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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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Metabolism of C5-DMB-Cer in wild-type and LY-A  cells in the presence or absence of brefeldin A. Wild-type CHO  (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml  brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C. After washing, cell monolayers were  further incubated in Nutridoma medium containing 0.34 mg/ml  BSA without or with 1 μg/ml brefeldin A for the indicated times  at 33°C. Cellular lipids were extracted and analyzed by TLC. The  formation of the DMB derivative of GlcCer was too low (<5%  of the level of C5-DMB-SM) to be determined accurately.
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Figure 6: Metabolism of C5-DMB-Cer in wild-type and LY-A cells in the presence or absence of brefeldin A. Wild-type CHO (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA without or with 1 μg/ml brefeldin A for the indicated times at 33°C. Cellular lipids were extracted and analyzed by TLC. The formation of the DMB derivative of GlcCer was too low (<5% of the level of C5-DMB-SM) to be determined accurately.

Mentions: For testing whether the behavior of C5-DMB-Cer in cells reflected the defect of SM synthesis in LY-A cells, the metabolic rate of C5-DMB-Cer to C5-DMB-SM was compared between wild-type and LY-A cells. Cells were preincubated with C5-DMB-Cer for 30 min at 4°C for transferring the probe to intracellular membranes and, after washing the extracellular C5-DMB-Cer, cells were incubated at 33°C for various times to start conversion of intracellular C5-DMB-Cer to C5-DMB-SM. During the chase, C5-DMB-Cer was metabolized to C5-DMB-SM in a time-dependent manner, resulting in conversion of 50% of the total lipidic fluorescence to C5-DMB-SM in wild-type cells 30 min after the chase, while the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was about half of the wild-type level (Fig. 6 A). In addition, the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was restored to the wild-type level by brefeldin A treatment (Fig. 6 B). Note that the level of total lipidic DMB fluorescence associated with cells was identical in the two cell types during the pulse and chase (data not shown). These results indicated that the behavior of C5-DMB-Cer at least partly reflects that of natural Cer in 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)

Metabolism of C5-DMB-Cer in wild-type and LY-A  cells in the presence or absence of brefeldin A. Wild-type CHO  (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml  brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C. After washing, cell monolayers were  further incubated in Nutridoma medium containing 0.34 mg/ml  BSA without or with 1 μg/ml brefeldin A for the indicated times  at 33°C. Cellular lipids were extracted and analyzed by TLC. The  formation of the DMB derivative of GlcCer was too low (<5%  of the level of C5-DMB-SM) to be determined accurately.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Metabolism of C5-DMB-Cer in wild-type and LY-A cells in the presence or absence of brefeldin A. Wild-type CHO (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA without or with 1 μg/ml brefeldin A for the indicated times at 33°C. Cellular lipids were extracted and analyzed by TLC. The formation of the DMB derivative of GlcCer was too low (<5% of the level of C5-DMB-SM) to be determined accurately.
Mentions: For testing whether the behavior of C5-DMB-Cer in cells reflected the defect of SM synthesis in LY-A cells, the metabolic rate of C5-DMB-Cer to C5-DMB-SM was compared between wild-type and LY-A cells. Cells were preincubated with C5-DMB-Cer for 30 min at 4°C for transferring the probe to intracellular membranes and, after washing the extracellular C5-DMB-Cer, cells were incubated at 33°C for various times to start conversion of intracellular C5-DMB-Cer to C5-DMB-SM. During the chase, C5-DMB-Cer was metabolized to C5-DMB-SM in a time-dependent manner, resulting in conversion of 50% of the total lipidic fluorescence to C5-DMB-SM in wild-type cells 30 min after the chase, while the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was about half of the wild-type level (Fig. 6 A). In addition, the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was restored to the wild-type level by brefeldin A treatment (Fig. 6 B). Note that the level of total lipidic DMB fluorescence associated with cells was identical in the two cell types during the pulse and chase (data not shown). These results indicated that the behavior of C5-DMB-Cer at least partly reflects that of natural Cer in 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