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Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae

Sukesh Chander Sharma
DOI: http://dx.doi.org/10.1111/j.1567-1364.2006.00149.x 1047-1051 First published online: 1 November 2006

Abstract

Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-to-phospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine, revealed that the amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg strains. The present findings suggest that adaptive responses in yeast cells with altered sterol structure are possibly manifested through changes in membrane lipid composition and fluidity, and not through transbilayer rearrangement of aminophospholipids.

Keywords
  • sterols
  • phospholipids
  • fluidity
  • membrane asymmetry
  • fluorescence polarization
  • Saccharomyces cerevisiae

Introduction

Plasma membranes are essential components of all cells and function to separate the cytoplasm from the environment. Sterols, especially free ones, are major constituents of many biological membranes; about 80% are found in the plasma membrane, where they perform bulk functions, contributing to fluidity and permeability, and also participate in the control of membrane-associated processes (Yeagle, 1985; Parks & Cassey, 1995; Daum, 1998; Umebayashi & Nakano, 2003). They control the mobility of phospholipid acyl chains, thicken the plasma membrane, and interact with the fatty acyl chains of phospholipids and proteins. Changes in sterol composition have been reported to alter the sensitivity of yeast cells to certain drugs (Zweytick, 2000). Overall, the physiologic role of sterols in yeast remains largely unknown. Recently, asymmetry and transbilayer movement of phospholipids of the plasma membrane has become an important topic in biochemical and biophysical research (Valachovic, 2001). Sterol mutants are very valuable tools for understanding sterol functions in yeast. Several studies have been performed on various sterol auxotrophs cultivated in medium supplemented with sterol(s) and unsaturated fatty acids under anaerobic conditions, which often lead to complex changes in sterol esterification patterns (Pomorski, 2001). However, nystatin-resistant mutants can be cultured under normal growth conditions. These strains were used in the present study to investigate the in vitro effects of altered sterol structures in erg mutants. The results obtained suggested that changes in sterol structure are manifested through phospholipid composition, sterol/phospholipid molar ratio and decreased membrane order, but not through asymmetric distribution of aminophospholipids in the plasma membrane.

Materials and methods

Yeast strains

The strains of Saccharomyces cerevisiae used in the present study are indicated in Table 1.

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Table 1

Saccharomyces cerevisiae strains used

Yeast strainsBiochemical lesionSterol(s) accumulated
Wild type (A184D)Ergosterol (ergosta-5,7,22-triene-3β-ol)
erg2Δ-8→-7 IsomeraseErgosta-5,8,22-trienol, ergosta-8,22-dien-3β-ol
erg3Δ-5→-6 DesaturaseErgosta-5,7,24(8)-trien-3β-ol
erg6C-24 MethyltransferaseZymosterol (cholesta-5,7,22,24-tetra-en-3β-ol)

Materials

Yeast extract, peptone, dextrose and Bacto agar were procured from Himedia Laboratories, Mumbai, India. Trinitrobenzene sulfonic acid (TNBS), ergosterol, fluorescamine, diphenylhexatriene (DPH), trimethylammonium (TMA)-DPH and phospholipid standards were obtained from Sigma Chemical Co. Silica gel G 60 thin-layer chromatography (TLC) aluminum-backed precoated plates were obtained from Merck, Darmstadt, Germany. All other reagents were of analytical grade, and solvents were distilled before use.

Growth conditions

Cells were grown in YPD medium containing yeast extract 1%, peptone 2% and dextrose 2% (pH 5.5) until midlogarithmic phase at 28°C and 180 r.p.m. Cells were harvested by centrifugation at 2000 g for 5 min at 4°C. The pellet was washed twice with distilled water and once with 0.1 M potassium phosphate buffer (pH 6.8) containing 1 mM EDTA.

TNBS labeling procedure

TNBS labeling was done according to the method of Cebron & Calderon (1991), with some modifications. Cells suspended in 0.1 M potassium phosphate buffer (pH 7.8) containing 0.5 mM MgCl2 were washed and then suspended in the same buffer containing 5 mM buffered TNBS, and immediately placed in a water bath maintained at 4°C for 30 min, with periodic gentle mixing in the dark. After incubation, cells were washed with cold potassium acetate buffer (pH 6.0) until no yellow color could be seen in the supernatant.

Fluorescamine labeling procedure

Cells in phosphate buffer (pH 7.8) containing 600 mM KCl were transferred to a 25-mL conical flask and cooled to 10°C, and 60 μmol of fluorescamine dissolved in dry dimethyl sulfoxide was added dropwise in the dark, with constant swirling. After 30 s, the reaction was terminated by adding an equal volume of (1 M) ammonia in the same buffer. The suspension was centrifuged at 2000 g for 5 min at 4°C, and the pellet was washed several times until the supernatant was free of color.

Lipid extraction, separation and analyses

The total lipids from the yeast cell pellet after TNBS and fluorescamine labeling, as well as of controls, were extracted with the Bligh and Dyer procedure (Bligh & Dyer, 1959) as described previously (Sharma, 2001), using chloroform/methanol/water 1 : 2 : 0.4 (v/v) containing 0.001% butylated hydroxytoluene. The mixture was vortexed several times. It was then centrifuged, the supernatant was collected, and the pellet was treated with solvent mixture again. Both fractions were pooled, and chloroform was added to obtain two phases. The lower chloroform phase containing lipid was evaporated under a stream of nitrogen. The residue was placed under vacuum in order to remove traces of moisture. The residue was dissolved in 100 μL of chloroform/methanol 1 : 1 (v/v) and was stored at −20°C until analysis. The phospholipids were separated by two-dimensional TLC, using chloroform/methanol/ammonia 65 : 35 : 5 (v/v) in the first dimension, and then chloroform/methanol/acetone/acetic acid/water 50 : 10 : 20 : 10 : 5 (v/v) in the second dimension. The spots were identified by known standards. The aminophospholipids that reacted with TNBS gave a yellow color. The fluorescamine-derivatized aminophospholipids were localized by immediate exposure to UV light. The iodine-stained (phosphorus-positive) spots were cut out and eluted with chloroform/methanol (1 : 1, v/v). The phosphorus estimated by the Ames procedure was multiplied by 25 to obtain the amount of phospholipids. Total sterols were estimated by the Sperry and Webb procedure (Sperry & Webb, 1950). For estimating esterified sterols, free sterols were precipitated by an ethanolic solution of digitonin (1%). Fatty acid analysis of the phospholipid fraction was performed by gas-liquid chromatography on a glass column containing 15% DEGS on 80-100 mesh chromsorb-W, as described previously (Sharma & Singh, 2000).

Fluidity measurements

Harvested cells were suspended in 0.1 M potassium phosphate buffer (pH 6.8) to a density of 500 μg dry weight mL−1. The cells were incubated at 10°C for 10 min with DPH or TMA-DPH in tetrahydrofuran to a final concentration of 0.5 μM. After incubation, cells were washed three times with same buffer. Fluorescence polarization was measured according to the method of Haggerty. (1978) on a Kontron SFM spectrofluorimeter with 10-nm bandwidth at 360 and 450 nm as excitation and emission wavelengths, respectively.

Results and discussion

The wild type and various erg mutants were grown on YPD medium until midlogarithmic phase. All strains gave similar yields, except for that of erg6, which was low. The total sterol(s) and phospholipid contents of all strains were measured. These values did not vary much on the basis of percentage of total lipid. The results support the findings of Parks and colleagues, who reported that sterol auxotrophs maintained their phospholipid contents unaltered when grown in exogenously added sterols (Low, 1985). However, there were some major changes observed in the individual phospholipids; the values are given in Table 2. The relative amounts of individual phospholipids did not differ between the cells with chemical labeling reagents and unlabeled cells. Phosphatidylcholine had the highest level, followed by phosphatidylinositol, phosphatidylethanolamine and phosphatidylserine. The phosphatidylcholine content was decreased in the erg3 mutant (35.80%) but was increased in the erg6 mutant (50.25%) as compared to the wild type (42.00%). The content of nonbilayer-forming lipid, i.e. phosphatidylethanolamine, showed a slight decrease in erg2, whereas in erg6 it was decreased to 15.25%, in comparison to 20.38% in the wild type. The content of another major lipid, phosphatidylinositol, showed minor increases in erg2 and erg6 cells as compared to controls. The anionic phospholipid phosphatidylserine showed an increase in erg2 and a low level in erg6. The present results support previous findings that sterol molecules play regulatory roles in the coordination of some selected biosynthetic reactions in yeast sterol auxotrophs (Low, 1985; Ramgopal, 1990). Moreover, it has recently been proposed that there is also self-regulation of the lipid content of membranes by nonbilayer lipids (Garab, 2000). Although extensive studies have shown that biological membranes are tightly regulated in their lipid content (de Kruijf, 1978; Williams, 2000), it is known that not only is the free sterol/phospholipid molar ratio an important factor that determines fluidity, but sterol structure also influences the phase properties of biological membranes (Arora, 2004). Therefore, it was worthwhile studying the effect of sterol structure with regard to membrane fluidity. These ratios were decreased in all mutants from 0.29 of control to 0.17, 0.20 and 0.23, respectively. The fatty acid profiles of phospholipid fractions did not show any major differences (data not given). Changes in the fluidity of plasma membranes were further studied using two fluorescent probes: (1) hydrophobic DPH, and (2) TMA-DPH, a tetramethylammonium derivative. The latter is a rigid molecule that is preferentially localized in the outer leaflet as a reporter. As DPH is a hydrophobic molecule, fluorescence microscopy was used to ensure that it did not enter the cells at the temperature under consideration. The values for anisotropies are given in Fig. 1. It is apparent that fluorescence anisotropy decreased with an increase in temperature, so the anisotropy value of both probes were lower in mutants than in wild-type cells, suggesting increased freedom in rotational movements due to decreased membrane order. The results are in contradiction to those with the other spin-labeled probe, 5-doxyl stearic acid, where greater membrane rigidity was observed in erg mutants and no lipid thermotropic transitions in auxotrophs (Lees, 1979; Low, 1985; Rodrigues, 1985).

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Table 2

Lipid composition of yeast cells after chemical labeling

StrainPhospholipidsPC/PE ratioFS/PL molar ratio
PCPEPEPEPIPSPS
Wild42.00 ± 1.8020.38 ± 2.152.21 ± 0.022.99 ± 0.0923.15 ± 1.3010.70 ± 0.552.020.29
erg235.80 ± 0.9818.65 ± 1.032.05 ± 0.022.30 ± 0.0428.20 ± 2.3314.25 ± 1.053.060.17
erg342.79 ± 1.2521.23 ± 1.892.30 ± 0.122.90 ± 0.1222.72 ± 2.159.93 ± 0.802.180.20
erg650.25 ± 2.0215.25 ± 1.023.25 ± 0.032.80 ± 0.7927.77 ± 1.237.78 ± 0.210.42 ± 0.052.390.23
  • * Labeling by trinitrobenzene sulfonic acid (TNBS).

  • Labeling by fluorescamine.

  • *** Labeling by TNBS.

  • + P<0.05.

  • The results are mean±SD of three independent experiments done in triplicate. The values were compared by Student's t-test.

  • PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; FS, free sterols.

Figure 1

Fluorescence anisotropies of wild type and sterol mutants of Saccharomyces cerevisiae. Open symbols indicate cells labeling with DPH while solid symbols indicate labeling with TMA-DPH as described in Materials and methods Vertical lines indicate standard deviation of three experiments.

Sterols have also been reported to alter not only flip–flop rates but also the transbilayer distribution of phospholipids (Williams, 2000; Leventis & Silvius, 2001). Several cell types, from mammals to yeast, exhibit a nonrandom distribution of phospholipids. In general, the aminophospholipids phosphatidylethanolamine and phosphatidylserine are sequestered to the inner leaflet. Any phosphatidylserine in the external leaflet is considered to constitute an apoptotic signal (Valachovic, 2001; Manno, 2002). As erg mutants differ in lipid composition and fluidity, the transbilayer distribution of aminophospholipids was studied by two chemical probes, i.e. TNBS and fluorescamine; these results are shown in Table 3. The values around 9–13% are in agreement with previous observations (Cebron & Calderon, 1991; Balasubramaniam & Gupta, 1996). The labeling of phosphatidylethanolamine with fluorescamine was slightly more extensive than that with TNBS. The amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg mutant strains, except in erg6. It was noticed that there was much greater labeling of phosphatidylethanolamine and very little of phosphatidylserine with these probes, because excess phosphatidylethanolamine and phosphatidylserine derivatization was not detected in this mutant. The erg6 mutants were reported to have altered permeability to several dyes, cations, and antibiotics (Bard, 1978; Welihinda, 1994; Zweytick, 2000). Fluorescamine, although it penetrates the cell membranes (Valtersson & Filipson, 1985), seems to be a much better probe due to its very short lifetime (milliseconds). However, unlike mammalian cells, yeast cells are capable of maintaining intracellular pH near 6.5, hence the probe is unlikely to react with amino groups. These present findings suggest that adaptive responses to altered sterol structure are manifested through changes in lipid composition and fluidity, but not through transbilayer rearrangements in aminophospholipids. Further work is in progress to characterize strains that accumulate one type of sterol only.

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Table 3

Summary of chemical labeling in the wild type and erg mutants

StrainPercentage labeling
PEPEPS
Wild9.78 ± 0.0512.36 ± 0.23
erg29.98 ± 0.1210.97 ± 0.12
erg39.75 ± 0.0811.11 ± 0.09
erg617.57 ± 1.2313.79 ± 0.125.12 ± 0.02
  • * Labeling by trinitrobenzene sulfonic acid (TNBS).

  • Labeling by fluorescamine.

  • The results are means±SD of three independent experiments done in triplicate.

  • PE, phosphatidylethanolamine; PS, phosphatidylserine.

Acknowledgements

I gratefully acknowledge the strains provided by Professor M. Bard of Indiana University. I also thank Panjab University and DST, New Delhi for providing partial financial support for attending the symposium. I am grateful to Dr S.K. Soni for his kind help in the preparation of this manuscript.

Footnotes

  • Editor: Rafael Sentandreu

References

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