STRUCTURAL BASIS OF ORGANELLE TETHERING

描述(申请人提供)：高尔基仪器被划分成相互关联的水池，产生堆叠。哺乳动物细胞中的多个堆栈移动到中心体周围位置，在那里相邻堆栈中的池子以同型方式融合(即顺式和顺式等)。以形成一个分隔的膜网络。堆叠成带状网络的横向连接提供了最佳的处理效率，并在细胞周期进展和伤口愈合中发挥作用。一个主要的目标是确定连接反应中特异性和调节的基础。这将提供对膜网络如何在细胞分裂过程中形成、区隔和遗传的机械理解。它还有望深入了解与激活与膜结合有关的膜系留、促进反式相互作用以及与圈套介导膜融合的协调有关的重大而令人烦恼的问题。我们的工作模型是，GM130通过C端的PDZ配体与GRASP65的PDZ2结构域结合，将GRASP65招募到顺式脑池。然后，相邻脑池上的GRASP65通过其PDZ1沟槽与TRAN相互作用，PDZ1沟槽通过GRASP65中存在的内部PDZ配体以同型方式结合。这提高了将膜靠近以进行诱捕接触和膜融合的效率和保真度。我们还假设，内侧池上的平行反应涉及Golgin 45和GRASP55同型相互作用对GRASP55的招募。因此，每个GRAP亚型中双PDZ相互作用的特异性建立并维持了分隔的膜网络。最后，有证据表明，信号通路调节这些相互作用，我们假设级联导致PLK1介导的GRAP磷酸化，导致内部拴系配体失活，从而断裂高尔基带，促进有丝分裂进入。这一模式的许多方面都是新颖的，如果得到证实，我们相信将在该领域具有开创性。作为提供严格和详细测试的一种手段，我们建议确定GRASP结构域的结构，这是两种异构体的保守部分，包含两个PDZ样结构域。然后，我们将使用点突变和三种分析方法对其结构/功能关系的特定假说进行测试：纯化蛋白相互作用，体内细胞器连接，以及击倒和拯救后的活体成像。这项工作将定义拴系和定位GRAP复合体到各自脑池的相互作用界面，揭示特异性在拴系不同GRAPP亚型的不同高尔基体池中的机制和功能作用，并揭示GRAPP结构域的磷酸化抑制机制。 与公共卫生相关：这项提议的目标是对有丝分裂调节的拴系反应的结构性理解，该反应将相邻的脑池连接起来，形成被称为高尔基带的细胞内膜网络。这项工作有望在至少两个具有根本意义的领域取得重要进展。第一种是膜系留。膜系留是膜运输中的一个基本反应，它增加了膜融合的保真度和效率。这些反应形成了建立和维持细胞内隔室的基础。第二个问题涉及到其中一个隔室--高尔基体是如何组织起来的，以及它如何支持其功能。高尔基体处理新合成的蛋白质和脂类，这些反应在预防和治疗人类疾病方面非常重要。膜转运缺陷是许多人类疾病的原因，我们对这些缺陷的分子基础的理解为未来有效的治疗铺平了道路。此外，在针对缺陷蛋白产品引起的多种疾病的治疗开发中，了解运输及其分泌室的建立是一个至关重要的问题，在这些缺陷蛋白产品中，这些蛋白依赖于诸如蛋白质折叠、质量控制、糖基化、蛋白分解激活和定位等分泌过程。这些疾病包括囊性纤维化、普里恩相关疾病、糖尿病和阿尔茨海默氏症，仅举几例。人类疾病也是由脑室功能本身的缺陷引起的。例如，糖链合成障碍是一个庞大且迅速增长的群体，越来越明显的是，主要缺陷可能是由内质网和高尔基体组成的膜运输系统中的糖链转移酶的运输和定位。

DESCRIPTION (provided by applicant): The Golgi apparatus is compartmentalized into cisternae that associate with one another generating stacks. The multiple stacks in mammalian cells move to a peri-centrosomal position where cisternae in neighboring stacks fuse in a homotypic fashion (i.e. cis with cis, etc.) to form a compartmentalized membrane network. The lateral linking of stacks into a ribbon-like network confers optimal processing efficiency and plays a role in cell cycle progression and wound healing. A major goal is to determine the basis for specificity and regulation in the linking reaction. This will provide mechanistic understanding of how membrane networks are formed, compartmentalized, and inherited during cell division. It also promises insight into significant, yet vexing, questions about membrane tethering related to activation of tethering upon membrane binding, promotion of trans interactions, and coordination with SNARE-mediate membrane fusion. Our working model is that GM130 recruits GRASP65 to cis cisternae using a C-terminal PDZ ligand to bind the PDZ2 domain of GRASP65. Then, GRASP65 on adjacent cisternae interact in trans via their PDZ1 grooves, which bind in a homotypic fashion via internal PDZ ligands present in GRASP65. This enhances efficiency and fidelity of bringing the membranes into close proximity for SNARE contacts and membrane fusion. We also hypothesize that a parallel reaction on medial cisternae involves recruitment of GRASP55 by golgin 45 and GRASP55 homotypic interactions. Thus, the specificity of dual PDZ interactions in each GRASP isoform establishes and maintains the compartmentalized membrane network. Finally, evidence suggests that signaling pathways regulate these interactions and we hypothesize that a cascade leads to PLK1-mediated GRASP phosphorylation causing inactivation of the internal tethering ligands to fragment the Golgi ribbon and promote mitotic entry. Many aspects of this model are novel and, if verified, we believe, would be groundbreaking in the field. As a means of providing rigorous and detailed tests we propose to determine the structure of the GRASP domain, which is the conserved part of the two isoforms and contains the two PDZ-like domains. We will then carryout tests of specific hypotheses regarding its structure/function relationship using point mutations and three types of assay: purified protein interactions, in vivo organelle tethering, and live imaging after knockdown and rescue. This work will define the interaction interfaces for tethering and for localizing the GRASP complexes to their respective cisternae, reveal the mechanism and functional role of specificity in tethering distinct Golgi cisternae with distinct GRASP isoforms, and uncover the mechanism of GRASP domain phospho-inhibition. PUBLIC HEALTH RELEVANCE: The goal of this proposal is a structural understanding of the mitotically regulated tethering reaction that links adjacent cisternae to form an intracellular membrane network known as the Golgi ribbon. This work promises important advances in at least two areas of fundamental significance. The first is membrane tethering. Membrane tethering is a fundamental reaction in membrane trafficking that increases fidelity and efficiency of membrane fusion. These reactions form the basis for establishment and maintenance of intracellular compartments. The second concerns how one of these compartments, the Golgi apparatus, is organized and how this supports its function. The Golgi processes newly synthesized proteins and lipids and these reactions are important in preventing and treating human disease. Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. Further, understanding trafficking and its establishment of secretory compartments is a vital concern in the development of therapeutics targeting the multitude of diseases that arise from defective protein products in which these proteins depend on secretory processes such as protein folding, quality control, glycosylation, proteolytic activation, and localization. Such diseases include cystic fibrosis, prion-related diseases, diabetes, and Alzheimer's disease, to name just a few. Human disease also arises from defects in compartment function itself. For example, disorders of glycan synthesis are a substantial and rapidly growing group and it is becoming increasingly evident that the primary defect can be in the transport and localization of the glycan transferases within the membrane trafficking system comprised by the endoplasmic reticulum and the Golgi apparatus.