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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 and C6-NBD-Cer in normal and ATP-depleted cells over time. Cells were preincubated  in Ham's F-12 with or without energy inhibitors (50 mM 2-deoxy- d-glucose and 5 mM NaN3) for 15 min at 33°C, then incubated  with 1.25 μM C5-DMB-Cer (A) or 5 μM C6-NBD-Cer (B) for 30  min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA with or  without the inhibitors for the indicated time at 33°C. Lipids extracted from the cells were separated by TLC, and the amounts  of C5-DMB-SM (solid lines in A), C6-NBD-SM (solid lines in B),  and C6-NBD-GlcCer (dotted lines in B) were determined. Formation of C5-DMB-GlcCer was too low to be quantified. Closed  circles, wild-type cells without inhibitors; open circles, wild-type  cells with inhibitors; closed squares, mutant LY-A cells without  inhibitors; open squares, mutant LY-A cells with inhibitors.
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Figure 9: Metabolism of C5-DMB-Cer and C6-NBD-Cer in normal and ATP-depleted cells over time. Cells were preincubated in Ham's F-12 with or without energy inhibitors (50 mM 2-deoxy- d-glucose and 5 mM NaN3) for 15 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer (A) or 5 μM C6-NBD-Cer (B) for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA with or without the inhibitors for the indicated time at 33°C. Lipids extracted from the cells were separated by TLC, and the amounts of C5-DMB-SM (solid lines in A), C6-NBD-SM (solid lines in B), and C6-NBD-GlcCer (dotted lines in B) were determined. Formation of C5-DMB-GlcCer was too low to be quantified. Closed circles, wild-type cells without inhibitors; open circles, wild-type cells with inhibitors; closed squares, mutant LY-A cells without inhibitors; open squares, mutant LY-A cells with inhibitors.

Mentions: Biochemical analysis showed that the ATP-depleted conditions caused an ∼50% reduction of the conversion rate of C5-DMB-Cer to C5-DMB-SM in wild-type cells (Fig. 9), indicating that the ATP-dependent redistribution of C5-DMB-Cer is responsible for synthesis of C5-DMB-SM in wild-type cells. The reduced rate in wild-type cells was almost identical to the rate in LY-A cells, which was not affected by ATP depletion (Fig. 9), being consistent with the similar level of DMB fluorescence in Golgi apparatus between ATP-depleted wild-type and normally cultured LY-A cells (Fig. 8, B and C). There was no difference in the total amount of lipidic DMB fluorescence associated with cells between the normal and ATP-depleted cells even after the 33°C chase (data not shown). Collectively, these results indicated that, in wild-type cells, redistribution of intracellular C5-DMB-Cer to the Golgi apparatus for C5-DMB-SM synthesis consists of an ATP-dependent pathway(s) and an ATP-independent (or less ATP-dependent) pathway(s), and that the ATP-dependent trafficking pathway of C5-DMB-Cer is almost completely impaired 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)

Metabolism of C5-DMB-Cer and C6-NBD-Cer in normal and ATP-depleted cells over time. Cells were preincubated  in Ham's F-12 with or without energy inhibitors (50 mM 2-deoxy- d-glucose and 5 mM NaN3) for 15 min at 33°C, then incubated  with 1.25 μM C5-DMB-Cer (A) or 5 μM C6-NBD-Cer (B) for 30  min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA with or  without the inhibitors for the indicated time at 33°C. Lipids extracted from the cells were separated by TLC, and the amounts  of C5-DMB-SM (solid lines in A), C6-NBD-SM (solid lines in B),  and C6-NBD-GlcCer (dotted lines in B) were determined. Formation of C5-DMB-GlcCer was too low to be quantified. Closed  circles, wild-type cells without inhibitors; open circles, wild-type  cells with inhibitors; closed squares, mutant LY-A cells without  inhibitors; open squares, mutant LY-A cells with inhibitors.
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Figure 9: Metabolism of C5-DMB-Cer and C6-NBD-Cer in normal and ATP-depleted cells over time. Cells were preincubated in Ham's F-12 with or without energy inhibitors (50 mM 2-deoxy- d-glucose and 5 mM NaN3) for 15 min at 33°C, then incubated with 1.25 μM C5-DMB-Cer (A) or 5 μM C6-NBD-Cer (B) for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA with or without the inhibitors for the indicated time at 33°C. Lipids extracted from the cells were separated by TLC, and the amounts of C5-DMB-SM (solid lines in A), C6-NBD-SM (solid lines in B), and C6-NBD-GlcCer (dotted lines in B) were determined. Formation of C5-DMB-GlcCer was too low to be quantified. Closed circles, wild-type cells without inhibitors; open circles, wild-type cells with inhibitors; closed squares, mutant LY-A cells without inhibitors; open squares, mutant LY-A cells with inhibitors.
Mentions: Biochemical analysis showed that the ATP-depleted conditions caused an ∼50% reduction of the conversion rate of C5-DMB-Cer to C5-DMB-SM in wild-type cells (Fig. 9), indicating that the ATP-dependent redistribution of C5-DMB-Cer is responsible for synthesis of C5-DMB-SM in wild-type cells. The reduced rate in wild-type cells was almost identical to the rate in LY-A cells, which was not affected by ATP depletion (Fig. 9), being consistent with the similar level of DMB fluorescence in Golgi apparatus between ATP-depleted wild-type and normally cultured LY-A cells (Fig. 8, B and C). There was no difference in the total amount of lipidic DMB fluorescence associated with cells between the normal and ATP-depleted cells even after the 33°C chase (data not shown). Collectively, these results indicated that, in wild-type cells, redistribution of intracellular C5-DMB-Cer to the Golgi apparatus for C5-DMB-SM synthesis consists of an ATP-dependent pathway(s) and an ATP-independent (or less ATP-dependent) pathway(s), and that the ATP-dependent trafficking pathway of C5-DMB-Cer is almost completely impaired 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