STRUCTURAL STUDY OF THE OUTER SHELL OF THE COPII VESICLE COAT

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这是旨在获得酵母COPII囊泡外壳结构信息的系列提案中的第三个。COPII是一个420 kDa的五聚体复合体，负责从内质网（ER）出芽运输囊泡并将特定的货物内容物运送到高尔基复合体。在第一个方案中，我们专注于COPII外壳的内壳，Sec23/24-Sar1复合体。我们能够确定三个Sec24与cargo和SNARE序列结合的复合物的结构（论文发表在Cell上）。在当前的提案中，我们关注的是COPII的外壳，由Sec13/31复合物组成。Sec 13/31在功能上（而非结构上）与网格蛋白是同源的——它在内质网膜上聚合COPII内壳并导致膜变形。因为，像网格蛋白一样，Sec13/31是一种能够寡聚的长而灵活的结构（如EM图像所示），所以完整的复合物不太可能结晶。因此，我们将蛋白质分解成我们认为功能上有趣的两个片段，这两个片段构成了我们在本次申请中提出的两个项目。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is the third in a series of proposals that aims at obtaining structural information on the yeast COPII vesicalar coat. COPII is a 420 kDa pentameric complex responsible for budding transport vesicles from the endoplasmic reticulum (ER) and delivering the specific cargo contents to the Golgi complex. In the first proposal we focused on the inner shell of the COPII coat, the Sec23/24-Sar1 complex. We were able to determine the structures of three complexes of Sec24 bond to cargo and SNARE sequences (paper published in Cell). In the current proposal we focus on the outer shell of COPII , comprising of the Sec13/31 complex. Sec 13/31 is functionally, but not structurally, homolosous to clathrin--it polymerizes the COPII inner shell on the ER membrane and causes membrane deformation. Since, like clathrin, Sec13/31 is a long, flexible structure capable of oligomerizing (as seen in EM images) it is unlikely that the intact complex can be crystallized. There fore, we have dessected the protein into waht we believe are the tow functionally interesting fragments, and these constitue the two projects that we propose in this application.