The Structure and Assembly of the COPII Coat

描述（由申请人提供）：COPII蛋白通过将蛋白质和脂质货物运输出内质网（ER），在早期分泌途径中发挥关键作用。COPII蛋白由Sar1（一种GTPase）、Sec23/24（货物选择和GAP活性）和Sec13/31（促进外壳组装）组成。它们共同形成一层吸收和浓缩货物的外膜，并逐渐将内质网膜变形成一个囊泡。最近，研究人员发现Sec13/31可以自组装成一个独特的立方体笼状结构。这些结构通过低温电子显微镜（cryoEM）和单粒子重建来解决，并对COPII包被囊泡组装的一些机制有了深入的了解。这些初步研究得到了由Sec13/31与Sec23/24自组装形成的COPII外壳的新结构的支持。这两种结构共同构成了解剖COPII蛋白在细胞中发挥功能的机制的基础。目前的提案旨在通过四个具体目标来解决COPII结构和组装的问题。目的1提出确定单个COPII包被和COPII包被囊泡的结构。这些结构将阐明货物与Sec23/24和Sec23/24与Sec13/31相互作用的方式，从而有助于了解货物指导适当大小囊泡组装的机制。在aim 2中，提出了确定COPII外壳与Sar1复合物的结构，这些研究将在分子水平上揭示Sar1如何参与启动COPII外壳的形成。在aim 3中探讨了系绳的分子机制，其中提出了确定COPII外壳与TRAPPI系绳蛋白Bet3复合物的结构。最后，目标4提出了在不同中间阶段开发中毒COPII笼组件的检测方法，并确定中间产物的结构。总之，这些研究将通过进一步了解COPII结构以及它们如何在细胞中组装来推动囊泡运输领域。反过来，这将有助于理解COPII蛋白在乳糜微粒滞留病和颅透镜缝发育不良等疾病中的作用，这两种疾病分别由Sar1和Sec23/24突变引起，以及表现为运输缺陷的疾病，如囊性纤维化。

DESCRIPTION (provided by applicant): COPII proteins play a critical role in the early secretory pathway by transporting protein and lipid cargo out of the endoplasmic reticulum (ER). The COPII proteins consist of Sar1 (a GTPase), Sec23/24 (cargo selection and GAP activity), and Sec13/31 (promotes coat assembly). Together, these form a coat that recruits and concentrates cargo and gradually deforms the ER membrane into a vesicle. Recently, it was found that Sec13/31 self-assembles into a unique cuboctahedron cage-like structure. These structures were solved with cryo-electron microscopy (cryoEM) and single particle reconstruction and yielded insights into some of the mechanisms by which COPII coated vesicles are assembled. These initial studies were bolstered with a new structure of a COPII coat formed from the self-assembly of Sec13/31 with Sec23/24. Together, these two structures form a foundation for dissecting the mechanisms by which the COPII proteins perform their functions in the cell. The current proposal seeks to address questions about COPII structure and assembly through four specific aims. Aim 1 proposes to determine the structures of individual COPII coats and COPII coated vesicles. These structures will shed light on the ways that cargo interacts with Sec23/24 and Sec23/24 interacts with Sec13/31, and thus contribute to a picture of the mechanisms by which cargo directs the assembly of vesicles of the proper size. In aim 2, it is proposed to determine the structure of the COPII coat in complex with Sar1, and these studies will reveal at a molecular level how Sar1 is involved in initiating the formation of the COPII coat. The molecular mechanisms of tethering are explored in aim 3, where it is proposed to determine the structure of the COPII coat in complex with the TRAPPI tether protein Bet3. Finally, aim 4 proposes to develop assays for poisoning COPII cage assembly at various intermediate stages and to determine the structures of the intermediates. Together, these studies will drive the vesicle transport field by furthering our understanding of COPII structures and how they are assembled in the cell. This, in turn, will aid in the understanding of the role COPII proteins play in diseases like chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which result from mutations in Sar1 and Sec23/24 respectively, and diseases that manifest as transport defects such as cystic fibrosis. PUBLIC HEALTH RELEVANCE: The COPII proteins are involved in the secretory pathway, which is a critical and fundamental pathway in eukaryotes such as humans. Two diseases, chylomicron retention disease and cranio-lenticulo sutural dysplasia, are associated with mutations in COPII proteins, and a host of diseases including cystic fibrosis result from mutations that cause the proteins to be retained in the ER. Understanding of the mechanisms by which cargo proteins interact with the COPII coat and how COPII coat assembles will help us understand the role COPII plays in these diseases and may help in identifying novel targets for therapeutics.