Cholesterol Transbilayer Distribution in Mammalian Cells: Mechanisms and Functions

Abstract

Phospholipids and proteins in the plasma membrane (PM) bilayer are well established to be both laterally and transversely asymmetric. However, cholesterol transbilayer distribution within PM remains inconclusive. A fluorescent sterol, dehydroergosterol (DHE), is well-documented to primarily reside in the inner leaflet of PM (∼80%). It remains unclear if native cholesterol shares this surprising asymmetry.The purpose of this study is to first determine cholesterol transbilayer distribution in PM. We developed a protocol that is capable of analyzing cholesterol in a leaflet-specific manner using β-cyclodextrin (βCD). In symmetric large unilamellar vesicles (LUVs), we found that cholesterol flip-flop is rapid at 37°C, leading to 100% extraction/exchange by βCD. However, at 0°C, βCD is only able to remove exactly 50% cholesterol, indicating a complete inhibition of cholesterol flip-flop. We then applied this protocol to erythrocytes and found that only 20-25% cholesterol is accessible by βCD at 0°C, although 100% is accessible at 37°C. Therefore, most cholesterol resides in the inner leaflet of PM in mammalian cells.We then investigated the role of phospholipid transbilayer asymmetry on cholesterol asymmetry. We found that, only in the asymmetric LUVs with long chain (22 carbon) sphingomyelin in the outer leaflet, could we observe cholesterol enrichment in the inner leaflet. Similar experiments with short chain (16 carbon) sphingomyelin and phosphatidylcholine (16:0/18:1) failed to influence cholesterol distribution.We therefore conclude that, like DHE, cholesterol is enriched in the inner leaflet of PM and that this asymmetry is regulated by the phospholipid asymmetry and, more specifically, by long chain sphingolipids. We suggest that the current lipid raft model may need to be revised to reflect this cholesterol asymmetry.