Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Impaired regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation in lovastatin-resistant cells.

L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.

Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells.

To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.

Neuronal ceroid lipofuscinosis (nclf), a new disorder of the mouse linked to chromosome 9.

The neuronal ceroid lipofuscinoses (NCLs) comprise a set of at least 6 distinct human and an unknown number of animal diseases characterized by storage of proteolipids in lysosomes of many cell types. By unknown mechanisms, this accumulation leads to or is associated with severe neuronal and retinal degeneration. The genes for 3 human NCLs, infantile, late infantile, and juvenile, have been cloned. The first murine form of NCL, the motor neuron degeneration (mnd) mouse, has been described and mapped to proximal Chromosome 8. Here we describe a second genetic variant of NCL in the mouse, neuronal ceroid lipofuscinosis, nclf. These mice exhibited a phenotype that was almost exactly the same as that observed in mnd/mnd mice. Homozygous nclf mice developed progressive retinal atrophy early in life and become paralyzed at around 9 months of age. They accumulated luxol fast blue staining material in cytoplasm of neurons and many other cell types. Ultrastructurally, affected lysosomes had a "finger print pattern" with membranous material arranged in "pentalaminar" patterns. Affected mice developed severe cerebral gliosis in late stages of their disease. They also had severe Wallerian degeneration of long tracts in spinal cord and brain stem, lesions that accounted for the distinctive upper motor neuron signs displayed by both nclf/nclf and mnd/mnd mice. By crossing nclf/nclf mice with CAST/Ei mice, linkage analysis of nclf with respect to SSLP markers was performed, showing that nclf is located on Chromosome 9 between D9Mit164 and D9Mit165, in a region that is homologous with human Ch 15q21, where the gene for one variant of late infantile NCL, CLN6, recently has been mapped. The genes for two proteolipids known to be stored in lysosomes of animals and people with NCL were also mapped in this study and found not to map to the mnd or nclf loci nor to any mouse locus homologous to any known human NCL disease locus.

Abnormalities in mitochondria-associated membranes and phospholipid biosynthetic enzymes in the mnd/mnd mouse model of neuronal ceroid lipofuscinosis.

Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248-7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190-11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes-CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase-were also reduced by > 50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b5, NADH:cytochrome b5 reductase and mitochondrial F1Fzero-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.

Compartmentation of cholesterol within the cell.

Mammalian cells tightly regulate their cholesterol content and the intracellular disposition of cholesterol. Most cellular free cholesterol resides in the plasma membrane, where it exists in lateral domains. Mechanisms governing cellular cholesterol levels and compartmentation are still largely unknown. In this review, we highlight recent studies documenting the compartmentation of cholesterol, especially those that have relevance to the regulation of cholesterol content.

Two related proteolipids and dolichol-linked oligosaccharides accumulate in motor neuron degeneration mice (mnd/mnd), a model for neuronal ceroid lipofuscinosis.

In this study, we show that two biochemical markers of neuronal ceroid lipofuscinoses (NCLs) are present in a mutant mouse (mnd/mnd) that exhibits symptoms of the disease. Subunit c of the mitochondrial F1F0-ATP synthase, a proteolipid that accumulates in storage bodies of most forms of NCL and several animal models, is dramatically increased in mnd/mnd mouse brain, kidney, liver, heart, and pancreas. Interestingly, another related proteolipid, subunit c of the vacuolar H(+)-ATPase, also accumulates in several mnd/mnd tissues. The molar ratio of the vacuolar subunit c to the F1F0 subunit c is approximately one to two in enriched storage bodies from brain. The relative accumulation of the vacuolar subunit c correlates with its abundance in normal tissues. It appears in decreasing amounts in brain, kidney, and liver and is not detected in heart or pancreas. Aged mice and two mutant mouse lines, juvenile bare (jb) and mucopolysaccharidosis, type VII (gusmps), did not accumulate either of these proteolipids. Dolichol-linked oligosaccharides also accumulate in NCLs and are increased 17-fold in mnd/mnd mouse brain. Thus, mnd/mnd mice seem to be an excellent model for NCLs since they not only share clinical signs and histopathology, but also two biochemical markers. The accumulation of the vacuolar subunit c in this model may prove to be a marker for distinguishing different forms of NCLs.

Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.

We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.

Evidence for isopentenyladenine modification on a cell cycle-regulated protein.

We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.

The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.

In mammalian cells, low density lipoprotein (LDL) is bound, internalized, and delivered to lysosomes where LDL-cholesteryl esters are hydrolyzed to unesterified cholesterol. The mechanisms of intracellular transport of LDL-cholesterol from lysosomes to other cellular sites and LDL-mediated regulation of cellular cholesterol metabolism are unknown. We have identified a pharmacological agent, U18666A (3-beta-[2-diethyl-amino)ethoxy]androst-5-en-17-one), which impairs the intracellular transport of LDL-derived cholesterol in cultured Chinese hamster ovary (CHO) cells. U18666A blocks the ability of LDL-derived cholesterol to stimulate cholesterol esterification, and to suppress 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL receptor activities. However, U18666A does not impair 25-hydroxycholesterol-mediated regulation of these processes. In addition, U18666A impedes the ability of LDL-derived cholesterol to support the growth of CHO cells. However, U18666A has only moderate effects on growth supported by non-lipoprotein cholesterol. LDL binding, internalization, and lysosomal hydrolysis of LDL-cholesteryl esters are not affected by the presence of U18666A. Analysis of intracellular cholesterol transport reveals that LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated CHO cells which results in impaired movement of LDL-derived cholesterol to other cell membranes.

The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts.

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts.

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver.

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.

Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) is a single polypeptide chain with two contiguous domains: a soluble domain (548 amino acids) that catalyzes the rate-controlling step in cholesterol synthesis and a membrane-bound domain (339 amino acids) that anchors the protein to the endoplasmic reticulum (ER). HMG CoA reductase is degraded at least 10-fold more rapidly than other ER proteins; degradation is accelerated in the presence of cholesterol. To understand this controlled degradation, we transfected reductase-deficient Chinese hamster ovary (CHO) cells with a plasmid expression vector containing a reductase cDNA that lacks the segment encoding the membrane domain. The plasmid produced a truncated reductase (37 kd smaller than normal) that was enzymatically active with normal kinetics; most of the truncated enzyme was found in the cytosol. The truncated enzyme was degraded one-fifth as fast as the holoenzyme; degradation was no longer accelerated by sterols. We conclude that the membrane-bound domain of reductase plays a crucial role in the rapid and regulated degradation of this ER 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.

Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells.

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.

Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.

Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hydroxycholesterol in UT-1 cells.

UT-1 cells are a clone of Chinese hamster ovary cells that were selected to grow in the presence of compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. These cells have 100- to 1,000-fold more immunoprecipitable reductase than normal. The enzyme activity is rapidly decreased when low density lipoprotein (LDL) or 25-hydroxycholesterol is added to the culture medium. In this current study, a quantitative immunoprecipitation assay was used to determine whether LDL and 25-hydroxycholesterol inhibit the synthesis or stimulate the degradation of reductase in UT-1 cells. Each of these agents inhibited the incorporation of [35S]methionine into immunoprecipitable reductase by more than 98%. Pulse-chase experiments showed that reductase was degraded with a half-life of 10-13 hr in UT-1 cells and that the rate of degradation of preformed enzyme was increased 3-fold by the addition of either LDL or 25-hydroxycholesterol. We conclude that the predominant mechanism by which LDL and 25-hydroxycholesterol decrease reductase activity in UT-1 cells is a profound suppression of synthesis of the enzyme.

Appearance of crystalloid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We have developed a line of Chinese hamster ovary cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the membrane-bound enzyme that controls cholesterol synthesis. This line, designated (UT-1, was obtained by stepwise adaptation of cells to growth in increasing concentrations of compactin, a competitive inhibitor of reductase. Reductase accounts for approximately 2% of total cell protein in UT-1 cells, as calculated from enzyme specific activity and by immunoprecipitation of reductase after growth of cells in [35S]methionine. After solubilization in the presence of the protease inhibitor leupeptin and electrophoresis in NaDodSO4/polyacrylamide gels, reductase subunits from UT-1 cells were visualized by immunoblotting as a single band (Mr = 62,000). To accommodate the increased amounts of reductase, UT-1 cells developed marked proliferation of tubular smooth endoplasmic reticulum (ER) membranes, as revealed by immunofluorescence and electron microscopy. The ER tubules were packed in crystalloid hexagonal arrays. When UT-1 cells were incubated with low density lipoprotein, reductase activity was suppressed by 90% in 12 hr and the crystalloid ER disappeared. UT-1 cells should be useful for studies of the regulation of reductase and also for studies of the synthesis and degradation of smooth ER.

Synthesis of delta 2-isopentenyl tRNA from mevalonate in cultured human fibroblasts.

Human fibroblasts are shown to incorporate [3H]-mevalonolactone into 3H-labeled delta 2-isopentenyl tRNA. This incorporation was observed in cells that were incubated with compactin (ML-236B), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme. A reductase that blocks mevalonate production by cells. When incubated with low concentrations of [3H]mevalonolactone in the presence of compactin and in the absence of exogenous cholesterol, the cells incorporated small amounts of [3H]mevalonolactone into delta 2-isopentenyl tRNA and large amounts into cholesterol. In the presence of low density lipoprotein, which serves as a source of cholesterol, the incorporation of [3H]mevalonolactone into cholesterol was reduced by 90% and the incorporation into delta 2-isopentenyl tRNA was stimulated by 10-fold. Thus, cultured mammalian cells are now known to use mevalonate for synthesis of three nonsterol products, ubiquinone, dolichol, and delta 2-isopentenyl tRNA, as well as for synthesis of cholesterol.