Eukaryotic cell plasma membranes posses an asymmetric transbilayer distribution of phospholipids. The choline-containing phospholipids, phosphatidylcholine and sphingomyelin are enriched in the outer monolayer, while the aminophospholipids, particularly phosphatidylserine (PS), are enriched on the cytoplasmic surface. The dissipation of this gradient and exposure of PS on the surface of the cell, induces the recognition and destruction of the cell by macrophages and, in blood cells, the activation of plasma clotting factors. The action of several lipid-specific transporters, including an inwardly-directed, aminophospholipid-specific, ATPase or “flippase,” outwardly-directed "floppases," and a bi-directlional, non-specific "scramblase", play important roles in the generation, maintenance and regulated dissipation of plasma membrane phospholipid asymmetry. The aminophospholipid flippase is highly selective for its lipid substrate. In the human erythrocyte, PS is the preferred substrate, requires energy in the form of ATP to catalyze lipid translocation, and is sensitive to inhibition by sulfhydryl reagents. Although phosphatidylethanolamine, and other PS analogs, are transported by the flippase, transport is much slower, but is also ATP dependent and sulfhydryl reagent sensitive. Perhaps the most distinguishing characteristic, however, is an absolute specificity for the sn-1,2 configuration of the glycerol moieity of PS, but a lack of stereochemical specificity for the serine headgroup. We, and others, have purified and characterized two candidate flippases: a PS-stimulated ATPase from human erythrocytes and a P4-ATPase (ATP8A1). These enzymes share the same exquisite structural specificity for PS transport observed in intact cells, which is distinct from other PS-binding proteins. Our current studies focus on using the characteristic lipid specificity of the PS flippase to determine the substrate binding site of ATP8A1 and elucidate the transport pathway through the protein, using molecular modeling and selective mutagenesis. Several models recently proposed by others for the mechanisms of lipid transport by P4-ATPases will be discussed and compared to our modeling and experimental data.

Substrate secificity of the plasma membrane phosphatidylserine flippase

249th National Meeting (2015)

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