Fast translocation of phosphatidylcholine to the outer membrane leaflet after its synthesis at the inner membrane surface in human erythrocytes

Abstract

The translocation rate of [14C]phosphatidylcholine to the outer membrane leaflet of human erythrocytes after its primary synthesis from lysophosphatidylcholine by acylation with 14C-labeled oleic or palmitic acid in the inner leaflet has been measured by following the time-dependent increase of cleavability of 14C-labeled phospholipids by external phospholipase A2 (5 min, 37 degrees C). Immediately after a short acylation time period of 10 min about 20% of the newly synthesized [14C]phosphatidylcholine are already detectable in the outer leaflet. After an incubation of 1 h at 37 degrees C following 10 min of acylation the fractions of labeled and native phosphatidylcholine accessible to the lipase are identical, which demonstrates that [14C]phosphatidylcholine has attained the same asymmetric distribution as its endogenous analogue. The calculated halftime of the outward translocation is about 20 min and its activation energy is low, 30 kJ/mol. Translocation is inhibited by a 5 min treatment with phenylglyoxal following acylation. A fast translocation is not observed for newly synthesized phosphatidylethanolamine. Results suggest a selective, protein-mediated outward translocation of newly synthesized phosphatidylcholine.