PS Membrane Asymmetry Influences the Folding and Insertion of a Transmembrane Helix

Abstract

The plasma membrane (PM) has an asymmetric distribution of lipids between the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM but can redistribute to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, membranes contain also proteins. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence and can experience topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at low pH, as its name implies, is a well-characterized model for studying these transitions. Although it is known that the inclusion of PS in symmetric vesicles affects the membrane insertion process of pHLIP by lowering the pH midpoint of insertion, it is unclear how PS asymmetry affects these topological transitions. Here, we studied pHLIP's topology using freely-floating asymmetric phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We found that the protocol to create asymmetric vesicles had to be modified due to the inclusion of PS. We used Annexin V labeled with an Alexa 568 fluorophore as a new way of quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets because of the asymmetric distribution of charged lipids. We thus conclude that lipid asymmetry can have consequences for the behavior of membrane-associated proteins. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important details of protein-membrane interactions.