The Role of Growth Temperature and Lipid Composition in Phase Separation of Yeast Vacuole Membranes

Abstract

Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into two coexisting liquid phases. This phenomenon provides a physical mechanism for lateral organization of biological membranes. Recent studies show that these domains play a key role in a central eukaryotic signaling pathway. Howthe cell adapts and tunes its vacuole membrane to invoke phase separation is still largely unknown. Here we examine how membrane composition changes due to two different environmental stimuli: growth temperature and nutrient depletion. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. We test this hypothesis by growing yeast at 25°C and 30°C and find that the yeast adapts to this change by dramatically shifting the demixing temperature of vacuole membranes (to 29.7°C and 42.7°C respectively). This stark change in vacuole miscibility temperatures scales with growth temperature both in live cells and in isolated vacuoles, showing that yeast alter their membrane compositions to maintain phase separation. We also directly investigate the role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. The yeast growth cycle starts with a logarithmic phase of rapid growth followed by a stationary phase in which glucose becomes scarce and vacuole membranes exhibit coexisting liquid domains. We isolate vacuoles from yeast in the logarithmic phase of growth, in which the membranes do not natively exhibit domains. Using fluorescence microscopy, we observe changes in vacuole phase separation as a function of ergosterol content. We find the surprising result that domains appear in vacuole membranes with reduced ergosterol.