PS membrane asymmetry influences the folding and insertion of a transmembrane helix

Abstract

The plasma membrane (PM) contains 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. The influence of PS asymmetry and its loss on membrane protein structure and organization have not been widely addressed. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence, which can enable topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at acidic pH -as its name implies, is a useful and 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 influences 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 developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa 568 fluorophore as a new probe to quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets caused by the asymmetric distribution of charged lipids between the leaflets. 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 aspects of protein-membrane interactions. 5 symmetric PS vesicles, our lab previously showed that both the insertion pK and the insertion depth in State II decreased with increasing PS concentration (27). We proposed that both observations can be explained by an unfavorable interaction between the negatively charged PS head group and the seven negative charges present on pHLIP at neutral pH (27). However, it is unknown how a more biologically faithful model system, one in which PS is enriched in the inner leaflet, would influence pHLIP insertion. To test this, we modified a technique for producing freely-floating vesicles in order to mimic the asymmetric distribution of PS in the PM (28). We found that PS asymmetry caused an increase in the midpoint of pHLIP insertion, suggesting that for a physiologically relevant transbilayer charge distribution-i.e., less negative charge in the outer compared to the inner leaflet-lowers the energetic barrier for insertion. This finding proposes a general role for PS asymmetry in promoting the folding of TM proteins. 6 Materials and MethodsMaterials. The lipids 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (POPCd31), 1palmitoyl-2-oleoyl-sn-glycero-3-pho...