Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae.

Saccharomyces cerevisiae GL7 cells require exogenous sterol and unsaturated fatty acid for growth. When grown in the presence of cholesterol or 7-dehydrocholesterol, the cells incorporated less saturated fatty acid into phospholipids than cells grown with ergosterol, stigmasterol, or beta-sitosterol as the sterol source. This lower saturated fatty acid content was most pronounced in phosphatidylethanolamine, slightly less so in phosphatidylcholine, and least evident in phosphatidylserine and phosphatidylinositol. Growing the cells with the various sterols did not affect the ratios of individual phospholipids. The ability of strain GL7 to use 7-dehydrocholesterol as the only sterol supplement for growth was dependent upon the nature of the unsaturated fatty acids added to the growth medium. In the presence of linoleic, linolenic, or a mixture of palmitoleic and oleic acids, excellent growth was observed with either ergosterol, cholesterol, or 7-dehydrocholesterol. However, when the medium was supplemented with either oleic or petroselenic acid, the cells grew more slowly (oleic) or much more poorly (petroselenic) with 7-dehydrocholesterol than with ergosterol. A specific relationship between sterol structure and membrane fatty acid composition in yeast cells is implied.     

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 effects on phospholipid biosynthesis in the yeast strain GL7

Cells of the yeast sterol auxotroph GL7 were grown on either ergosterol or cholesterol to mid-logarithmic phase and total membrane fractions prepared. Activities of phospholipid biosynthetic enzymes in the two cell types were determined. The rates of phosphatidyl-ethanolamine-phosphatidyl-choline-N-methyl transferase and acyl-CoA-alpha-glycerol-3-phosphate transcylase were significantly greater in ergosterol-grown than in cholesterol-grown cells. These reactions were also inhibited by the polyene antibiotic filipin. By contrast the activities of long-chain fatty acyl-CoA synthetase, CTP-phosphatidate-cytidyl transferase, phosphatidylserine decarboxylase and of phosphatidylinositol synthetase were identical in the two (ergosterol and cholesterol) cultures and unaffected by filipin. The ergosterol effect on phosphatidyl-ethanolamine N-methyl transferase was greatest in cells harvested in early log phase, intermediate in the mid-log phase cells, and not significant in stationary phase cells.     

Plasma-Membrane lipid composition and ethanol tolerance inSaccharomyces cerevisiae

Populations of cells suspended anaerobically in buffered (pH 4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration.     

The effect of altered ergosterol content on the transport of various amino acids in Candida albicans

Candida albicans cells have low levels of ergosterol when grown in ascorbic acid-supplemented media. When cells are grown in hydroquinone-supplemented media, the ergosterol levels became higher as compared to normal cells. The uptake of lysine, glycine, glutamic acid, proline, methionine and serine is reduced in hydroquinone-supplemented cells. In contrast to hydroquinone-supplemented cells, the rate and level of accumulation of these amino acids are higher in ascorbic acid-supplemented cells. Nystatin-resistant isolates of C. albicans with low ergosterol contents also exhibit an increased rate and level of accumulation of these amino acids. The uptake of phenylalanine and leucine remained unaffected by such a change in ergosterol levels brought about by different supplementation of the media. The results demonstrate a correlation between ergosterol levels and amino acids uptake. Contrary to various reports, the rate of K⁺ efflux does not seem to correlate with the amino acid uptake in C. albicans cells.     

Complex formation between solanaceous steroidal glycoalkaloids and free sterols in vitro

Both the steroidal glycoalkaloid mixture from potato (α-solanine and α-chaconine) and pure α-tomatine are able to complex with the sterols cholesterol, sitosterol, stigmasterol, campesterol and ergosterol in vitro. The sterol-complexing ability of tomatine was greater than that of the potato alkaloids and more akin to that of the steroidal saponin, digitonin. With all three compounds, cholesterol was the least-readily bound sterol while binding to other sterols was of a similar order. Complex formation with tomatine was not markedly influenced by temperature, and with the aglycone tomatidine did not appear to occur at all.     

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.     

Growth of Candida albicans in the presence of hydrocarbons: a correlation between sterol concentration and hydrocarbon uptake

Summary Candida albicans KTCC 89062 grown on n-alkanes showed higher levels of sterol content as compared to glucose-grown cells. Certain sterols, such as lanosterol, were significantly reduced in cells grown on n-alkanes, while others, such as ergosterol, increased in these cells. Sterol fractions declined as the chain length of the n-alkanes increased. Ergosterol supplementation of the chemically defined medium showed an increase in the uptake of dodecane (C₁₂) by cells grown on such medium. Increase in the concentration of ergosterol supplementation resulted in an increase in C₁₂ uptake. The uptake of C₁₂ was not stimulated by ergosterol supplementation in the case of non-viable yeast cells.     

How cholesterol interacts with proteins and lipids during its intracellular transport

Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein–lipid and protein–protein interactions. Similarly, membrane lipids and their physico-chemical properties directly affect cholesterol partitioning and thereby contribute to the highly heterogeneous intracellular cholesterol distribution. Movement of cholesterol in cells is mediated by vesicle trafficking along the endocytic and secretory pathways as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol–lipid and sterol–protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics in membranes and explain how such models are related to sterol flux between organelles. An overview of various sterol-transfer proteins is given, and the physico-chemical principles of their function in non-vesicular sterol transport are explained. We also discuss selected experimental approaches for characterization of sterol–protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how specific protein–lipid and protein–protein interactions help overcoming the extremely low water solubility of cholesterol, thereby controlling intracellular cholesterol movement. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.     

Effect of exogenous steroids on sterol synthesis in L-cell mouse fibroblasts

A number of steroids have been tested in an L-cell tissue culture system to determine their effects on cellular sterol biosynthesis and cellular growth. Cholesterol, desmosterol, lathosterol, 7-dehydrocholesterol, and cholestanone reduce de novo synthesis and produce only limited toxicity at high concentrations of exogenous sterol. Considerable cellular toxicity is observed when cells are grown in the presence of coprostanol and Delta(4)-cholestenone. No marked effect on either cell growth or sterol biosynthesis is produced by cholestanol, beta-sitosterol, stigmasterol, campesterol, ergosterol, cholesteryl oleate, or cholestane.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

The Effect of Specific Sterols on Cell Size and Fatty Acid Composition of Tetrahymena pyriformis W

The size and fatty acid composition of Tetrahymena pyriformis W cells were influenced by the provision of a nutritional supplement of ergosterol, cholesterol, or tetrahymanol, but not of 20-isocholesterol. Ergosterol and cholesterol addition led to a reduction in cellular volume, an increase in glycerophospholipid saturated fatty acid content, and an increase in palmitoleic acid and its metabolic products when compared to unsupplemented controls. Tetrahymanol supplementation resulted in an increase in cellular volume, a decrease in saturated fatty acid content, and a reduction in palmitoleic acid and derivatives. 20-Isocholesterol was accumulated by the cells; however, this compound had no effect on any of the parameters followed in this investigation and had only a small depressant effect on tetrahymanol biosynthesis. Ergosterol and cholesterol had the same impact on the ciliates, even though the ergosterol-supplemented cells contained approximately three times as much free sterol as did cholesterol-grown cells. The amount of the free cholesterol and metabolic products in supplemented cultures was similar to the amount of tetrahymanol present in control cultures. This observation suggests that the cells recognize qualitative differences among the various polycyclic alcohols rather than responding to the amount of sterol present. Increased cellular levels of tetrahymanol led to a response unlike that of the true sterols, which again suggests that the high degree of specificity depends on the structure of the added polycyclic alcohol. The changes in fatty acid composition may be required to maintain proper interaction of the polar lipids and the polycyclic alcohols to give an appropriate degree of membrane fluidity.     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K⁺ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).     

Multiple functions for sterols in Saccharomyces cerevisiae

Analyses with a yeast sterol auxotroph indicated that there are at least four different levels of function for sterol which have been designated sparking, critical domain, domain and bulk. Growth of yeast sterol auxotrophs on cholestanol is precluded unless minute amounts of ergosterol are available. We have designated this phenomenon the sparking of growth, in which cholestanol satisfies an overall membrane sterol requirement and ergosterol fulfills a high specificity sparking function. The critical domain role for sterol is observed under conditions of lanosterol supplementation where low levels of ergosterol (10-times those necessary for sparking on cholestanol) are required for growth. The sterol functions designated domain and bulk are illustrated by assessing cellular free sterol levels and plasma membrane properties of a sterol auxotroph after growth on different concentrations of exogenously supplied sterol. Plasma membranes isolated from auxotrophs grown on domain or bulk levels of sterol underwent no lipid thermotropic transitions, while plasma membranes from cells grown on critical domain levels of sterol underwent a lipid thermotropic transition, when analyzed by steady-state fluorescence anisotropy.     

Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.     

LM cell growth and membrane lipid adaptation to sterol structure

Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.     

Role of ergosterol in growth inhibition of Saccharomyces cerevisiae by syringomycin E

The antifungal activity of the lipodepsipeptide syringomycin E from Pseudomonas syringae pv. syringae is modulated by sterols. To study the requirement of the predominant fungal sterol, ergosterol, in syringomycin E action, the sterol composition of Saccharomyces cerevisiae sterol auxotroph strain FY-14 was modified and sensitivity to syringomycin E examined. Cells containing solely ergosterol, cholesterol, β-sitosterol or stigmasterol were sensitive to syringomycin E with the latter two being the most sensitive. Cells containing growth-promoting cholesterol were the most sensitive and those with growth-promoting ergosterol the least sensitive. It is concluded that sensitivity to syringomycin E is modulated by growth-promoting sterols and does not necessarily require ergosterol.     

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.