Enzymology of Golgi Stack Formation

高尔基体堆栈形成的酶学

• 批准号: 6912726
• 负责人: VIVEK MALHOTRA
• 金额: $ 32.49万
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6912726
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; cell component structure /function; electron microscopy; guanosine triphosphate; intracellular membranes; laboratory mouse; laboratory rabbit; membrane biogenesis; membrane fusion; protein binding; protein degradation; protein kinase; protein purification; protein sequence; protein transport; protein tyrosine kinase; tissue /cell culture; yeast two hybrid system

DESCRIPTION (provided by applicant): We have identified a serine threonine kinase called protein kinase D (PKD) as an essential component in the reactions leading to the fission of transport carriers from the TGN. When the kinase activity of PKD is compromised the transport carriers form from the TGN but fail to undergo fission. The cargo containing carriers grow as a result into large tubes. PKD is recruited to specific regions of the Trans Golgi Network (TGN) via the first cysteine rich domain (C1a). Cia binds to the TGN via Diacyiglycerol (DAG). Reducing the cellular levels of DAG inhibit PKD recruitment to the TGN and protein transport to the cell surface is blocked. Our aim is understand the mechanism by which PKD cycles on and off the TGN. We will identify components involved in the binding and the release of PKD from the TGN. We will reconstitute the generation of PKD containing tubes from the TGN in permeabilized cells. This is aimed towards the purification of components that are involved in the formation of the TGN derived transport carriers. We have generated a cell line, in which we can accumulate the PKD containing tubes and then cause their dissociation by shifting to a permissible temperature. This approach will be used to isolate the PKD containing TGN derived transport carriers. This will help reveal the molecular composition of the TGN derived transport carriers. In addition, we will provide a function for another isoform of PKD3, which we propose, is involved in transport from Golgi to the ER. We will formally test the hypothesis that the organization of the Golgi stacks is regulated through TGN. Vesiculation of the TGN causes vesiculation of the early Golgi cisternae by uncontrolled generation of COPI vesicle. Our studies will reveal novel insights into the process by which transport carriers form from the TON and how a regulated production of these carriers is essential for maintaining the overall Golgi organization.

描述（由申请人提供）：我们已经确定了一种称为蛋白激酶D（PKD）的丝氨酸苏氨酸激酶是导致TGN转运载体裂变的反应中的重要组成部分。当PKD的激酶活性受到损害时，TGN的传输载体形成但未能进行裂变。含有载体的货物随之生长成大管。 PKD通过第一个半胱氨酸富域（C1A）招募到反式高尔基网络（TGN）的特定区域。 CIA通过二酰甘油（DAG）与TGN结合。降低DAG的细胞水平抑制PKD募集到TGN，并将蛋白质转运到细胞表面。我们的目的是了解PKD在TGN上循环和关闭的机制。我们将确定与TGN结合和释放涉及的组件。我们将重建透明细胞中含有TGN的管子的PKD产生。这旨在纯化与TGN衍生的运输载体形成有关的组件。我们已经生成了一个细胞系，其中我们可以积聚包含管的PKD，然后通过转移到允许的温度来使其解离。这种方法将用于隔离含有TGN衍生的运输载体的PKD。这将有助于揭示TGN衍生的传输载体的分子组成。此外，我们将为PKD3的另一种同工型提供一个函数，我们提出，该功能参与了从高尔基体到ER的运输。我们将正式检验以下假设：高尔基堆栈的组织通过TGN调节。 TGN的囊泡导致早期高尔基水库的囊泡通过不受控制的Copi囊泡。我们的研究将揭示有关从吨的运输载体形成的过程以及这些载体的监管生产对于维持整个高尔基人组织至关重要的过程的新见解。