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

我们制备了一种与胆固醇结合的细胞溶血素的非溶细胞性衍生物θ-毒素，作为胆固醇的探针，并证明了该探针(BC-θ；酶刻痕和生物素化的θ-毒素)选择性地与完整细胞富含胆固醇的膜区中的胆固醇结合。通过电子显微镜分析，我们发现BCθ结合在洗涤剂不溶低密度组分(RAFT组分)中的一组膜上。结果表明，RAFT组分中含有不同种类的膜。因此，我们进一步分离了BC-θ结合膜并对其进行了表征。从BCθ结合的Jurkat T细胞中制备RAFT组分，并使用亲和素磁珠将其分离成BCθ结合的膜和未结合的膜。BCθ结合膜的胆固醇含量高于未结合组分。Flotillin是一种典型的RAFT标记蛋白，而RAFT组分中的Flotillin和Src家族蛋白激酶，如Lck和Fyn，几乎都是从BCθ结合组分中回收的。在参与T细胞信号转导的分子中，从BCθ结合组分中恢复了LAT，而从未结合组分中恢复了CD3和ZAP70。这些结果表明，RAFT组分在分子组成上是不均匀的，BCθ可用于RAFT组分中高胆固醇膜的分离和定位分子的检测。

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.