Enzymology of Golgi Stack Formation

描述(由申请人提供)：我们已经鉴定出一种名为蛋白激酶D(PKD)的丝氨酸苏氨酸激酶是导致TGN运输载体分裂的反应中的重要组成部分。当PKD的激酶活性受损时，运输载体从TGN形成，但不能发生分裂。结果，装载货物的承运人长成了大管子。PKD通过第一个富含半胱氨酸的结构域(C1a)被招募到Trans Golgi Network(TGN)的特定区域。CIA通过二酰甘油(DAG)与TGN结合。降低细胞中DAG的水平可抑制PKD向TGN的募集，阻止蛋白质向细胞表面的运输。我们的目的是了解PKD在TGN上和TGN外循环的机制。我们将确定与PKD从TGN结合和释放有关的组件。我们将在通透性细胞中重建含有TGN的PKD管的生成。这是为了提纯参与形成TGN衍生运输载体的组分。我们已经创造了一种细胞系，在其中我们可以积累含有管子的PKD，然后通过移动到允许的温度来导致它们解离。这种方法将被用于分离含有TGN来源的运输载体的PKD。这将有助于揭示TGN衍生运输载体的分子组成。此外，我们还将提供另一种PKD3亚型的功能，该亚型参与从高尔基体到内质网的运输。我们将正式测试高尔基体堆栈的组织是通过TGN监管的假设。TGN的囊泡化通过不受控制的COPI囊泡的产生导致早期高尔基体池的囊泡化。我们的研究将揭示运输承运人从吨形成的过程，以及这些承运人的规范生产如何对维持整个高尔基组织至关重要。

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.