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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 brefeldin A on metabolic labeling of lipids  with [14C]serine in wild-type and LY-A cells. Cells were preincubated in Nutridoma medium containing 1 μg/ml brefeldin A for  30 min at 33°C and then labeled with [14C]serine 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. Circles, phosphatidylserine; squares, phosphatidylethanolamine, in the panel  named PS & PE.
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Figure 5: Effect of brefeldin A on metabolic labeling of lipids with [14C]serine in wild-type and LY-A cells. Cells were preincubated in Nutridoma medium containing 1 μg/ml brefeldin A for 30 min at 33°C and then labeled with [14C]serine 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. Circles, phosphatidylserine; squares, phosphatidylethanolamine, in the panel named PS & PE.

Mentions: After pretreatment with 1 μg/ml brefeldin A for 30 min at 33°C, cells were labeled with [14C]serine in the presence of brefeldin A, and the labeling rates of lipids were compared between wild-type and LY-A cells. Under conditions without brefeldin A, the rate of SM synthesis in LY-A cells was only 10% of the wild-type level, while the labeling rates of other lipids in LY-A cells were comparable to the wild-type levels (Fig. 1). In contrast, under brefeldin A–treated conditions, the rate of SM synthesis in LY-A cells was quite similar to the wild-type level, which was about threefold higher than the rate in brefeldin A–untreated wild-type cells, while labeling rates of Cer, GlcCer, phosphatidylserine, and phosphatidylethanolamine were also similar between wild-type and LY-A cells (Fig. 5). In addition, we used [3H]sphingosine and [3H]dihydrosphingosine for monitoring the rate of Cer-to-SM conversion and observed restoration of SM synthesis in LY-A cells to the wild-type levels under brefeldin A–treated conditions (data not shown). These results demonstrated that LY-A cells had the Golgi apparatus–localized SM synthase normally and suggested that LY-A cells were defective in translocation of Cer from the ER to the Golgi apparatus.


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 brefeldin A on metabolic labeling of lipids  with [14C]serine in wild-type and LY-A cells. Cells were preincubated in Nutridoma medium containing 1 μg/ml brefeldin A for  30 min at 33°C and then labeled with [14C]serine 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. Circles, phosphatidylserine; squares, phosphatidylethanolamine, in the panel  named PS & PE.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Effect of brefeldin A on metabolic labeling of lipids with [14C]serine in wild-type and LY-A cells. Cells were preincubated in Nutridoma medium containing 1 μg/ml brefeldin A for 30 min at 33°C and then labeled with [14C]serine 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. Circles, phosphatidylserine; squares, phosphatidylethanolamine, in the panel named PS & PE.
Mentions: After pretreatment with 1 μg/ml brefeldin A for 30 min at 33°C, cells were labeled with [14C]serine in the presence of brefeldin A, and the labeling rates of lipids were compared between wild-type and LY-A cells. Under conditions without brefeldin A, the rate of SM synthesis in LY-A cells was only 10% of the wild-type level, while the labeling rates of other lipids in LY-A cells were comparable to the wild-type levels (Fig. 1). In contrast, under brefeldin A–treated conditions, the rate of SM synthesis in LY-A cells was quite similar to the wild-type level, which was about threefold higher than the rate in brefeldin A–untreated wild-type cells, while labeling rates of Cer, GlcCer, phosphatidylserine, and phosphatidylethanolamine were also similar between wild-type and LY-A cells (Fig. 5). In addition, we used [3H]sphingosine and [3H]dihydrosphingosine for monitoring the rate of Cer-to-SM conversion and observed restoration of SM synthesis in LY-A cells to the wild-type levels under brefeldin A–treated conditions (data not shown). These results demonstrated that LY-A cells had the Golgi apparatus–localized SM synthase normally and suggested that LY-A cells were defective in translocation of Cer from the ER to the Golgi apparatus.

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