MEMBRANE FUSION ATPASES AND THE GOLGI APPARATUS

膜融合ATP酶和高尔基体

• 批准号: 6636401
• 负责人: GRAHAM B WARREN
• 金额: $ 29.43万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636401
• 关键词: Golgi apparatus; adenosinetriphosphatase; cell cycle; cell free system; enzyme mechanism; laboratory rabbit; laboratory rat; liposomes; membrane fusion; microinjections; phosphorylation; recombinant proteins; syntaxin

The dramatic change in Golgi morphology during mitosis in animal cells is part of the process that ensures inheritance of this organelle through successive generations. During prophase, the Golgi ribbon breaks down into dispersed stacks which fragment into clusters of vesicles and tubules. During telophase this process is reversed and a Golgi ribbon is formed in each daughter cell. Using a cell-free assay that reconstitutes many aspects of this process we have been able to identify NSF and p97 as two of the ATPases needed to reassemble cisternae from mitotic Golgi fragments. Both ATPases act through the Golgi t-SNARE, syntaxin 5, to rebuild Golgi cisternae, but the underlying mechanism is different. The aim of this project is to work out precisely how these two ATPases operate and co-operate to rebuild Golgi cisternae and how this process is regulated during mitosis. The specific aims are: 1. To work out the mechanism of action of p97. To test the hypothesis that p97 breaks up t-t SNARE complexes just as NSF breaks up v-t SNARE complexes. We will identify precisely which Golgi SNARES are involved and which other components are needed to separate and prime the SNARES. Eventually we will reconstitute this process using recombinant and purified proteins incorporated into liposomes. 2. To study the mitotic regulation of Syntaxin 5 and p97. We will build on our preliminary observations that mitotic syntaxin 5 is part of a new complex. We will map the phosphorylation sites that permit incorporation into this complex and identify the other proteins in it. We will explore the idea that inactivation of p97 during mitosis is caused by phosphorylation of p47 and consequent dissociation from p97. 3. To elucidate the respective roles played by p97 and NSF in the reassembly of the Golgi. We will test the idea that p97 assembles the cores whereas NSF assembles the rims using our cell-free assay and micro-injection into mitotic cells. We will determine whether p97 can stitch Golgi stacks together to give a polarised ribbon and analyse the consequences of disrupting this ribbon in vivo.

动物细胞有丝分裂过程中高尔基体形态的急剧变化是确保该细胞器通过连续几代人遗传的过程的一部分。 在预言期间，高尔基丝带分解成分散的堆栈，它们碎片成囊泡和小管的簇。 在末期期间，此过程被逆转，并且在每个子细胞中形成高尔基丝带。 使用无细胞的测定，我们能够将NSF和p97识别为从有丝分裂高尔基片段重新组装蓄积的两个ATPases。 两种ATPases通过高尔基T-snare（语法5）起作用，以重建高尔基甲壳虫，但基本机制是不同的。该项目的目的是确切地确定这两个ATP酶如何运作和合作以重建高尔基水甲壳虫，以及在有丝分裂过程中如何调节这一过程。 具体目的是：1。确定p97的作用机理。 为了测试p97分解T-T SNARE复合物的假设，就像NSF分解V-T SNARE复合物一样。 我们将准确确定涉及哪些高尔基虫，以及需要哪些其他组件来分离和灌注蜗杆。 最终，我们将使用掺入脂质体中的重组和纯化的蛋白质来重建这一过程。 2。研究语法5和p97的有丝分裂调节。 我们将基于初步观察，即有丝分裂语法5是新综合体的一部分。我们将绘制允许掺入该复合物并识别其中其他蛋白质的磷酸化位点。 我们将探讨这样一种观念，即在有丝分裂过程中p97的灭活是由p47的磷酸化引起的，因此与p97解离。 3。阐明P97和NSF在高尔基体重新组装中扮演的各个角色。 我们将测试p97组装核心的想法，而NSF则使用无细胞测定法和微注射到有丝分裂细胞的情况下组装了轮辋。 我们将确定p97是否可以将高尔基体堆叠在一起以产生极化的色带并分析破坏体内这种色带的后果。