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 [(3)H]ceramide that had been produced from [(3)H]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [(3)H]ceramide-to-[(3)H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [(3)H]ceramide-to-[(3)H]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 [(3)H]ceramide-to-[(3)H]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 Golgi apparatus were merged, no longer required cytosol for conversion of [(3)H]ceramide to [(3)H]SM. These results indicated that the assay of [(3)H]ceramide-to-[(3)H]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 [(3)H]ceramide to [(3)H]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.
Funakoshi
T
T
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Yasuda
S
S
Fukasawa
M
M
Nishijima
M
M
Hanada
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002518:Ceramides / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism; D009154:Mutation; D013107:Sphingolipids / Q000378:metabolism; D013109:Sphingomyelins / Q000096:biosynthesis*; D013110:Sphingosine / Q000378:metabolism
D002518:Ceramides; D005963:Glucosylceramides; D013107:Sphingolipids; D013109:Sphingomyelins; D000255:Adenosine Triphosphate; D013110:Sphingosine