Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts.

We have previously shown that a fluorescent derivative of phosphatidic acid, 1-acyl-2-[N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl]phosphatidic acid (C6-NBD-PA) is rapidly transferred from liposomes to Chinese hamster fibroblasts at 2 degrees C, resulting in intense labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope, but not the plasma membrane. During this labeling, C6-NBD-PA is metabolized predominantly to fluorescent diacylglycerol (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study we investigated the mechanism by which C6-NBD-PA enters cells and is translocated to intracellular membranes at low temperature. (i) When hydrolysis of C6-NBD-PA to diacylglycerol was prevented by using a nonhydrolyzable fluorescent phosphonate analog, intense labeling of the plasma membrane occurred but fluorescent lipid did not enter the cytoplasm of cells. (ii) Experiments using C6-NBD-PA and cells prelabeled with 32Pi demonstrated that some of the fluorescent diacylglycerol was rephosphorylated at 2 degrees C. (iii) When cells were treated with 1,3-[palmitoyl, N-(4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl]-glycerophosphate, the lipid was dephosphorylated to 1,3-diacylglycerol but its rephosphorylation could not be detected. Nevertheless, rapid labeling of cytoplasmic membranes occurred. (iv) Formation of fluorescent diacylglycerol at the plasma membrane by treatment of cells with fluorescent phosphatidylcholine followed by phospholipase C at 2 degrees C resulted in strong labeling of intracellular membranes. Based on these results, a working model is presented for the uptake and intracellular translocation of phosphatidic acid involving formation of diacylglycerol at the plasma membrane followed by its transbilayer movement, facilitated translocation to intracellular membranes, and rephosphorylation.