(Received for publication, April 27, 1995)
From the
We have previously shown that the membrane domain of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from hamster
contains all of the sequences required for both localization to the
endoplasmic reticulum and regulated degradation of the enzyme. It has
been reported that the enzymatic activity and mRNA levels of HMG-CoA
reductase from sea urchin embryos cultured in the presence of
regulators were unchanged compared to levels in control embryos
(Woodward, H. D., Allen, M. C., and Lennarz, W. J.(1988) J. Biol.
Chem. 263, 18411-18418). This observation led us to
investigate the possibility that the sea urchin enzyme is not subject
to regulated protein turnover. Interestingly, the sea urchin enzyme
shares 62% amino acid sequence identity with the hamster enzyme in the
membrane domain and shares similar predicted topological features. In
the current studies we have compared the degradation phenotypes of the
sea urchin HMG-CoA reductase and the hamster HMG-CoA reductase in
Chinese hamster ovary cells to further elucidate the role of the
membrane domain in enzyme degradation in response to physiological
regulators. To accomplish this, we constructed sea urchin HMGal
(uHMGal), the structural equivalent of hamster HMGal (httMGal), which
has the sea urchin HMG-CoA reductase membrane domain fused to Escherichia coli -galactosidase. The uHMGal was stably
expressed in CHO cells, and we found that the degradation of uHMGal is
not accelerated by sterols, and even in the absence of sterols, it is
less stable than hHMGal. We also constructed chimeric hamster/sea
urchin HMGal molecules to investigate which amino acid sequences from
the hamster enzyme are sufficient to confer sterol-regulated
degradation upon the sea urchin enzyme. Our results identify the second
membrane-spanning domain of hamster enzyme as important for the
regulated degradation of HMG-CoA reductase.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) ()reductase catalyzes the production of mevalonate, a key
intermediate in the synthesis of sterols and non-sterol isoprenoid
metabolites important for many cellular functions. When cells are
starved for mevalonate-derived metabolites, the amount of HMG-CoA
reductase is elevated about 200-fold over the basal level. This is
achieved by a combination of enhanced transcription of the gene,
enhanced translation of the mRNA, and reduced degradation of the
enzyme(1) . The increase in enzyme levels can be reversed
within a few hours by adding mevalonate or sterols to the culture
medium(1) . Much attention has been focused on elucidating the
mechanisms of these regulated processes, including efforts to
understand the degradation process by which levels of HMG-CoA reductase
are regulated in response to the concentration of products in the
cholesterol biosynthetic pathway.
Mammalian HMG-CoA reductase is an integral membrane protein of the endoplasmic reticulum (ER). The structure of HMG-CoA reductase can be divided into two domains. The C-terminal two-thirds of the polypeptide chain extends into cytosol of the cell and contains the catalytic activity. The N-terminal one-third of the polypeptide chain, not required for catalytic activity, contains alternating hydrophobic and hydrophilic segments that traverse the ER membrane eight times(2) . When the sequences encoding the membrane domain are deleted from a cDNA, and the resulting shortened cDNA expressed in the Chinese hamster ovary (CHO) cells, the truncated reductase, which is catalytically active, is found in the cytosol and is no longer subject to regulated degradation in the presence of mevalonate and sterols(3) . Deletion of two membrane-spanning sequences in the middle of the membrane domain abolishes sterol- and nonsterol-accelerated turnover of the enzyme as well(4) .
In
another case, HMGal, a mutant form of hamster HMG-CoA reductase, in
which the entire catalytic domain is replaced with E. coli -galactosidase, still displays rapid degradation in response
to the regulatory molecules, mevalonate, and sterols(5) . These
observations demonstrate that the membrane domain of reductase contains
all of the information required for not only ER localization but also
regulated degradation of the enzyme.
The primary structures of HMG-CoA reductase have been deduced from cDNA sequences from many eukaryotes including yeast(6) , schistosomes(7) , plants(8, 9) , insects(10, 11) , sea urchin(12) , frog(13) , and mammals(14, 15) . Knowledge of the amino acid sequence of several HMG-CoA reductases has substantially aided the investigation of the structural basis for the regulated degradation.
It has been reported that the activity and mRNA level of HMG-CoA reductase from sea urchin embryos cultured in the presence of mevalonate or sterols were unchanged as compared to control embryos (12) . This observation led us to investigate the possibility that sea urchin enzyme is not subject to regulation, including protein turnover. Interestingly, the sea urchin enzyme shares 62% amino acid sequence identity and 75% similarity with the hamster enzyme in the membrane domain and shares similar predicted topological features (Fig.1). Therefore, sea urchin HMG-CoA reductase serves as a natural source of mutations for study of the membrane domain of the hamster enzyme.
Figure 1: Model of the topological structure of the HMGals. For the model of the membrane domain of HMGals, amino acid residues are shown in the single-letter code, and each rectangle represents a putative membrane span. In the uHMGal sequence, amino acid residues identical to hHMGal are indicated by open circles, conserved amino acids by gray circles, and nonconserved amino acids by solid circles.
In current studies we have compared the degradation
phenotypes of the sea urchin HMG-CoA reductase and the hamster HMG-CoA
reductase both expressed in CHO cells, to elucidate further the role of
the membrane domain in enzyme degradation in response to physiological
regulators. To accomplish this, we constructed sea urchin HMGal
(uHMGal) the structural equivalent of hamster HMGal (hHMGal), which has
the sea urchin HMG-CoA reductase membrane domain connected to E.
coli -galactosidase. This uHMGal was stably expressed in CHO
cells, and we measured half-lives in the absence or presence of
sterols. We found that the degradation of uHMGal is not accelerated by
sterols, and even in the absence of sterols, it is less stable than
hHMGal. We also constructed chimeric hamster/sea urchin HMGal mutants
to investigate which amino acid sequences from the hamster enzyme are
sufficient to confer sterol-regulated degradation to the sea urchin
enzyme. Our results identify the second membrane-spanning domain of
hamster enzyme as important for the regulated degradation of HMG-CoA
reductase.
We used a replica plate procedure that allowed us to screen for the
cells whose -galactosidase activity is high and, at the same time,
preserve a ``replica'' of those colonies. G418-resistant
colonies were screened for highest
-galactosidase activity by
using the replica plating procedure described by Esko and Raetz (25) with modifications. Briefly, 150-200 colonies/100
15-mm tissue culture dish were overlaid with a polyester-PeCap
HD7-17 filter membrane, a paper filter, and a layer of glass
beads. The cells were allowed to grow through the filter membrane for
7-10 days before they were screened for
-galactosidase
activity. The filter was carefully removed from the master dish and
washed with phosphate-buffered saline (PBS) briefly. The medium in the
master dish was replaced with fresh medium, and the cells were
maintained by further incubation at 37 °C. The filter was overlaid
on a paper filter (Whatman, no. 42) previously soaked with PBS
containing X-Gal (1 mg/ml), which is a membrane-permeable substrate for
-galactosidase, and incubated for several hours at 37 °C.
After staining with X-Gal, blue colonies were isolated from the master
dish.
Figure 2:
The uHMGal degradation is not regulated.
CHO cells expressing either hHMGal or uHMGal were grown in 24-well
plates with MEM supplemented with 5% lipid poor serum, 0.25 mg/ml G418,
1 µM compactin, and 0.1 mM mevalonate (panel
A). 2.5 µM 25-hydroxycholesterol was added (gray
bars) for an additional 20-h period. The specific
-galactosidase activity is presented as mean values of triplicate
assays. These values were normalized 100% activity of control cells
that received no addition (open bars). The error bars represent the standard error of the mean, which did not exceed 6%
for any of the values displayed. In panel B, cells expressing
either hHMGal (circles) or uHMGal (triangles) were
metabolically labeled for 0.5 h with Tran
S-label®,
and the cells were either collected (t = 0) or chased
in the absence (open symbols) or presence of 2.5 µM 25-hydroxycholesterol (closed symbols). At the indicated
times, lysates were prepared, and each fusion protein was
immunoprecipitated from the lysates and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis followed by fluorography.
The fluorograms were quantified using a Molecular Imager
(Bio-Rad).
Using a pulse-chase protocol, we determined the half-lives of these two fusion proteins to confirm that the half-life of uHMGal is not regulated by 25-hydroxycholesterol. As shown in Fig.2B, with the hHMGal, 25-hydroxycholesterol accelerates the degradation of the hHMGal protein, reducing the half-life from about 4.6 to 2.0 h. 25-Hydroxycholesterol did not reduce the half-life of uHMGal, which is shorter than the half-life of hHMGal.
These results reveal that the sea urchin HMG-CoA reductase is not subject to regulated degradation, when expressed in the CHO cells.
Figure 3:
ALLN blocks uttMGal degradation. Cells
expressing uHMGal were metabolically labeled for 0.5 h with
TranS-label® and chased for 4 h without or with 2.5
µM 25-hydroxycholesterol in the absence or presence of 0.2
mM ALLN. At the indicated time, lysates were prepared, and
uHMGal was immunoprecipitated from the lysates and analyzed by
SDS-polyacrylamide gel electrophoresis followed by
fluorography.
In order to determine the localization of
uHMGal, we stained the CHO cells expressing the uHMGal with X-Gal as a
substrate as described previously(23) . The in situ -galactosidase activity staining pattern of uHMGal is the
same as that of hHMGal (data not shown), indicating that the uHMGal is
properly localized to the ER.
Since the uHMGal is structurally quite similar to hHMGal, localized correctly and the degradation is sensitive to ALLN, it is likely that the same cellular components are responsible for the degradation of both the hHMGal and uHMGal in the CHO cells.
In order to more finely determine what primary
structures within the hamster HMG-CoA reductase are critical in
conferring the regulated phenotype, we first constructed two chimeric
HMGal mutants in which each half of the membrane domain from hamster
enzyme was swapped with the homologous domain from sea urchin enzyme.
The amino acid sequence comparison shows that the C-terminal half of
the membrane domain (amino acid residues 161-338) of the sea
urchin enzyme, containing membrane spans 5 through 8, shares
significantly less homology with the C-terminal half of hamster HMG-CoA
reductase (Fig.1). We have designated the hamster/sea urchin
chimera 5-8 uHMGal as a mutant in which membrane spans 1 through
4 of the hamster enzyme were fused to membrane spans 5 through 8 of the
sea urchin membrane domain (Table1). In 1-4 uHMGal the
opposite construct of the 5-8 uHMGal membrane spans 1 through 4
are from sea urchin enzyme while 5 through 8 are from hamster enzyme.
The effects of 25-hydroxycholesterol on the steady-state levels of
-galactosidase activities are shown in Fig.4. In the
chimeric mutant 5-8 uHMGal,
-galactosidase activity was
reduced by 25-hydroxycholesterol to similar levels as observed with
hHMGal. Half-lives of the 5-8 uHMGal were also determined either
in the absence or presence of 25-hydroxycholesterol (Table2).
The basal half-life was about 5.2 h which is similar to that of hHMGal.
When cells were incubated with 25-hydroxycholesterol, the half-life was
reduced to about 3.5 h. This 30% reduction of the half-life can
approximately explain the loss of
-galactosidase activity observed
in steady state experiments. On the other hand, the steady-state
-galactosidase activity of 1-4 uHMGal was quite reproducibly
increased to about 140% by the addition of 25-hydroxycholesterol rather
than decreased. When the half-life of 1-4 uHMGal was determined
it was found to be about 0.42 h which is extremely short. We did not
observe acceleration of the degradation in the presence of
25-hydroxycholesterol (Table2). This phenotype is similar to
that of the uHMGal, but it is important to note that the half-life of
uHMGal has the potential to be accelerated since 1-4 uHMGal has a
substantially shorter half-life.
Figure 4:
The effect of 25-hydroxycholesterol on
hHMGal, uHMGal, 1-4 uHMGal, and 5-8 uHMGal. Cells
expressing hHMGal, uHMGal, 1-4 uHMGal, and 5-8 uHMGal were
grown and treated with 2.5 µM 25-hydroxycholesterol for 20
h as described in Fig.1. and under ``Experimental
Procedures.'' The specific -galactosidase activity was
measured and calculated as described in Fig.1. The error
bars represent the standard error of the mean, which did not
exceed 8% for any of the values displayed.
Together, these results indicate that the N-terminal half of the membrane domain of the hamster HMG-CoA reductase can confer regulation to the sea urchin enzyme. Amino acid differences in the N-terminal half of the membrane domain of the sea urchin enzyme appear to be responsible both for the lability of the enzyme and the lack of regulation.
Figure 5:
The effect of 25-hydroxycholesterol on
hHMGal, 3-8 uHMGal, 2-8 uHMGal, and 1,3-8 uHMGal.
Cells expressing hHMGal, 3-8 uHMGal, 2-8 uHMGal, and
1,3-8 uHMGal were grown and treated with 2.5 µM
25-hydroxycholesterol for 20 h as described in Fig.1. and under
``Experimental Procedures.'' The specific -galactosidase
activity was measured and calculated as described in Fig.1. The error bars represent the standard error of the mean, which did
not exceed 10% for any of the values
displayed.
These results indicated that the sequences responsible for the
regulated phenotype are located within membrane spans 1 and 2 and
probably in membrane span 2. To determine whether the critical
sequences for the regulatory response are located solely in membrane
span 2 or in both membrane spans 1 and 2, we also created a chimeric
mutant, 1,3-8 uHMGal, in which only the second membrane-spanning
domain of the sea urchin enzyme was replaced by the corresponding
region of the hamster enzyme. -Galactosidase activity of
1,3-8 uHMGal, like that of the hamster enzyme, was decreased to
about 50% by 25-hydroxycholesterol, suggesting that the second
membrane-spanning domain alone is sufficient to confer the regulated
phenotype onto the non-regulated sea urchin enzyme.
Phenotypes of
the next group of chimeric mutants support the results described above.
In the second group of mutants, which were reciprocal constructs
complementary to the first group of mutants, portions of the sea urchin
membrane domain were substituted into the hamster enzyme to determine
whether sequences from the sea urchin enzyme could abolish the
regulated phenotype of hHMGal. As shown in Fig.6, substituting
the first membrane-spanning domain of the hamster enzyme with that from
the sea urchin enzyme (1 uHMGal) did not affect the regulated
phenotype. However, when both membrane spans 1 and 2 (1-2
uHMGal), or membrane span 2 (2 uHMGal) from the sea urchin enzyme, were
introduced into the hamster HMGal 25-hydroxycholesterol had essentially
no effect on steady-state -galactosidase activity.
Figure 6:
The effect of 25-hydroxycholesterol on
hHMGal, 1 uHMGal, 1-2 uHMGal, and 2 uHMGal. Cells expressing
hHMGal, 1 uHMGal, 1-2 uHMGal, and 2 uHMGal were grown and treated
with 2.5 µM 25-hydroxycholesterol for 20 h as described in Fig.1. and under ``Experimental Procedures.'' The
specific -galactosidase activity was measured and calculated as
described in Fig.1. The error bars represent the
standard error of the mean, which did not exceed 11% for any of the
values displayed.
Half-lives of these chimeric mutant proteins were determined using the pulse-chase protocol. The data are summarized in Table2. As shown in Table 2, half-life data reflect the steady-state phenotype of the chimeric mutants with the exception of 3-8 uHMGal. However, when second membrane-spaning domain of hamster enzyme is introduced into the sea urchin enzyme, its basal degradation rate is about 3.5 h, substantially longer than uHMGal and is accelerated by 25-hydroxysterol. This result reflects the steady-state experiment. Replacing the first membrane-spanning domain did not cause the regulated phenotype of the 2-8 uHMGal mutant. This result is also consistent with the steady-state experiment. Unexpectedly, in the case of 3-8 uHMGal, the basal half-life of the chimeric HMGal was significantly shorter compared to that of hamster enzyme, and its half-life was not accelerated by 25-hydroxycholesterol. These results do not agree with the activity measurements described above. The basal half-life of the mutant is probably too short to be accurately measured using our pulse-chase protocol. For the activity measurements, the 20-h cumulative effect of any difference in half-life may cause the difference in steady-state levels.
The degradation rate of the 1
uHMGal mutant, which shows regulated steady-state -galactosidase
activity, is accelerated by 25-hydroxycholesterol reducing its
half-life from 2.5 to 1.1 h. This chimeric fusion protein is
significantly less stable than the hamster HMGal, but the degradation
of this mutant protein and the response of its
-galactosidase
activity to 25-hydroxycholestrol are essentially similar to that of the
hamster enzyme, suggesting that the mutations within the first
membrane-spanning domain do not seem to be critical for the regulated
degradation of HMG-CoA reductase. In the case of the 1-2 uHMGal
mutant, the basal half-life was quite short, about 1.6 h, and this
chimeric mutant lost the regulated degradation by 25-hydroxycholesterol
as seen in steady-state
-galactosidase measurements. On the other
hand, the membrane span 2 (2 uHMGal mutant) replacement alone which
also resulted in a non-regulated mutant form of hHMGal in the
steady-state measurements has a relatively longer basal half-life, 7.0
h, compared to that of hamster enzyme, but 25-hydroxycholesterol failed
to enhance the rate of degradation in this mutant.
These results suggest that the sequences within the first membrane-spanning domain are responsible for lability of the enzyme and that the amino acid differences in membrane span 2 between hamster and sea urchin enzymes are involved in the regulatory response.
Together, these results strongly suggest that membrane span 2 of hamster HMG-CoA reductase plays an important role in regulated degradation.
The current results show that membrane span 2 of the hamster enzyme is important for regulated degradation. Olender and Simoni (2) have shown that the first membrane-spanning domain functions as a signal sequence to direct enzyme localization to the ER. Another question that arose was whether the first and second membrane spanning regions alone were sufficient to confer the regulated phenotype to the enzyme. In order to answer this question, we constructed a deletion mutant where the region encoding membrane spans 3 through 8 was deleted. When this mutant protein was expressed in CHO cells it was rapidly degraded, and we could not observe regulated degradation phenotype using either steady-state measurements and pulse-chase experiments (data not shown). These results suggested that the whole multiple membrane-spanning domain is required for regulated degradation and that the degradation system may recognize the tertiary structure of the membrane domain of the enzyme.
The ER membrane protein HMG-CoA reductase contains several
stretches of hydrophobic residues in its N-terminal domain which
topologically are highly conserved between species. The multiple
membrane-spanning domain of HMG-CoA reductase has been shown to be both
necessary and sufficient to confer sterol- and non-sterol-accelerated
degradation upon the enzyme(3, 5) . Our focus on
studying the role of the hydrophobic membrane domain in regulated
degradation is based on the hypothesis that pathway regulatory
molecules like sterols, which are themselves hydrophobic, might
interact directly with HMG-CoA reductase via the hydrophobic regions of
the membrane domain. To test this hypothesis, we have conducted a
systematic mutagenesis study of the membrane domain of hamster HMG-CoA
reductase in an attempt to localize the subregion(s) within the
membrane domain which are required for regulated degradation. In
previous studies we have replaced, individually, all eight predicted
membrane spans of hamster HMG-CoA reductase with the first membrane
span from bacteriorhodopsin(23) . The phenotypes of these
replacement mutants indicated that several structural components of the
membrane domain are required for the regulated degradation of HMG-CoA
reductase. These results also indicated that the entire membrane domain
of HMG-CoA reductase is not required for the protein's regulated
degradation and that it should be possible to identify components of
the membrane domain which are necessary for regulated degradation. In
order to more precisely define the subregions or residues essential for
enhanced degradation in response to regulatory molecules, we first
tested which individual subregions were necessary and are sufficient to
confer regulation. To accomplish this, we introduced the subregions of
interest into a heterologous protein and tested the degradation
phenotypes. The choice of a heterologous protein is difficult in the
case of a multiple membrane-spanning protein. We chose HMG-CoA
reductase from another species, sea urchin, based on reports that the
activity of this enzyme is non-responsive to mevalonate and sterols
when tested in early sea urchin embryos(12) . We examined the
membrane domain of sea urchin HMG-CoA reductase using a
-galactosidase fusion protein. When we tested this fusion protein
for regulated degradation in transfected CHO cells, the results, shown
in Fig.2, indicated that the degradation of uHMGal is not
regulated by exogenous sterols even though uHMGal is properly localized
to the ER in this heterologous system. Therefore, we can use the sea
urchin enzyme to serve as a source of sequences for identifying
subregions from hamster enzyme that confer regulated degradation. To
define more precisely the structural features of membrane domain
responsible for regulated degradation, we constructed hamster/sea
urchin chimeric mutants and tested their phenotypes in CHO cells. When
membrane span 2 of hamster and sea urchin enzymes were exchanged, their
degradation phenotypes were also exchanged. 2 uHMGal is the mutant
where membrane span 2 of the regulated hamster enzyme was replaced with
the corresponding region of the non-regulated sea urchin enzyme. The
half-life of 2 uHMGal is not accelerated by exogenous sterols,
suggesting that membrane span 2 may be involved in sterol signaling.
The reciprocal mutant, 1,3-8 uHMGal where membrane span 2 of the
hamster enzyme is introduced into the sea urchin enzyme results in a
regulated phenotype. These results clearly show that the membrane span
2 of the hamster enzyme is important for the regulated degradation of
HMG-CoA reductase. The amino acid sequence in this region shows that
membrane span 2 from the sea urchin enzyme has relatively less identity
(46%) with that of the hamster enzyme than in other membrane spans,
however the membrane span 2 is still highly homologous, with 75%
similarity (Fig.1). One can speculate that sterols accelerate
HMG-CoA reductase turnover by a direct interaction with the second
membrane-spanning domain. To test this hypothesis, further dissection
of the second membrane-spanning domain is required.