ARCHITECTURE AND FUNCTION OF THE GOLGI APPARATUS

高尔基体的结构和功能

• 批准号: 6490220
• 负责人: GRAHAM B WARREN
• 金额: $ 29.73万
• 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: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490220
• 关键词: Golgi apparatus; HeLa cells; SDS polyacrylamide gel electrophoresis; cell cycle; electron microscopy; fluorescence microscopy; guanosinetriphosphatases; immunoelectron microscopy; laboratory rat; mass spectrometry; molecular stacking; polymerase chain reaction; protein binding; protein kinase; protein reconstitution; protein structure function; protein transport; regulatory gene; synthetic peptide; tissue /cell culture; vesicle /vacuole

The unique and complex architecture of the Golgi apparatus has been conserved through most of eukaryotic evolution but the role it plays in the functioning of this organelle is far from clear. One reason has been the lack of knowledge concerning those structural proteins responsible for generating and maintaining the central architectural feature of the Golgi apparatus, the stacks of closely-apposed and flattened cisternal membranes with associated transport vesicles. By exploiting the dramatic transformation of the Golgi during mitosis in animal cells, we have identified a complex of two proteins that play a crucial role in Golgi architecture. GRASP65 is involved in stacking cisternae whereas GM130 mediates the docking (or tethering) or transport vesicles with cisternal rims. Our aim is to work our precisely how GRASP65 and GM130 carry out their functions and to identify other proteins that help them determine Golgi structure. These proteins will also be used as tools to manipulate Golgi architecture and so help us understand its relationship to Golgi function. The specific aims of this proposal are: 1. To work out how GRASP65 stacks cisternae and whether additional proteins are required. If GRASP65 only stacks a subset of Golgi cisternae then the stacking mechanism of other cisternae will be studied. 2. To disrupt the architecture of the Golgi in vivo using GRASP65 antibodies and fragments as tools and to determine the effects on a variety of Golgi functions. 3. To elucidate the mechanism of vesicle tethering and the roles played by GM130, Giantin and p115. Biochemical and microscopic approaches will be used to work out the sequence of events that assemble and disassemble tethering complexes and to identify and characterise those GTPases and kinases that regulate this process. 4. To determine whether a template is needed for the reassembly of the Golgi at the end of mitosis and during biogenesis by studying cytoplasts that lack a Golgi apparatus.

高尔基体独特而复杂的结构在真核生物进化的大部分过程中得到了保留，但它在该细胞器功能中所扮演的角色还远不清楚。原因之一是缺乏对那些负责产生和维持高尔基体中心结构特征的结构蛋白的了解，高尔基体是紧密排列且扁平的脑池膜与相关运输囊泡的堆叠。通过利用动物细胞有丝分裂过程中高尔基体的巨大转变，我们发现了两种蛋白质的复合物，它们在高尔基体结构中发挥着至关重要的作用。 GRASP65 参与池的堆叠，而 GM130 介导囊泡与池边缘的对接（或束缚）或运输。我们的目标是准确研究 GRASP65 和 GM130 如何发挥其功能，并鉴定其他有助于它们确定高尔基体结构的蛋白质。 这些蛋白质还将用作操纵高尔基体结构的工具，从而帮助我们了解其与高尔基体功能的关系。 该提案的具体目标是： 1. 弄清楚GRASP65如何堆叠水池以及是否需要额外的蛋白质。 如果GRASP65仅堆叠高尔基池的一个子集，那么将研究其他池的堆叠机制。 2. 使用GRASP65抗体和片段作为工具破坏体内高尔基体的结构，并确定对各种高尔基体功能的影响。 3. 阐明囊泡束缚机制以及GM130、Giantin和p115的作用。 将使用生化和微观方法来确定组装和分解束缚复合物的事件序列，并识别和表征调节该过程的 GTP 酶和激酶。 4. 通过研究缺乏高尔基体的细胞质，确定有丝分裂末期和生物发生过程中高尔基体的重组是否需要模板。