COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p
COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p
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DOI：
10.1091/mbc.7.11.1815
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发表时间：
1996-11-01
影响因子：
3.3
通讯作者：
Kaiser, CA
中科院分区：
文献类型：
--
作者：
Gimeno, RE;Espenshade, P;Kaiser, CA
在内质网 (ER) 处形成 COPII 包被的囊泡需要将五种胞质外壳蛋白（Sec23p、Sec24p、Sec13p、Sec31p 和 Sar1p）组装到膜上。第六个囊泡外壳成分 Sec16p 与 ER 膜紧密相连，并被提议充当可溶性外壳蛋白膜关联的支架。我们之前表明 Sec23p 与 Sec16p 的 C 端区域结合。在这里，我们使用双杂交和共沉淀测定来证明必需的 COPII 蛋白 Sec24p 与 Sec16p 的中心区域结合。体外重建与纯化重组蛋白的结合表明 Sec24p 与 Sec16p 中心结构域的相互作用不依赖于 Sec23p 的存在。然而，Sec23p 促进 Sec24p 与 Sec16p 的结合，并且这三种蛋白质可以在体外形成三元复合物。 Sec24p 的截短表明 Sec24p 的 N 端和 C 端区域显示出不同的结合特异性。 C 末端与 Sec16p 的中心结构域结合，而 Sec24p 的 N 末端与 Sec16p 的中心结构域和 Sec23p 结合。这些发现将与 Sec16p 的结合定义为 Sec24p 的新功能，并支持 Sec16p 组织 COPII 外套组装的想法。
Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.