Enzymology of Golgi Stack Formation

描述（由申请人提供）：需要膜分裂以将装满货物的运输载体与母体隔室分离。我们特别感兴趣的裂变运输载体的transGolgi网络（TGN）的细胞表面途径。我们的工作假设是，一类特殊的蛋白质被招募来调节TGN的脂质组成，以产生局部和短暂的裂变活动。我们发现三聚体G蛋白亚基GB-γ在TGN中产生二酰甘油（DAG）。DAG激活TGN结合的蛋白激酶Ceta并募集蛋白激酶D（PKD）。PKCeta磷酸化以激活PKD。这些组分的失活导致细胞表面预定的货物在附着于TGN的大小管中积累。另一方面，它们的过度激活使TGN囊泡化。这些成分符合预期的参与膜裂变的成分的标准。我们的新数据表明，TGN中GB-γ依赖性DAG的产生是通过激活和募集磷脂酶（33，（aim#1）。因此产生的DAG在分裂后通过二酰基甘油激酶（DGK）0的PKD依赖性活化代谢（aim#2）。此外，我们的研究结果揭示了PKD结合蛋白（称为yusukin），我们建议调节PKD依赖性DGK 0激活的时间。这基本上防止了DAG的过早消耗，这将在运输载体形成过程中抑制裂变。该提议的前3个目的描述了将加强PLCB 3、yusukin和DGK 0在TGN至细胞表面转运载体的PKD依赖性裂变中的参与的实验。我们已经筛选了果蝇基因组的SiRNA和确定130个运输组件。我们的目的（#4）是从这个库中鉴定出那些在哺乳动物细胞中特异性参与PKD依赖性膜分裂的蛋白。目的#5描述了使用纯化的组分和大鼠肝脏高尔基体膜在体外重建膜分裂的实验。生物化学和形态学程序将用于监测蛋白质的连续募集，以及导致膜分裂的事件中修饰脂质如DAG和磷脂酰肌醇-4-磷酸（PIP）的产生。我们的发现将揭示生长因子、激素和神经肽的重要受体被转运到细胞表面的机制。这些组分的不适当递送是导致细胞生长和分化缺陷的事件的主要原因之一。

DESCRIPTION (provided by applicant): Membrane fission is required to dissociate cargo filled transport carriers from the maternal compartment. We are specifically interested in the fission of transport carriers of the trans Golgi network (TGN) to the cell surface pathway. Our working hypothesis is that a special class of proteins are recruited to modulate lipid composition of TGN to generate a localized and transient fission activity. We have found that trimeric G protein subunits GB-gamma generate diacylglycerol (DAG) in the TGN. DAG activates the TGN bound protein kinase Ceta and recruits protein kinase D (PKD). PKCeta phosphorylates to activate PKD. Inactivation of these components causes accumulation of cell surface destined cargo in large tubules attached to the TGN. Their overactivation, on the other hand, vesiculates the TGN. These components fit the criteria expected of components involved in membrane fission. Our new data suggests that GB-gamma dependent DAG production in the TGN is through activation and recruitment of phospholipase (33,(aim#1). DAG generated as a result is metabolized, post-fission, by PKD dependent activation of diacylglycerol kinase(DGK)0 (aim#2). ln addition, our results have revealed a PKD binding protein (called yusukin), which we propose regulates the timing of PKD dependent activation of DGK0. This essentially prevents premature consumption of DAG, which would inhibit fission in the midst of transport carrier formation. The first 3 aims of this proposal describe experiments that will strengthen the proposed involvement of PLCB3, yusukin and DGK0 in PKD dependent fission of TGN to cell surface transport carriers. We have screened the drosophila genome by SiRNA and identified 130 transport components. Our aim (#4) is to identify from this pool, those specifically involved in PKD dependent membrane fission in mammalian cells. Aim #5 describes experiments to reconstitute membrane fission in vitro using purified components and rat liver Golgi membranes. Biochemical and morphological procedures will be used to monitor sequential recruitment of proteins, and generation of modified lipids such as DAG and phosphatidylinositol-4-phosphate (PIP) in events leading to membrane fission. Our findings will reveal the mechanism by which important receptors for growth factors, hormones and neuropeptides are transported to the cell surface. Inappropriate delivery of these components is 1 of the major causes of events leading to defects in cell growth and differentiation.