Glycobiology Advance Access originally published online on April 21, 2004
Glycobiology 2004 14(8):701-712; doi:10.1093/glycob/cwh085
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Glycobiology vol. 14 no. 8 © Oxford University Press 2004; all rights reserved.
Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity
3 Department of Laboratory Medicine & Pathobiology, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada; 4 Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada; and 5 Division of Immunity, Infection, Injury and Repair, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada
Received on March 3, 2004; revised on April 6, 2004; accepted on April 8, 2004
In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to <50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1–containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.
1 Present address: Department of Anaesthesia Research, St. Michael's Hospital, 30 Bond Street, Room 9-015, Queen Wing, Toronto, Ontario M5B 1W8, Canada
2 To whom correspondence should be addressed; e-mail: cling{at}sickkids.on.ca
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