LAUSR

Significance Membrane proteins that translocate various compounds across biological membranes play important roles in maintaining intracellular homeostasis in cells. Scramblases are membrane proteins that translocate phospholipids between the inner and outer leaflets of the plasma membrane. However, the mechanisms through which these scramblases translocate phospholipids carrying a hydrophilic residue across the hydrophobic membranes are still unclear. Here, we developed a microsystem containing lipid-bilayer membrane arrays with asymmetrically distributed fluorescent phospholipids. Using this system, we measured phospholipid transport mediated by the TMEM16F scramblase at the single-molecule level, thereby enabling characterization of the biophysical features of phospholipid transport. Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.