Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.

The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (Liu et al., 2017). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.