Structure and function of cholesterol-enriched membrane microdomains (lipid rafts)

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，并证明探针（BCθ）在完整细胞的富含胆固醇的膜结构域选择性地结合胆固醇。基于在洗涤剂不溶性低密度部分（筏分数）中回收的BCθ结合膜亚群的发现，我们从Jurkat T细胞的筏分数中分离出BCθ结合膜。bc θ结合膜亚群的胆固醇/磷脂（C/P）摩尔比（~ 1.0）远高于筏形分数中bc θ未结合的亚群（~ 0.3）。它不仅含有筏标记GM1和flotillin，还含有一些t细胞受体（TCR）信号分子，包括Lck、Fyn和LAT。此外，TCR信号级联的抑制分子Csk和PAG也包含在bc θ-结合膜中。另一方面，CD3和Zap70定位于bc θ-未结合膜，在非刺激条件下与其他TCR信号分子分离。然而，在TCR的刺激下，部分CD3和Zap70被招募到bc θ结合的膜上。用于脂筏制备的Triton X-100浓度既不影响C/P比，也不影响bc θ-结合膜的蛋白质组成。这些结果表明，我们的方法可用于分离特定的富含胆固醇的T细胞膜结构域，该结构域可能是控制TCR信号级联的核心结构域。

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) binds selectively to cholesterol in cholesterol-rich membrane domains of intact cells. Based on the finding that BCθ binds to a subpopulation of membranes among those recovered in detergent-insoluble, low-density fractions (raft fractions),' we isolated BCθ-bound membranes from the raft fractions of Jurkat T cells. The BCθ-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (〜1.0) than the BCθ-unbound population in raft fractions (〜0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signaling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signaling cascade, are also contained in the BCθ-bound membranes. On the other hand, CD3 and Zap70 are localized in the BCθ-unbound membranes, segregated from other TCR signaling molecules under non-stimulated conditions. However, upon stimulation of TCR, portions of CD3 and Zap70 are recruited to the BCθ-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCθ-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T cells, which could be a core domain controlling the TCR signaling cascade.