Phase separation in 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. Budding yeast is currently the only known living, unperturbed cell system in which reversible, micron-scale phase separation of a membrane has been clearly observed. In this dissertation, I show how the yeast cell adapts and tunes its vacuole membrane to invoke phase separation. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. I measure miscibility transition temperatures of vacuole membranes and find that they scale linearly with the temperature cells are grown at. Then, I purify yeast vacuoles in two growth stages, one in which vacuole membranes phase separate, and the other in which the membranes do not, and, I analyze the lipidome for the membrane. I find that the lipid profile of the vacuole membrane changes dramatically through the growth stages. Melting temperatures have been measured (or can be estimated) for about half of the lipids in vacuole membranes. For this set of lipids, I observe that membranes that phase separate have slightly higher melting temperatures. Overall, this research demonstrates that yeast physiologically adapt to maintain proximity to the transition and identifies key molecular players for membrane phase separation in living cells.