UT-1 Cells Research Articles

Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase: EVIDENCE FOR REQUIREMENT OF A GERANYLGERANYLATED PROTEIN

In mammalian cells, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between β-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR.

Common SNPs in HMGCR in Micronesians and Whites Associated With LDL-Cholesterol Levels Affect Alternative Splicing of Exon13

Background- Variation in LDL-cholesterol (LDL-C) among individuals is a complex genetic trait involving multiple genes and gene-environment interactions. In a genome-wide association study (GWAS) to identify genetic variants influencing LDL-C in an isolated population from Kosrae, we observed associations for SNPs in the gene encoding 3hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR). Three of these SNPs (rs7703051, rs12654264, and rs3846663) met the statistical threshold of genome-wide significance when combined with data from the Diabetes Genetics Initiative GWAS. We followed up the association results and identified a functional SNP in intron13 (rs3846662), which was in linkage disequilibrium with the SNPs of genome-wide significance and affected alternative splicing of HMGCR mRNA. In vitro studies in human lymphoblastoid cells demonstrated that homozygosity for the rs3846662 minor allele was associated with up to 2.2-fold lower expression of alternatively spliced HMGCR mRNA lacking exon13, and minigene transfection assays confirmed that allele status at rs3846662 directly modulated alternative splicing of HMGCR exon13 (42.9+/-3.9 versus 63.7+/-1.0%Deltaexon13/total HMGCR mRNA, P=0.02). Further, the alternative splice variant could not restore HMGCR activity when expressed in HMGCR deficient UT-2 cells. We identified variants in HMGCR that are associated with LDL-C across populations and affect alternative splicing of HMGCR exon13.

Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition.

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.

Role of peroxisomes in isoprenoid biosynthesis.

Our group and others have recently demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biosynthesis that previously were considered to be cytosolic or located in the endoplasmic reticulum (ER). Peroxisomes have been shown to contain HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, and FPP synthase. Four of the five enzymes required for the conversion of mevalonate to FPP contain a conserved putative PTS1 or PTS2, supporting the concept of targeted transport into peroxisomes. To date, no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein, and which is localized exclusively to peroxisomes, to facilitate our studies on the function, regulation, and structure of the peroxisomal HMG-CoA reductase. This cell line was obtained by growing UT2 cells (which lack the ER HMG-CoA reductase) in the absence of mevalonate. The surviving cells exhibited a marked increase in a 90-kD HMG-CoA reductase that was localized exclusively to peroxisomes. The wild-type CHO cells contain two HMG-CoA reductase proteins, the well-characterized 97-kD protein localized in the ER, and a 90-kD protein localized in peroxisomes. We have also identified the mutations in the UT2 cells responsible for the lack of the 97-kD protein. In addition, peroxisomal-deficient Pex2 CHO cell mutants display reduced HMG-CoA reductase levels and have reduced rates of sterol and nonsterol biosynthesis. These data further support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis.

Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells

UT2 cells are a mutant clone of Chinese hamster ovary (CHO) cells that are deficient in the 97 kDa endoplasmic reticulum (ER) 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein. The analysis of UT2 cell cDNA and genomic DNA has led to the identification of two novel point mutations in intronic sequences of the ER HMG-CoA reductase gene. One mutation identified at the +1 position (G → A) of the 5′ splice site of exon 11–12 junction was shown to cause exon 11 skipping which resulted in the insertion of premature stop codons. We also identified a second mutation at the +5 position (G → A) of the 5′ splice site in the intron spanning exons 13 and 14. Furthermore, the data indicate that the two mutations in the reductase gene are present on the same allele. As demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) of UT2 cell mRNA, the mutations produce aberrant spliced messages. If the aberrant messages were translated, truncated proteins of 44 kDa or 66 kDa would be predicted. More importantly, these truncated proteins would be expected not to have catalytic activity. In addition, we have also recently demonstrated that the UT2 cells express a 90 kDa HMG-CoA reductase protein that is localized exclusively in peroxisomes, and is up-regulated when the cells are grown in the absence of added mevalonate. Thus, the mutations identified in the ER reductase gene in UT2 cells indicate that neither a 97 kDa nor a 90 kDa reductase protein can be produced from this gene. —Engfelt, W. H., K. R. Masuda, V. G. Paton, and S. K. Krisans. Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells. J. Lipid. Res. 1998. 39: 2182–2191.

Characterization of UT2 Cells

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [3H]GG-OH is actively incorporated into UT-2 proteins. Prominent [3H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [3H]geranylgeranylated cysteine residues. Even though [3H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.

The Effect of Sterols and Brefeldin A on Protein Degradation in UT-1 Cells

UT-1 cells, a mutant Chinese hamster ovary (CHO) cell line induced to produce an abundance of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), were used to determine the effects of sterols and brefeldin A on the degradation of this enzyme. Brefeldin A has been shown to cause retention of proteins in and relocation of proteins to the endoplasmic reticulum (ER). UT-1 cells were incubated with (a) sterols only (12 μg/ml cholesterol and 0.2 μg/ml 25-hydroxycholesterol), (b) sterols and brefeldin A (0.5 μg/ml), and (c) brefeldin A only. Western blot analysis showed that incubation with sterols and brefeldin A decreased HMGR levels more slowly than incubation with sterols alone over the first 24-36 h of incubation; however, the rates were not significantly different. By 48 h of incubation, HMGR had decreased to a level comparable to that found when cells were incubated in sterols only. Incubation with brefeldin A alone did not cause a decrease in HMGR over the same 48-h time period. HMGR was undetectable in parental CHO cells under all of the conditions described. Indirect immunofluorescence microscopy revealed a pattern of tight, perinuclear staining with sterol incubation. After 48 h in sterols, HMGR staining was uniformly decreased throughout the cytoplasm. This change in staining pattern is also observed during incubation of UT-1 cells with sterols and brefeldin A. Incubation for 48 h with brefeldin A alone had no effect on the tight perinuclear pattern originally observed. Diffuse, faint staining of CHO cells under all conditions served as a negative control. The results of these experiments indicated that brefeldin A, and therefore retention of proteins in the ER, does not interfere with the degradation of HMG CoA reductase. Despite the presence of brefeldin A, sterol-mediated dispersal and degradation of the crystalloid ER (CER) continued in UT-1 cells. Lack of brefeldin A sensitivity implied that the mechanism for CER dissolution was distinct from previously described mechanisms for ER to Golgi transport.

HMG‐CoA reductase mRNA in the post‐implantation rat embryo studied by in situ hybridization

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the rate limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Inhibitors of HMG-CoA reductase are teratogenic in vivo and induce neural tube defects in rat embryo culture, effects which appear unrelated to cholesterol deficiency. This study is the first to localize HMG-CoA reductase mRNA by in situ hybridization (ISH). Expression of reductase mRNA was examined in post-implantation rat embryos, and for control purposes in rat liver and UT-1 cells, using a digoxigenin-11 (dig-11) labelled cRNA probe. Eighteen-day fetal liver showed heavy but patchy hybridization, and adult rat liver showed strong hybridization only on some periportal hepatocytes, which was absent in livers of fasted animals. UT-1 cells stimulated to overexpress HMG-CoA reductase mRNA were strongly positive with the same probe. Control hybridizations with sense strand RNA probe, or with cRNA probe on pre-RNased tissue were negative. Strong hybridization signal for HMG-CoA reductase mRNA was observed in all tissues of the post-implantation rat embryo, from egg cylinder to 30 somite stages (7 to 12 days). Heavy signal was noted in primitive ectoderm and neural tube. The wide embryonic and extraembryonic distribution and abundance of HMG-CoA reductase mRNA may reflect developmental requirements for products of the mevalonate pathway, e.g., isoprenoids for post-translational farnesylation of p21ras.

The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum of UT-1 cells.

We have investigated the role of the smooth endoplasmic reticulum (SER) of UT-1 cells in the biogenesis of the glycoprotein (G) of vesicular stomatitis virus (VSV). Using immunofluorescence microscopy, we observed the wild type G protein in the SER of infected cells. When these cells were infected with the mutant VSV strain ts045, the G protein was unable to reach the Golgi apparatus at 40 degrees C, but was able to exit the rough endoplasmic reticulum (RER) and accumulate in the SER. Ribophorin II, a RER marker, remained excluded from the SER during the viral infection, ruling out the possibility that the infection had destroyed the separate identities of these two organelles. Thus, the mechanism that results in the retention of this mutant glycoprotein in the ER at 39.9 degrees C does not limit its lateral mobility within the ER system. We have also localized GRP78/BiP to the SER of UT-1 cells indicating that other mutant proteins may also have access to this organelle. Upon incubation at 32 degrees C, the mutant G protein was able to leave the SER and move to the Golgi apparatus. To measure how rapidly this transfer occurs, we assayed the conversion of the G protein's N-linked oligosaccharides from endoglycosidase H-sensitive to endoglycosidase H-resistant forms. After a 5-min lag, transport of the G protein followed first order kinetics (t1/2 = 15 min). In contrast, no lag was seen in the transport of G protein that had accumulated in the RER of control UT-1 cells lacking extensive SER. In these cells, the transport of G protein also exhibited first order kinetics (t1/2 = 17 min). Possible implications of this lag are discussed.

Purified crystalloid endoplasmic reticulum from UT-1 cells contains multiple proteins in addition to 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The crystalloid endoplasmic reticulum (ER) of UT-1 cells is a specialized smooth ER that houses 3-hydroxy-3-methylglutaryl-CoA reductase, a membrane protein that regulates endogenous cholesterol synthesis. The biogenesis of this ER is coupled to the over production of 3-hydroxy-3-methylglutaryl-CoA reductase. To understand better this membrane system and the relationship between the synthesis of a membrane protein and the formation of membrane, we have purified the crystalloid ER. Purified crystalloid ER did not contain significant amounts of membrane derived from the Golgi apparatus, mitochondria, or plasma membrane. Approximately 24% of the protein in this organelle corresponded to 3-hydroxy-3-methylglutaryl-CoA reductase; however, at least eight other proteins were detected by gel electrophoresis. One of these proteins (Mr 73,000) was as abundant as reductase. These results suggest that the biogenesis of this ER involves the coordinate synthesis of multiple membrane and content proteins.

Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells.

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA.

We here report the isolation and nucleotide sequencing of a full-length 3.3-kilobase cDNA for the cytoplasmic form of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a regulated enzyme in the cholesterol biosynthetic pathway. The cDNA was isolated from UT-1 cells, a compactin-resistant line of Chinese hamster ovary cells. UT-1 cells produce large amounts of mRNA for HMG-CoA synthase and the next enzyme in the pathway, HMG-CoA reductase, as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. The identity of the cDNA for HMG-CoA synthase was confirmed through comparison of the NH2-terminal amino acid sequence predicted from the cDNA with that determined chemically from the purified enzyme. Anti-peptide antibodies directed against the amino acid sequence predicted from the cDNA precipitated HMG-CoA synthase activity from liver cytoplasm. The feeding of cholesterol to hamsters led to a decrease of more than 85% in the amount of mRNA for HMG-CoA synthase and HMG-CoA reductase in hamster liver. These data indicate that the mRNAs for cytoplasmic HMG-CoA synthase and for HMG-CoA reductase, two sequential enzymes in the cholesterol biosynthetic pathway, are coordinately regulated by cholesterol.

Multiple mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase determined by multiple transcription initiation sites and intron splicing sites in the 5'-untranslated region.

The current studies show that mRNAs with 16 different 5'-untranslated regions (varying in length from 68 to 670 nucleotides) are produced from the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in hamster UT-1 cells. This complex pattern of mRNAs results from a combination of multiple transcription initiation sites and multiple 5' splice donor sites for the intron in the 5'-untranslated region of the gene. Analysis of the multiple mRNAs was made possible by a modification of the S1 nuclease technique in which we used a series of progressively truncated uniformly labeled, single-stranded [32P]DNA probes in addition to the usual end-labeled 32P-probes. All of the reductase mRNAs are diminished when UT-1 cells are incubated with sterols, indicating that all of them are subject to coordinate control.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

HMG CoA reductase: A negatively regulated gene with unusual promoter and 5′ untranslated regions

The rate-limiting enzyme of cholesterol biosynthesis, HMG CoA reductase, is controlled by negative feedback regulation of transcription. We have isolated the reductase gene from a bacteriophage λ genomic library prepared from hamster UT-1 cells. The 25 kilobase gene is split into 20 exons. The 5′ untranslated and promoter regions differ from those of previously characterized genes. The 5′ untranslated region encompasses as many as 670 nucleotides; contains up to eight AUG codons upstream of the codon used to initiate translation; and has multiple transcription initiation sites as determined by S1 nuclease mapping and primer extension analysis. The promoter region lacks a characteristic TATA box and CCAAT box; is rich in G+C residues (65%); and contains repeat sequences homologous to the 21 base pair repeats of the SV40 promoter. These unusual features may be relevant to the mechanism of expression of “housekeeping” genes, particularly those that are subject to negative feedback regulation.

Increase in membrane cholesterol: a possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells.

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.

3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides.

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) is an abundant protein of the crystalloid endoplasmic reticulum of UT-1 cells, a line of cultured hamster cells that over-produces the reductase as a result of gene amplification. In the current studies, we show that reductase in UT-1 cells is a glycoprotein. The solubilized enzyme (Mr = 97,000) from UT-1 cells, Chinese hamster ovary cells, and rat liver was adsorbed quantitatively and specifically to concanavalin A-Sepharose. UT-1 cells incorporated [1,6-3H]glucosamine into the reductase; after release with endo-N-acetylglucosaminidase H most of the radioactivity was found in N-linked "high-mannose" chains, including Man6(GlcNAc)2, Man7(GlcNAc)2, and Man8(GlcNAc)2. The carbohydrate of the reductase was localized to a 30- to 35-kilodalton fragment that was separable proteolytically from a cytoplasmic 53-kilodalton fragment that contained the active site of the enzyme. We conclude that 3-hydroxy-3-methylglutaryl-CoA reductase is a transmembrane glycoprotein with an active site facing the cytoplasm and a carbohydrate-bearing site oriented toward the lumen of the endoplasmic reticulum.