Waheed A A, Shimada Y, Heijnen H F, Nakamura M, Inomata M, Hayashi M, Iwashita S, Slot J W, Ohno-Iwashita Y
Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4926-31. doi: 10.1073/pnas.091090798. Epub 2001 Apr 17.
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 (BCtheta) 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 BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta 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 BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.
越来越多的证据表明，质膜中存在富含鞘脂和胆固醇的微结构域（脂筏）。特定蛋白质在这些膜结构域中组装，并在信号转导和许多其他细胞活动中发挥作用。胆固醇耗竭会导致脂筏相关蛋白的解离，这表明胆固醇在脂筏的结构维持和功能中起着至关重要的作用。然而，目前还没有工具可用于检测和监测活细胞中的脂筏胆固醇。在这里，我们表明，产气荚膜梭菌溶血素O（θ毒素）的蛋白酶切割和生物素化衍生物（BCtheta）选择性地结合完整细胞中富含胆固醇的微结构域，这些结构域符合脂筏的标准。在用BCtheta孵育后，我们在蔗糖梯度上对未处理和经Triton X-100处理的血小板匀浆进行了分级分离。BCtheta主要定位于漂浮的低密度组分（FLDF）中，其中胆固醇、鞘磷脂和Src家族激酶含量丰富。免疫电子显微镜显示，BCtheta与FLDF中的一部分囊泡结合。用环糊精使血小板中的胆固醇耗竭35%，同时FLDF中的胆固醇减少76%，几乎完全消除了BCtheta与FLDF的结合。在有或没有胆固醇耗竭的人表皮样癌A431细胞中，BCtheta和制霉菌素的染色模式表明，BCtheta与细胞表面的特定膜结构域结合，而制霉菌素的结合对细胞胆固醇是无差别的。此外，BCtheta的结合不会对细胞膜造成任何损伤，这表明BCtheta是检测活细胞中膜脂筏的有用探针。
