Enzymology of Golgi Stack Formation

描述（由申请人提供）：需要膜裂变来将装满货物的运输载体从母舱分离。我们特别感兴趣的是反高尔基网络（TGN）到细胞表面途径的转运载体的裂变。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中产生二酰基甘油（DAG）。DAG激活TGN结合蛋白激酶Ceta并募集蛋白激酶D （PKD）。PKCeta磷酸化激活PKD。这些成分的失活导致细胞表面货物在附着于TGN的大管中积累。另一方面，它们的过度激活会使TGN形成囊泡。这些成分符合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的产生是通过磷脂酶的激活和募集（33,(aim#1)）。由此产生的DAG在裂变后通过PKD依赖的二酰基甘油激酶（DGK）0的激活代谢（目的#2）。此外，我们的研究结果揭示了PKD结合蛋白（称为yusukin），我们认为它调节PKD依赖性DGK0激活的时间。这从根本上防止了DAG的过早消耗，DAG会在转运载体形成过程中抑制裂变。本提案的前3个目标描述了将加强PLCB3， yusukin和DGK0参与PKD依赖性TGN向细胞表面运输载体的裂变的实验。我们通过SiRNA筛选果蝇基因组，鉴定出130个转运组分。我们的目标（#4）是从这个库中识别出那些在哺乳动物细胞中特异性参与PKD依赖性膜裂变的细胞。Aim #5描述了使用纯化组分和大鼠肝脏高尔基体膜在体外重建膜裂变的实验。生化和形态学程序将用于监测在导致膜裂变的事件中蛋白质的连续募集和修饰脂质的产生，如DAG和磷脂酰肌醇-4-磷酸（PIP）。我们的发现将揭示生长因子、激素和神经肽等重要受体转运到细胞表面的机制。这些成分的不适当传递是导致细胞生长和分化缺陷的主要原因之一。

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.