Enzymology of Golgi Stack Formation

描述（由申请人提供）：我们已经确定了一种丝氨酸苏氨酸激酶，称为蛋白激酶D (PKD)，是导致TGN运输载体裂变的反应的重要组成部分。当PKD的激酶活性受损时，转运载体从TGN形成，但不能进行裂变。装载货物的载体因此成长为巨大的管道。PKD通过第一富半胱氨酸结构域（C1a）被招募到Trans高尔基体网络（TGN）的特定区域。Cia通过二甘油酯（DAG）与TGN结合。降低DAG的细胞水平会抑制PKD向TGN的募集，从而阻断蛋白质向细胞表面的转运。我们的目标是了解PKD在TGN上和TGN下循环的机制。我们将识别与PKD从TGN的结合和释放有关的成分。我们将从渗透细胞的TGN中重建含有PKD的管。这是针对成分的纯化，参与形成的TGN衍生的运输载体。我们已经产生了一个细胞系，我们可以在其中积累含有PKD的管，然后通过转移到允许的温度使它们分离。这种方法将用于分离含有TGN衍生转运载体的PKD。这将有助于揭示TGN衍生转运载体的分子组成。此外，我们将提供PKD3的另一种异构体的功能，我们认为它参与了从高尔基体到内质网的运输。我们将正式测试高尔基堆叠的组织是通过TGN调节的假设。通过不受控制的COPI囊泡的产生，TGN的囊泡引起早期高尔基池的囊泡。我们的研究将揭示运输载体从TON形成的过程的新见解，以及这些载体的受控生产如何对维持整个高尔基体组织至关重要。

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.