Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin—Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-α (PKC-α) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5′-[γ-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3–4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-α binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.
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February 2002
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Research Article|
January 25 2002
Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin–Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor
Abdelkarim ABOUSALHAM;
Abdelkarim ABOUSALHAM
1
∗Department of Biochemistry, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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Tom C. HOBMAN;
Tom C. HOBMAN
†Department of Cell Biology, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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Jay DEWALD;
Jay DEWALD
∗Department of Biochemistry, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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Michael GARBUTT;
Michael GARBUTT
†Department of Cell Biology, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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David N. BRINDLEY
David N. BRINDLEY
2
∗Department of Biochemistry, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
2To whom correspondence should be addressed (e-mail david.brindley@ualberta.ca)
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Publisher: Portland Press Ltd
Received:
September 27 2001
Revision Received:
November 02 2001
Accepted:
November 15 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 361 (3): 653–661.
Article history
Received:
September 27 2001
Revision Received:
November 02 2001
Accepted:
November 15 2001
Citation
Abdelkarim ABOUSALHAM, Tom C. HOBMAN, Jay DEWALD, Michael GARBUTT, David N. BRINDLEY; Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin–Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor. Biochem J 1 February 2002; 361 (3): 653–661. doi: https://doi.org/10.1042/bj3610653
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