Phospholipid transfer protein-mediated incorporation and subcellular distribution of exogenous phosphatidylcholine and spingomyelin in cultured neuroblastoma cells

Murine neuroblastoma cells (strain N1E-115) were incubated with 2-[fatty acyl-¹⁴C]acylphosphatidylcholine/sphingomyelin or phosphatidylcholine/[choline-³H]sphingomyelin liposomes (1:1, mol/mol; 1.2 μmol total lipid/mg cell protein) in the presence of partially purified rat liver phospholipid transfer protein (2.5 mg/ml), cytochalasin B (50 μM) and 2-deoxyglucose (50 mM) for 10 min. Washed cells were chased for periods of up to 45 min at 37°C with medium containing transfer protein and unlabeled liposomes. Total transfer protein-dependent incorporation of [¹⁴C]phosphatidylcholine ([¹⁴C]PC) and [³H]sphingomyelin was 136.7 ± 26.5(n = 5) and 23.7 ± 5.4(n = 6) nmol/mg protein per 10 min incubation, respectively, (mean ± S.D.). Incorporation of [¹⁴C]PC into the mitochondrial membrane fraction was 128-fold greater (nmol/mg protein) than incorporation of [³H]sphingomyelin. In contrast, incorporation of [³H]sphingomyelin into a fraction enriched in plasma membrane and into microsomes was 1.4- and 2.6-fold greater, respectively, than incorporation of [¹⁴C]PC. During the chase periods, the specific activities of total cellular phospholipids decreased as intact [¹⁴C]PC and [³H]sphingomyelin accumulated in the culture medium. In the case of cells labeled with [¹⁴C]PC, the effect was due primarily to a decrease in the amount of labeled phospholipid in the mitochondrial fraction; in the case of cells labeled with [³H]sphingomyelin, the decrease in activity was greatest in microsomal and plasma membrane phospholipids. The rate and extent of non-endocytotic incorporation of exogenous phosphatidylcholine into the cell membrane of cultured neuroblastoma cells, and its subsequent subcellular disposition, is different from that of exogenous sphingomyelin. Whereas PC is evidently incorporated into and turned over most rapidly in fraction enriched in mitochondrial membranes, sphingomyelin appears to be preferentially incorporated into microsomal and plasma membrane.