Abstract
We have measured the movement of newly synthesized phosphatidylethanolamine (PE) molecules from sites of intracellular synthesis to the plasma membrane in cultured V79 Chinese hamster fibroblasts. Plasma membrane PE was distinguished from intracellular PE by its derivatization with an amino-reactive reagent, trinitrobenzene sulfonic acid, under nonpermeating conditions. Within minutes after the addition of radiolabeled precursors of PE to the culture medium, radiolabeled PE appeared at the plasma membrane. The fraction of radiolabeled PE molecules appearing at the plasma membrane increased rapidly over a 2-h period and then increased very slowly for several days to a constant specific radioactivity. By measuring the release of radiolabeled secretory proteins, we determined that the transport of newly synthesized proteins to the cell surface occurred more slowly than the transport of PE. Preincubation of cells with either cytochalasin B, cytochalasin D, colchicine, oncobendazole, sodium azide, 2-deoxyglucose, dinitrophenol, p-trifluoromethoxyphenylhydrazone, or monensin did not block the transport of de novo synthesized PE; however, incubation of cells in culture medium at 2 degrees C effectively halted the appearance of new PE molecules at the plasma membrane. When cells which had been incubated at 2 degrees C were warmed, PE molecules from intracellular PE pools once again began to appear at the plasma membrane. These results suggest that the rapid transport of newly synthesized PE molecules to the plasma membrane occurs by a mechanism independent of that used for the transport of newly synthesized proteins.
Highlights
We have measured the movement of newly synthe- elles [6]
Rate of PE Movement to the Plasma Membrane-To determine how rapidly newly synthesized P E was transported to the plasma membrane,cells were incubated for various times with ['Hlethanolamine and the fractionof ['HIPE appearing at theplasma membranewas determined. [3H]PEwas present at theplasma membrane almost immediately after the addition of ['Hlethanolamine to the culturemedium (Fig. 5)
Thispaperpresents two importantobservations.First, within minutes of their synthesis, P E molecules can be detected at the plasma membrane
Summary
Identification of Plasma Membrane PE"v79 fibroblasts rapidly incorporated radiolabeled ethanolamine into PE (Fig. amount of radioactivity present in the different lipid species was 1).The incorporation occurred without an appreciable lag and determined by liquid scintillation counting [19, 20]. When cells were incubated at 2 "Cwith 2-15 mM TNBS, only a small fraction of the cellular PE was derivatized similar reactions carried out at37 "C resulted in the trinitrophenylation of large amounts of cellular PE (data nosht own). This temperature-dependent labeling phenomenon is characteristic of a cell type impermeable to TNBS altow temperature [4,5, 31]. We have expressed the "i a amount of radiolabeled PE derivatized by the reaction with
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