Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts.
Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.
Nelson
K L
KL
Department of Biochemistry and Microbiology, University of Victoria, Box 3055, Victoria, British Columbia V8W 3P6, Canada.
Buckley
J T
JT
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000187:drug effects*; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D013109:Sphingomyelins / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects; D013997:Time Factors; D047392:beta-Cyclodextrins
D001427:Bacterial Toxins; D003505:Cyclodextrins; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D007473:Ion Channels; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D052899:Pore Forming Cytotoxic Proteins; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C032676:aerolysin; D002784:Cholesterol; D000469:Alkaline Phosphatase