Regulatory mechanisms that control vesicle secretion at the endoplasmic reticulum

描述(申请人提供)：这项建议的长期目标是确定在早期分泌途径中调节细胞器空间分布的分子机制，并确定这种结构对细胞生长和发育过程中正常的膜运输的重要性。大多数用于分泌的生物合成货物分子都是在内质网(ER)的特定亚域内开始它们的旅程的，这些亚域被称为内质网出口部位。在这些位置，首先产生COPII包被的囊泡，将货物包装到ER-Golgi中间隔室(ERGIC)，ERGIC是并列在ER出口部位的稳定细胞器。COPII外壳由两个多聚体蛋白复合体Sec23/24和Sec13/31和小的GTPase Sar1组成。虽然这些因素足以在体外从化学定义的膜中重建囊泡萌发，但还需要更多的蛋白质来促进COPII囊泡的生物发生和细胞内的顺行运输。这项建议侧重于TFG的作用，这是一种后生动物特有的蛋白质，需要强劲地将COPII外壳亚单位招募到ER出口部位。根据我们的初步结果，我们假设TFG在ER/ERGIC界面形成一个高度调控的网络，促进COPII涂层的稳定性和囊泡的外泄。重要的是，TFG的突变与进行性神经退行性疾病有关，这表明COPII介导的转运在维持神经元功能方面发挥了作用。我们提出了体内和体外相结合的方法来实现我们的目标，利用在人类细胞中开发的分析方法和建立的生化方法来研究早期分泌途径组件的结构和功能。利用电子断层摄影术，我们最近确定了模式生物秀丽线虫生殖细胞早期分泌途径的结构。我们的发现揭示了一个电子致密网络的存在，其中充满了TFG分子，它包围了ER出口位点和ERGIC膜之间的区域。我们推测，这个网络在COPII动力学的调节中起作用，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表明，TFG在控制早期分泌途径的结构和功能方面发挥了保守的作用。此外，我们最近使用冷冻电子显微镜和小角X射线散射定义了TFG的结构模型，这使得我们提出了TFG在ER和ERGIC膜之间的界面上的作用的可测试模型。这一建议的具体目的是：1)确定TFG在ER/ERGIC界面组装和拆解的结构基础；2)确定TFG在COPII介导的囊泡运输中的作用；3)确定TFG促进神经元维持的机制。总之，这项提案中概述的实验将为早期分泌途径的组织如何促进COPII包裹的囊泡中新合成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动态平衡所必需的。

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.