Phospholipid Translocation as Driver of Cholesterol (Re)Distribution

Abstract

The asymmetric distribution of the major phospholipid classes between the two leaflets of the mammalian plasma membrane (PM) has been known for almost 50 years. In contrast, the interleaflet distribution of cholesterol, the single most abundant component of the PM, has been actively debated but remains unclear. Using advanced enzyme-assisted lipidomics, we performed a detailed quantification of the phospholipid distribution in the two PM leaflets of red blood cells. These measurements revealed a significant imbalance in the total abundance of phospholipids, with the cytoplasmic leaflet containing ∼50% more phospholipids than the exoplasmic leaflet. Because the total area of the two leaflets in a bilayer must be equalized, another lipid component is likely asymmetrically distributed to buffer the area difference. Cholesterol, a lipid capable of rapid flip-flop and comprising a large fraction of the bilayer, may perform this balancing act by enriching in the exoplasmic leaflet. Furthermore, due to its ability to rapidly flip between leaflets, cholesterol may dynamically buffer PM properties in response to changes in the phospholipid leaflet composition. Thus, we hypothesize that cholesterol redistribution relieves the tension arising from phospholipid flip-flop and test this hypothesis both computationally and experimentally. Coarse-grained and all-atom molecular dynamics simulations of biomimetic bilayers reveal that cholesterol indeed changes its distribution in response to phospholipid flip-flop and by doing so, reduces the accumulated leaflet tension. To understand and fully characterize the resulting changes in bilayer properties, we further analyze the distinct effects of phospholipid and cholesterol flip-flop on the bilayer elastic moduli, spontaneous curvature and acyl chain order parameters. These findings are complemented with experimental measurements of dynamics in the morphology and physical properties of model membranes with and without cholesterol. Thus, our studies provide mechanistic insights into the mechanisms of cholesterol's distribution in a biological membrane.