Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane

Abstract

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A 2 (PLA 2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP 2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15–20% after 60 min incubation with PLA 2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA 2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA 2 hydrolyzed approx. 20% of PI, PIP 2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP 2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA 2 led to a decrease in the intensity of anti-PIP 2 staining. These results demonstrate that significant fractions of PIP 2, PI and PA are localized on the outer surface of the erythrocyte membrane.