title: pH-independent retrograde targeting of glycolipids to the Golgi complex.
abstract: A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood. In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells. The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid "rafts" and that internalization is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.
authors.affiliation: Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: F B
authors.initials: FB
authors.lastname: Schapiro
authors.forename: C
authors.initials: C
authors.lastname: Lingwood
authors.forename: W
authors.initials: W
authors.lastname: Furuya
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D003603:Cytotoxins / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli; D045683:Furin; D005677:G(M1) Ganglioside / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D022622:Shiga Toxin 1; D013381:Subtilisins / Q000378:metabolism; D014281:Trihexosylceramides / Q000378:metabolism; D014709:Vero Cells; D014734:Vibrio cholerae
chemical_list: D001427:Bacterial Toxins; D002966:Clathrin; D003603:Cytotoxins; D004768:Enterotoxins; D006017:Glycolipids; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; C037420:stN protein, Vibrio cholerae; D005677:G(M1) Ganglioside; D003571:Cytochalasin B; C018549:globotriaosylceramide; D013381:Subtilisins; D045683:Furin