Supplement: GOLGI BIOGENESIS AND FUNCTION

项目总结 高尔基复合体是细胞内转运、糖基化和分选的中心膜细胞器。 膜蛋白和分泌蛋白。高尔基体的基本结构是一堆扁平的池子，但如何 这种结构是在细胞分裂过程中组装和遗传的，人们对此知之甚少。在过去几年里，我们 已经开发出一种独特的多学科方法，利用生物化学、细胞生物学、电子显微镜、 最近，蛋白质组学和糖组学，结合一种新的体外重建试验，提供了一种 高尔基体构造形成和功能的力学解释。我们发现GRASP55和GRASP65 通过形成有丝分裂调节的反式-核糖核酸，在高尔基体结构的形成中发挥互补和重要的作用 将高尔基膜堆叠在一起的低聚物。用GRASP55/65作为操作高尔基堆栈的工具 形成，我们提供了第一个证据，高尔基堆积阻碍蛋白质运输，以确保准确 翻译后修饰。我们推测GRAP蛋白在高尔基体中起着重要作用 通过齐聚和与其他蛋白质的相互作用形成结构，从而 调节蛋白质的运输和糖基化。这项建议是我们先前建议的合理延续 研究评估高尔基体结构是如何组装的，以及为什么它对蛋白质运输很重要， 糖基化和分选。具体目标是：1)鉴定和表征新的GRASP65相互作用 调节高尔基体结构形成的蛋白质。我们的初步数据显示，细胞内含有蛋白质 促进GRASP65寡聚的间期细胞胞浆。我们已经确定了20种相互作用的蛋白质 我们将用GRASP65描述它们在高尔基结构形成中的作用。这也将有助于我们 了解抓握如何按照先前的报告执行多种功能。2)确定结构-- 高尔基体在蛋白质转运、糖基化和分选中的功能关系。我们会操纵高尔基人 结构，通过使用最近开发的CRISPR/Cas9技术敲除GRASP55/65，以及通过 在细胞中表达GRASP55/65突变体以确定高尔基体破坏和修复的后果 关于蛋白质的运输、修饰和分泌。值得注意的是，高尔基体结构和功能的改变 与多种人类疾病有关，包括癌症、自身免疫性疾病、病毒感染和 亨廷顿病和阿尔茨海默氏症。高尔基缺陷可能会影响贩运、分拣和修改 大量的蛋白质，并在细胞内和细胞表面引起全局性影响，危及 细胞功能的多样性。更好地理解高尔基体构造的形成及其与其本质的关系 在了解细胞在人类疾病中的作用之前，细胞功能是必需的。我们建议的研究将 确定高尔基体破坏对蛋白质运输和加工的影响，从而提供 关于正常情况下高尔基体的作用以及高尔基体之间关系的基本信息 缺陷与疾病发病机制。

PROJECT SUMMARY The Golgi complex is a central membrane organelle for intracellular trafficking, glycosylation and sorting of membrane and secretory proteins. The basic structure of the Golgi is a stack of flattened cisternae, but how this structure is assembled and inherited during cell division is poorly understood. In the last few years, we have developed a unique multidisciplinary approach employing biochemistry, cell biology, electron microscopy, and more recently proteomics and glycomics, combined with a novel in vitro reconstitution assay, to provide a mechanistic explanation for Golgi structure formation and function. We found that GRASP55 and GRASP65 play complementary and essential roles in Golgi structure formation by forming mitotically regulated trans- oligomers that hold the Golgi membranes into stacks. Using GRASP55/65 as tools to manipulate Golgi stack formation, we provided the first evidence that Golgi stacking impedes protein trafficking to ensure accurate post-translational modifications. We hypothesize that the GRASP proteins play essential roles in Golgi structure formation through oligomerization and interaction with other proteins, which subsequently regulates protein trafficking and glycosylation. This proposal is a logical continuation of our previous studies to assess how the Golgi structure is assembled and why it is important for protein trafficking, glycosylation and sorting. The specific aims are: 1) Identify and characterize novel GRASP65 interacting proteins that regulate Golgi structure formation. Our preliminary data showed that there are proteins in the interphase cell cytosol that enhance GRASP65 oligomerization. We have identified 20 proteins that interact with GRASP65 and we will characterize their roles in Golgi structure formation. This will also help us understand how GRASPs perform multiple functions as previously reported. 2) Determine the structure- function relationship of the Golgi in protein trafficking, glycosylation, and sorting. We will manipulate the Golgi structure by knocking out GRASP55/65, using the recently developed CRISPR/Cas9 technique, and by expressing GRASP55/65 mutants in cells to determine the consequences of Golgi destruction and restoration on protein trafficking, modifications and secretion. Significantly, alterations in Golgi structure and function have been associated with a variety of human diseases, including cancer, autoimmune disease, viral infections, and Huntington's and Alzheimer's diseases. Golgi defects may affect the trafficking, sorting and modification of a large number of proteins and cause global effects inside the cell and on the cell surface that compromise a variety of cellular functions. A better understanding of Golgi structure formation and the relationship to its vital cellular function is required before its role in human disease can be understood. Our proposed study will determine the consequence of Golgi destruction on protein trafficking and processing, and thus provide fundamental information on the role of the Golgi under normal conditions and the relationship between Golgi defects and disease pathogenesis.