The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (L_d) and/or liquid ordered (L_o) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within L_d domains. However, the ILVs that form are rich in L_o lipids. We interpret this surprising observation as preferential recruitment of the L_o phase beneath the ESCRT complex due to its increased rigidity, where the L_d phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. L_d domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.