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(Received for publication, September 20, 1995; and in revised form, October 26, 1995) From the
We have recently demonstrated that mevalonate kinase and
farnesyl diphosphate (FPP) synthase are localized predominantly in
peroxisomes. This observation raises the question regarding the
subcellular localization of the enzymes that catalyze the individual
steps in the pathway between mevalonate kinase and FPP synthase
(phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and
isopentenyl diphosphate isomerase). These enzyme are found in the
100,000
Recently, it has been demonstrated by our group and others that
peroxisomes contain a number of enzymes involved in cholesterol
biosynthesis that previously were considered to be cytosolic or located
exclusively in the endoplasmic reticulum. Peroxisomes have been shown
to contain acetoacetyl-CoA thiolase(1, 2) ,
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) ( The demonstration that mevalonate kinase
and FPP synthase are localized predominantly in peroxisomes (8, 9) raises the question regarding the localization
of the enzymes that catalyze the individual steps in the pathway
between mevalonate kinase and FPP synthase (phosphomevalonate kinase,
mevalonate diphosphate decarboxylase, and isopentenyl diphosphate
isomerase). Based on results obtained from fractionation studies, these
enzymes are believed to be localized in the cytosol. However, recent
data have shown that the activities of these enzymes as well as
mevalonate kinase and FPP synthase are significantly reduced in liver
tissue obtained from patients with peroxisome-deficient diseases
(Zellweger syndrome and neonatal adrenoleukodystrophy), thus indicating
a peroxisomal localization(9) . We have routinely employed
three different methods to study subcellular localization of proteins:
(i) analytical subcellular fractionation and measurements of enzyme
activities, (ii) immunoblotting of the protein in the isolated
fractions with a monospecific antibody, and (iii) immunoelectron and
immunofluorescence microscopy. In the studies demonstrating the
peroxisomal localization of mevalonate kinase and FPP
synthase(8, 9) , it was shown that analytical
subcellular fractionation of liver and measurements of enzyme
activities are not sufficient to determine intracellular localization
due to the release of these enzymes in the cytososlic fraction from
peroxisomes during the isolation of the organelle. Immunoelectron and
immunofluorescence microscopy studies with specific antibodies to these
enzymes were critical in determining the correct subcellular
localization. To our knowledge antibodies to phosphomevalonate kinase,
mevalonate diphosphate decarboxylase, and isopentenyl diphosphate
isomerase are not currently available. Hence, in order to study the
subcellular localization of these enzymes, we have selected to use
permeabilized cells, which retain their organelle integrity yet lack
cytosolic components. Permeabilized cells have been used successfully
in a number of different studies dealing with subcellular function and
localization.
To test whether only the plasma membrane was disrupted and the cell
organelles remained intact, we determined various marker enzyme
activities in both permeabilized and intact cells. Table 1illustrates that there was no significant difference
between the two groups in the total activity per plate of esterase
(marker enzyme for endoplasmic reticulum) and catalase (marker enzyme
for peroxisomes), whereas phosphoglucose isomerase (a marker enzyme for
cytosolic fraction) was measurable in the control cells and was not
detectable in the permeabilized cells. Furthermore, the protein
concentration in permeabilized cells was approximately 30-50%
less than that of intact cells. This corresponds to the protein content
of cell cytosol. However, to further demonstrate that the peroxisomal,
ER and mitochondrial compartments of cells remain intact after
permeabilization while the cytosolic contents disappear, we employed an
additional method. Control and permeabilized cells grown on coverslips
were treated in parallel with antibodies to various marker enzymes as
described under ``Experimental Procedures.'' Fig. 1illustrates the immunofluoresence pattern obtained for
cytosol, peroxisomes, ER, and mitochondria in control cells and
digitonin permeabilized cells. Panel A in Fig. 1shows
cytosolic labeling in intact CV-1 cells, whereas the permeabilized CV-1
cells in panel B are devoid of cytosolic labeling. The bright
fluorescence in the center of the cell is due to autofluorescence of
the nucleus. In panels C and D, the cells were
labeled for peroxisomal proteins using an antibody made against the
peroxisomal targeting signal (SKL at the C terminus)(20) . A
uniform punctate pattern characteristic of peroxisomal labeling is
observed in both the intact cells (panel C) as well as in the
permeabilized cells (panel D). Additionally, the pattern of ER
labeling is similar in control cells (panel E) and
permeabilized cells (panel F). Cells in panels G (control) and H (permeabilized) demonstrate that the
mitochondrial membrane also remains intact during selective
permeabilization with digitonin. Taken together, these results
demonstrate that the plasma membrane of CV-1 cells can be selectively
permeabilized with low concentrations of digitonin, resulting in the
release of cytosolic proteins while maintaining organelle integrity.
Figure 1:
Selective permeabilization of the
plasma membrane releases cytosolic components but maintains the
integrity of subcellular organelles. Control and permeabilized cells
grown on coverslips were treated in parallel with antibodies to various
marker enzymes as described under ``Experimental
Procedures.'' Panels A, C, E, and G illustrate control cell labeling; panels B, D, F, and H represent labeling of permeabilized cells. Panel A shows cytosolic labeling in intact cells; panel B illustrates the absence of cytosolic labeling in permeabilized
cells. The bright fluorescence in the center of the cell is due to
autofluorescence of the nucleus. In panels C and D,
the cells were labeled for peroxisomal proteins, in panels E and F for ER proteins, and in panels G and H for mitochondrial proteins.
Figure 2:
The release of catalase activity from
peroxisomes as a function of time after selective permeabilization of
the plasma membrane. Cells were permeabilized and then assayed for
catalase activity in the presence and absence of 0.1% Triton X-100. The
graph represents mean ± S.E. values of three sets of
experiments. Values are expressed as percent of total catalase activity
at each time point as measured in the presence of Triton
X-100.
Results of
these experiments are shown in Table 3. The mean value for
control cells was 4287 dpm/plate and for permeabilized cells 3026
dpm/plate. These two means are not significantly different. No
conversion of mevalonate to cholesterol was observed if ATP and/or
NADPH were omitted from the reaction buffer (data not shown). These
results indicate that the cytosolic fraction of cells is not necessary
for the biosynthesis of cholesterol from mevalonate.
In summary, the
current report demonstrates that mevalonate kinase, phosphomevalonate
kinase, and mevalonate diphosphate decarboxylase activities in extracts
prepared from intact cells are equal to those of selectively
permeabilized cells that lack cytosolic enzymes. We also demonstrate
structure-linked latency of phosphomevalonate kinase and mevalonate
diphosphate decarboxylase that is consistent with a peroxisomal
localization of these enzymes. These results in combination with the
previous observation that mevalonate kinase and FPP synthase are
predominantly localized to peroxisomes(8, 9) , suggest
that all of the cholesterogenic enzymes involved in the conversion of
mevalonate to FPP are localized to the peroxisome. This conclusion is
further supported by the direct finding that cholesterol biosynthesis
from mevalonate can occur in selectively permeabilized cells lacking
cytosolic components and indirectly by the previous observation that
all the required enzymes for the conversion of mevalonate to FPP are
significantly reduced in tissue obtained from patients with Zellweger
syndrome and neonatal adrenoleukodystrophy(9) . The
isoprenoid biosynthetic pathway is unrivaled in nature for the chemical
diversity of the compounds it produces. FPP is a key intermediate that
serves as a substrate for a number of critical branch-point enzymes
including the synthesis of squalene, cholesterol, farnesylated and
geranylgeranylated proteins, dolichols, coenzyme Q, and the isoprenoid
moiety of heme a. Thus, the regulation and levels of FPP are
important since large perturbations in FPP could alter the flux of
isoprenoid compounds down the various branches of the pathway. If
indeed, the majority of the cell's FPP is produced in the
peroxisomes, this means that FPP and/or farnesol has to be transported
out of peroxisomes for further metabolism. Since phosphorylated
products of mevalonate are not able to cross the peroxisomal membrane,
it is likely that FPP may also be impermeable. Therefore, is FPP first
converted to farnesol in the peroxisome, and then is freely diffusible
out of the organelle? Or is there a transport/binding protein that
facilitates the movement of these intermediates? What determines where
FPP is utilized? What regulates FPP conversion to farnesol and farnesol
conversion to dicarboxylic acids? These are important questions that
need to be addressed in order to understand the regulation of FPP/and
or farnesol. It is significant to note two recent studies
demonstrating the potential importance of farnesol in regulation of
cellular function (21, 22) . In the first study,
farnesol has been identified as the non-sterol derivative that can
initiate and promote the degradation of HMG-CoA reductase in
permeabilized cells(21) . In the second study, an orphan
nuclear receptor named farnesoid X-activated receptor (FXR) is
described that is activated by farnesol(22) . Thus, FXR
provides an example of a vertebrate transcription factor that is
regulated by an intracellular metabolite (farnesol) and may indicate
the existence of a novel vertebrate-signaling pathway(22) . The
FXR target genes remain to be identified.
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1784-1788
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CONVERSION OF MEVALONATE TO FARNESYL DIPHOSPHATE OCCURS IN THE
PEROXISOMES (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
g supernatant fraction of cells or tissues and
have been considered to be cytoplasmic proteins. In the current
studies, we show that the activities of mevalonate kinase,
phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are
equal in extracts prepared from intact cells and selectively
permeabilized cells, which lack cytosolic enzymes. We also demonstrate
structure-linked latency of phosphomevalonate kinase and mevalonate
diphosphate decarboxylase that is consistent with a peroxisomal
localization of these enzymes. Finally, we show that cholesterol
biosynthesis from mevalonate can occur in selectively permeabilized
cells lacking cytosolic components. These results suggest that the
peroxisome is the major site of the synthesis of FPP from mevalonate,
since all of the cholestrogenic enzymes involved in this conversion are
localized in the peroxisome.
)synthase(3) , HMG-CoA
reductase(4, 5, 6) , mevalonate
kinase(7, 8) , and most recently farnesyl diphosphate
(FPP) synthase(9) . Both mevalonate kinase and FPP synthase
seem to be localized predominantly, if not exclusively, to peroxisomes (8, 9) .
Materials
Biochemicals were purchased from
Sigma. (RS)-[5-
H]Mevalonic acid, (R)-[2-
C]mevalonic acid-5-phosphate, (R)-[5-H]mevalonic acid-5-diphosphate,
[1-C]isopentenyl diphosphate,
[1-H]farnesyl diphosphate,
[26-C]cholesterol, and (R)-mevalonic
acid-5-phosphate were purchased from American Radiolabeled Chemicals
Inc. AG1-X8 200-400 mesh formate resin was purchased from
Bio-Rad. All cell culture media and sera were purchased from Life
Technologies, Inc. Monkey kidney cells (CV-1) were obtained from
American Type Culture Collection.Cell Permeabilization
CV-1 cells were seeded at a
density of 4.0 10
on 60-mm plates and grown to 70%
confluence in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum. Care was taken to seed cells in a manner that
resulted in all plates per given experiment to have the same number of
cells. This was important since enzyme activities and rates of
cholesterol synthesis were determined per plate. As indicated in
individual experiments, cells were transferred to media containing
lipoprotein-deficient serum 24 h prior to the experiment. The day of
the experiment, the media was aspirated off the plates and the plates
were then washed twice with ice cold KH buffer (50 mM HEPES,
110 mM KOAc, pH 7.2). The plates were then transferred to ice
and the cells were incubated for 5 min in KHM buffer containing 20
µg/ml digitonin, 20 mM HEPES, 110 mM KOAc, and 2
mM MgOAc, pH 7.2. The digitonin solution was then aspirated
off the cells, and the cells were washed twice with ice-cold KH buffer
and subsequently allowed to incubate in KH buffer for 30
min(10) . This procedure allows the cells to remain attached to
the plates. Control cells were treated similarly and washed with the
same buffer lacking digitonin.Measurement of Cholesterol Synthesis
CV-1 cells
were grown to 80% confluence on 160-mm plates in standard media, and
transferred to media containing lipoprotein-deficient serum 24 h prior
to use. The day of the experiment, the cells were permeabilized as
described and each plate was scraped into 3 ml of reaction buffer. The
reaction buffer consisted of the following: 100 mM phosphate
buffer, 4 mM MgCl
, 1 mM dithiothreitol,
15 mM ATP, and 5 mM NADPH, pH 7.4. Control cells were
scraped into 3 ml of reaction buffer and gently homogenized by hand in
a glass homogenizer by three up and down strokes. Subsequently, 10
µCi of (RS)-[5-H]mevalonic acid in
100 mM phosphate buffer, pH 7.4, was added to each reaction
and incubated for 4 h at 37 °C. The reaction was terminated by
adding 3 ml of 60% KOH (w/v) and saponified for 1 h at 70 °C. After
saponification, internal [26-C]cholesterol
standard was added to each tube and extraction and separation of the
non-saponifiable lipids was performed as
described(11, 12) . The sterol fraction was separated
by thin layer chromatography (hexane:diethyl ether, 50:50). The
recovery of cholesterol averaged 60-80% and was well resolved
from squalene and lanosterol.Mevalonate Kinase Assay
The reaction buffer
consisted of 100 mM phosphate buffer, 4 mM MgCl, 1 mM dithiothreitol, 1 mM EDTA, pH 7.4. (RS)-[5-H]Mevalonic
acid was added at a specific activity of 625 dpm/nmol. Reactions were
run on tissue culture plates by adding 800 µl of reaction buffer
containing 50,000 dpm of substrate and incubating at 37 °C for 10,
20, or 30 min. Reactions were terminated by scraping cells into
Eppendorf tubes and boiling the sample for 3 min.
[1-C]Isopentenyl diphosphate was added as an
internal standard to each sample, and the products were separated on
DEAE-cellulose (Cl form)
columns(8, 13) .
Phosphomevalonate Kinase Assay
The incubation
conditions were the same as for mevalonate kinase except that (R)-[2-C]mevalonic acid-5-phosphate was
added at a specific activity of 6,000 dpm/nmol and 50,000 dpm per assay
was used. The products were loaded on AG1-X8 formate resin column (3
ml) and washed with 50 ml of 4 N formic acid to elute first
the substrate and subsequently with 50 ml of 0.8 M ammonium
formate in 4 N formic acid to elute the phosphorylated
products(9, 14) .Mevalonate Diphosphate Decarboxylase Assay
The
incubation conditions were the same as for mevalonate kinase except
that (R)-[5-H]mevalonic
acid-5-diphosphate at 50,000 dpm/pmol/assay was used. The products were
loaded on AG1-X8 formate resin column (3 ml) and washed with 50 ml of
0.4 M ammonium formate in 4 N formic acid and
subsequently with 50 ml of 0.8 M ammonium formate in 4 N formic acid(9, 14) .Isopentenyl Diphosphate Isomerase Assay
The
reaction buffer consisted of the following: 10 mM HEPES, 10
mM KF, 0.01% bovine serum albumin, 1 mM MgCl, and 10 mM 
-mercaptoethanol. To
each sample, 790 µl of reaction buffer was added and allowed to
incubate at 37 °C for at least 2 min. The
[1-C]isopentenyl diphosphate substrate was then
added at a concentration of 16,000 dpm/nmol in 10 µl. The samples
were incubated for 5 min at 37 °C; the reaction was stopped by
adding 2 ml of 25% HCl in methanol and incubating another 10 min at 37
°C. [1-H]Farnesyl diphosphate standard was
then added to each sample. The samples were transferred from the tissue
culture plates to glass tubes, extracted with 6 ml of hexane, vortexed,
and centrifuged. The hexane phase was transferred to a scintillation
vial and counted(15) .Latency of Catalase in Permeabilized Cells
Cells
were permeabilized as described above and assayed for catalase activity
in the presence or absence of 0.1% Triton X-100 on 60-mm tissue culture
plates for the indicated time intervals. The mixture was then
transferred to cuvettes, and absorbance was read as described for the
measurement of catalase(16) .Marker Enzyme Assays
The activity of
phosphoglucose isomerase was measured according to the method of
Noltmann et al.(17) , and esterase was determined
according to the method of Beaufay et al.(18) .
Protein concentration was determined by the BCA method (Pierce) using
bovine serum albumin as a standard.Immunofluorescence
CV-1 cells were grown on
coverslips, and individual coverslips of control and permeabilized
cells were treated in parallel with antibodies to various marker
enzymes as described(8) . For cytosolic labeling, the cells
were treated with sheep anti-fatty acid synthase antibody (1:20
dilution) and subsequently with secondary fluorescein conjugate-labeled
donkey anti-sheep antibody (1:100 dilution). For peroxisomal proteins
the cells were treated with rabbit anti-SKL antibody (1:200 dilution),
and for endoplasmic reticulum labeling, rabbit antibody against HMG-CoA
reductase (1:30 dilution) was used. For mitochondrial distribution, a
rabbit anti-cytochrome c oxidase antibody (1:25 dilution) was
used. The secondary label for the peroxisomal, ER, and mitochondrial
labels was Texas Red-labeled goat anti-rabbit IgG antibody (1:100
dilution).
Selective Permeabilization of the Plasma Membrane and
Release of Cytosolic Components
Recently, several laboratories
have been successful in permeabilizing cells in a manner that allows
the cell organelles to remain intact while the plasma membrane is
disrupted(10, 19) . Digitonin permeabilizes cells by
complexing with cholesterol. Since the ER membranes, peroxisomal
membranes, and most other cell organelles are almost devoid of
cholesterol, digitonin complexes almost exclusively with the plasma
membrane. The main consequence of permeabilizing cells is the loss of
cytosolic components, while leaving the organelles virtually intact.
Validation of the Experimental Model
In order to
obtain information on the functional characteristics of the peroxisomal
membrane and to investigate how long the peroxisomes would maintain
their integrity, the latency of catalase was measured as a function of
time in permeabilized cells. Fig. 2shows that catalase remains
up to 70% latent (i.e. inside the peroxisomes) during the
first 10 min after cell permeabilization. After 30 min, less than 5% of
catalase activity is latent. Based on these results, an incubation
period of 10 min (or less) was selected for all subsequent experiments
dealing with latency determinations.
Total Activity and Latency Determinations of Mevalonate
Kinase, Phosphomevalonate Kinase, and Mevalonate Diphosphate
Decarboxylase in Control and Permeabilized Cells
In order to
determine if mevalonate kinase, phosphomevalonate kinase, and
mevalonate diphosphate decarboxylase are present in the cytosol or in a
membrane-bound organelle, the activities of these enzymes were
determined in control and in permeabilized cells in the presence and
absence of 0.1% Triton X-100. Table 2illustrates the results.
The activities of all three enzymes were similar in control cells and
in permeabilized cells treated with Triton X-100. These results suggest
that these enzymes are not predominantly found in the cytosol, since
the activities were not increased in control cells containing cytosolic
proteins. Similar levels of all three enzyme activities were measured
in control cells in the presence of Triton X-100 or in the absence in
cells disrupted by homogenization. However, with permeabilized cells in
the absence of Triton X-100, the activities of phosphomevalonate kinase
and mevalonate diphosphate decarboxylase were significantly lower.
These results suggest that the substrates for these two enzymes are not
freely permeable across the organelle membrane, whereas the activity of
mevalonate kinase was the same in permeabilized cell in the presence or
absence of Triton X-100, indicating that the substrate for this enzyme
is freely permeable through the peroxisomal membrane. We were unable to
measure any isopentenyl diphosphate isomerase activity in these cells
(data not shown).
Measurement of Rate of Cholesterol Synthesis in Control
and Permeabilized Cells
If indeed the enzymes required for
conversion of mevalonate to FPP (i.e. mevalonate kinase,
phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and
isopentenyl diphosphate isomerase) are not present in the cytosol, then
permeabilized and control cells would be expected to have similar rates
of cholesterol synthesis, using mevalonate as substrate.
)
We thank A. Sreedhar for providing technical expertise
for the immunofluoresence studies.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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