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.

我们发现了一种新型化学物质伊利马醌，可以使高尔基体产生囊泡 膜并解聚微管，而其他细胞内 细胞器和细胞骨架元素不受影响。移除后 IQ，微管和高尔基复合体迅速重新组装 他们的原始形式。在此组装过程中，VGM（用于多孔 高尔基膜）首先融合形成高尔基池堆叠。这 该过程不需要微管，也可以在 16 时发生 摄氏度，但温度不低于 16 摄氏度。然后堆叠在 微管的存在和 37°C 下的孵化聚集为 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 的高尔基体堆叠。 该堆栈组件具有 对 ATP、GTP 水解、16-37 ℃ 孵育的强制要求 C 度和胞质蛋白。我们有一个独特的机会来隔离 催化 VGM 之间融合并提供 用于逐步组装高尔基体堆栈的结构支架。 我们想要表征堆栈组装过程中的中间体， 定义在功能上活跃的最小组装结构 囊泡蛋白运输。我们将分离胞质 GAF（对于高尔基体 将 VGM 组装成堆栈所需的组装系数） 水池。我们想要分离 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，例如 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是否 修饰具有功能意义，酶是什么 负责细胞周期期间这些修饰的调节。 因此，通过一系列纯化的 GAF、抗体和 cDNA 的组合 通过功能分析，我们希望阐明堆栈的机制 高尔基池维持在间期细胞中，并构建在每个细胞中 细胞分裂后的子细胞。这是以前从未尝试过的 因此有望揭示参与调节的新蛋白质 高尔基体堆栈的三维组织及其意义 该组织参与囊泡蛋白质运输。