A mechanism for the in vitro stimulation of adrenal cortisol biosynthesis by epidermal growth factor

EGF stimulates adrenal steroidogenesis in ewes and in ovine adrenal slices. In vitro, The stimulation is blocked by the cholesterol synthesis inhibitors compactin and AY 9944. EGF stimulates the incorporation of [14C]acetate into cholesterol. EGF increases the activity of the rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase. EGF has no effect on the levels of any intermediates involved in the conversion of pregnenolone to cortisol, although ACTH produced changes consistent with 17 alpha-hydroxylase activation. We propose that EGF increases adrenal cortisol synthesis in vitro by a stimulation of cholesterol precursor biosynthesis mediated through activation of HMG CoA reductase.     

Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain

Sterols accelerate degradation of the ER enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), which catalyzes a rate-controlling step in cholesterol biosynthesis. This degradation contributes to feedback inhibition of synthesis of cholesterol and nonsterol isoprenoids. Here, we show that degradation of HMG CoA reductase is accelerated by the sterol-induced binding of its sterol-sensing domain to the ER protein insig-1. Accelerated degradation is inhibited by overexpression of the sterol-sensing domain of SREBP cleavage-activating protein (SCAP), suggesting that both proteins bind to the same site on insig-1. Whereas insig-1 binding to SCAP leads to ER retention, insig-1 binding to HMG CoA reductase leads to accelerated degradation that is blocked by proteasome inhibitors. Insig-1 appears to play an essential role in the sterol-mediated trafficking of two proteins with sterol-sensing domains, HMG CoA reductase and SCAP.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

Synthesis of sterols and 5-lipoxygenase products are required for the G1-S phase transition of interleukin-2-dependent lymphocyte proliferation

A murine killer T cell line, G-CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL-2), was used to analyze the mechanism of IL-2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL-2. Compactin, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate-limiting step of the sterol synthesis), inhibited the IL-2-induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL-2 action. The IL-2-induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5-lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5-lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5-lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL-2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.     

The effect of mevinolin on cytosolic acetoacetyl coenzyme a thiolase activity in Chinese hamster ovary fibroblasts

The effects of mevinolin on cytosolic acetoacetyl CoA thiolase activity were studied in wild type Chinese hamster ovary fibroblasts and in CHO cells adapted to growth in high levels of mevinolin. Acetoacetyl CoA thiolase, HMG CoA synthase and HMG CoA reductase activities were elevated in the mevinolin resistant line, KH 2.0. Thiolase activity was also increased when wild type cells were incubated for 5 days with 1 micron mevinolin. These results are consistent with the hypothesis that the regulation of the first three enzymes in the cholesterol biosynthetic pathway is mediated at least in part via a common mechanism.     

The effect of factors released from the tumor-transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts

Quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis after addition of conditioned media from spontaneously tumor-transformed 3T3 cells (3T6-cells) or from SV-40-transformed 3T3 cells (SV-3T3 cells). The conditioned media were found to stimulate both the chromosome cycle (i.e., DNA synthesis and cell division) and the growth cycle (i.e., cellular enlargement). Furthermore, addition of conditioned media to quiescent 3T3 cells increased the activity of HMG CoA reductase--an enzyme previously proposed to exercise some control on cell proliferation in 3T3 cells (Larsson and Zetterberg: J. Cell. Physiol. 129:99-102, 1986. The increased activity of HMG CoA reductase after treatment with tumor cell conditioned media was correlated to the stimulatory effects on DNA synthesis. By treating 3T3 cells stimulated to resume proliferation by addition of conditioned media with mevinolin (a competitive inhibitor of HMG CoA reductase) the activity of HMG CoA reductase as well as the DNA synthesis and cell division were efficiently inhibited. In contrast, HMG CoA activity was not coupled to the cellular enlargement. Therefore, it is proposed that one set of factors present in tumor cell conditioned media preferentially stimulates the chromosome cycle by increasing the HMG-CoA reductase activity, whereas another set of factors is responsible for growth in cell size. Both types of factors are required for balanced growth.     

Studies on the catalytic site of rat liver HMG-CoA reductase: interaction with CoA-thioesters and inactivation by iodoacetamide

The localization of reactive cysteines and characterization of the HMG-CoA binding domain of rat liver HMG-CoA reductase were studied using iodoacetamide (IAAD) and short-chain acyl-CoA thioesters. Freeze-thaw-solubilized HMG-CoA reductase is irreversibly inactivated by IAAD with a second order rate constant of 0.78 M-1 sec-1 at 37 degrees C and pH 7.2. This IAAD inactivation is slowed down by pretreatment of the enzyme with disulfides, indicating that inactivation of HMG-CoA reductase occurs mainly through alkylation of specific cysteine residues in the protein. The substrate HMG-CoA, but not NADP(H), effectively protects the reductase from IAAD inactivation. When both HMG-CoA and NADP(H) are present, the reductase is inactivated by IAAD at a rate much faster than the inactivation in the presence of HMG-CoA alone. Of the two moieties of the HMG-CoA thioester, the CoA moiety confers protection from IAAD inactivation whereas HMG is totally ineffective. A series of CoA-thioesters of mono- and dicarboxylic acids of various size were tested for their effect on the activity of HMG-CoA reductase. The CoA analog, desulfo-CoA (des-CoA), and all CoA-thioesters of monocarboxylic acids of up to 6 carbons in length exhibit mixed-type inhibition of reductase activity. The competitive inhibition constants (Ki) for these compounds vary between 1 and 2 mM, whereas the noncompetitive component (K'i) is relatively constant (540 +/- 20 microM). As the acyl chain length increases beyond 6 carbons, the thioesters of monocarboxylic acids become more potent and acquire the characteristics of pure noncompetitive inhibitors. In contrast, the monothioesters of dicarboxylic acids are pure competitive inhibitors with Ki values which are similar to the Ki values of the corresponding thioesters of monocarboxylates. HMG does not affect reductase activity in concentrations of up to 2 mM, yet it greatly enhances the inhibition of the enzyme by des-CoA. Specifically, HMG affects only the Ki value of des-CoA by decreasing it from 1030 microM to 280 microM. The results indicate that reactive cysteine(s) are localized in the catalytic site of HMG-CoA reductase. Within the active site, these cysteines are closely associated with and probably participate in the binding of the CoA moiety of the substrate HMG-CoA. The results are also consistent with the existence of a noncatalytic hydrophobic site in HMG-CoA reductase.     

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.     

Cell cycle-specific growth inhibition of human breast cancer cells induced by metabolic inhibitors

Proliferation of exponentially growing breast cancer cells (lineHs578T) was blocked specifically in G₁ by 3-hydroxy-3-methylglutarylCoenzyme A (HMG CoA) reductase inhibition, as well as by inhibitionof N-linked glycosylation. As a consequence of these inhibitoryconditions, the cells were synchronized in the G₁ stage of thecell cycle. The similarities in the kinetic responses pointto the possibility that the two different types of metabolicinhibitions block cell cycle progression by common mechanisms.One possibility is that the inhibition of HMG CoA reductaseactivity also leads to a depressed rate of N-linked glycosylation,which in turn may constitute the critical event for cell cycleprogression and cell growth. In order to investigate whetherthis relationship exists in breast cancer cells, cells synchronizedin G₁ by mevinolin (an inhibitor of HMG CoA reductase) wereused. Upon addition of mevalonate, whose endogenous synthesisis catalysed by HMG CoA reductase, the cells entered S phaseafter a 4 h pre-replicative period. Mevalonate stimulation alsoled to a rapid and substantial increase in N-linked glycosylation,measured by determining the uptake of radioactive glucosamine.This metabolic event was found to be of critical importancefor the initiation of DNA synthesis. However, as soon as thecells had entered S phase, they were independent of the levelof N-linked glycosylation. breast cancer cells glycosylation HMG CoA reductase     

Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

MDCK Cells seeded on extracellular matrix- (ECM) coated dishes and exposed to medium supplemented with high-density lipoproteins (HDLs, 750 micrograms protein/ml) and transferrin (10 micrograms/ml) have a proliferative rate, final cell density, and morphological appearance similar to those of cells grown in serum-supplemented medium. The mitogenic stimulus provided by HDLs is not limited by the initial cell density at which cultures are seeded, nor is it limited in time, since cells grown in medium supplemented with transferrin and HDLs grew to at least 50 generations. The presence of HDLs in the medium is required in order for cells to survive, since cells actively proliferating in the presence of medium supplemented with HDLs and transferrin begin to die within 2 days after being transferred to medium supplemented only with transferrin. Low-density lipoprotein (LDL) is mitogenic for MDCK cells when present at low concentrations (from 2.5 to 100 micrograms protein/ml). Above 100 micrograms protein/ml, LDL is cytotoxic and therefore cannot support cell proliferation at an optimal rate. The mitogenic effect of HDLs is also observed when cells are maintained on fibronectin-coated dishes. However, the proliferative rate of the cells is suboptimal and cultures cannot be passaged on this substrate indefinitely, as they can be on ECM-coated dishes. A close association between the ability of HDLs to support cell proliferation and their ability to induce the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is observed. HMG CoA reductase activity is 18 times higher (70 pmoles/min/10(6) cells) in proliferating cells than in confluent, nondividing cells (4 pmoles/min/10(6) cells). The HMG Coa reductase activity of sparse cells is more sensitive to induction by HDLs (eight-fold higher than control cells) than is the enzyme activity of confluent cells (two-fold higher than control levels). The dose-response relationship between the abilities of HDLs to support proliferation and to induce HMG CoA reductase activity are similar. The time course of the stimulation of proliferation and the increase in enzyme activity of sparse, quiescent cells after exposure to HDLs are parallel. The HMG CoA reductase activity of sparse MDCK cells is induced six-fold by exposure to compactin, a competitive inhibitor of HMG CoA reductase. This induction of HMG CoA reductase is prevented by mevalonic acid, not affected by LDL, and synergistically enhanced by simultaneous exposure to HDLs. HDLs effect a rescue from the cytotoxic effect of compactin, whereas LDL does not.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of reduced phytoalexin production on the resistance of upland cotton (Gossypium hirsutum) to verticillium and fusarium wilts

The effect on disease development of inhibiting the production of the sesquiterpenoid phytoalexin hemigossypol (HG) in cotton resistant to both verticillium and fusarium wilts was investigated. Inhibition was achieved by treating the plants with the sodium salt of compactin, a competitive inhibitor of hydroxy-methylglutaryl (HMG) CoA reductase. Compactin treatment (150 μg litre^-1) reduced HG production by a mean of 48%. The enzyme inhibitor did not mimic symptoms in uninfected plants or significantly reduce the ability of the conidia of either Fusarium oxysporum f.sp. vasinfectum or Verticillium dahliae to germinate. Treatment of infected plants with compactin resulted in a breakdown of resistance to verticillium wilt but not to fusarium wilt. These results support the view that HG production is the primary mechanism of resistance to verticillium wilt, but not to fusarium wilt.     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.     

Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast.

The mechanism of action of yeast beta-hydroxy-beta-methylglutaryl-coenzyme A reductase has been investigated through kinetic studies on the oxidation of mevaldate by nicotinamide adeninine dinucleotide phosphate (NADP) in the presence of coenzyme A (CoA) and on the reduction of mevaldate by reduced NADP (NADPH) in the absence of presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, coenzyme A and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. These results suggest that CoA is an allosteric activator for the second reductive step and that it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the beta-hydroxy-beta-methylglutaryl-coenzymeA and the first NADPH. The binding of NADPH enhances the binding of the beta-hydroxy-beta-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. Thus, the replacement of NADP by NADPH after the first reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the second reductive step leave the enzyme before the release of the second NADP. This release of the last product is probably the rate-limiting step for the overall process.     

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).     

The nature of inhibition of 3-hydroxy-3-methylglutaryl CoA reductase by garlic-derived diallyl disulfide

A concentration dependent inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase was found on preincubation of microsomal preparations with diallyl disulfide, a component of garlic oil. This inhibited state was only partially reversed even with high concentrations of DTT. Glutathione, a naturally occurring reducing thiol agent, was ineffective. The substrate, HMG CoA, but not NADPH, was able to give partial protection for the DTT-dependent, but not glutathione-dependent activity. The garlic-derived diallyl disulfide is the most effective among the sulfides tested for inhibition of HMG CoA reductase. Formation of protein internal disulfides, inaccessible for reduction by thiol agents, but not of protein dimer, is likely to be the cause of this inactivation.     

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.     

Location and regulation of early enzymes of sterol biosynthesis in yeast.

Acetoacetyl CoA thiolase and 3-hydroxy-3-methylglutaryl (HMG) CoA synthase were found almost entirely in the cytosol of Saccharomyces cerevisiae, whereas HMG CoA reductase was found almost entirely in mitochondria and further located in the matrix. Formation of all three enzymes was inhibited by cycloheximide, but not by chloramphenicol, indicating that they were synthesized in the cytosol. In anaerobically growing cells the levels of acetoacetyl CoA thiolase and HMG CoA synthase were decreased by ergosterol, whereas HMG CoA reductase levels were affected only slightly, suggesting that in yeast the enzymes responsible for synthesis of HMG CoA were regulated by ergosterol. Aerobically growing cells were essentially impermeable to ergosterol and cholesterol, whereas those growing anaerobically and requiring sterols were readily permeable. Mutants blocked in ergosterol formation were also permeable to sterols under aerobic conditions.