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. 2010 Sep 24;285(39):30224-32.
doi: 10.1074/jbc.M110.123554. Epub 2010 Jul 20.

Yeast lipids can phase-separate into micrometer-scale membrane domains

Christian Klose  1 , Christer S Ejsing, Ana J García-Sáez, Hermann-Josef Kaiser, Julio L Sampaio, Michal A Surma, Andrej Shevchenko, Petra Schwille, Kai Simons
Affiliations

Yeast lipids can phase-separate into micrometer-scale membrane domains

Christian Klose et al. J Biol Chem. .

Abstract

The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although there is biochemical evidence for lipid raft-dependent protein and lipid sorting in the yeast Saccharomyces cerevisiae, direct evidence for an interaction between yeast sphingolipids and the yeast sterol ergosterol, resulting in membrane domain formation, is lacking. Here we show that model membranes formed from yeast total lipid extracts possess an inherent self-organization potential resulting in liquid-disordered-liquid-ordered phase coexistence at physiologically relevant temperature. Analyses of lipid extracts from mutants defective in sphingolipid metabolism as well as reconstitution of purified yeast lipids in model membranes of defined composition suggest that membrane domain formation depends on specific interactions between yeast sphingolipids and ergosterol. Taken together, these results provide a mechanistic explanation for lipid raft-dependent lipid and protein sorting in yeast.

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Figures

FIGURE 1.
FIGURE 1.
GUVs from total lipid extracts show micrometer-scale phase separation. DiD (0.1 mol %) was used as marker for the Ld phase (red), whereas BODIPY-cholesterol (0.1 mol %) distributes uniformly in the bilayer (green). A, DiD is excluded from certain areas of the GUV as revealed by confocal fluorescence microscopy (scale bars = 10 μm). B, autocorrelation curves obtained by two-focus scanning FCS. Curves for the Ld phase (red) were obtained from areas of the GUVs that were labeled with DiD (0.1 mol %). Curves for the Lo phase were obtained from areas excluding DiD. Curves were recorded by detecting BODIPY-cholesterol (0.01 mol %). C, the diffusion coefficients calculated were D(Lo) = 0.13 (±0.02) and D(Ld) = 3.1 (±0.2) (± S.E.; n ≥ 28).
FIGURE 2.
FIGURE 2.
Total lipid extracts from sphingolipid metabolism mutants exhibit altered membrane properties. A, C-laurdan spectroscopy with LUVs from total lipid extracts. LUVs from extracts of the sphingolipid metabolism mutants sur2Δ (GP = 0.1; ±0.006) and elo3Δ (GP = 0.043; ±0.003) have a reduced order as compared with wild type (GP = 0.133; ±0.002). Error bars indicate S.E. (n = 3). The differences are statistically significant (p < 0.05 for sur2Δ versus wild type and p < 0.01 for elo3Δ versus wild type). B, GUVs formed from the same extracts as in A visualized by confocal fluorescence microscopy. Membranes were labeled with the Ld phase marker DiD (0.1 mol %). Arrowheads indicate membrane domains that exclude DiD. No phase separation could be observed in GUVs made from sur2Δ and elo3Δ extracts. Scale bars: wild type = 10 μm; sur2Δ = 20 μm; elo3Δ = 10 μm.
FIGURE 3.
FIGURE 3.
Phase transition temperature of IPC. Phase transition temperatures (Tm) were determined by DPH fluorescence anisotropy measurements as described under “Experimental Procedures.” The Tm of IPC was determined to be 53.4 °C (±0.2; S.E.; red). As a control, the Tm of C18-SM was determined (Tm = 44.0 °C; ±0.1; S.E.; black). (n = 3).
FIGURE 4.
FIGURE 4.
IPC and ergosterol form ordered membranes. Membrane order was measured by C-laurdan spectroscopy of LUVs consisting of binary and ternary equimolar lipid mixtures. Error bars indicate S.E. (n ≥ 3). SM = C18-SM; chol = cholesterol; erg = ergosterol; PI = yeast PI; PC = palmitoyl-oleyl phosphatidylcholine.
FIGURE 5.
FIGURE 5.
Phase separation of GUVs containing IPC/yeast PI/ergosterol. A, GUVs produced from equimolar mixtures of IPC, yeast PI, and ergosterol show micrometer-scale phase separation as observed by confocal fluorescence microscopy. DiD (0.1 mol %) was used as a marker for the Ld phase, whereas BODIPY-cholesterol (0.1 mol %) labels both the Ld and the Lo phase. DiD is excluded from parts of the GUVs (scale bar = 10 μm). B, autocorrelation curves obtained by two-focus scanning FCS. Curves for the Ld phase (red) were obtained from areas of the GUVs that were labeled with DiD (0.1 mol %). Curves for the Lo phase were obtained from areas excluding DiD. Curves were recorded by detecting BODIPY-cholesterol (0.01 mol %). C, the diffusion coefficients calculated were D(Lo) = 0.35 (±0.06) and D(Ld) = 2.2 (±0.2) (± S.E.; n ≥ 4).
FIGURE 6.
FIGURE 6.
Membrane order of GUVs containing IPC/yeast PI/ergosterol as determined by C-laurdan microscopy. GUVs as in Fig. 5 were labeled with 0.05 mol % Rh-DOPE as a marker for the Ld phase, stained with C-laurdan, and imaged by two-photon fluorescence microscopy. A, GUVs show phase separation as indicated by the exclusion of Rh-DOPE from parts of the GUVs (scale bar = 10 μm). The false-colored GP image indicates differences in membrane order of the two domains. The color bar indicates the GP values. B, C-laurdan GP values as sampled from the GP images. Error bars indicate S.E., n = 3.
FIGURE 7.
FIGURE 7.
Structural differences between yeast and mammalian sphingolipids and sterols. Depicted are the structures of the major IPC species (IPC 18:0;3/26:0;1), C18-SM (SM), ergosterol, and cholesterol. Differences are highlighted and described in the text.
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