ENZYMOLOGY OF GOLGI STACK FORMATION

高尔基体堆栈形成的酶学

• 批准号: 2444791
• 负责人: VIVEK MALHOTRA
• 金额: $ 20.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444791
• 关键词: Golgi apparatus; cell cycle; chimeric proteins; electron microscopy; high performance liquid chromatography; immunoelectron microscopy; laboratory mouse; laboratory rabbit; membrane structure; monoclonal antibody; posttranslational modifications; protein degradation; protein sequence; protein structure; protein transport; reagent /indicator; ultraviolet spectrometry; vesicle /vacuole

We have identified a novel chemical ilimaquinone that vesiculates Golgi membranes and depolymerizes microtubules, while other intracellular organelles and cytoskeletal elements remain unaffected. Upon removal of IQ, both the microtubules and the Golgi complex rapidly reassemble to their original form. During this assembly process VGMs (for vesiculated Golgi membranes) first fuse to forms stacks of Golgi cisternae. This process does not require microtubules and can also take place at 16 degrees C but not at temperatures below 16 degrees C. The stacks then in the presence of microtubules and incubation at 37 degrees C congregate as a complex in the pericentriolar region. We have reconstituted the assembly of Golgi stacks from VGMs in semi-intact cells. This stack assembly has an obligate requirement for ATP, hydrolysis of GTP, incubation at 16-37 degrees C and cytosolic proteins. We have a unique opportunity to isolate the proteins that catalyze the fusion between VGMs and provide a structural scaffold for the step wise assembly of Golgi stacks. We want to characterize intermediates in the stack assembly process and define the minimum assembled structure that is functionally active in the vesicular protein transport. We will Isolate cytosolic GAFs (for Golgi Assembly Factors) required for the assembly of VGMs into stacks of cisternae. We want to isolate the mitotic counterparts of GAFs to test whether these proteins undergo any posttranslational modifications such as phosphorylation/dephosphorylation in association with cell-cycle events. The questions we want to address are whether these posttranslational modifications have a functional significance and what are the enzymes responsible for the regulation of these modifications during cell-cycle. Therefore, with a battery of purified GAFs, antibodies and cDNAs combined with a functional assay we want to elucidate the mechanism by which stacks of Golgi cisternae are maintained in interphase cells and built in each daughter cell after cell-division. This has never been attempted before, and therefore promises to reveal novel proteins involved in regulating the three dimensional organization of Golgi stacks and the significance of this organization in vesicular protein transport.

我们已经确定了一种新的化学物质， 膜和解聚微管，而其他细胞内 细胞器和细胞骨架元件不受影响。一旦移除 IQ，微管和高尔基复合体迅速重组， 他们的原始形式。在该组装过程中，VGM（用于囊泡化的 高尔基膜）首先融合形成堆叠的高尔基池。这 这个过程不需要微管，也可以在16 但不能在低于16摄氏度的温度下。堆栈然后在 微管的存在和在37摄氏度下的孵育聚集， 一种位于中心粒周围区域的复合体。我们重组了议会 半完整细胞中VGM的高尔基体堆积。 该堆叠组件具有 对ATP、GTP水解、16-37 ℃孵育的强制性要求 度C和胞质蛋白质。我们有一个独一无二的机会 催化VGM之间的融合并提供 逐步组装高尔基体的结构支架。 我们希望表征电池组组装过程中的中间体， 定义最小的组装结构，在功能上是活跃的， 囊泡蛋白质转运我们将分离细胞质GAF（对于高尔基体 装配系数），将VGM装配成 蓄水池我们想分离出GAF的有丝分裂对应物来测试 这些蛋白质是否经历任何翻译后修饰， 与细胞周期事件相关的磷酸化/去磷酸化。 我们要解决的问题是这些翻译后的 修饰具有功能意义， 负责在细胞周期中调节这些修饰。 因此，用一组纯化的GAF、抗体和cDNA组合， 通过功能分析，我们希望阐明堆叠的机制， 高尔基体池维持在间期细胞中， 细胞分裂后的子细胞。这是以前从未尝试过的， 因此有望揭示新的蛋白质参与调节 高尔基体的三维结构及其意义 这种组织在囊泡蛋白质运输中的作用。