title: Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts.
abstract: Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.
authors.affiliation: Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, Japan. iwashita@tmig.or.jp
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Ohno-Iwashita
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Shimada
authors.forename: A Abdul
authors.initials: AA
authors.lastname: Waheed
authors.forename: Masami
authors.initials: M
authors.lastname: Hayashi
authors.forename: Mitsushi
authors.initials: M
authors.lastname: Inomata
authors.forename: Megumi
authors.initials: M
authors.lastname: Nakamura
authors.forename: Mikako
authors.initials: M
authors.lastname: Maruya
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwashita