Intracellular glycosylphosphatidylinositols accumulate on endosomes: toxicity of alpha-toxin to Leishmania major.
Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.
Zheng
Zhifeng
Z
Department of Cellular Biology, The University of Georgia, 724 Biological Sciences, Athens, GA 30602, USA.
Tweten
Rodney K
RK
Mensa-Wilmot
Kojo
K
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D001665:Binding Sites; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007202:Indicators and Reagents / Q000378:metabolism; D018320:Leishmania major / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D017830:Octoxynol / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D012118:Resorcinols / Q000378:metabolism
D001427:Bacterial Toxins; D003902:Detergents; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D007202:Indicators and Reagents; D015800:Protozoan Proteins; D012118:Resorcinols; C064270:hemolytic toxin, Clostridium septicum; C005282:orcinol; D017830:Octoxynol; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania