Flippase

Flippases are transmembrane lipid transporter proteins located in the cell membrane. They are responsible for aiding the movement of phospholipid molecules between the two layers, or leaflets, that compose the membrane (transverse diffusion, also known as a "flip-flop" transition). Flippases move lipids to the cytosolic layer, usually from the extracellular layer. Floppases do the opposite, moving lipids to the extracellular layer. Both flippases and floppases are powered by ATP hydrolysis and are either P4-ATPases or ATP-Binding Cassette transporters. Scramblases are energy-independent and transport lipids in both directions.^[1][2][3]

In organisms, the cell membrane consists of a phospholipid bilayer. In the bilayer, the phospholipid molecule is movable. These movements are categorized into two types, lateral movements and transverse movements (also called Flip-Flop). The first is the lateral movement, where the phospholipid moves horizontally on the same side of the membrane. Lateral movement is very fast, with an average speed of up to 2 mm per second.^[4] Transverse movement is the movement of phospholipid molecule from one side of the membrane to the other. Transverse movement is very slow, and without the assistance of enzymes, it may only occur even once a month.^[4] The reason is that the polar head groups of phospholipid molecule cannot pass easily through the hydrophobic center of the bilayer, limiting their diffusion in this dimension.

Although Flip-Flop is slow, This movement is necessary to continue their normal function of growth and mobility.^[5] The possibility of active maintenance of an asymmetric distribution of molecules in the phospholipid bilayer was predicted in the early 1970s by Mark Bretscher.^[6]  Asymmetry molecule of membrane has been proved to related to the broad physiological implications of lipid asymmetry, from cell shape determination to critical signaling processes like blood coagulation and apoptosis.^[7] Many cells maintain asymmetric distributions of phospholipids between their cytoplasmic and exoplasmic membrane leaflets. The loss of asymmetry, in particular the appearance of the anionic phospholipid phosphatidylserine on the exoplasmic face, can serve as an early indicator of apoptosis^[8] and as a signal for efferocytosis.^[9]

Lipid transporters transport or flip lipids across the bilayers. There exist three major classes of Lipid Transporters:

1. P-type Flippase
2. ABC Flippase
3. Scramblases

P-type Flippase and ABC Flippase are energy-dependent (ATP) enzyme that can create lipid asymmetry and transport specific lipids. Scramblases are energy-independent enzyme that can dissipate lipid asymmetry and have a broad lipid specificity.^[11] Flippases belong to P-type Flippase^[10] and it moves lipids from the exoplasmic to the cytosolic face.^[11]

P4-type flippase contains a large transmembrane segment and two major subunits, a catalytic domain called the alpha-subunit and an accessory domain named beta-subunit.^[5] Transmembrane segments contain 10 transmembrane alpha helices and this domain together with beta-subunit plays important role in stability, localization and recognition of substrate (lipid) of flippase.^[5] Alpha-subunits include A, P and N domains and each of them corresponds to a different function of flippase. A-domain is an actuator segment of flippase that facilitates phospholipid binding through conformational change of the complex, although it does not bind the phospholipid itself. P-domain is responsible for binding phosphate, a product of ATP hydrolysis. The next domain is N-domain, whose job is to bind to substrate (ATP).^[5] Finally, a C-terminal autoregulatory domain has been identified, whose function differs between yeast and mammalian P4-type flippases.^[12]

In order to bind specific lipid on the outer layer of membrane, P4-type flippase needs to be phosphorylated by ATP on its P-domain. After ATP hydrolysis and phosphorylation, P4-type flippases undergo conformational change from E1 to E2 (E1 and E2 stand for different conformations of flippases).^[5] Further conformational change is induced by the binding of a phospholipid, resulting in the E2Pi.PL conformation.^[12] The flippase in its E2 conformation can then be dephosphorylated at its P-domain, allowing the lipid to be transported to the inner layer of membrane, where it diffuses away from the flippase. As the phospholipid dissociates from the complex, a conformational change on flippase occurs from E2 back to E1 readying it for the next cycle of lipid transportation.^[5]

The A-domain binds to the N-domain after that domain releases ADP. The A-domain can bind to the N-domain by a TGES four-amino-acid motif when the P-domain is phosphorylated. The release of ADP from the N-domain transitions the complex from the E1P-ADP state to the E2P state, which might be further stabilized by binding of the C-terminal regulatory domain. Binding of a phospholipid to the first two transmembrane segments induces a conformational change that rotates the A domain outward by 22 degrees, allowing dephosphorylation of the P domain. Dephosphorylation of the P-domain is energetically coupled to translocation of the polar phospholipid head across the membrane leaflets.^[12]

• Scramblase