title: Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine & Pathobiology, University of Toronto, 555 University Avenue, Toronto, Ontario, Canada.
authors.forename: Aye Aye
authors.initials: AA
authors.lastname: Khine
authors.forename: Patty
authors.initials: P
authors.lastname: Tam
authors.forename: Anita
authors.initials: A
authors.lastname: Nutikka
authors.forename: Clifford A
authors.initials: CA
authors.lastname: Lingwood
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000378:metabolism* / Q000633:toxicity; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D016923:Cell Death; D002470:Cell Survival; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D005372:Filipin / Q000378:metabolism* / Q000633:toxicity; D006056:Golgi Apparatus / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D022622:Shiga Toxin 1 / Q000378:metabolism* / Q000633:toxicity; D014281:Trihexosylceramides / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D051242:Caveolin 1; D022461:Caveolins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D020126:Brefeldin A; C018549:globotriaosylceramide; D005372:Filipin