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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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SM synthase activity in wild-type and LY-A cell lysates. (A) The dependence of C5-DMB-SM formation on a dose  of C5-DMB-Cer. Cell lysate (100 μg protein) was incubated in an  SM synthase assay mixture containing the indicated amounts of  C5-DMB-Cer for 30 min. (B) Time course of C5-DMB-SM formation. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing 10 μM C5-DMB-Cer for the indicated times. C5-DMB-SM produced was determined as described  in Materials and Methods. Open symbols, wild-type CHO lysate;  closed symbols, mutant LY-A lysate.
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Figure 4: SM synthase activity in wild-type and LY-A cell lysates. (A) The dependence of C5-DMB-SM formation on a dose of C5-DMB-Cer. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing the indicated amounts of C5-DMB-Cer for 30 min. (B) Time course of C5-DMB-SM formation. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing 10 μM C5-DMB-Cer for the indicated times. C5-DMB-SM produced was determined as described in Materials and Methods. Open symbols, wild-type CHO lysate; closed symbols, mutant LY-A lysate.

Mentions: To identify the mechanism underlying the defect in conversion of Cer to SM in LY-A cells, we examined SM synthase activity in cell lysates by using C5-DMB-Cer, a fluorescent Cer analogue, as the substrate. LY-A cells showed essentially the same level of SM synthase activity as wild-type cells, as there was no difference in the dependence of C5-DMB-SM formation on C5-DMB-Cer concentration or in the time course of C5-DMB-SM formation between wild-type and LY-A cells (Fig. 4, A and B). Similarly, when C6-NBD-Cer or N-[14C]palmitoyl-sphingosine was used as the substrate, no difference in activity of SM synthase between the two cell types was observed (data not shown).


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)

SM synthase activity in wild-type and LY-A cell lysates. (A) The dependence of C5-DMB-SM formation on a dose  of C5-DMB-Cer. Cell lysate (100 μg protein) was incubated in an  SM synthase assay mixture containing the indicated amounts of  C5-DMB-Cer for 30 min. (B) Time course of C5-DMB-SM formation. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing 10 μM C5-DMB-Cer for the indicated times. C5-DMB-SM produced was determined as described  in Materials and Methods. Open symbols, wild-type CHO lysate;  closed symbols, mutant LY-A lysate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: SM synthase activity in wild-type and LY-A cell lysates. (A) The dependence of C5-DMB-SM formation on a dose of C5-DMB-Cer. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing the indicated amounts of C5-DMB-Cer for 30 min. (B) Time course of C5-DMB-SM formation. Cell lysate (100 μg protein) was incubated in an SM synthase assay mixture containing 10 μM C5-DMB-Cer for the indicated times. C5-DMB-SM produced was determined as described in Materials and Methods. Open symbols, wild-type CHO lysate; closed symbols, mutant LY-A lysate.
Mentions: To identify the mechanism underlying the defect in conversion of Cer to SM in LY-A cells, we examined SM synthase activity in cell lysates by using C5-DMB-Cer, a fluorescent Cer analogue, as the substrate. LY-A cells showed essentially the same level of SM synthase activity as wild-type cells, as there was no difference in the dependence of C5-DMB-SM formation on C5-DMB-Cer concentration or in the time course of C5-DMB-SM formation between wild-type and LY-A cells (Fig. 4, A and B). Similarly, when C6-NBD-Cer or N-[14C]palmitoyl-sphingosine was used as the substrate, no difference in activity of SM synthase between the two cell types was observed (data not shown).

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