Characterization of mammalian COG complex-interacting Golgi trafficking machinery

项目摘要/摘要 细胞内膜转运调节细胞内蛋白质和脂类的运输。这个过程是双向的。 由顺行(分泌)和逆行(内吞)分支组成。细胞内膜运输是 进化保守，其机制是模块化的，功能上相同的组件运行在不同的 贩卖人口的步骤。因此，详细了解贩运的一个步骤将有助于理解整个 胞内膜转运过程。 保守的寡聚体高尔基体(COG)复合体作为囊状系绳在高尔基体内运输。高尔基人是 蛋白质翻译后修饰的中心枢纽，主要是糖基化。因此，委员会的主要工作是 COG是将回收驻留的酶和货物受体的小泡拴在一起。为了实现其职能，COG与 有圈套、RAB和其他系绳，但对这些相互作用的详细理解是我们提出的一个谜 通过成对探测COG/合作伙伴的相互作用、它们的动力学并通过定义它们的分子环境来解决 通过邻近标记研究。COG的耗尽导致特定转运中间体的积累-COG 复杂依赖(CCD)小泡，很可能代表循环高尔基酶的一大类高尔基小泡 和货物受体。 COG亚基突变导致先天性糖基化紊乱(CDG)II型，属于 一组常染色体隐性遗传性多系统疾病，具有几种可区分的症状，包括 发育缺陷和小头畸形。这些缺陷通常伴随着神经和肝脏损害。 CoG-CDG缺陷是在患者的成纤维细胞中进行的研究，这些成纤维细胞并不代表受影响最严重的组织；更灵活的 细胞基础模型将有助于我们在开发治疗这种疾病的方法方面取得进展。 我们假设，揭示COG/合作伙伴相互作用的分子基础将有助于破译 气泡对接平台的装配/拆卸机理以及对不同人群的详细分析 Ccd囊泡将揭示其特定的来源、萌芽和拴系机制以及一套完整的蛋白质，这些蛋白质 以依赖COG的方式传输流量。基于细胞的COG-CDG模型的开发将使我们能够测试 不需要患者参与的不同COG突变的影响，并为发展为 治疗方案。为了验证这一假设，首先，我们将在COG和其关键字之间的分子相互作用中表征 配对蛋白(Aim1)。接下来，我们将使用降糖辅助的COG耗竭来积累、纯化和表征CCD 囊泡(AIM2)。最后，我们将开发和表征一个新的基于IPSC的COG CDG细胞模型(目标3)。 成功实现这些目标将提供对COG复杂功能的机械性理解，表征 COG依赖的转运中间体，并创建一组携带人类COG突变的等基因干细胞系。 此外，这些结果对于从功能上理解高尔基体动态和囊泡贩运是必要的。 将军。

PROJECT SUMMARY/ABSTRACT Intracellular membrane trafficking mediates the intracellular delivery of proteins and lipids. The process is bidirectional and consists of the anterograde (secretory) and retrograde (endocytic) branches. Intracellular membrane trafficking is evolutionary conserved, and its machinery is modular, with functionally homologous components operating on different trafficking steps. Therefore, a detailed understanding of one trafficking step will help in understanding the entire intracellular membrane trafficking process. The Conserved Oligomeric Golgi (COG) complex operates as a vesicular tether for intra-Golgi trafficking. The Golgi is the central hub for protein posttranslational modifications, mostly glycosylation. Consequently, the primary job of the COG is to tether vesicles that recycle resident enzymes and cargo receptors. To achieve its function, the COG interacts with SNAREs, Rabs, and other tethers, but the detailed understanding of these interactions is an enigma that we propose to solve by pairwise probing of COG/partner interactions, their kinetics, and by defining their molecular environment through proximity-labeling studies. Depletion of COG causes accumulation of specific transport intermediates – COG complex dependent (CCD) vesicles that are likely to represent a major class of Golgi vesicles that recycle Golgi enzymes and cargo receptors. Mutations in COG subunits result in congenital disorders of glycosylation (CDG) type II category, which belong to a group of autosomal recessive multi-systemic disorders with several distinguishable symptoms that include global developmental defects and microcephaly. These deficits are often accompanied by neurological and liver impairment. COG-CDG defects are studied in patients’ fibroblasts, which do not represent the most affected tissues; a more flexible cell base model will benefit our progress in developing a cure for this disorder. We hypothesize that a revealing of the molecular basis of COG/partner interactions will help in deciphering the mechanisms of assembly/disassembly of vesicle docking platforms and that a detailed analysis of different populations of CCD vesicles will uncover their specific origin, budding and tethering machinery and a complete set of proteins that traffic in a COG-dependent manner. The development of cell-based models for COG-CDGs will allow us to test the effects of different COG mutations without the need for patient involvement and pave the way for the development of treatment protocols. To test this hypothesis, first, we will characterize in molecular interactions between COG and its key partner proteins (Aim1). Next, we will use a degrone-assisted COG depletion to accumulate, purify and characterize CCD vesicles (Aim2). Finally, we will develop and characterize a novel iPSC-based cellular model for COG CDGs (Aim 3). Success in accomplishing these aims will provide a mechanistic understanding of COG complex function, characterize COG-dependent trafficking intermediates, and create a set of isogenic stem cell lines bearing human COG mutations. Moreover, these results will be necessary for a functional understanding of Golgi dynamic and vesicular trafficking in general.