Phospholipid Preparations to Characterize Protein–Lipid Interactions In Vitro

The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein–lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.