Gimeno R E, Espenshade P, Kaiser C A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Mol Biol Cell. 1996 Nov;7(11):1815-23. doi: 10.1091/mbc.7.11.1815.
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.
在内质网（ER）上形成COPII被膜小泡需要五种胞质被膜蛋白Sec23p、Sec24p、Sec13p、Sec31p和Sar1p组装到膜上。第六种小泡被膜成分Sec16p与内质网膜紧密相连，并被认为作为可溶性被膜蛋白膜结合的支架。我们之前发现Sec23p与Sec16p的C末端区域结合。在此我们使用双杂交和共沉淀分析来证明必需的COPII蛋白Sec24p与Sec16p的中央区域结合。用纯化的重组蛋白进行体外结合重建表明，Sec24p与Sec16p中央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，并且这三种蛋白在体外可以形成三元复合物。Sec24p的截短表明Sec24p的N末端和C末端区域表现出不同的结合特异性。C末端与Sec16p的中央结构域结合，而Sec24p的N末端既与Sec16p的中央结构域结合，也与Sec23p结合。这些发现将与Sec16p的结合定义为Sec24p的一种新功能，并支持Sec16p组织COPII被膜组装的观点。
