STRUCTURAL BASIS OF ORGANELLE TETHERING

描述（由申请人提供）：高尔基体被划分成池，池之间相互关联，产生堆栈。哺乳动物细胞中的多个堆叠移动到中心体周围的位置，在那里相邻堆叠中的池池以同型方式融合（即顺式与顺式等），形成一个区隔化的膜网络。堆叠的横向连接成带状网络提供了最佳的加工效率，并在细胞周期进程和伤口愈合中发挥作用。一个主要的目标是确定连接反应的特异性和调节的基础。这将提供细胞膜网络在细胞分裂过程中是如何形成、区隔化和遗传的机制理解。它还有望深入了解有关膜系栓的重要但令人烦恼的问题，这些问题与膜结合时系栓的激活、促进反式相互作用以及与snare介导的膜融合协调有关。我们的工作模型是GM130利用c端PDZ配体结合GRASP65的PDZ2结构域将GRASP65招募到顺式池中。然后，相邻池上的GRASP65通过其PDZ1凹槽进行反式相互作用，PDZ1凹槽通过GRASP65中存在的内部PDZ配体以同型方式结合。这提高了将膜带入近距离接触和膜融合的效率和保真度。我们还假设内侧池的平行反应涉及通过golgin 45和GRASP55同型相互作用募集GRASP55。因此，每个GRASP异构体中双PDZ相互作用的特异性建立并维持了区隔化的膜网络。最后，有证据表明信号通路调节这些相互作用，我们假设一个级联导致plk1介导的GRASP磷酸化，导致内部捆绑配体失活，从而使高尔基带片段化并促进有丝分裂进入。该模型的许多方面都是新颖的，如果得到验证，我们相信将在该领域具有开创性。作为一种提供严格和详细测试的手段，我们建议确定GRASP结构域的结构，它是两个同工异构体的保守部分，包含两个pdz样结构域。然后，我们将使用点突变和三种类型的检测方法对其结构/功能关系的特定假设进行测试：纯化蛋白相互作用、体内细胞器系固以及敲除和挽救后的实时成像。这项工作将定义拴住和定位到各自池的相互作用界面，揭示特异性在拴住具有不同GRASP异构体的高尔基池中的机制和功能作用，揭示GRASP结构域磷酸化抑制的机制。

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.