Early changes in membrane permeability, production of oxidative burst and modification of PAL activity induced by ergosterol in cotyledons of Mimosa pudica

Ergosterol (a fungal membrane component) was shown to induce transient influx of protons and membrane hyperpolarization in cotyledonary cells of Mimosa pudica L. By contrast, chitosan (a fungal wall component with known elicitor properties) triggered membrane depolarization. In the processes induced by ergosterol, a specific desensitization was observed, since cells did not react to a second ergosterol application but did respond to a chitosan treatment. This comparative study correspondingly shows that ergosterol and chitosan were perceived in a distinct manner by plant cells. Generation of O2*-, visualized by infiltration with nitroblue tetrazolium, was displayed in organs treated with ergosterol and chitosan. This AOS production was preceded by an increase in activity of NADPH oxidase measured in protein extracts of treated cotyledons. In all the previously described processes, cholesterol had no effect, thereby indicating that ergosterol specifically induced these physiological changes known to participate in the reaction chain activated by characteristic elicitors. Contrary to chitosan, ergosterol did not greatly activate secondary metabolism as shown by the small change in content of free phenolics and by the low modification in activity of PAL, the key enzyme of this metabolic pathway. Therefore, future studies have to clarify the signalling cascade triggered by ergosterol recognition.     

Mechanisms of sterol uptake and transport in yeast

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Susceptibilities of phospholipid vesicles containing different sterols to amphotericin B-loaded lysophosphatidylcholine micelles

To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

Filipin is a reliable in situ marker of ergosterol in the plasma membrane of germinating conidia (spores) of Penicillium discolor and stains intensively at the site of germ tube formation

Filipin, a widely used fluorescent sterol marker is also a potent antibiotic. In this study we address the reliability of filipin as a monitor of ergosterol in fungal cells. A revised staining protocol was developed to minimize any biological effect of the compound. Germinating conidia of Penicillium discolor stained with filipin, displayed a fluorescent cap at the location of germ tube appearance and formation. During germ tube emergence, the fluorescent intensity of the cap increased. This was confirmed by HPLC as an increase of the net cellular ergosterol content. Filipin staining is absent during early germination, while FM dyes, similar molecules, stain the plasma membrane after 1 h. This indicates that the conidial cell wall is no barrier for filipin. To evaluate if filipin does bind ergosterol in situ, natamycin, more specific to ergosterol, was added before filipin staining. This resulted in a marked decrease in fluorescence indicating high ergosterol levels. This was characterized further in ergDelta-mutant cells of Saccharomyces cerevisiae containing altered sterols. Here ergosterol containing cells showed a high fluorescence decrease. Taken together, these data suggest that filipin monitors an ergosterol-enriched cap in germinating conidia at the site of germ tube formation. Furthermore, the sterol-rich cap decreases and reappears after a period of actin disruption. Myriocin that affects sphingolipid synthesis results in an increase of cellular ergosterol and overall filipin fluorescence, but not at the ergosterol cap, where fluorescence is significantly lowered. In conclusion, in this work we have demonstrated an effective revised method for ergosterol staining with filipin and demonstrated its specificity in both Penicillium and Saccharomyces.     

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.     

Perception of Fungal Sterols in Plants (Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells)

Suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) reacted to spores and spore exudates of the pathogen Cladosporium fulvum with a rapid, transient alkalinization of their growth medium that resembled the previously described alkalinization response elicited by chitin fragments (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316) and was likewise inhibited by the protein kinase inhibitor K-252a. However, the spore factor recognized by the cells differed from chitin fragments in that it was butanol soluble and active in cells refractory to stimulation by chitin fragments. The spore factor was purified and identified as ergosterol, the main sterol of most higher fungi. With pure ergosterol, half-maximal induction was reached at about 10 pm. After treatment with ergosterol, tomato cells became refractory to a subsequent stimulation by C. fulvum and vice versa, indicating that ergosterol was the principal component of the spores recognized by the plant cells. Most other sterols were inactive, including cholesterol, a range of animal steroid hormones, and all natural plant sterols tested, except for stigmasterol, which was about 106 times less active than ergosterol. Our data demonstrate that tomato cells perceive ergosterol with a selectivity and sensitivity that resembles the perception of steroid hormones in animals.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.     

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.     

Effects of the detergent sucrose monolaurate on binding of amphotericin B to sterols and its toxicity for cells

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The cytotoxicity of AmB is related to its binding to membrane sterols and its clinical usefulness is based on its greater affinity to ergosterol, the fungal sterol, compared to the mammalian cell sterol, cholesterol (1-3). Here we report that sucrose monolaurate (L.S.) decreased the binding of AmB to cholesterol without interfering with its binding to ergosterol. Furthermore, the toxicity of AmB for mouse erythrocytes (RBC) and cultured mouse fibroblasts, L-929, cells was significantly decreased by low concentrations of L.S., whereas under the same conditions, its toxicity for Candida albicans was unaffected. We observed a very good correlation between the spectroscopic and cell studies. The results reported here on the effects of L.S. on the selectivity of AmB toxicity for fungal cells compared to animal cells and the relative nontoxic nature of sugar esters suggest a potential for compounds of this type to enhance the therapeutic index of AmB.     

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.     

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.     

Lipids of a citric-acid-producing Aspergillus niger strain grown in copper- and in manganese-supplemented media

A citric-acid-producing Aspergillus niger strain was cultivated in conditions favouring citric acid biosynthesis and in conditions hindering it. During both extreme processes, the mycelia were analysed for their lipid content, individual lipid classes, the content of sterols and free fatty acids. Since phospholipids, especially phosphatidylcholine and sterols, play an essential role in membrane permeability one can conclude that the differences observed substantially contribute to citric acid excretion into fermentation media. The difference in sterol composition was the most pronounced. Citric-acid-excreting mycelia contained lower quantities of sterols and ergosterol was the only component. A. niger mycelia grown in conditions hindering citric acid accumulation contained higher amounts of sterols with ergosterol as the main component and six other sterol components representing a minor amount.Offprint requests to: K. Jernejc     

Lipids in roots of Pinus sylvestris seedlings and in mycelia of Pisolithus tinctorius during ectomycorrhiza formation: changes in fatty acid and sterol composition

The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24‐ethyllanosta‐8,24(24′)‐diene‐3beta,22zeta‐diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant‐specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.