Limits...
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.

Show MeSH

Related in: MedlinePlus

ER-to-Golgi apparatus trafficking of GPI-anchored  or membrane-spanning glycoprotein in wild-type and LY-A cells.  Cells were pulsed for 15 min with [35S]methionine/cysteine and  chased for the indicated time. Immunoprecipitates were treated  with Endo H for 16 h at 37°C and separated by SDS-PAGE as  described in Materials and Methods. (A and C) Wild-type CHO  cell derivatives expressing PLAP and PLAP-HA, respectively;  (B and D) mutant LY-A cell derivatives expressing PLAP and  PLAP-HA, respectively. R, Endo H–resistant form; S, Endo  H–sensitive form; C, cleaved form by Endo H.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132924&req=5

Figure 11: ER-to-Golgi apparatus trafficking of GPI-anchored or membrane-spanning glycoprotein in wild-type and LY-A cells. Cells were pulsed for 15 min with [35S]methionine/cysteine and chased for the indicated time. Immunoprecipitates were treated with Endo H for 16 h at 37°C and separated by SDS-PAGE as described in Materials and Methods. (A and C) Wild-type CHO cell derivatives expressing PLAP and PLAP-HA, respectively; (B and D) mutant LY-A cell derivatives expressing PLAP and PLAP-HA, respectively. R, Endo H–resistant form; S, Endo H–sensitive form; C, cleaved form by Endo H.

Mentions: To address the question of whether ER-to-Golgi apparatus trafficking of proteins was affected in LY-A cells, we determined the acquisition rate of Endo H resistance, a well-accepted determinant for arrival in the medial Golgi apparatus (Kornfeld and Kornfeld, 1985). For this, we used wild-type and LY-A cells constitutively expressing GPI-anchored alkaline phosphatase (PLAP) or chimeric alkaline phosphatase (PLAP-HA) having a membrane-spanning region in place of the GPI anchor. PLAP and PLAP-HA in these cells were pulse-labeled with [35S]methionine/cysteine for 15 min and chased for various time at 33°C. Then, extracts from the cells were immunoprecipitated with anti-PLAP antibody, treated with Endo H, and separated by SDS-PAGE for detection of the Endo H–sensitive and –resistant forms. From the data shown in Fig. 11, the acquisition rates of Endo H resistance of not only PLAP but also PLAP-HA were quite similar (t1/2 = ∼30 min) between wild-type and LY-A cells. Thus, it was likely that the machinery for ER-to-Golgi apparatus trafficking of glycoproteins operated normally in 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)

ER-to-Golgi apparatus trafficking of GPI-anchored  or membrane-spanning glycoprotein in wild-type and LY-A cells.  Cells were pulsed for 15 min with [35S]methionine/cysteine and  chased for the indicated time. Immunoprecipitates were treated  with Endo H for 16 h at 37°C and separated by SDS-PAGE as  described in Materials and Methods. (A and C) Wild-type CHO  cell derivatives expressing PLAP and PLAP-HA, respectively;  (B and D) mutant LY-A cell derivatives expressing PLAP and  PLAP-HA, respectively. R, Endo H–resistant form; S, Endo  H–sensitive form; C, cleaved form by Endo H.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: ER-to-Golgi apparatus trafficking of GPI-anchored or membrane-spanning glycoprotein in wild-type and LY-A cells. Cells were pulsed for 15 min with [35S]methionine/cysteine and chased for the indicated time. Immunoprecipitates were treated with Endo H for 16 h at 37°C and separated by SDS-PAGE as described in Materials and Methods. (A and C) Wild-type CHO cell derivatives expressing PLAP and PLAP-HA, respectively; (B and D) mutant LY-A cell derivatives expressing PLAP and PLAP-HA, respectively. R, Endo H–resistant form; S, Endo H–sensitive form; C, cleaved form by Endo H.
Mentions: To address the question of whether ER-to-Golgi apparatus trafficking of proteins was affected in LY-A cells, we determined the acquisition rate of Endo H resistance, a well-accepted determinant for arrival in the medial Golgi apparatus (Kornfeld and Kornfeld, 1985). For this, we used wild-type and LY-A cells constitutively expressing GPI-anchored alkaline phosphatase (PLAP) or chimeric alkaline phosphatase (PLAP-HA) having a membrane-spanning region in place of the GPI anchor. PLAP and PLAP-HA in these cells were pulse-labeled with [35S]methionine/cysteine for 15 min and chased for various time at 33°C. Then, extracts from the cells were immunoprecipitated with anti-PLAP antibody, treated with Endo H, and separated by SDS-PAGE for detection of the Endo H–sensitive and –resistant forms. From the data shown in Fig. 11, the acquisition rates of Endo H resistance of not only PLAP but also PLAP-HA were quite similar (t1/2 = ∼30 min) between wild-type and LY-A cells. Thus, it was likely that the machinery for ER-to-Golgi apparatus trafficking of glycoproteins operated normally in 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