Reconstitution of ATP- and cytosol-dependent transport of de Novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells

Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [H-3]ceramide that had been produced from [H-3]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [H-3]ceramide-to-[H-3]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [H-3]ceramide-to-[H-3]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [H-3]ceramide-to-[H-3] SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgis apparatus were merged, no longer required cytosol for conversion of [H-3]ceramide to [H-3]SM. These results indicated that the assay of [H-3]ceramide-to-[H-3]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [H-3]ceramide to [H-3]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.