No abstract

Membranes of vacuoles, the lysosomal organelles of Saccharomyces cerevisiae (budding yeast), undergo extraordinary changes during the cell’s normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and vacuole membranes phase separate into micrometer-scale domains of two liquid phases. Recent studies suggest that these domains promote yeast survival by organizing membrane proteins that play key roles in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether cells regulate phase transitions in response to new physical conditions and how this occurs. Here, we measure transition temperatures and find that after an increase of roughly 15 °C, vacuole membranes appear uniform, independent of growth temperature. Moreover, populations of cells grown at a single temperature regulate this transition to occur over a surprisingly narrow temperature range. Remarkably, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. We isolate vacuoles from yeast during the rapid stage of growth, when their membranes do not natively exhibit domains. Ergosterol is the major sterol in yeast. We find that domains appear when ergosterol is depleted, contradicting the prevalent assumption that increases in sterol concentration generally cause membrane phase separation in vivo, but in agreement with previous studies using artificial and cell-derived membranes.

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Recent studies suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a key role in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether yeast stringently regulate the phase transition and how they respond to new physical conditions. Here, we measure transition temperatures - an increase of roughly 15°C returns vacuole membranes to a state that appears uniform across a range of growth temperatures. We find that broad populations of yeast grown at a single temperature regulate the transition to occur over a surprisingly narrow temperature range. Moreover, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. Specifically, we test how levels of ergosterol, the main sterol in yeast, induce or eliminate membrane domains. We isolate vacuoles from yeast during their rapid stage of growth, when their membranes do not natively exhibit domains. We find that membrane domains materialize when ergosterol is depleted, contradicting the assumption that increases in ergosterol cause membrane phase separation in vivo, and in agreement with prior studies that use artificial and cell-derived membranes.

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micron-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (~50%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These results fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.

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...