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Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.

Fukasawa M, Nishijima M, Hanada K - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

ABSTRACT
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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Metabolic labeling of sphingolipids with [3H]sphingosine in wild-type and LY-A cells. Cells were incubated with 1 μM  [3H]sphingosine for the indicated times at 33°C. Radioactivity of  each labeled lipid was determined as described in Materials and  Methods. Open symbols, wild-type CHO cells; closed symbols,  mutant LY-A cells; So, sphingosine.
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Figure 2: Metabolic labeling of sphingolipids with [3H]sphingosine in wild-type and LY-A cells. Cells were incubated with 1 μM [3H]sphingosine for the indicated times at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. Open symbols, wild-type CHO cells; closed symbols, mutant LY-A cells; So, sphingosine.

Mentions: A defect in SM formation might result in some repression of de novo sphingoid base formation. Thus, to prevent such possible feedback repression from affecting Cer-to-SM conversion by metabolic labeling, we used [3H]sphingosine as an alternative precursor for monitoring SM synthesis, since it has been shown that CHO cells are able to utilize exogenously supplied sphingosine for synthesis of Cer and complex sphingolipids (Hanada et al., 1992). When cells were labeled with 1 μM [3H]sphingosine at 33°C for various times up to 2 h, wild-type and LY-A cells similarly accumulated free [3H]sphingosine, with a plateau level at 15–30 min after incubation, and showed almost equal levels of the total radioactivity of cell-associated lipids (Fig. 2), indicating that the two cell types had an equal efficiency of sphingosine uptake. Under these conditions, formation of radioactive SM in LY-A cells was <30% of the wild-type level throughout the incubation, whereas formations of Cer, GlcCer, and GM3 were slightly higher in LY-A cells than in wild-type cells (Fig. 2). When [3H]dihydrosphingosine was used as another precursor, we obtained results similar to those obtained with [3H]sphingosine (data not shown).


Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.

Fukasawa M, Nishijima M, Hanada K - J. Cell Biol. (1999)

Metabolic labeling of sphingolipids with [3H]sphingosine in wild-type and LY-A cells. Cells were incubated with 1 μM  [3H]sphingosine for the indicated times at 33°C. Radioactivity of  each labeled lipid was determined as described in Materials and  Methods. Open symbols, wild-type CHO cells; closed symbols,  mutant LY-A cells; So, sphingosine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132924&req=5

Figure 2: Metabolic labeling of sphingolipids with [3H]sphingosine in wild-type and LY-A cells. Cells were incubated with 1 μM [3H]sphingosine for the indicated times at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. Open symbols, wild-type CHO cells; closed symbols, mutant LY-A cells; So, sphingosine.
Mentions: A defect in SM formation might result in some repression of de novo sphingoid base formation. Thus, to prevent such possible feedback repression from affecting Cer-to-SM conversion by metabolic labeling, we used [3H]sphingosine as an alternative precursor for monitoring SM synthesis, since it has been shown that CHO cells are able to utilize exogenously supplied sphingosine for synthesis of Cer and complex sphingolipids (Hanada et al., 1992). When cells were labeled with 1 μM [3H]sphingosine at 33°C for various times up to 2 h, wild-type and LY-A cells similarly accumulated free [3H]sphingosine, with a plateau level at 15–30 min after incubation, and showed almost equal levels of the total radioactivity of cell-associated lipids (Fig. 2), indicating that the two cell types had an equal efficiency of sphingosine uptake. Under these conditions, formation of radioactive SM in LY-A cells was <30% of the wild-type level throughout the incubation, whereas formations of Cer, GlcCer, and GM3 were slightly higher in LY-A cells than in wild-type cells (Fig. 2). When [3H]dihydrosphingosine was used as another precursor, we obtained results similar to those obtained with [3H]sphingosine (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus