Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures

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.

Remodeling of yeast vacuole membrane lipidomes from the log (1-phase) to stationary stage (2-phases)

“…Ergosterol is the major sterol in yeast, and it has been suggested that phase separation of vacuole membranes could be due to an increase in ergosterol (8). An increase in ergosterol would be consistent with a report that the ratio of filipin staining of sterols in vacuole versus plasma membranes is higher in the stationary stage than the log stage, although vacuole and plasma membrane levels were not measured independently (10, 15). It would also be consistent with the expectation that esterified sterols in lipid droplets are mobilized in the stationary stage by lipophagy, which is required for the maintenance of phase separation in the vacuole membrane (17, 21, 33, 37).…”

“…An obvious candidate for a bait protein was Vph1, which has high expression levels in the log stage (8, 31). Vph1 has been extensively used for visualizing vacuole domains (5, 7–10, 15), and was previously used by us to demonstrate the utility of MemPrep for isolating vacuole membranes in the log stage (25). However, in the stationary stage, expression levels of Vph1 are lower (Fig.…”

“…As a result, in the stationary stage, the vacuole membrane contains micron-scale domains that are enriched in particular lipids and proteins (6–9). This phase transition is reversible, with a transition temperature roughly 15°C above the yeast’s growth temperature (10). Recent studies suggest that phase-separated domains in vacuole membranes regulate the metabolic response of yeast to nutrient limitation through the TORC1 pathway, which regulates protein synthesis, autophagy, lipophagy, and other processes (8, 9, 11–14).…”

“…We expect the lipidome to be important because the few mutations that are known to perturb phase separation of the vacuole involve lipid trafficking and metabolism (8, 9, 15). We expect observable changes in the lipidome because log-stage vacuole membranes do not separate over large shifts in temperature from 30°C to 5°C, whereas stationary stage vacuole membranes do phase separate, even when they are grown over a range of growth temperatures (10). However, we do not necessarily expect the changes driving phase separation to be large.…”

“…Multicomponent model membranes containing ergosterol can separate into coexisting liquid phases (16). Moreover, phase separation in isolated vacuoles is reversed through changes in ergosterol levels (8, 10, 17). Klose et al .…”