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. 2011 Feb 18;286(7):5043-54.
doi: 10.1074/jbc.M110.154435. Epub 2010 Dec 2.

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts

Francisco Aresta-Branco  1 , André M Cordeiro, H Susana Marinho, Luísa Cyrne, Fernando Antunes, Rodrigo F M de Almeida
Affiliations

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts

Francisco Aresta-Branco et al. J Biol Chem. .

Abstract

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

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Figures

FIGURE 1.
FIGURE 1.
Plasma membrane of S. cerevisiae contains highly ordered (gel-like) lipid domains. The long component lifetime (A) and normalized amplitude (B) and the mean fluorescence lifetime (C) of t-PnA were obtained from the fluorescence intensity decay of the probe at 24 °C as described under “Experimental Procedures.” The analysis was performed for WT (BY4147) intact cells and spheroplasts (SP) in mid-exponential phase, in the isolated plasma membrane fraction (PM), and in liposomes reconstituted from the lipids extracted from the isolated plasma membrane of WT cells (PM lipids). The long component is also shown for liposomes reconstituted from total lipid extracts (A). The values are the mean ± S.D. of at least four independent experiments. *, p < 0.001 versus WT cells; **, p < 0.01 versus WT cells; ***, p < 0.05 versus WT cells.
FIGURE 2.
FIGURE 2.
Rigidity and abundance of gel domains in the plasma membrane of S. cerevisiae may change with mutations involving sterol, sphingolipid, and GPI anchor biosynthetic pathways. The long component lifetime (A) and normalized amplitude (B) and the mean fluorescence lifetime (C) of t-PnA were obtained from the fluorescence intensity decay of the probe at 24 °C as described under “Experimental Procedures.” The analysis was performed for mid-exponential phase WT (BY4741), erg6Δ, scs7Δ, and per1Δ, WT (RH3435), and erg2Δerg6Δ cells. The values are the mean ± S.D. of at least four independent experiments. *, p < 0.001 versus WT cells; ***, p < 0.05 versus WT cells.
FIGURE 3.
FIGURE 3.
Global order of the cell membrane system of S. cerevisiae is higher in spheroplasts and deletion mutant strains of sphingolipid and sterol metabolism than in WT intact cells. The DPH steady-state fluorescence anisotropy at 24 °C was obtained as described under “Experimental Procedures” for WT (BY4741) intact cells and spheroplasts (SP) and for erg6Δ, scs7Δ, and per1Δ intact cells in mid-exponential phase. The values are the mean ± S.D. of at least five independent experiments. *, p < 0.001 versus WT cells.
FIGURE 4.
FIGURE 4.
Lipids from the plasma membrane of S. cerevisiae and phytoceramide form gel domains at physiological temperatures. A, phytoceramide, but not ergosterol, when mixed with POPC promotes the formation of a gel phase and the appearance of a long component >30 ns in t-PnA fluorescence decay: long component lifetime τi of t-PnA fluorescence intensity decay incorporated in MLVs composed of mixtures of POPC/phytoceramide (gray circles) and POPC/ergosterol (open circles) at 24 °C (see inset for normalized amplitudes). B, steady-state fluorescence anisotropy of t-PnA incorporated in MLVs of POPC/phytoceramide (80:20 mol/mol) as a function of temperature. C, ergosterol at high concentrations abolishes the phytoceramide-enriched gel phase: long component of t-PnA fluorescence decay at 24 °C incorporated in MLVs composed of 1:5 (mol/mol) mixtures of phytoceramide/(POPC + ergosterol) in varying proportions. The dashed line indicates the 30-ns value above which gel domains are known to be present. D, plasma membrane lipids of S. cerevisiae undergo a gel/fluid transition at high temperatures: steady-state fluorescence anisotropy of t-PnA labeling liposomes reconstituted from isolated plasma membrane lipid extracts of mid-exponential WT (BY4147) cells as a function of temperature. E, steady-state fluorescence anisotropy of t-PnA (black bars) versus DPH (white bars) for WT cells in mid-exponential phase and liposomes reconstituted from isolated plasma membrane lipid extracts (PM lipids) and from total lipid extracts of WT (BY4147) cells. A and C, X stands for mole fraction; the lines are merely to guide the eye; in τi, i = 3 or 4, depending on the mixture. B and D, straight lines are linear fits to the data points. They are used to determine the initial and the final temperatures of the gel/fluid transition, pointed by the arrows, from the intercept of the line with the steepest slope with the lines at lower and at higher temperatures, respectively. All panels: the values are the mean ± S.D. of at least four independent experiments. *, p < 0.001 versus t-PnA; **, p < 0.001 versus WT cells; ***, p < 0.05 versus t-PnA; #, p < 0.05 versus WT cells.
FIGURE 5.
FIGURE 5.
Ergosterol is not required for the presence of gel domains formed from S. cerevisiae plasma membrane lipids. The long component lifetime of t-PnA fluorescence intensity decay at 24 °C (mean ± S.D.) is shown for liposomes reconstituted from lipids extracted from the isolated plasma membrane (PM) fraction of WT (BY4741) cells in mid-exponential phase, untreated (n = 5) or treated (n = 3) with methyl-β-cyclodextrin, as described under “Experimental Procedures.”
SCHEME 1.
SCHEME 1.
Depiction of the proposed model explaining the inverse relationship between the abundance of highly ordered sphingolipid-enriched domains and the global order of the membrane. Top represents the lipid components of the plasma membrane of WT or per1Δ cells, and the bottom may represent the plasma membrane of WT spheroplasts or the cell membrane system of erg6Δ or scs7Δ intact cells. Sphingolipids are indicated with their polar heads in gray and glycerophospholipids in black. Top, high abundance of sphingolipid-enriched domains in the plasma membrane, with concomitant sphingolipid depletion in the remainder of the plasma membrane and/or other membranes. Bottom, decreased gel domain abundance implies that the sphingolipids are more scattered through the disordered domains of the plasma membrane and/or intracellular membranes, leading to an increased global order of the cell membrane system.
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