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Glycobiology Advance Access published online on April 21, 2004
Glycobiology, doi:10.1093/glycob/cwh085
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Received March 3, 2004
Revised April 6, 2004
Accepted April 8, 2004

ORIGINAL ARTICLES

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

Aye Aye Khine 1, Patty Tam 1, Anita Nutikka 2, Clifford A. Lingwood 3*

1 Department of Laboratory Medicine & Pathobiology, University of Toronto, Ontario, Canada
2 Division of Immunity, Infection, Injury and Repair, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada
3 Department of Laboratory Medicine & Pathobiology, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada; Division of Immunity, Infection, Injury and Repair, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada

* To whom correspondence should be addressed. E-mail: cling{at}sickkids.on.ca.


  Abstract

In verotoxin 1 (VT1) sensitive cells, globotriaosyl ceramide(Gb3) bound VT1 is endocytosed, and transported retrogradely to the Golgi/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 3hr exposure to <50 ng/mlVT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 hr incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae dependent) route for cytotoxicity becomes significant. Brefeldin A, which prevents Golgi-dependant 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 non-toxic 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 signalling. While cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin1 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 non-interconverting pools of membrane Gb3.

Key words: Cholesterol, Endocytosis, Glycolipid enriched microdomains-rafts, Golgi-Endoplasmic reticulum, Retrograde transport


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