MEMBRANE FUSION ATPASES AND THE GOLGI APPARATUS

膜融合ATP酶和高尔基体

基本信息

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
项目类别：
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

项目摘要

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.

动物细胞有丝分裂期间高尔基体形态的巨大变化是确保该细胞器世代遗传的过程的一部分。在前期，高尔基带分解成分散的堆叠，再分裂成囊泡和小管簇。在末期，这个过程被逆转，并且在每个子细胞中形成高尔基带。使用重构该过程许多方面的无细胞测定，我们已经能够将 NSF 和 p97 鉴定为从有丝分裂高尔基体片段重新组装池所需的两种 ATP 酶。两种 ATP 酶均通过高尔基体 t-SNARE（突触蛋白 5）发挥作用，重建高尔基体池，但潜在机制不同。该项目的目的是准确弄清楚这两种 ATP 酶如何运作和合作重建高尔基体池，以及在有丝分裂过程中如何调节这一过程。具体目标是： 1. 阐明p97的作用机制。检验 p97 分解 t-t SNARE 复合物就像 NSF 分解 v-t SNARE 复合物的假设。我们将准确识别涉及哪些高尔基体圈套以及分离和启动圈套所需的其他组件。最终，我们将使用掺入脂质体的重组和纯化蛋白质来重建这一过程。 2. 研究Syntaxin 5和p97的有丝分裂调控。我们将基于我们的初步观察，即有丝分裂突触蛋白 5 是新复合体的一部分。我们将绘制允许掺入该复合物的磷酸化位点并鉴定其中的其他蛋白质。我们将探讨有丝分裂期间 p97 失活是由 p47 磷酸化以及随后与 p97 解离引起的想法。 3.阐明p97和NSF在高尔基体重组中各自的作用。我们将使用我们的无细胞测定和显微注射到有丝分裂细胞中来测试 p97 组装核心而 NSF 组装边缘的想法。我们将确定 p97 是否可以将高尔基体堆叠缝合在一起以产生偏振带，并分析在体内破坏该带的后果。