Reconstitution approaches for understanding cellular lipid dynamics

Lipid transport is essential for membrane biogenesis and maintenance. Newly synthesized phospholipids are produced on the cytosolic leaflet of the endoplasmic reticulum (ER). Redistribution of lipids in cells is achieved by lipid scramblases across the lipid bilayer or alternatively, by lipid transport proteins (LTPs) to other organelles. Despite their important functions, the molecular identities and mechanisms of these proteins are only now emerging. Here, we describe reconstitution approaches with purified proteins and artificial membranes to study lipid dynamics in vitro, including a fluorescence-based assay to identify lipid scrambling activity and a fluorescence resonance energy transfer (FRET)-based assay to assess protein-mediated lipid transfer between liposomes or between liposomes and lipid monolayers. Together, these methods enable mechanistic studies of cellular lipid dynamics regulated by proteins.