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

我们制备了一种非细胞溶解衍生物的perfringolysin O (θ-toxin)，一种胆固醇结合的细胞溶解素，作为胆固醇探针，并证明了探针（BCθ；蛋白酶缺口和生物素化的θ-toxin）在完整细胞的富含胆固醇的膜结构域中选择性地结合胆固醇。通过电子显微镜分析，我们发现BCθ与在洗涤剂不溶性低密度馏分（筏馏分）中回收的膜亚群结合。结果表明，筏状馏分中含有不均匀的膜群。因此，我们进一步分离了bc θ-结合膜并对其进行了表征。从bc θ结合的Jurkat T细胞制备筏形分数，并使用亲和素磁珠将其分离到bc θ结合和未结合的膜中。bc θ结合膜的胆固醇含量高于未结合膜。筏子馏分中典型的筏子标记蛋白Flotillin和src家族蛋白激酶，如Lck和Fyn，几乎全部从bc θ-结合馏分中回收。在参与t细胞信号传导的分子中，LAT是从bc θ-结合的部分中回收的，而CD3和Zap70是从未结合的部分中回收的。这些结果表明筏状馏分在分子组成上是不均匀的，并揭示了BCθ对筏状馏分中富含胆固醇的膜的亚分馏和检测其定位的分子是有用的。

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.