The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.     

Characteristics of sterol uptake in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted. When cultured on 11 micrograms of sterol ml-1 or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated. Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium. The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration. The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability. The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.     

Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue.

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.     

Anaerobiosis induces complex changes in sterol esterification pattern in the yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.     

Regulation by heme of sterol uptake in Saccharomyces cerevisiae

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.     

Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.     

The synthesis and esterification of cholesterol by hepatocytes and H35 hepatoma cells are independent of the level of nonspecific lipid transfer protein

The level of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) is 16-fold lower in the Reuber H35 hepatoma cells as compared to the hepatocytes in culture (49 and 810 ng of protein per mg of 105000 X g supernatant protein, respectively). In order to establish whether there is a relationship between the level of nonspecific transfer protein and intracellular cholesterol metabolism, we have determined the biosynthesis and esterification of cholesterol in these hepatoma cells and hepatocytes. Both types of cells incorporated [3H]mevalonate into cholesterol and cholesterol ester. Incubation of both cell types with [3H]cholesterol in the medium resulted in a time-dependent uptake and subsequent conversion into cholesterol ester. In both instances, the amount of 3H label incorporated into cholesterol per mg of cellular protein was about 2-fold higher for the hepatoma cells. The kinetics of esterification of endogenously synthesized cholesterol were similar for both hepatoma cells and hepatocytes. Esterification of cholesterol derived from the medium proceeded 2-times faster in the hepatoma cells than in the hepatocytes. From the kinetics of cholesterol esterification we conclude that cells do not discriminate between cholesterol synthesized de novo and cholesterol derived from the medium. In addition, the proposition that the nonspecific lipid transfer protein is involved in cholesterol synthesis and esterification is not substantiated by this study.     

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.     

On the role of the sterol hydroxyl group in membranes.

The adequacy of sterol derivatives containing a blocked 3-hydroxyl group for sustaining the growth of two sterol auxotrophs has been investigated. Mycoplasma capricolum, a cholesterol-requiring bacterium, grows nearly as well on media supplemented with cholesteryl methyl ether or cholesteryl acetate as on free cholesterol. The two derivatives are recovered unchanged from the bacterial cells. Similarly, cholesteryl methyl ether or ergosteryl methyl ether replace cholesterol or ergosterol as sterol sources for a yeast mutant, strain GL7, defective in 2,3-oxidosqualene-lanosterol cyclization. During aerobic or semianaerobic growth, yeast cells demethylate some of the cholesteryl methyl ether to free cholesterol. However, cells growing on cholesterol methyl ether under strict anaerobic conditions do not produce free sterol. The bearing of these results on the postulated requirement of a free sterol hydroxyl group for membrane function is discussed. Sterol esterification does not appear to be essential for the two microbial systems.     

Stimulation of esterified cholesterol accumulation in tissue culture cells exposed to high density lipoproteins enriched in free cholesterol.

Human high density lipoprotein enriched in free cholesterol was obtained by exposing the lipoprotein to lipid dispersions having a free cholesterol/lecithin molar ratio greater than two. The metabolism of cholesterol was studied in tissue culture cells exposed to normal and cholesterol-enriched lipoproteins. Incubation of Fu5-AH rat hepatoma cells in medium containing cholesterol-enriched lipoprotein resulted in the accumulation of cellular cholesterol whereas normal high density lipoprotein produced no change in cellular content. The accumulated sterol was recovered primarily as esterified cholesterol and was derived almost entirely from lipoprotein free cholesterol. The esterification of incorporated free cholesterol and the cellular cholesterol content were directly related to the molar ratio of free cholesterol to phospholipid in the lipoprotein and to the concentration of lipoprotein in the culture medium. Isotopic experiments utilizing lipoprotein labeled with 125I or [4-14C]cholesteryl oleate demonstrated that a large fraction of the cholesterol incorporated from lipoprotein enriched in free cholesterol occurred by mechanisms that did not result in lipoprotein internalization and degradation. The response of other tissue culture cells to cholesterol/phospholipid dispersions is presented. The data indicate that the lipid composition of a lipoprotein can regulate free cholesterol uptake and esterification as well as cellular cholesterol content.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

The side-chain cleavage of cholesterol sulfate--I. The effect of adrenodoxin on the binding of cholesterol sulfate to cytochrome P-450scc

Difference spectroscopy was used to measure the binding of cholesterol sulfate (CS) to cytochrome P-450scc. The uncomplexed cytochrome and the complex of the cytochrome with adrenodoxin (ADX) were both titrated with CS in order to test whether ADX increased the affinity of the cytochrome for the sterol sulfate. The addition of ADX to the cytochrome had different effects on the binding of the sterol sulfate depending on several factors including: (1) The method of preparation of the cytochrome P-450scc, (2) The concentration of cytochrome P-450scc, (3) The method by which CS was suspended in aqueous solution, and (4) Whether or not the solutions of cytochrome contained non-ionic detergents. The results of this study suggest that the method of isolation of cytochrome P-450scc, and non-ionic detergents, greatly modulate the apparent affinity of cytochrome P-450scc for CS. In the absence of detergents the addition of adrenodoxin to dilute solutions of cytochrome P-450scc appears to enhance only slightly (1- to 2-fold) the affinity of the cytochrome for the sterol sulfate.     

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.     

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.     

The effect of phospholipids on the in vitro polymerization of rat brain tubulin

The association of brain tubulin, as measured by the temperature-dependent development of turbidity at 350 nm, is greatly stimulated by the detergent Nonidet P-40 in crude extracts of rat brain tissue. Stimulation of turbidity development is also obtained with partially purified rat brain tubulin treated with Nonidet or other detergents, or preincubated with phospholipase C or D; treatment with bovine pancreatic phospholipase A₂ produces an inhibition. Exogenous phospholipids, diglycerides, other related derivatives, and lipophilic extracts of tubulin and brain supernatants can also alter the turbidity development. In addition, microtubules arising from tubulin obtained in the presence of Tween-20 or Nonidet P-40 exhibit a 50 and 100% increased specific viscosity, respectively, over that of tubulin prepared in the absence of detergent or in the presence of Kyro or Triton N-101. The effectiveness of these detergents in removing phospholipids from tubulin preparations follows a similar pattern: Nonidet P-40 removes 80%, Tween-20 removes 50%, and Kyro or Triton N-101 removes none. The total mass of microtubule formed, as determined by sedimentation, is the same regardless of the effect of the detergents on the viscosity. The microtubules obtained in the presence of Nonidet P-40 have a normal appearance when examined by electron microscopy, and their composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is indistinguishable from that of standard tubulin, especially with regard to the minor protein bands always present in the tubulin preparations. The results obtained suggest that the phospholipids associated to brain tubulin preparations might have a role in determining the association of tubulin and/or the final dimensions of the assembled microtubules.