The Role of Ergosterol in Phase Separation of Yeast Vacuole Membranes

Abstract

Bovine brain cerebroside (z75% galactosyl) has been studied by differential scanning calorimetry (DSC) in mixtures with cholesterol (Chol) and/or brain ceramide, the latter containing mostly C18:0 fatty acid. Complementing previous studies we have focused our attention on mixtures in which the cerebroside is predominant. Pure, fully hydrated cerebroside exhibits an ordereddisordered thermotropic transition centred at 64.8 C. In binary mixtures with Chol (up to 30 mol% Chol) DSC reveals good mixing in that range of concentrations, and a cerebroside behaviour similar to that of sphingomyelin (SM) or DPPC in the presence of Chol: the transition temperature hardly changes, but the transition widens until it becomes hardly detectable at 30 mol% Chol. Mixtures with 0-40 mol% brain ceramide show also good mixing, with a gradual increase in transition temperature as increasing amounts of ceramide are added. Mixtures with phospholipids (brain SM, Tm z 35 C, and egg PC, Tm z-15 C ) display a rather similar behaviour, with a progressive widening of the endotherm and a decrease in the transition temperature of the mixture. A ternary system composed of cerebroside:ceramide:Chol (54:23:23 mol ratio) appears to be a homogeneous gel phase with a transition to a fluid phase centred at z 67 C. The calorimetric data are supported by Laurdan measurements of bilayer order as a function of T. Partial phase diagrams of cerebroside:Chol and cerebroside:ceramide have been obtained from the above data. To date, the only type of membrane in a living cell that is known to phase separate into large (micron-scale) domains is that of the yeast vacuole. In contrast, within plasma membranes, the distribution of lipids and proteins appears to be heterogeneous on short time and length scales. In model membranes containing ternary or quaternary mixtures of lipids, the presence of submicron domains has been inferred by indirect methods such as FRET, neutron diffraction, and NMR. In cases when indirect methods rely on model-dependent assumptions, it is powerful to directly image the membranes. However, direct imaging of submicron domains presents significant experimental challenges. Here, we present two novel cryo electron microscopy methods to directly image domains in submicron vesicles containing ternary and quaternary lipid mixtures. First, we use a probe-free method to image domains that are thicker than the surrounding membrane. To do so, we leverage existing ternary phase diagrams (assessed via fluorescence microscopy) and AFM data to identify lipid types and ratios likely to produce thickness mismatches = 1 nm. Second, we label domains in quaternary mixtures of lipids with an electron-dense protein probe, and we image the distribution of this probe. 394-PosBiological membranes are asymmetric such that lipid compositions in the inner and outer leaflet of the lipid bilayer are different. While the outer leaflet contains lipid-mixtures containing high-Tm (lipid gel-to liquid melting temperature) lipids, low-Tm lipids and cholesterol, which allow...