Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC theta) of perfringolysin O (theta -toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC theta on a sucrose gradient. BC theta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC theta binds to a subpopulation of vesicles in FLDF, Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC theta binding to FLDF. The staining patterns of BC theta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC theta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC theta binding does not cause any damage to cell membranes, indicating that BC theta is a useful probe for the detection of membrane rafts in living cells.