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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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Possible models for ER-to-Golgi apparatus trafficking  of Cer responsible for SM and GlcCer biosynthesis. For ER-to-Golgi apparatus trafficking pathways of Cer, at least two distinct  pathways are proposed to exist; one is ATP-dependent (black arrows) and another is ATP-independent (or less ATP-dependent)  (white arrows). Mutant LY-A cells have a defect in the ATP-dependent pathway. (A) A portion of Cer produced on the cytosolic surface at the ER is rapidly internalized to the lumenal side,  and the lumenal Cer is delivered to the Golgi apparatus via an  ATP-dependent pathway for SM synthesis, while Cer remaining  at the cytosolic side of the ER is delivered to the cytosolic side of  the Golgi apparatus via an ATP-independent pathway for  GlcCer and SM synthesis. (B) Regardless of the Cer topology in  the ER membrane, a portion of newly synthesized Cer is delivered to the Golgi apparatus via an ATP-dependent pathway,  which is specifically directed to the Golgi subcompartment where  SM synthase is localized, while another portion of Cer is delivered to more various subcompartments of the Golgi apparatus  via an ATP-independent pathway and can be converted to both  SM and GlcCer. Ser, serine; PalCoA, palmitoyl CoA; PM,  plasma membrane.
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Figure 12: Possible models for ER-to-Golgi apparatus trafficking of Cer responsible for SM and GlcCer biosynthesis. For ER-to-Golgi apparatus trafficking pathways of Cer, at least two distinct pathways are proposed to exist; one is ATP-dependent (black arrows) and another is ATP-independent (or less ATP-dependent) (white arrows). Mutant LY-A cells have a defect in the ATP-dependent pathway. (A) A portion of Cer produced on the cytosolic surface at the ER is rapidly internalized to the lumenal side, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent pathway for GlcCer and SM synthesis. (B) Regardless of the Cer topology in the ER membrane, a portion of newly synthesized Cer is delivered to the Golgi apparatus via an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while another portion of Cer is delivered to more various subcompartments of the Golgi apparatus via an ATP-independent pathway and can be converted to both SM and GlcCer. Ser, serine; PalCoA, palmitoyl CoA; PM, plasma membrane.

Mentions: Two hypothetical models may be conceivable to explain the preferential delivery of Cer to SM synthase by the ATP-dependent mechanism. In one model (Fig. 12 A), a portion of newly synthesized Cer molecules is rapidly internalized to the lumenal side of the ER, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent and vesicle-mediated pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent (or less ATP-dependent) pathway for GlcCer synthesis. In another model (Fig. 12 B), regardless of the Cer topology in the ER membrane, a portion of the Cer molecules enters an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while Cer molecules associated with an ATP-independent (or less ATP-dependent) pathway have a broader specificity for their destination and are eventually accessible to both GlcCer and SM synthases. If LY-A cells have a deficiency in an intra–Golgi apparatus transport of Cer, it may be conceivable that the ATP-dependent step for specific delivery to the SM synthase– localizing compartment is located at the intra–Golgi apparatus transport of Cer.


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)

Possible models for ER-to-Golgi apparatus trafficking  of Cer responsible for SM and GlcCer biosynthesis. For ER-to-Golgi apparatus trafficking pathways of Cer, at least two distinct  pathways are proposed to exist; one is ATP-dependent (black arrows) and another is ATP-independent (or less ATP-dependent)  (white arrows). Mutant LY-A cells have a defect in the ATP-dependent pathway. (A) A portion of Cer produced on the cytosolic surface at the ER is rapidly internalized to the lumenal side,  and the lumenal Cer is delivered to the Golgi apparatus via an  ATP-dependent pathway for SM synthesis, while Cer remaining  at the cytosolic side of the ER is delivered to the cytosolic side of  the Golgi apparatus via an ATP-independent pathway for  GlcCer and SM synthesis. (B) Regardless of the Cer topology in  the ER membrane, a portion of newly synthesized Cer is delivered to the Golgi apparatus via an ATP-dependent pathway,  which is specifically directed to the Golgi subcompartment where  SM synthase is localized, while another portion of Cer is delivered to more various subcompartments of the Golgi apparatus  via an ATP-independent pathway and can be converted to both  SM and GlcCer. Ser, serine; PalCoA, palmitoyl CoA; PM,  plasma membrane.
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Related In: Results  -  Collection

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Figure 12: Possible models for ER-to-Golgi apparatus trafficking of Cer responsible for SM and GlcCer biosynthesis. For ER-to-Golgi apparatus trafficking pathways of Cer, at least two distinct pathways are proposed to exist; one is ATP-dependent (black arrows) and another is ATP-independent (or less ATP-dependent) (white arrows). Mutant LY-A cells have a defect in the ATP-dependent pathway. (A) A portion of Cer produced on the cytosolic surface at the ER is rapidly internalized to the lumenal side, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent pathway for GlcCer and SM synthesis. (B) Regardless of the Cer topology in the ER membrane, a portion of newly synthesized Cer is delivered to the Golgi apparatus via an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while another portion of Cer is delivered to more various subcompartments of the Golgi apparatus via an ATP-independent pathway and can be converted to both SM and GlcCer. Ser, serine; PalCoA, palmitoyl CoA; PM, plasma membrane.
Mentions: Two hypothetical models may be conceivable to explain the preferential delivery of Cer to SM synthase by the ATP-dependent mechanism. In one model (Fig. 12 A), a portion of newly synthesized Cer molecules is rapidly internalized to the lumenal side of the ER, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent and vesicle-mediated pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent (or less ATP-dependent) pathway for GlcCer synthesis. In another model (Fig. 12 B), regardless of the Cer topology in the ER membrane, a portion of the Cer molecules enters an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while Cer molecules associated with an ATP-independent (or less ATP-dependent) pathway have a broader specificity for their destination and are eventually accessible to both GlcCer and SM synthases. If LY-A cells have a deficiency in an intra–Golgi apparatus transport of Cer, it may be conceivable that the ATP-dependent step for specific delivery to the SM synthase– localizing compartment is located at the intra–Golgi apparatus transport of Cer.

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