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.     

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.     

Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Molecular and Cellular Biochemistry - Within the last few years considerable evidence has accumulated which indicates that changes in HMG-CoA reductase are due primarily, if not solely, to changes...     

3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease. Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo, we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells. While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin. Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage.     

Improved assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Two improvements are described for the assay of HMG CoA reductase. These are a simple synthesis of the substrate precursor HMG-3-(14)C anhydride and a double-label ((14)C and (3)H) method for determining the amount of mevalonate-3-(14)C that is formed from the substrate.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

Isoflavones inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase in vitro

Isoflavones identified as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in soybean paste were assayed using the catalytic portion of Syrian hamster HMG-CoA reductase, and the kinetic values were measured using HMG-CoA and NADPH. The inhibition of HMG-CoA reductase by these inhibitors was competitive with HMG-CoA and noncompetitive with NADPH. Ki values for genistein, daidzein, and glycitein were 27.7, 49.5, and 94.7 microM, respectively.     

Genetic and biochemical analysis of the transmembrane domain of Arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase

We have examined the amino terminal membrane anchoring domain of Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmg1p), a key enzyme of the isoprenoid biosynthetic pathway. Using both in vitro and in vivo approaches, we have analyzed a series of recombinant derivatives to identify key structural elements which play a role in defining Hmg1p transmembrane topology. Based on our results, we have proposed a topological model for Hmg1p in which the enzyme spans the lipid bilayer twice. We have shown the two transmembrane segments, designated TMS1 and TMS2, to be structurally and functionally inequivalent in their ability to direct the targeting and orientation of reporter proteins. Furthermore, we provide evidence indicating both the extreme amino terminal end and carboxyl terminal domain of the protein reside in the cytosol. This model therefore provides a key basis for the future examination of the role of the transmembrane domain in the targeting and regulation of Hmg1p in plant cells. J. Cell. Biochem. 65:443–459. © 1997 Wiley-Liss Inc.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

The effect of glucagon on the synthesis and degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Incubation of rat hepatocytes with glucagon results in a time- and dose-dependent decrease in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We demonstrate, using immunoprecipitation of radiolabeled enzyme, that 10 nM glucagon inhibits the synthesis of the enzyme by approximately 50%, but that the apparent rate of degradation of the enzyme is not affected by the hormone. We also demonstrate that the intact reductase polypeptide contained phosphoserine. We conclude that glucagon inhibits the activity of the reductase by inhibition of enzyme synthesis.     

Clinical pharmacology of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

In this article, de novo cholesterol synthesis, its inhibition by HMG-CoA reductase inhibitors (statins) and clinical pharmacology aspects of the statins have been reviewed. Statins are available in both active and pro-drug forms. Their affinity to bind and subsequently to inhibit HMG-CoA reductase activity is approximately 3 orders of magnitude higher than that of natural substrate (HMG-CoA). All members of this group of lipid-lowering agents are, to a varying degree, absorbed from the gut. However, their bioavailability depends on their lipophobicity and their concomitant use with meals. The interaction between HMG-CoA reductase inhibitors and other lipid-lowering agents has been reviewed in more detail. One major side-effect of lipid-lowering combination therapy is myopathy with or without rhabdomyolysis. Combination of statins with gemfibrozil seems to increase risk of this adverse event, particularly in patients with renal impairment, more than combination with other lipid-lowering agents. Combination therapy with other agents including anticoagulants, antihypertensive, anti-inflammatory, oral hypoglycemic and antifungal agents as well as beta-blockers, H2 blockers, cyclosporine and digoxin has been also reviewed. The pleiotropic non-lipid lowering properties of statins and their effects on the quality of lipoprotein particles, the activities of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase as well as their possible synergistic effects with n-3 fatty acids, phytosterols, vitamin E and aspirin in reducing cardiovascular events warrant further investigation.     

Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver

A procedure for the purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP⁺ oxidoreductase (CoA-acylating); EC 1.1.1.34] from rat liver microsomes has been developed. The enzyme preparations obtained by this procedure have specific activities of 16 to 23 μmol of mevalonate formed per minute per milligram of protein. These enzyme preparations were judged to be homogeneous on the basis of comigration of enzyme activity and protein on polyacrylamide gels.     

Microtubules and the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells.