Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex.
Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.
Tomita
Toshio
T
Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan. tomita@biochem.tohoku.ac.jp
Noguchi
Kayoko
K
Mimuro
Hitomi
H
Ukaji
Fumio
F
Ito
Kiyoshi
K
Sugawara-Tomita
Noriko
N
Hashimoto
Yohichi
Y
D000818:Animals; D002784:Cholesterol / Q000737:chemistry; D004285:Dogs; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects; D006736:Horses; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009183:Mycotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry; D020076:Pleurotus / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012756:Sheep; D013109:Sphingomyelins / Q000378:metabolism / Q000494:pharmacology*
D006454:Hemoglobins; D006460:Hemolysin Proteins; D008081:Liposomes; D009183:Mycotoxins; D010743:Phospholipids; D013109:Sphingomyelins; D002784:Cholesterol; D011188:Potassium