Phospholipid Flippase Activity In Giant Plasma Membrane Vesicles And Rat Basophilic Leukemia-2H3 (Rbl-2H3) Cells

Abstract

Phospholipid flippase proteins regulate plasma membrane phospholipid asymmetry. Characterization of these transmembrane proteins is an ongoing area of research, as the mechanisms by which flippases function is poorly understood and the identification of proteins that exhibit flippase activity is incomplete. We have investigated flippases activity in our giant plasma membrane vesicles (GPMVs) and intact RBL-2H3 cells. Results from our fluorescence quenching assays provide insight on flippase kinetics as well as flippase mediated phospholipid distribution across the membrane bilayer. With fluorescent annexin V binding assays, we also demonstrate that RBL-2H3 cells can be stimulated or perturbed in various ways that promote flippase mediated phosphatidylserine (PS) exposure on the cell surface. In particular, activated RBL-2H3 cells display distinctly punctate PS exposure, while cholesterol depleted cells exhibit sparse PS exposure on the membrane extracellular leaflet. Cells treated with the cysteine modification reagent Nethylmaleimide (NEM) exhibit more uniform PS exposure at the cell surface, consistent with NEM-sensitive flippases. These studies further characterize the sensitivities of plasma membrane flippases and may potentially aid in the identification of additional proteins with flippase functionality.