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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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Effect of ATP depletion on metabolism of sphingolipids labeled by [3H]sphingosine in wild-type and LY-A cells. (Top  and middle) After ATP depletion of cells as described in the legend to Fig. 9, wild-type CHO (WT) and mutant LY-A cells were  incubated with 1 μM [3H]sphingosine for 30 min at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. The values of [3H]lactosylceramide and  [3H]GM3 were both <10% of those of [3H]GlcCer. (Bottom) For  the estimation of specific effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases, the corrected values  of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer are  shown as percentages of the value of normal cultured wild-type  cells. The values of NBD-SM and -GlcCer formation are from  the data shown in Fig. 9 B. N, normal culture conditions; D, ATP-depleted conditions; So, sphingosine.
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Figure 10: Effect of ATP depletion on metabolism of sphingolipids labeled by [3H]sphingosine in wild-type and LY-A cells. (Top and middle) After ATP depletion of cells as described in the legend to Fig. 9, wild-type CHO (WT) and mutant LY-A cells were incubated with 1 μM [3H]sphingosine for 30 min at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. The values of [3H]lactosylceramide and [3H]GM3 were both <10% of those of [3H]GlcCer. (Bottom) For the estimation of specific effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases, the corrected values of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer are shown as percentages of the value of normal cultured wild-type cells. The values of NBD-SM and -GlcCer formation are from the data shown in Fig. 9 B. N, normal culture conditions; D, ATP-depleted conditions; So, sphingosine.

Mentions: We next examined the effect of ATP depletion on conversion of natural Cer to SM by metabolic labeling with [3H]sphingosine. ATP depletion of wild-type cells resulted in the reduction of the [3H]SM formation to the level of LY-A cells, which was not affected by ATP depletion (Fig. 10), being consistent with the results of the pulse–chase experiments using C5-DMB-Cer (Fig. 9 A). Although the [3H]SM formation in wild-type cells under the ATP-depleted conditions was only 30% of the normal level, the [3H]GlcCer formation under the ATP-depleted conditions was ∼65% of the normal level both in wild-type and LY-A cells (Fig. 10). Considering that GlcCer formation appeared to be partially inhibited by a reduction of the UDP-glucose level under the ATP-depletion conditions, effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases could be estimated by the corrected values of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer, respectively, although we did not eliminate the possibility that access of C6-NBD-Cer to SM and GlcCer synthases was not completely ATP independent. The corrected values suggested that the influence of ATP depletion on [3H]Cer trafficking caused ∼70% and ∼20% inhibition of [3H]SM and [3H]GlcCer formation, respectively (Fig. 10, bottom). ATP depletion did not reduce the level of [3H]sphingosine associated with cells and did cause an increase in the level of [3H]Cer along with the striking decrease in [3H]SM, ruling out the possibility that the decreased formation of [3H]SM under the ATP-depleted conditions was due to a decrease in cellular uptake of [3H]sphingosine or formation of [3H]Cer from [3H]sphingosine. These results demonstrated evidence that trafficking of natural Cer from the ER to the site for SM synthesis is mainly ATP dependent and that this ATP-dependent trafficking of Cer is defective in LY-A cells.


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)

Effect of ATP depletion on metabolism of sphingolipids labeled by [3H]sphingosine in wild-type and LY-A cells. (Top  and middle) After ATP depletion of cells as described in the legend to Fig. 9, wild-type CHO (WT) and mutant LY-A cells were  incubated with 1 μM [3H]sphingosine for 30 min at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. The values of [3H]lactosylceramide and  [3H]GM3 were both <10% of those of [3H]GlcCer. (Bottom) For  the estimation of specific effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases, the corrected values  of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer are  shown as percentages of the value of normal cultured wild-type  cells. The values of NBD-SM and -GlcCer formation are from  the data shown in Fig. 9 B. N, normal culture conditions; D, ATP-depleted conditions; So, sphingosine.
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Figure 10: Effect of ATP depletion on metabolism of sphingolipids labeled by [3H]sphingosine in wild-type and LY-A cells. (Top and middle) After ATP depletion of cells as described in the legend to Fig. 9, wild-type CHO (WT) and mutant LY-A cells were incubated with 1 μM [3H]sphingosine for 30 min at 33°C. Radioactivity of each labeled lipid was determined as described in Materials and Methods. The values of [3H]lactosylceramide and [3H]GM3 were both <10% of those of [3H]GlcCer. (Bottom) For the estimation of specific effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases, the corrected values of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer are shown as percentages of the value of normal cultured wild-type cells. The values of NBD-SM and -GlcCer formation are from the data shown in Fig. 9 B. N, normal culture conditions; D, ATP-depleted conditions; So, sphingosine.
Mentions: We next examined the effect of ATP depletion on conversion of natural Cer to SM by metabolic labeling with [3H]sphingosine. ATP depletion of wild-type cells resulted in the reduction of the [3H]SM formation to the level of LY-A cells, which was not affected by ATP depletion (Fig. 10), being consistent with the results of the pulse–chase experiments using C5-DMB-Cer (Fig. 9 A). Although the [3H]SM formation in wild-type cells under the ATP-depleted conditions was only 30% of the normal level, the [3H]GlcCer formation under the ATP-depleted conditions was ∼65% of the normal level both in wild-type and LY-A cells (Fig. 10). Considering that GlcCer formation appeared to be partially inhibited by a reduction of the UDP-glucose level under the ATP-depletion conditions, effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases could be estimated by the corrected values of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer, respectively, although we did not eliminate the possibility that access of C6-NBD-Cer to SM and GlcCer synthases was not completely ATP independent. The corrected values suggested that the influence of ATP depletion on [3H]Cer trafficking caused ∼70% and ∼20% inhibition of [3H]SM and [3H]GlcCer formation, respectively (Fig. 10, bottom). ATP depletion did not reduce the level of [3H]sphingosine associated with cells and did cause an increase in the level of [3H]Cer along with the striking decrease in [3H]SM, ruling out the possibility that the decreased formation of [3H]SM under the ATP-depleted conditions was due to a decrease in cellular uptake of [3H]sphingosine or formation of [3H]Cer from [3H]sphingosine. These results demonstrated evidence that trafficking of natural Cer from the ER to the site for SM synthesis is mainly ATP dependent and that this ATP-dependent trafficking of Cer is defective in LY-A cells.

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