Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.
Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.
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DOI：
10.1074/jbc.m808387200
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发表时间：
2009-04-10
期刊：
影响因子：
--
通讯作者：
Roberts LM
中科院分区：
文献类型：
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作者：
Mayerhofer PU;Cook JP;Wahlman J;Pinheiro TT;Moore KA;Lord JM;Johnson AE;Roberts LM
在哺乳动物细胞内吞后，异二聚体植物毒素蓖麻毒素被转运到内质网（ER），在内质网中，蓖麻毒素A链（RTA）必须穿过内质网膜到达其核糖体底物。通过凝胶过滤层析、沉淀、荧光、荧光共振能量转移和圆二色，我们发现荧光标记和未标记的RTA都能与内质网微粒体膜和带负电荷的脂质体结合。RTA在0-30°C下与膜结合，使某些RTA残基暴露于双分子层的非极性脂质核心，而蛋白质的二级结构几乎没有变化。然而，当温度升高时，RTA中的主要结构重排发生。在37°C时，膜结合毒素失去了一些螺旋内容物，其C端靠近膜表面，并插入双分子层。然后RTA稳定地结合在膜上，因为它不能用碳酸盐提取。结构变化的强烈温度依赖性与脂质相变化不一致，因为荧光检测到的膜迁移率在30至37°C之间几乎没有变化。相反，结构重排可能先于或启动毒素通过内质网膜到细胞质的逆转录。RTA中这些变化对温度的强烈依赖性进一步表明，它们在植物毒素的哺乳动物靶点中最容易发生。
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 °C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 °C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 °C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.