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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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Intracellular distribution of DMB fluorescence  in wild-type and LY-A cells  over time at 33°C after labeling with C5-DMB-Cer. Wild-type CHO (A, C, and E) and  mutant LY-A (B, D, and F)  cells were incubated with  1.25 μM C5-DMB-Cer for 30  min at 4°C, washed, further  incubated in Nutridoma medium containing 0.34 mg/ml  BSA for 0 (A and B), 15 (C  and D), or 30 (E and F) min  at 33°C, and photographed.  All photomicrographs were  exposed and printed identically. Bar, 10 μm.
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Figure 7: Intracellular distribution of DMB fluorescence in wild-type and LY-A cells over time at 33°C after labeling with C5-DMB-Cer. Wild-type CHO (A, C, and E) and mutant LY-A (B, D, and F) cells were incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C, washed, further incubated in Nutridoma medium containing 0.34 mg/ml BSA for 0 (A and B), 15 (C and D), or 30 (E and F) min at 33°C, and photographed. All photomicrographs were exposed and printed identically. Bar, 10 μm.

Mentions: Distribution patterns of intracellular DMB fluorescence were compared between LY-A and wild-type cells under the pulse–chase conditions. After prelabeling of cells with C5-DMB-Cer at 4°C, intracellular fluorescence was distributed throughout intracellular membranes including the ER in both wild-type and LY-A cells (Fig. 7, A and B). After the chase at 33°C for 15–30 min, most of the intracellular fluorescence in wild-type cells accumulated in the perinuclear region corresponding to the Golgi complex (Fig. 7, C and E), in agreement with previous studies (Pagano et al., 1991; Ktistakis et al., 1995). Interestingly, accumulation of intracellular fluorescence in the Golgi region was lower in LY-A cells than wild-type cells (Fig. 7, D and F vs. C and E). The lower level of the Golgi fluorescence in LY-A cells was not due to an enhanced secretion of intracellular DMB fluorescence to the medium, because there was no significant difference in the total amount of DMB lipids associated with cells between the two cell types during the pulse and chase periods (data not shown). These observations also provided evidence that LY-A cells are defective in ER-to-Golgi apparatus trafficking 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)

Intracellular distribution of DMB fluorescence  in wild-type and LY-A cells  over time at 33°C after labeling with C5-DMB-Cer. Wild-type CHO (A, C, and E) and  mutant LY-A (B, D, and F)  cells were incubated with  1.25 μM C5-DMB-Cer for 30  min at 4°C, washed, further  incubated in Nutridoma medium containing 0.34 mg/ml  BSA for 0 (A and B), 15 (C  and D), or 30 (E and F) min  at 33°C, and photographed.  All photomicrographs were  exposed and printed identically. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Intracellular distribution of DMB fluorescence in wild-type and LY-A cells over time at 33°C after labeling with C5-DMB-Cer. Wild-type CHO (A, C, and E) and mutant LY-A (B, D, and F) cells were incubated with 1.25 μM C5-DMB-Cer for 30 min at 4°C, washed, further incubated in Nutridoma medium containing 0.34 mg/ml BSA for 0 (A and B), 15 (C and D), or 30 (E and F) min at 33°C, and photographed. All photomicrographs were exposed and printed identically. Bar, 10 μm.
Mentions: Distribution patterns of intracellular DMB fluorescence were compared between LY-A and wild-type cells under the pulse–chase conditions. After prelabeling of cells with C5-DMB-Cer at 4°C, intracellular fluorescence was distributed throughout intracellular membranes including the ER in both wild-type and LY-A cells (Fig. 7, A and B). After the chase at 33°C for 15–30 min, most of the intracellular fluorescence in wild-type cells accumulated in the perinuclear region corresponding to the Golgi complex (Fig. 7, C and E), in agreement with previous studies (Pagano et al., 1991; Ktistakis et al., 1995). Interestingly, accumulation of intracellular fluorescence in the Golgi region was lower in LY-A cells than wild-type cells (Fig. 7, D and F vs. C and E). The lower level of the Golgi fluorescence in LY-A cells was not due to an enhanced secretion of intracellular DMB fluorescence to the medium, because there was no significant difference in the total amount of DMB lipids associated with cells between the two cell types during the pulse and chase periods (data not shown). These observations also provided evidence that LY-A cells are defective in ER-to-Golgi apparatus trafficking 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