Lipid Phase Separation Induced by the Apolar Polyisoprenoid Squalane Demonstrates Its Role in Membrane Domain Formation in Archaeal Membranes

Abstract

Archaea synthesize methyl-branched, ether phospholipids, which confer the archaeal membrane exceptional physicochemical properties. A novel membrane organization was proposed recently to explain the thermal and high pressure tolerance of the polyextremophilic archaeon Thermococcus barophilus. According to this theoretical model, apolar molecules could populate the midplane of the bilayer and could alter the physicochemical properties of the membrane, among which is the possibility to form membrane domains. We tested this hypothesis using neutron diffraction on a model archaeal membrane composed of two archaeal diether lipids with phosphocholine and phosphoethanolamine headgroups in the presence of the apolar polyisoprenoid squalane. We show that squalane is inserted in the midplane at a maximal concentration between 5 and 10 mol % and that squalane can modify the lateral organization of the membrane and induces the coexistence of separate phases. The lateral reorganization is temperature-and squalane concentration-dependent and could be due to the release of lipid chain frustration and the induction of a negative curvature in the lipids.

Apolar Polyisoprenoids Located in the Midplane of the Bilayer Regulate the Response of an Archaeal-Like Membrane to High Temperature and Pressure

“…The spectra for the membrane containing h-squalane and d-squalane overlap fairly well except within the region corresponding to the midplane of the bilayer (−10 to 10 Å) where the sample containing d-squalane shows an excess of scattering density. This indicates that squalane is located in the midplane of the bilayer and is in agreement with the findings of Salvador-Castell et al (2020b). In this work we see that this localization of squalane is seen in both Phase LT and Phase HT at low (20 bar) and high (1,000 bar) pressure.…”

“…The formation of a new phase, Phase HT , is most likely due to the presence of lipids with different preferred curvatures within the membrane as seen by Salvador-Castell et al (2020b). The bilayer is made up of two phytanyl lipids with different polar head groups (DoPhPC and DoPhPE) and the apolar isoprenoid squalane.…”

“…Here we show that squalane is also capable of promoting the formation of Phase HT at high pressures. Previously at ambient pressure, it was shown that the phase coexistence and lateral organization within a DoPhPC:DoPhPE membrane was squalane dependent leading to the hypothesis that squalane may promote the formation of membrane domains (Salvador-Castell et al, 2020b). That would mean that the ability of squalane to favor phase separation at high pressures, may indicate that squalane could also promote domain formation in archaea that live at such high-pressure conditions.…”

“…In order to explain the ability of T. barophilus to live at high temperature in presence of bilayer forming lipids, it was hypothesized that apolar lipids sit at the midplane of the bilayer and provide enhanced stability (Cario et al, 2015). The ability of apolar lipids such as squalane to localize to the midplane of the lipid bilayer has been confirmed in model membranes of both bacteriallike and archaea-like lipids (Hauß et al, 2002;Salvador-Castell et al, 2020b). The presence of apolar molecules is capable of modulating membrane physicochemical properties, for example, the presence of squalane at the bilayer midplane has been shown to increase permeability to water and decrease proton permeability (Haines, 2001).…”

“…The presence of apolar molecules is capable of modulating membrane physicochemical properties, for example, the presence of squalane at the bilayer midplane has been shown to increase permeability to water and decrease proton permeability (Haines, 2001). Apolar molecules at the midplane have been shown to increase the tendency toward negative membrane curvature by reducing packing frustration (Salvador-Castell et al, 2020a,b) and to play a role in phase separation and domain formation (Gilmore et al, 2013;Salvador-Castell et al, 2020b). Such isoprenoid hydrocarbons are found in all archaea (Langworthy et al, 1982) suggesting this hypothesis could be extended to explain adaptation to high temperature and pressure in other archaea processing lipid bilayers.…”