Dr. Akira Endo (Tokyo Noko University, Tokyo).
Anti-calnexin polyclonal antibodies were kindly provided by Dr. John
Bergeron (McGill University, Montreal) and Dr. Ron Kopito (Stanford
University). ALLN and thapsigargin were obtained from Calbiochem (San
Diego, CA). All other reagents were obtained from Sigma.Cell Culture
Chinese hamster ovary (CHO) cells
were maintained as monolayers in MEM supplemented with 5% fetal calf
serum. Squalene synthase-deficient CHO (SSD) cells were maintained in
the same medium containing 10% fetal calf serum. Unless otherwise
indicated, medium was replaced 24 h before experiments with MEM
containing 5% lipid poor serum, prepared by the method of Rothblat et al.(20) , supplemented with 10 µM compactin and 100 µM mevalonate.Measurement of Protein Degradation in Vivo by
Pulse-Chase
Cells grown in 60-mm dishes were washed with 4 ml of
phosphate-buffered saline (PBS) and starved for 1 h in 1 ml of
methionine-free MEM/lipid-poor serum followed by a 30-min labeling
period in 0.3 ml of starvation medium containing 333 µCi/mmol
Tran[S]label. This medium was removed, and chase
medium (MEM/lipid-poor serum containing 2 mM cold methionine)
was applied to cells. At various times during the chase period, cells
were washed three times with ice-cold PBS and scraped from the dish in
0.7 ml of ice-cold solubilization buffer (50 mM Tris-Cl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 2 µg/ml calpain inhibitor I). Lysates were
centrifuged at 16,000 
g for 30 min at 4 °C.
Supernatants were removed, and aliquots were taken for total labeled
protein quantitation (see below). Supernatants were incubated overnight
with 2 µl of anti-reductase antibody, which was prepared against
synthetic peptides corresponding to the membrane domain of
reductase(21) . Next 20 µl of protein A-Sepharose was added
to adsorb immunoprecipitated protein, and then samples were centrifuged
2 min at 16,000 g, and pellets were rinsed with
solubilization buffer followed by 10 mM Tris-Cl, pH 7.5, 0.1%
Nonidet P-40. Pellets were dissolved in loading buffer (62.5 mM Tris, pH 6.8, 8 M urea, 0.25% bromphenol blue, 15% SDS,
20% glycerol, 25 mg/ml dithiothreitol), incubated 30 min at 37 °C,
and subjected to 5-15% gradient polyacrylamide gel
electrophoresis (PAGE) for 16 h. The gel was fixed in a 45% methanol,
10% acetic acid solution, impregnated with Enlightning, dried under
vacuum, and either exposed to x-ray film at -80 °C for
quantitation by an LKB Ultroscan XL laser densitometer or quantified
using a Bio-Rad Molecular Imager with Molecular Analyst software. g, and then supernatants were removed and diluted 10-fold with
HBS containing 1% Triton X-100. Samples were precleared with protein
A-Sepharose and then incubated overnight with 10 µl of
anti-calnexin antibody at 4 °C. Adsorbance of immunoprecipitated
protein was done as in the above procedure, except that washes were
done with HBS containing 1% Triton X-100 and 0.1% SDS.Quantitation of Total Radiolabeled Protein
From
each sample in the pulse-chase procedure, 20 µl was spotted onto a
1-in
piece of Whatman 3MM paper. These were dried
thoroughly and then dropped into boiling 5% trichloroacetic acid for 5
min followed by immersion in cold trichloroacetic acid and then cold
95% ethanol. Papers were again dried thoroughly, immersed in Cytoscint
scintillation fluid, and counted for radioactivity using a Beckman LS
7500.Measurement of Protein Degradation in Permeabilized
Cells
Cell permeabilization was described by Meigs and Simoni (13) and modified by Correll et al.(19) to
the following procedure. Briefly, following metabolic labeling of cell
proteins, cells were digitonin-permeabilized in suspension and
incubated at 37 °C with or without farnesol and with an
NADPH-generating system. After various times of incubation, samples
were diluted with solubilization buffer (see above), and
immunoprecipitation and PAGE were performed as described above.Immunoblotting
Proteins were separated by
5-15% gradient PAGE for 16 h and then transferred to a
nitrocellulose membrane using a Hoefer TE Series blot transfer unit for
4 h at 300 mA. The membrane was stained with the appropriate primary
antibody (anti-reductase or anti-calnexin), washed, and then stained
with alkaline phosphatase-conjugated secondary antibody. After
additional washes, chemiluminescent substrate (Bio-Rad) was added, and
membranes were exposed to x-ray film for 5-30 min. Bands were
quantified by densitometry.Lipid Saponification and Farnesol Extraction
For
each experimental condition we used ten 150-mm plates of cells grown to
near confluency. Cells were washed with PBS, harvested with trypsin,
and resuspended in fresh MEM/lipid-poor serum. Cell suspensions were
placed in 40-ml Pyrex glass centrifuge tubes and centrifuged at 800
g for 5 min at 4 °C, and then pellets were washed
with PBS and resuspended in 3 ml of PBS. A small aliquot was removed
for cell number estimation using a Coulter counter and a hemacytometer
and also for protein determination by the method of Lowry et al.(22) . 0.2 µCi of [H]farnesol
was added as an internal standard to determine recovery, and 1/100 of
volume was immediately removed for scintillation counting. Next 300
µl of 10 N sodium hydroxide was added, samples were frozen
overnight and then thawed, and 15 ml of a 4:1 mixture of
ethanol:hexanes was added and mixed well. Samples were then heated to
80 °C for 1 h to saponify lipids. Samples were evaporated to
3-4 ml under N, and then 10 ml water and 10 ml
diethyl ether were added and mixed vigorously. After phases separated
the ether was removed to another tube. This extraction was repeated
twice more, and then 1 g of anhydrous sodium sulfate was added to the
pooled ether to remove any traces of aqueous solution. Ether was
filtered through cotton and then evaporated under N to
roughly 0.5 ml. Methanol was added to 10 ml and then evaporated to less
than 400 µl under N. Methanol was added to bring volume
to 400 µl, and 1/100 of volume was removed for scintillation
counting (Beckman LS-7500) to determine recovery. Samples were stored
at -20 °C under N.Thin Layer
Chromatography
[H]farnesyl pyrophosphate,
either subjected to the above saponification procedure or not, as well
as [H]farnesol were spotted onto a 250 µM Silica Gel G plate, and chromatography was performed using a 4:1
mixture of hexanes:ethyl acetate. The plate was dried, and 1-cm
increments were scraped into scintillation vials and counted for
radioactivity.High Performance Liquid Chromatography
Extractions
from the above saponification procedure and farnesol standards were
analyzed by reverse phase HPLC using a Hewlett Packard Aminoquant 1090
Series II, complete with a diode array detector for UV light absorbance
analysis and outfitted with an Altex Ultrasphere C
column
(particle size, 5 µm; internal diameter, 4.6 250 mm).
Acetonitrile was used as mobile phase with a flow rate of 2 ml/min. For
each run, 25 µl of sample was injected, and column output was
detected by UV absorbance at 194 nm. Peaks of absorbance were
integrated and quantified using the Aminoquant data analysis software
and compared with standard curves.
Effect of Farnesol on Degradation of HMG-CoA Reductase
in Vivo
Previous work from our laboratory revealed that added
farnesyl acetate and farnesyl ethyl ether caused accelerated
degradation of reductase in intact CHO cells(17) , but no
similar effect was found at the concentrations of farnesol used in that
study (approximately 5, 50, and 100 µM). In the current
study we tested a wider range of farnesol concentrations and found that
as the concentration is raised above 20 µM but kept well
below the toxic level of 50 µM, added farnesol causes an
acceleration of reductase turnover. As shown in Fig. 1,
pulse-chase studies revealed that addition of 30 µM farnesol causes accelerated reductase degradation, with a
reduction in half-life from about 9.8 to 5.2 h in six experiments. This
effect is comparable with the acceleration induced by added mevalonate
under the same experimental conditions (12, 23) and
suggests that farnesol, as a natural isoprenoid pathway product, could
be a mevalonate-derived, nonsterol regulatory molecule involved in
accelerated degradation of reductase.
Sensitivity of Farnesol-induced Degradation to Inhibitors
of Mevalonate-induced Degradation
To begin to test the
hypothesis of farnesol as the nonsterol mevalonate-derived regulator
involved in reductase degradation, we utilized inhibitors that are
known to blunt the accelerated degradation caused by added mevalonate.
If farnesol is a mevalonate-derived regulatory molecule, one would
expect that the effect of farnesol would exhibit sensitivity to these
agents as well. For these experiments we tested ALLN, an inhibitor of
cysteine proteases and the proteasome, and thapsigargin, a
Ca
perturbant that inhibits the endoplasmic reticulum
Ca ATPase transmembrane pump. Both of these agents
have been shown to inhibit the effect of mevalonate on reductase
degradation(24, 25) . As can be seen in Fig. 2,
the regulatory effect of farnesol is inhibited by ALLN and by
thapsigargin, both used at concentrations known to inhibit
mevalonate-induced degradation.
Sensitivity of Farnesol-induced Degradation to a Mutation
in Squalene Synthase
Our laboratory previously characterized a
mutant CHO cell line lacking the enzyme squalene synthase(26) ,
which catalyzes the condensation of two farnesyl pyrophosphate
molecules to form squalene, which is the first step of the sterologenic
branch of the mevalonate pathway. Consistent with this previous study,
we found that these SSD cells failed to exhibit accelerated degradation
of reductase after mevalonate addition (Fig. 3). This cell line
thus provided another useful criterion for testing the physiological
relevance of the effect of farnesol. As also shown in Fig. 3,
the accelerated degradation of farnesol was likewise inhibited in SSD
cells as compared with wild type cells. The sensitivity of both
mevalonate and farnesol regulation to this mutation lends further
credence to the hypothesis that farnesol is a mevalonate-derived
nonsterol regulator.
Requirement of Farnesol and Sterols in Causing Regulated
Degradation
The primary reason for using SSD cells in this study
was as a criterion to determine whether farnesol shows the
characteristics expected of a putative mevalonate-derived regulator
(see Fig. 3). However, some of our findings in SSD cells had
interesting and important implications regarding the issue of a
synergistic requirement between the nonsterol and sterol regulators
involved in accelerated reductase degradation. Although it is generally
well accepted that the sterol component cannot cause accelerated
degradation without the nonsterol component(14, 15) ,
previous reports have disagreed whether the nonsterol component
reciprocally requires the sterol component. Our group (15) and
others (8) have reported that the sterol product is not
required, citing that inhibitors within the sterol branch of the
isoprenoid pathway do not prevent a mevalonate-induced response. On the
contrary, Correll and Edwards (16) have found that inhibitors
in the sterol branch do block the effect of mevalonate, suggesting that
the sterol component is required. Since the characterization of the SSD
mutation(26) , it has been clear that added mevalonate has a
stunted regulatory effect in these cells. In agreement with Bradfute et al.(26) , we found that exogenous
25-hydroxycholesterol triggers accelerated reductase degradation in SSD
cells to the same degree as in wild type cells (data not shown). This
is not surprising, given that all isoprenoid metabolites prior to
squalene synthase, including the putative nonsterol regulator, would be
expected to accumulate in SSD cells and provide the added sterol
component with its necessary nonsterol complement. However, we wished
to determine whether exogenous farnesol could amplify the accelerated
degradation caused by exogenous 25-hydroxycholesterol in SSD cells. We
therefore tested a range of concentrations of this regulatory sterol
and found that its effect begins to lose potency below 0.1
µM. But interestingly, as shown in Fig. 4, farnesol
acts synergistically with 25-hydroxycholesterol to cause a greater
acceleration of reductase degradation, which became clear at sterol
concentrations of 0.1 µM or less. Based on these results,
we conclude that the nonsterol regulatory product does require sterols
in order to cause regulated degradation of reductase. Furthermore, this
cooperativity of added farnesol and added sterols in inducing
accelerated reductase degradation further supports the hypothesis of
farnesol as a nonsterol regulatory molecule.
Change in Intracellular Farnesol Levels Following
Mevalonate Addition
Although our findings indicate that farnesol
satisfies a variety of criteria one would expect of a putative
mevalonate-derived nonsterol regulator, we felt it important to
determine whether intracellular levels of farnesol increased after
mevalonate addition as would be required. For this purpose we utilized
HPLC to analyze the farnesol levels in cells treated with 20 mM mevalonate. As shown in Fig. 5, farnesol levels in both CHO
and SSD cells increased by approximately 4.5-fold after 3 h of
mevalonate treatment. In a separate experiment, a 3-h treatment with 50
µM compactin caused farnesol levels in CHO cells to drop
43% (data not shown). To ensure that the detected levels of farnesol
were not partially due to hydrolysis of endogenous farnesyl
pyrophosphate during the saponification procedure, we repeated the
experiments with radiolabeled farnesyl pyrophosphate added to the cell
suspension before saponification of lipids. Thin layer chromatography
revealed that just after saponification, all radioactivity comigrated
with farnesyl pyrophosphate and not farnesol, and after subsequent
ether extraction essentially no radioactivity was present in the
extract (data not shown). Although these findings demonstrate that
intracellular levels of farnesol increase after the addition of
exogenous mevalonate and decrease after blockage of mevalonate
production, they admittedly reveal only a correlation between farnesol
levels and reductase degradation rate not a cause and effect
relationship. However, we feel they are an important contribution to
the accumulating body of evidence suggesting that farnesol is a
regulator of reductase degradation.
Degradation of Reductase in Vitro
We found
farnesol to cause an extremely rapid loss of reductase in vitro (data not shown), as was reported by Correll et
al.(19) . However, this abrupt farnesol-induced loss of
reductase protein (t
, approximately 1.5 h) was
anomalously rapid when compared with farnesol-induced degradation in vivo (t, approximately 5 h, see Fig. 1), and therefore led us to question the physiological
significance of this result. The finding that Triton X-100, a nonionic
detergent, also caused very rapid disappearance of reductase in
vitro (data not shown) further suggested that the effect caused by
farnesol in this permeabilized cell system might not be physiological.
Farnesol-induced Detergent Insolubility of HMG-CoA
Reductase and Calnexin in Permeabilized Cells
We also utilized a
CHO cell line stably transfected with a plasmid encoding the protein
HMGal. HMGal is a chimera comprised of the transmembrane regulatory
domain of reductase fused to Escherichia coli 
-galactosidase, and it has been shown to exhibit regulated
turnover in the same manner as endogenous
reductase(12, 27) . As can be seen in Fig. 7A, farnesol caused a rapid loss of HMGal protein in vitro. Surprisingly, however, farnesol did not cause a
corresponding loss of -galactosidase activity in the permeabilized
cells, as shown in Fig. 7B. In an attempt to resolve
these paradoxical findings, we noted that in the -galactosidase
activity determinations the total cell lysate was assayed, whereas in
the pulse-chase procedure the lysates were centrifuged at a low speed
to remove unlysed cells, nuclei, and detergent-insoluble material prior
to immunoprecipitation of reductase. Therefore, following this
centrifugation step we assayed the resulting pellet and the supernatant
for HMGal activity and found that a significant amount of HMGal
activity was detectable in the pellet (Fig. 7C). The
amount of activity in the pellet was found to increase with time and
with increasing concentrations of farnesol (data not shown). An
immunoblot analysis of the two fractions verified that farnesol caused
a significant fraction of reductase protein to appear in the pellet and
further revealed that a significant fraction of calnexin protein was
rendered insoluble by farnesol (Fig. 8). By comparison, we
tested lysates from cells treated with farnesol in vivo, and
in these virtually all of the HMGal activity was detected in the
supernatant and none was detected in the pellet (data not shown),
demonstrating that the in vivo effect of farnesol is distinct
from the apparently nonphysiological effect responsible for the in
vitro results.
-galactosidase activity by addition of 1
mg/ml o-nitrophenyl--galactopyranoside, followed by
colorimetric assay at 420 nm using a Beckman DU-64 spectrophotometer. C, results of cell lysate fractionations from samples at above
times and conditions are shown. Solubilized cell samples (see
``Experimental Procedures'') were centrifuged at 16,000
g for 30 min, and supernatants and pellets were
separated. For -galactosidase activity determination, pellets were
resuspended in solubilization buffer to a volume equal to supernatants,
and each was assayed for HMGal activity as described in B. FARN, farnesol.
g for 30 min, and then pellets and supernatants were
separated. For immunoblotting, pellets were directly treated with
loading buffer, whereas supernatants were treated with 70% acetone to
precipitate proteins, and then this pellet was dissolved in loading
buffer. Gel electrophoresis, blot transfer, and antibody staining (with
either reductase or calnexin primary antibodies) were performed as
described under ``Experimental
Procedures.''
)-galactosidase; MEM,
minimum essential medium; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; HPLC, high performance liquid
chromatography; ALLN, N-acetyl-Leu-Leu-norleucinal.
We are grateful to Dr. Christopher Silva for his
assistance with HPLC and thin layer chromatography.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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