Single-Molecule Analysis of Phospholipid Scrambling by TMEM16F

Abstract

In eukaryotic cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of plasma membranes. In various biological processes, this asymmetric distribution is disrupted by phospholipid scramblases, e.g., TMEM16F or Xkr-8, that translocate phospholipids bidirectionally between leaflets; however, despite their importance, the biophysical features of the scrambling reaction remain elusive due to technical difficulties to quantify the scrambling activities in vitro. To address this issue, we in this study developed a novel microsystem containing membrane bilayers with asymmetrically distributed phospholipids, and first succeeded to measure the phospholipid scrambling of TMEM16F at single-molecule level, which offered key benefits over macroscopic assay methods as it enabled characterization of the biophysical features of scrambling activities. The single molecule observation, combined with thermodynamic analysis, revealed that TMEM16F transports 4.5 × 104 lipids s−1 at 25 °C, with activation free energy (ΔG‡) of 47 kJ mol−1. Notably, ΔG‡ for spontaneous lipid flip-flop across membrane bilayers is about 100 kJ mol−1, showing that TMEM16F significantly reduces the activation free energy to achieve the efficient phospholipid scrambling. Importantly, the microsystem we have designed could also be used to investigate phospholipid transport by other membrane proteins, e.g., flippase and floppase, which would significantly impact in cell membrane biology.