LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse–chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.
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22 February 1999
Article|
February 22 1999
Genetic Evidence for ATP-dependent Endoplasmic Reticulum-to-Golgi Apparatus Trafficking of Ceramide for Sphingomyelin Synthesis in Chinese Hamster Ovary Cells
Masayoshi Fukasawa,
Masayoshi Fukasawa
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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Masahiro Nishijima,
Masahiro Nishijima
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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Kentaro Hanada
Kentaro Hanada
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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Masayoshi Fukasawa
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
Masahiro Nishijima
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
Kentaro Hanada
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
Address correspondence to Kentaro Hanada, Ph.D., Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111, ext. 2126. Fax: 81-3-5285-1157. E-mail: [email protected]
Received:
September 24 1998
Revision Received:
December 22 1998
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1999
J Cell Biol (1999) 144 (4): 673–685.
Article history
Received:
September 24 1998
Revision Received:
December 22 1998
Citation
Masayoshi Fukasawa, Masahiro Nishijima, Kentaro Hanada; Genetic Evidence for ATP-dependent Endoplasmic Reticulum-to-Golgi Apparatus Trafficking of Ceramide for Sphingomyelin Synthesis in Chinese Hamster Ovary Cells . J Cell Biol 22 February 1999; 144 (4): 673–685. doi: https://doi.org/10.1083/jcb.144.4.673
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