©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Dissection of the Role of the Membrane Domain in the Regulated Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (*)

(Received for publication, April 27, 1995)

Hidetoshi Kumagai Kristin T. Chun (§) Robert D. Simoni (¶)

From theDepartment of Biological Sciences, Stanford University, Stanford, California 94305-5020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-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.


INTRODUCTION

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) (^1)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 beta-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 beta-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.


EXPERIMENTAL PROCEDURES

Materials

All materials, unless specified, were obtained from commercial sources. 25-Hydroxycholesterol, DL-mevalonolactone, and 5-bromo-4-chloro-3-indoyl-beta-galactoside (X-Gal) were purchased from Sigma. Compactin was a genenous gift of A. Endo, Tokyo Noko University, Tokyo. Acetyl-leucyl-leucyl-norleucinal, (ALLN, calpain inhibitor I) was from Calbiochem-Novabiochem Corp. Minimum essential medium (MEM) without methionine and cysteine, and TranS-label® metabolic labeling reagent (specific activity >1000 Ci/mmol) were from ICN Biochemicals Inc. Anti-beta-galactosidase monoclonal antibody was purchased from Promega Corp. Anti-HMG-CoA reductase polyclonal antibody against synthetic peptides in the membrane domain of hamster HMG-CoA reductase was used(17) . Protein A-Sepharose CL-4B was purchased from Pharmacia Biotech Inc. Oligonucleotides were synthesized using an Applied Biosystems 3806 DNA synthesizer. Plasmid pZ3 containing a cDNA insert encoding sea urchin HMG-CoA reductase was a gift from H. Woodward and W. Lennarz(12) . Lipofectin® was obtained from Life Technologies, Inc. Polyester-PeCap HD7-17 membranes were purchased from Tetko Inc, and glass beads were from Baxter Diagnostics Inc.

Cell Culture

Chinese hamster ovary (CHO-K1) cells transfected with the plasmid pSV2-HMGal (5) were routinely grown as monolayers in MEM supplemented with 0.25 mg/ml active Geneticin® (G418) and either 5% fetal calf serum or 5% lipid-poor serum which was prepared by the method of Rothblat et al.(18) . In this study cells were grown for 24 h in medium supplemented with 5% lipid poor serum, 1 µM compactin, and 0.1 mM mevalonate before the experiments. This pretreatment was sufficient to maximize beta-galactosidase activity of HMGal and increase the protein amounts of HMGal and HMG-CoA reductase about 4-5-fold(19) .

beta-Galactosidase Assay

beta-Galactosidase activity was measured as described previously(5) . Protein concentrations were determined by the method of Lowry et al.(20) . Specific activity was expressed as OD420 nm/(h mg of protein).

Determination of Half-lives of Chimeric HMGal Mutants

The half-lives of HMGal and each chimeric HMGal mutant were measured as described previously(19) .

Construction of Hamster/Sea Urchin Chimeric Genes

The construction of the chimeric HMGal mutants is listed in Table1. For construction of those mutants, single-stranded DNAs were prepared from pEMBL9 (21) containing the cDNAs of either truncated hamster or sea urchin HMG-CoA reductase and used for oligonucleotide-directed mutagenesis (Amersham Corp.). For each chimeric mutant, oligonucleotide-directed mutagenesis was performed to create novel restriction endonuclease sites, which were utilized for the initial screening of mutant cDNAs, in the gene near the regions which encode the putative ends of a membrane-spanning domain of HMG-CoA reductase gene. To avoid extra mutations except the protein sequences from sea urchin enzyme, the unique restriction endonuclease sites were carefully designed by using silent mutations in the hHMGal. The identity of each mutant was confirmed by dideoxy sequencing of single- and double-stranded DNA with Sequenase® (United States Biochemicals Corp). After substitution of cDNA fragments encoding putative membrane-spanning domains between hamster and sea urchin enzymes, those chimeric hamster/sea urchin HMGal cDNAs were subcloned into the expression vector pcDLSRalpha-296 (22) for expression studies as described(5, 23) .



Transfection and Expression of Fusion Genes in CHO-K1 Cells

Plasmids encoding the fusion genes were co-transfected along with pSV2neo (24) into CHO cells. Transfection was conducted by Lipofectin® reagent according to the manufacturer's protocol. 10 µg of pcDL-SRalpha296 containing the cDNA of different HMGal species and 0.5 µg of pSV2neo were co-transfected into subconfluent monolayers of CHO cells. Transfectants were selected with 0.5-0.75 mg/ml of active G418, and resistant colonies with the highest beta-galactosidase activity were isolated using the replica plating procedure as described below. These selected cells were maintained in MEM supplemented with 5% fetal calf serum and 0.25 mg/ml active G418.

Isolation of Cells Expressing Elevated Levels of beta-Galactosidase

Although we attempted to optimize the transfection conditions, the resulting populations of transfected cells usually did not express sufficient fusion proteins to characterize the phenotype. We therefore had to isolate clones with elevated expression.

We used a replica plate procedure that allowed us to screen for the cells whose beta-galactosidase activity is high and, at the same time, preserve a ``replica'' of those colonies. G418-resistant colonies were screened for highest beta-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 beta-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 beta-galactosidase, and incubated for several hours at 37 °C. After staining with X-Gal, blue colonies were isolated from the master dish.


RESULTS

The Degradation of uHMGal Is Not Regulated

Woodward et al. (12) have shown that sea urchin HMG-CoA reductase activity was expressed during embryonic development, and its levels were not influenced by mevalonate and sterols, unlike mammalian HMG-CoA reductase, suggesting that the sea urchin HMG-CoA reductase is not subject to regulated degradation in sea urchin embryos. To determine if sea urchin HMG-CoA reductase is subject to regulated degradation, we examined the phenotype of uHMGal expressed in a heterologous system, CHO cells, and compared the results to those obtained with hHMGal. Steady-state levels of hHMGal and uHMGal were determined by measuring beta-galactosidase activity. For each cell line expressing either hHMGal or uHMGal, the beta-galactosidase-specific activity was measured in cells growing in the presence of 2.5 µM 25-hydroxycholesterol and compared to the activity measured in untreated cells. The difference between the amount of activity in treated and untreated cells mirrors the difference between the half-lives of the fusion protein under the different conditions(19) . As seen in Fig.2A, the beta-galactosidase activity of hHMGal was reduced to about 30-40% of control levels upon treatment with 25-hydroxycholesterol. In contrast, steady-state levels of the uHMGal were insensitive to 25-hydroxycholesterol treatment, suggesting that the sea urchin enzyme is not subject to regulated degradation.


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 beta-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 TranS-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.

The Degradation of Both Hamster and Sea Urchin HMGals Occurs in the Same Cellular Component

Since the degradation of uHMGal is relatively rapid, we wanted to determine that it was being degraded by the normal cellular machinery responsible for regulated degradation of the hamster protein or whether the protein was degraded by a non-physiological mechanism. We have previously shown that ALLN, a cysteine protease inhibitor, strongly blocks both the basal and the accelerated degradation of hamster HMG-CoA reductase and hHMGal(26) . Cells expressing uHMGal were subjected to pulse-chase analysis without or with 2.5 µM 25-hydroxycholesterol in the absence or presence of 0.2 mM ALLN. As can be seen in Fig.3, in both cases, with or without 25-hydroxycholesterol, ALLN blocked uHMGal turnover. These results are comparable to those obtained previously with hHMGal(26) .


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 beta-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.

The N-Terminal Half of the Membrane Domain of Hamster HMG-CoA Reductase Confers the Regulated Phenotype onto Sea Urchin HMG-CoA Reductase

It is somewhat surprising that the degradation of uHMGal is not regulated because, based on the amino acid sequence comparison, the membrane domain of sea urchin HMG-CoA reductase shares 62% amino acid sequence identity and 75% similarity with that of hamster reductase. The sea urchin HMG-CoA reductase can thus serve as a natural source of mutations for studying the membrane domain of the hamster enzyme. Using an approach where a specific domain of one protein is replaced with the homologous domain of another protein, we constructed chimeric hamster/sea urchin HMGal mutants to study which regions are essential for regulated degradation. We have previously shown using chimeras of hHMGal and bacteriorhodopsin that certain subregions of the hamster HMG-CoA reductase membrane domain are necessary for regulated degradation. This work suggested that the C-terminal half of the membrane domain which includes the region of membrane spans 5 through 8 (numbering is from the N terminus) plays a prominent role in regulated degradation based on membrane span replacement studies. When the fifth or sixth membrane spans is substituted with the first membrane span of bacteriorhodopsin, the mutant forms of the HMGal lost both sterol- and non-sterol-accelerated degradation. When the seventh and eighth membrane spans were replaced, HMGal stability is dramatically changed(23) . These results, however, represent the consequences of gross changes in the structure of HMG-CoA reductase.

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 beta-galactosidase activities are shown in Fig.4. In the chimeric mutant 5-8 uHMGal, beta-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 beta-galactosidase activity observed in steady state experiments. On the other hand, the steady-state beta-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 beta-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.

The Second Membrane Spanning Domain Is Important for the Regulated Degradation of HMG-CoA Reductase

Having shown that membrane spans 1 through 4 of the hamster enzyme confer regulated degradation onto the unregulated sea urchin enzyme, we attempted to further define the critical regions by constructing additional chimeras and characterizing their degradation phenotypes (Fig.5). In the first group of chimeric mutants, where the first two membrane-spanning domains of sea urchin enzyme were replaced with the corresponding regions of the hamster enzyme (3-8 uHMGal), steady-state beta-galactosidase activity was decreased by 25-hydroxycholesterol, similar to the decrease in hHMGal activity by 25-hydroxycholesterol. However, the replacement of the first membrane-spanning domain (2-8 uHMGal) was not sufficient to confer the regulated phenotype to the sea urchin enzyme based on the steady-state measurements.


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 beta-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. beta-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 beta-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 beta-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 beta-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 beta-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 beta-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.


DISCUSSION

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 beta-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.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant HL26502 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202-5122.

To whom correspondence should be addressed: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Tel.: 415-725-4817; Fax: 415-725-5807.

^1
The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; ALLN, acetyl-leucyl-leucyl-norleucinal; X-Gal, 5-bromo-4-chloro-3-indoyl-beta-galactoside; HMGal, a fusion protein consisting of the membrane domain and most of the linker domain of Syrian hamster HMG-CoA reductase fused to Escherichia coli beta-galactosidase; MEM, minimum essential medium; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We are grateful to Kentaro Hanada for helpful advice and discussions during the course of this work. We also thank the members of the Simoni laboratory for critically reviewing this manuscript.


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