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型的化学化学ilimaquinone，该化学物质囊化了高尔基 膜和解聚微管，而其他细胞内 细胞器和细胞骨架元素仍然不受影响。去除 智商，微管和高尔基络合物都快速重新组装 他们的原始形式。在此组装过程中，VGM（用于囊泡 高尔基膜）首先融合形成一堆高尔基水库。这 过程不需要微管，也可以在16岁时进行 c的学位，但在低于16度的温度下。 微管的存在和37度C聚集的孵育为 丁香三元区域的复合物。我们已经重构了大会 半独立性细胞中VGM的高尔基堆积。 这个堆栈组件有 ATP的强制性要求，GTP水解，在16-37中孵育 C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A的蛋白质 高尔基堆积的阶梯式组件的结构支架。 我们想在堆栈组装过程中表征中间体，并且 定义在功能上有效的最小组装结构 囊泡蛋白转运。我们将分离胞质GAF（对于高尔基 组装因子）将VGM组装成堆栈需要 水库。我们想隔离GAF的有丝分裂对应物进行测试 这些蛋白质是否经过任何翻译后修饰，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 修改具有功能意义，哪些是酶 负责调节细胞周期期间这些修饰。 因此，用一电纯化的GAF，抗体和cDNA组合 使用功能测定，我们要阐明堆叠的机制 高尔基蓄水池的硫磺位于间相细胞中并内置 细胞划分后的女儿细胞。以前从未尝试过 因此，有望揭示参与调节的新型蛋白质 高尔基堆栈的三维组织 该组织在囊泡蛋白运输中。