A photophysical study of the polyene antibiotic filipin: Self-aggregation and filipin–ergosterol interaction

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25°C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE–filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin–filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.     

Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.     

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.     

Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.     

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.     

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.     

Dehydroergosterol structural organization in aqueous medium and in a model system of membranes.

The aggregation of delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol or DHE), a fluorescent analog of cholesterol, was studied by photophysical techniques. It was concluded that the aqueous dispersions of DHE consist of strongly fluorescent microcrystals, and no evidence for self-quenching in micellar-type aggregates was found. The organization of DHE in model systems of membranes (phospholipid vesicles) is strongly dependent on the vesicle type. In small unilamellar vesicles, no evidence for aggregation is obtained, and the fluorescence anisotropy is rationalized on the basis of a random distribution of fluorophores. On the contrary, in large unilamellar vesicles (LUVs), a steeper concentration depolarization was observed. To explain this, a model that takes into account transbilayer dimer formation was derived. This was further confirmed from observation of excitonic absorption bands of 22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-23,24-bisnor- 5-cholen-3 beta-ol (NBD-cholesterol) in LUV, which disappear upon sonication. It is concluded that, in agreement with recent works, sterol aggregation is a very efficient process in large vesicles (and probably in natural membranes), even at very low concentrations (approximately 5 mol%).     

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.     

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.     

Permeabilizing action of filipin III on model membranes through a filipin-phospholipid binding.

The binding of the pentaene antibiotic filipin to egg-yolk phosphatidylcholine (EPC) and dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles, has been studied by ultraviolet (UV) absorption and circular dichroism (CD). A stoichiometry of one molecule of filipin for five molecules of phospholipid was demonstrated by CD when phospholipids were in fluid phase. The similarity of the CD spectra with EPC and DMPC established a similar filipin-phospholipid assemblage in both membranes. We therefore postulated that filipin incorporation leads to the formation of gel-like domains in fluid EPC membranes as previously demonstrated for fluid DMPC membranes (Milhaud, J., Mazerski J., Bolard, J. and Dufoure, E.J. (1989) Eur. Biophys. J. 17, 151-158). The release of fluorescent probes (carboxyfluorescein (CF) or calcein (CC)), entrapped in EPC small unilamellar vesicles (SUV), due to the action of filipin, was followed by fluorescence and CD measurements concomitantly. The following observations were made. (1) The percentage of released probe, as a function of the filipin/phospholipid molar ratios, was the same whether or not membranes contained cholesterol. (2) The permeabilization of vesicles proceeded concomitantly with filipin-phospholipid binding while filipin-cholesterol binding leveled off. (3) The release of the content of vesicles occurred by an all-or-none mechanism leaving the depleted vesicles intact. From these observations and from the previous structural findings, a new interpretation of the action of filipin is proposed. Precluding any disruptive effect, inducement of permeability would result from the high intrinsic permeability of the interfacial region at the boundaries of the gel-like domains corresponding to the filipin-phospholipid aggregates. Additionally, we obtained the permeability coefficients for the anionic forms of CC and CF across EPC SUV, 0.6.10(-10) cm s-1 and 2.10(-10) cm s-1, respectively, as compared to 2.5.10(-14) cm s-1 for the counterion Na+ (Hauser, H, Oldani, D. and Phillips, M.C. (1973) Biochemistry 12, 4507-4517).     

Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols

The interaction of the polyene antibiotic, filipin, with individual or mixed plant sterols (stigmasterol, sitosterol, campesterol and 24-methylpollinastanol) incorporated into large unilamellar vesicles (LUV) of soybean phosphatidylcholine (PC) as well as the filipin interaction with purified membrane fractions from maize roots containing these sterols was investigated by ultraviolet (UV) absorption and and circular dichroism (CD) spectroscopy. With both types of membrane preparation, dramatic changes in the UV absorption and CD spectra of the antibiotic were evidenced. When LUV containing stigmasterol, sitosterol and/or campesterol were incubated with low filipin concentrations (i.e., for filipin/sterol molar ratios (rst) lower than 1), CD signal characteristic of the formation of filipin-sterol complexes were observed. At higher rst values, the filipin-sterol interaction was shown to be in competition with a filipin-phospholipid interaction. With 24-methylpollinastanol-containing LUV, the filipin-phospholipid interaction was detected even at rst values lower than 1, which suggests a lower affinity of filipin for this sterol and emphasizes the structural differences between delta 5-sterols and 9 beta,19-cyclopropylsterols. With sterol-free soybean PC LUV, a filipin-phospholipid interaction could also be evidenced. With maize root cell membranes containing either delta 5-sterols or 9 beta,19-cyclopropylsterols, CD spectra similar to those obtained in the presence of LUV having these sterols as components were observed. Thus, the protein component of the membranes does not appear to be an important feature.     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence.

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol.

The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton interaction; the observed splitting profile indicates that the chromophores are in a stacked arrangement (parallel transition dipoles). For filipin incorporated in lipid bilayers, the sterol is able to induce the same type of aggregate, at variance with nystatin.     

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.     

Filipin labeling in Chlamydomonas gametes exposes site-specific lipid specializations.

A thin section study of mating Chlamydomonas cell wall-less CW 15 mating type plus (mt+) and mating type minus (mt-) gametes utilized filipin. The results show extensive labeling of mt+ and mt- plasma membranes. No labeling was seen on the mating structure membranes of activated mt+ or mt- gametes. These results indicate that differences exist between the plasma membrane and the mating structure membrane of gametes. If filipin is specific for the 3-beta-OH sterol, ergosterol and/or other Chlamydomonas sterols, then these results imply that the fusing mating structure membranes may be altered or reduced in sterol content. Such lipid specializations may increase local membrane fluidity and thereby facilitate the site-specific cell fusion associated with mating Chlamydomonas gametes.     

Rapid transbilayer movement of the fluorescent sterol dehydroergosterol in lipid membranes

This study establishes a new assay for measuring the transbilayer movement of dehydroergosterol (DHE) in lipid membranes. The assay is based on the rapid extraction of DHE by methyl-beta-cyclodextrin (M-CD) from liposomes. The concentration of DHE in the liposomal membrane was measured by using fluorescence resonance energy transfer (FRET) from DHE to dansyl-phosphatidylethanolamine, which is not extracted from liposomes by M-CD. The method was applied to small (SUV) and large (LUV) unilamellar vesicles of different compositions and at various temperatures. From the kinetics of FRET changes upon extraction of DHE from membranes, rates of M-CD mediated extraction and flip-flop of DHE could be deduced and were found to be dependent on the physical state of the lipid phase. For egg phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the liquid-crystalline state, halftimes of extraction and transbilayer movement were <5 s and approximately 20-50 s, respectively, at 10 degrees C. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine-SUV being in the gel state at 10 degrees C, the respective halftimes were 28 s and 5-8 min. Surprisingly, DHE could not be extracted from LUV consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. This might be an indication of specific interactions between DHE molecules in membranes depending on the phospholipid composition of the membrane.     

Susceptibilities of phospholipid membranes containing cholesterol or ergosterol to gramicidin and its derivative incorporated in lysophospholipid micelles

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.     

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.     

Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin. A fluorescence study

Competition studies between cholesterol and ergosterol were carried out to gain insight into the binding interactions between nystatin and these sterols. Lipid vesicles were prepared with mixtures of palmitoyloleoylphosphocholine/ergosterol/cholesterol, and both sterol molar ratio and total content were varied. The inhibitory effect of cholesterol toward the ergosterol ability to induce the formation of long-lived fluorescent antibiotic species was used to detect nystatin-cholesterol interactions. It was found that the key factor controlling nystatin photophysical properties in the ternary lipid mixtures was their ergosterol/cholesterol molar ratio and not their overall sterol content. Moreover, permeabilization studies showed that nystatin was able to form pores in all the mixed vesicles, but the initial rate of pore formation was also dependent on the ergosterol/cholesterol molar ratio. Our data show that ergosterol is displaced by competing cholesterol, indirectly confirming cholesterol's ability to coassemble with nystatin. The distinct spectroscopic properties emphasize the different molecular architecture adopted by nystatin-cholesterol and -ergosterol complexes, and therefore are relevant to understanding the interaction of the antibiotic with membranes.     

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.