ABSTRACT Membranes are molecular interfaces that insulate cells from external stresses, compartmentalize the cytoplasm, and control the flow of nutrients and information 1 . These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two leaflets of living bilayers 2,3 . Previous models of biomembrane structure and function have rested upon the implicit assumption that the two membrane leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PM). Using quantitative lipidomics, we discovered that cytoplasmic leaflets of human erythrocyte PMs have &gt;50% overabundance of phospholipids compared to exoplasmic leaflets. We show that this phospholipid imbalance is enabled by an asymmetric interleaflet distribution of cholesterol 4,5 , which rapidly redistributes to buffer leaflet stresses. Asymmetric phospholipid abundance and composition combine to enrich cholesterol in the exoplasmic PM leaflet. Through a combination of experimental and computational approaches we demonstrate how these lipid distributions impart unique functional characteristics to PMs, including low permeability, surprisingly fast cholesterol diffusion 6 , and resting tension in the cytoplasmic monolayer that regulates protein localization. Our observations of these previously overlooked aspects of membrane asymmetry represent an evolution of classic paradigms 1,7 of biomembrane structure and physiology.

Cell Membranes Sustain Phospholipid Imbalance Via Cholesterol Asymmetry