The asymmetric organization of phospholipids in biological membranes is created and maintained by a combination of vectoral lipid synthesis, slow spontaneous transbilayer movement and the action of a variety of phospholipid transporters. These transporters comprise three distinct functional classes. Flippases catalyze ATP-dependent import of phospholipids and belong to the P-type superfamily. Floppases are ATP-dependent exporters of the ABC transporter family. Scramblases facilitate the bidirectional dissipation of the asymmetry gradient, often upon cell activation. The regulated disruption of phosphatidylserine (PS) asymmetry by scramblase activation and flippase inhibition results in PS exposure on the cell surface. This early indicator of apoptosis is a recruitment mechanism for engulfment and digestion by macrophages. The simplicity, limited number of transporters, and minimal lipid metabolism of the human red blood cell has made it a useful system for the study of lipid transporters. As a result, red blood cell PS flippase activity has been well characterized. Transport of PS to the cytofacial surface of the plasma membrane is MgATP dependent, is sensitive to vanadate, Ca2+, and sulfhydryl modification and is highly selective for the structure of the lipid substrate. Modification of the amine, carboxyl, or phosphate groups of PS diminishes transport significantly. An important recognition element is the stereochemistry of the C2 carbon of the glycerol backbone; only the sn-1,2 isomer is a productive substrate. Purification of the enzyme responsible for flippase activity based on vanadate-sensitive, and stereoselective, PS-stimulated ATPase activity has yielded a fraction enriched in an 85-90 kDa protein. The prominent ATPase in this preparation is the ABC-transporter ABCB6, a mitchondrial importer of the heme precursor coproporphyrin III. Other ATPases also copurify with PS-stimulated ATPase activity, including the P4-ATPases ATP8A1 and ATP11C. Studies by our group and others have shown that recombinant ATP8A1 possesses PS-specific ATPase activity similar to the red blood cell flippase and, when reconstituted into liposomes, supports flippase activity. Recent studies have also reported that disruption of ATP11C expression results in physiological responses consistent with defective PS flippase activity. Evidence supporting a role for ABCB6 and the P4-ATPases in the activites of the PS flippase and the purfied PS-stimulated ATPase will be discussed.

Phospholipid flippases of the human erythocyte

253rd National Meeting (2017)

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