Regulation of phospholipid distribution in the lipid bilayer by flippases and scramblases.

Cellular membranes function as permeability barriers that separate cells from the external environment or partition cells into distinct compartments. These membranes are lipid bilayers composed of glycerophospholipids, sphingolipids and cholesterol, in which proteins are embedded. Glycerophospholipids and sphingolipids freely move laterally, whereas transverse movement between lipid bilayers is limited. Phospholipids are asymmetrically distributed between membrane leaflets but change their location in biological processes, serving as signalling molecules or enzyme activators. Designated proteins - flippases and scramblases - mediate this lipid movement between the bilayers. Flippases mediate the confined localization of specific phospholipids (phosphatidylserine (PtdSer) and phosphatidylethanolamine) to the cytoplasmic leaflet. Scramblases randomly scramble phospholipids between leaflets and facilitate the exposure of PtdSer on the cell surface, which serves as an important signalling molecule and as an 'eat me' signal for phagocytes. Defects in flippases and scramblases cause various human diseases. We herein review the recent research on the structure of flippases and scramblases and their physiological roles. Although still poorly understood, we address the mechanisms by which they translocate phospholipids between lipid bilayers and how defects cause human diseases.

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure.

The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the flippase activity normally counteracted Xkr8's ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and flippase down-regulation.

Single-molecule analysis of phospholipid scrambling by TMEM16F.

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.

The tertiary structure of the human Xkr8-Basigin complex that scrambles phospholipids at plasma membranes.

Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.

Flippase and scramblase for phosphatidylserine exposure.

In various biological processes, phosphatidylserine (PtdSer) that is normally sequestered to the inner leaflet of the plasma membrane (PM) is exposed to the cell surface. When platelets are activated, they expose PtdSer to activate the blood-clotting factors. Cells undergoing apoptosis and senescent neutrophils expose PtdSer that is recognized as an 'eat me' signal by phagocytes for clearance. The PtdSer-exposure and its internalization are mediated by phospholipid scramblases and flippases, respectively. Both have recently been molecularly identified, and their functional mechanism and physiological roles are being elucidated.