Regulatory mechanisms that control vesicle secretion at the endoplasmic reticulum

描述（由申请人提供）：本提案的长期目标是确定早期分泌途径中调节细胞器空间分布的分子机制，并确定该结构在细胞生长和发育过程中对正常膜运输的重要性。大多数用于分泌的生物合成货物分子在内质网（ER）的特定亚域内开始其旅程，称为内质网出口位点。在这些位置，首先产生copii包被的囊泡，包装货物以运输到内质网-高尔基中间区室（ERGIC），稳定的细胞器与内质网出口位置并置。COPII外壳由两个多聚体蛋白复合物Sec23/24和Sec13/31以及小的GTPase Sar1组成。尽管这些因素足以在体外从化学定义的膜中重建囊泡出芽，但还需要额外的蛋白质来促进COPII囊泡的生物发生和细胞内的顺行运输。这一建议的重点是TFG的作用，TFG是一种后生动物特异性蛋白，是COPII外壳亚基在内质网出口位点的强大募集所必需的。基于我们的初步结果，我们假设TFG在ER/ERGIC界面形成了一个高度调控的网络，促进了COPII涂层的稳定性和囊泡的排出。重要的是，TFG突变与进行性神经退行性疾病有关，表明copii介导的转运在维持神经元功能中的作用。我们建议结合体内和体外方法来实现我们的目标，利用在人类细胞中开发的检测方法和建立的生物化学方法来研究早期分泌途径成分的结构和功能。利用电子断层扫描，我们最近定义了模式生物秀丽隐杆线虫生殖细胞早期分泌通路的结构。我们的发现揭示了一个充满TFG分子的电子密集网络的存在，它包含了内质网出口位点和ERGIC膜之间的区域。我们假设该网络在调节COPII动力学中起作用，并有助于维持早期分泌途径的组织。以类似的方式，我们在人类细胞中的初步研究已经证明了TFG在控制早期分泌途径结构和功能方面的保守作用。此外，我们最近使用低温电子显微镜和小角度x射线散射定义了TFG的结构模型，这使我们提出了TFG在ER和ERGIC膜之间界面作用的可测试模型。本提案的具体目标是：1)确定TFG在ER/ERGIC界面组装和拆卸的结构基础，2)定义TFG对copii介导的囊泡运输的贡献，以及3)定义TFG有助于神经元维持的机制。总之，本提案中概述的实验将为早期分泌途径的组织如何促进copii包被囊泡中新合成的货物的快速顺行运输提供基本的新见解，这是正常人类发育和神经元稳态所必需的。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER), known as ER exit sites. At these locations, COPII-coated vesicles are first generated, packaging cargoes for transport to ER- Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to ER exit sites. The COPII coat is composed of two multimeric protein complexes, Sec23/24 and Sec13/31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII vesicle biogenesis and anterograde transport in cells. This proposal focuses on the role of TFG, a metazoan-specific protein required for the robust recruitment of COPII coat subunits to ER exit sites. Based on our preliminary results, we hypothesize that TFG forms a highly regulated meshwork at the ER/ERGIC interface that facilitates COPII coat stability and vesicle egress. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, suggesting a role for COPII-mediated transport in maintaining neuron function. We propose a combination of in vivo and in vitro approaches to achieve our aims, taking advantage of assays developed in human cells and biochemical methodologies established to study the structure and function of early secretory pathway components. Using electron tomography, we recently defined the architecture of the early secretory pathway in germ cells derived from the model organism C. elegans. Our findings revealed the presence of an electron-dense meshwork, filled with molecules of TFG, which encompasses the region between ER exit sites and ERGIC membranes. We hypothesize that this meshwork functions in the regulation of COPII dynamics and helps to maintain the organization of the early secretory pathway. In a similar fashion, our preliminary studies in human cells have demonstrated a conserved role for TFG in controlling early secretory pathway architecture and function. Additionally, we have recently defined a structural model for TFG using cryo-electron microscopy and small angle X-ray scattering, which has led us to propose a testable model for the role of TFG at the interface between ER and ERGIC membranes. The specific aims of this proposal are to: 1) determine the structural basis for TFG assembly and disassembly at the ER/ERGIC interface, 2) define the contributions of TFG to COPII-mediated vesicle transport, and 3) define mechanisms by which TFG contributes to neuronal maintenance. Together, the experiments outlined in this proposal will provide fundamental new insights into how the organization of the early secretory pathway promotes the rapid anterograde transport of newly synthesized cargoes in COPII-coated vesicles, which is necessary for normal human development and neuronal homeostasis.