Reconstitution and Assay of Biogenic Membrane-Derived Phospholipid Flippase Activity in Proteoliposomes

Abstract

Newly synthesized phospholipids in biogenic (self-synthesizing) membranes, such as the eukaryotic endoplasmic reticulum (ER) and bacterial cytoplasmic membrane (bCM), are initially located in the cytoplasmic leaflet of the membrane bilayer. In order to populate the exoplasmic leaflet of the bilayer to allow uniform bilayer growth, phospholipids have to be translocated (flipped) to the opposite membrane leaflet. Flipping does not occur spontaneously; in artificial bilayers, liposomal systems, and certain biomembranes, transverse movement of phospholipids, i.e., spontaneous transfer of a phospholipid from one side of the membrane to the other, occurs only very slowly—if at all. Nevertheless, it is clear from a number of studies that transbilayer movement of phospholipids occurs rapidly in the ER and bCM by a bidirectional, diffusion process facilitated by specific membrane proteins and requiring no metabolic energy. The latter observation largely rules out conventional activities of the ABC family of transporters, which are involved in energy-coupled, vectorial transport of solutes and some phospholipids. Although certain α-helical membrane-spanning peptides appear able to promote transbilayer movement of some classes of phospholipid in synthetic membranes (1,2), it is generally hypothesized that specific proteins, called flippases, are required for phospholipid flipping in the ER and bCM (1,3–9).