Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature

SHEETZ AND SINGER have proposed that the actions of certain amphipathic drugs in producing curvature of the membrane of human red blood cells (RBCs) could be explained by assuming that these drugs selectively entered one of the leaflets of the lipid bilayer, thus causing lateral expansion of that leaflet relative to the other1. The membrane was therefore forced to curve in order to accommodate this extra material and the direction of this curvature was predictable, leading to production of either stomatocytes or echinocytes. Membrane curvature, leading to the formation of stomatocytes or echinocytes, can also be produced by subjecting human RBCs to phospholipase C attack2–5. In this report we attempt to explain these phospholipase C-induced changes in terms of Sheetz and Singer's model, taking into account the observation that 1,2-diacylglycerol (the product of phospholipase C action), unlike phospholipids, can migrate rapidly across the membrane bilayer. Rapid transbilayer migration of 1, 2-diacylglycerol has previously been inferred from the observed increase in the rate of synthesis of phosphatidate in intact RBCs that have been exposed to phospholipase C: this phosphatidate is formed from diacylglycerol and cytosolic ATP by diacylglycerol kinase, presumably at the inner surface of the membrane2. We have now used this procedure, in which the endogenous diacylglycerol kinase of the erythrocyte is used to trap 1, 2-diacylglycerol appearing on the inner surface of the red cell membrane, to compare the time courses of phospholipase C-catalysed production of diacylglycerol and of the appearance of phosphatidate, and thus obtain an estimate of the rate of transbilayer migration of 1, 2-diacylglycerol.