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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 redistribution of  intracellular fluorescence in  wild-type and LY-A cells labeled with C5-DMB-Cer or  C6-NBD-Cer. 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–D)  or 5 μM C6-NBD-Cer (E–H)  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 15 min at 33°C,  and photographed. (A and E)  Wild-type cells without inhibitors; (B and F) mutant LY-A  cells without inhibitors; (C  and G) wild-type cells with  inhibitors; (D and H) mutant  LY-A cells with inhibitors.  For each fluorescent probe,  all photomicrographs were  exposed and printed identically. Bar, 10 μm.
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Figure 8: Effect of ATP depletion on redistribution of intracellular fluorescence in wild-type and LY-A cells labeled with C5-DMB-Cer or C6-NBD-Cer. 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–D) or 5 μM C6-NBD-Cer (E–H) 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 15 min at 33°C, and photographed. (A and E) Wild-type cells without inhibitors; (B and F) mutant LY-A cells without inhibitors; (C and G) wild-type cells with inhibitors; (D and H) mutant LY-A cells with inhibitors. For each fluorescent probe, all photomicrographs were exposed and printed identically. Bar, 10 μm.

Mentions: For examination of the energy dependence of intracellular redistribution of C5-DMB-Cer, cells were pretreated with or without energy inhibitors (50 mM 2-deoxy-d-glucose and 5 mM NaN3) for 15 min at 33°C, incubated with C5-DMB-Cer for 30 min at 4°C, washed, and chased for 15 min at 33°C in the presence or absence of the energy inhibitors. Measurements of intracellular ATP showed that wild-type and LY-A cells had the same ATP levels under the inhibitor-minus control conditions, and ATP levels in both cell types were reduced by >95% in the presence of the inhibitors. ATP depletion did not affect the level and distribution of C5-DMB-Cer during the prelabeling of cells at 4°C; DMB fluorescence was distributed to various intracellular membranes (data not shown), as seen under the normal conditions (Fig. 7, A and B). However, after the chase at 33°C, the level of DMB fluorescence accumulated into the Golgi region in wild-type cells was strikingly lower under the ATP-depleted conditions than under the control conditions, while in LY-A cells ATP depletion did not appreciably affect the Golgi level of DMB fluorescence (Fig. 8, A–D). Consequently, ATP depletion reduced the DMB fluorescence level associated with the Golgi apparatus in wild-type cells to the level of 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 redistribution of  intracellular fluorescence in  wild-type and LY-A cells labeled with C5-DMB-Cer or  C6-NBD-Cer. 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–D)  or 5 μM C6-NBD-Cer (E–H)  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 15 min at 33°C,  and photographed. (A and E)  Wild-type cells without inhibitors; (B and F) mutant LY-A  cells without inhibitors; (C  and G) wild-type cells with  inhibitors; (D and H) mutant  LY-A cells with inhibitors.  For each fluorescent probe,  all photomicrographs were  exposed and printed identically. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: Effect of ATP depletion on redistribution of intracellular fluorescence in wild-type and LY-A cells labeled with C5-DMB-Cer or C6-NBD-Cer. 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–D) or 5 μM C6-NBD-Cer (E–H) 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 15 min at 33°C, and photographed. (A and E) Wild-type cells without inhibitors; (B and F) mutant LY-A cells without inhibitors; (C and G) wild-type cells with inhibitors; (D and H) mutant LY-A cells with inhibitors. For each fluorescent probe, all photomicrographs were exposed and printed identically. Bar, 10 μm.
Mentions: For examination of the energy dependence of intracellular redistribution of C5-DMB-Cer, cells were pretreated with or without energy inhibitors (50 mM 2-deoxy-d-glucose and 5 mM NaN3) for 15 min at 33°C, incubated with C5-DMB-Cer for 30 min at 4°C, washed, and chased for 15 min at 33°C in the presence or absence of the energy inhibitors. Measurements of intracellular ATP showed that wild-type and LY-A cells had the same ATP levels under the inhibitor-minus control conditions, and ATP levels in both cell types were reduced by >95% in the presence of the inhibitors. ATP depletion did not affect the level and distribution of C5-DMB-Cer during the prelabeling of cells at 4°C; DMB fluorescence was distributed to various intracellular membranes (data not shown), as seen under the normal conditions (Fig. 7, A and B). However, after the chase at 33°C, the level of DMB fluorescence accumulated into the Golgi region in wild-type cells was strikingly lower under the ATP-depleted conditions than under the control conditions, while in LY-A cells ATP depletion did not appreciably affect the Golgi level of DMB fluorescence (Fig. 8, A–D). Consequently, ATP depletion reduced the DMB fluorescence level associated with the Golgi apparatus in wild-type cells to the level of 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