Molecular analysis of membrane microdomains (lipid rafts) with a new cholesterol-binding probe

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We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol, and demonstrated that the probe (BCθ; protease-nicked and biotinylated θ-toxin) binds selectively to cholesterol in cholesterol-rich membrane domains of intact cells. By electron microscopic analysis, we showed that BCθ binds to a subpopulation of membranes among those recovered in detergent-insoluble, low-density fractions (raft fractions). The results indicate that raft fractions contain heterogeneous populations of membranes. Therefore we further isolated BCθ-bound membranes and characterized them. Raft fractions were prepared from BCθ-bound Jurkat T cells and fractionated into BCθ-bound and -unbound membranes using avidin-magnet beads. BCθ-bound membranes have higher cholesterol contents than unbound fractions. Flotillin, a typical raft marker protein, and Src-family protein kinases, such as Lck and Fyn, in raft fractions were almost exclusively recovered from BCθ-bound fractions. Among molecules that participate in T-cell signaling, LAT was recovered from BCθ-bound fractions, while CD3 and Zap70 were recovered from unbound fractions. These results indicate that raft fractions are heterogeneous in molecular composition, and reveal that BCθ is useful for the subfractionation of cholesterol-rich membranes in raft fractions and for detecting molecules localized in them.