Absence of transbilayer diffusion of spin-labeled sphingomyelin on human erythrocytes. Comparison with the diffusion of several spin-labeled glycerophospholipids.

We have measured the transbilayer diffusion at 4 degrees C of spin labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid in the human erythrocyte membrane. Measurements were also carried out in ghosts, released without ATP, and on large unilamellar vesicles made with total lipid extract. As reported previously (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), the amino phospholipids are rapidly transported from the outer to the inner leaflet on fresh erythrocytes, whereas phosphatidylcholine diffuses slowly. We now show that phosphatidic acid behaves like phosphatidylcholine: approximately 10% is internalized in 5 h at 4 degrees C. Under the same experimental conditions, no inward transport of sphingomyelin can be detected. In ghosts resealed without ATP, all glycerophospholipids tested diffuse slowly from the outer to the inner leaflet (approx. 10% in 5 h) while no transport of sphingomyelin is seen. Finally in lipid vesicles, the inward diffusion of all glycerophospholipids is less than 2% in 5 h and a very small transport of sphingomyelin can be measured. These results confirm the existence of a selective inward aminophospholipid transport of fresh erythrocytes and suggest a slow and passive diffusion of all phospholipids on ghosts, resealed without ATP, as well as on lipid vesicles.