Sterol–lipids enable large-scale, liquid–liquid phase separation in bilayer membranes of only two components

Despite longstanding excitement and progress toward understanding liquid–liquid phase separation in natural and artificial membranes, fundamental questions have persisted about which molecules are required for this phenomenon. Except in extraordinary circumstances, the smallest number of components that has produced large-scale, liquid–liquid phase separation in bilayers has stubbornly remained at three: a sterol, a phospholipid with ordered chains, and a phospholipid with disordered chains. This requirement of three components is puzzling because only two components are required for liquid–liquid phase separation in lipid monolayers, which resemble half of a bilayer. Inspired by reports that sterols interact closely with lipids with ordered chains, we tested whether phase separation would occur in bilayers in which a sterol and lipid were replaced by a single, joined sterol–lipid. By evaluating a panel of sterol–lipids, some of which are present in bacteria, we found a minimal bilayer of only two components (PChemsPC and diPhyPC) that robustly demixes into micron-scale, liquid phases. It suggests an additional role for sterol–lipids in nature, and it reveals a membrane in which tie-lines (and, therefore, the lipid composition of each phase) are straightforward to determine and will be consistent across multiple laboratories.