| Abstract: | The internalization of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine following their insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes containing the fluorescent lipid 1,2-(palmitoyl-N-4-nitrobenzo-2-oxa-1,3-diazole-amino-caproyl) phosphatidylserine [palmitoyl-C6-NBD)-PS), were incubated with monolayer cell cultures at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells occurred, resulting in prominent labeling of the plasma membrane. However, if the cells were washed and warmed to 7 degrees C for 30 min, the (palmitoyl-C6-NBD)-PS also labeled numerous intracellular membranes. Evidence is presented suggesting that this internalization was not due to endocytosis, but was the result of transmembrane movement of the (palmitoyl-C6-NBD)-PS at the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. This transmembrane movement was reversibly inhibited by depletion of cellular ATP levels and was blocked by treatment with structural analogs of the lipid or by pretreatment of cells with glutaraldehyde or N-ethyl-maleimide. A fluorescent analog of phosphatidylethanolamine [palmitoyl-C6-NBD)-PE), which also exhibits transmembrane movement at the plasma membrane at 7 degrees C (Sleight, R. G., and Pagano, R. E. (1985) J. Biol. Chem. 260, 1146-1154), was further studied. Its transmembrane movement was also inhibited by depletion of cellular ATP levels, or by pretreatment of cells with N-ethylmaleimide. The transmembrane movement of the fluorescent phosphatidylserine and phosphatidylethanolamine analogs was inhibited when the unnatural D-isomers of these lipids were used, further suggesting that this process was stereospecific and therefore likely to have been protein-mediated. |
