Enzymology of Golgi Stack Formation

高尔基体堆栈形成的酶学

• 批准号: 7138890
• 负责人: VIVEK MALHOTRA
• 金额: $ 39.34万
• 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 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7138890
• 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): 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.

描述（由申请人提供）：需要膜裂变才能将装满货物的运输载体与母体隔室解离。我们特别对将反式高尔基网络（TGN）的传输载体裂变到细胞表面途径感兴趣。我们的工作假设是，募集了一类特殊的蛋白质来调节TGN的脂质组成，以产生局部和瞬时裂变活性。我们发现三聚体G蛋白亚基GB-GAMMA在TGN中产生二酰基甘油（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。这些成分的灭活导致细胞表面注定货物在附着在TGN上的大小管中的积累。另一方面，它们过度活化会囊括TGN。这些成分符合涉及膜裂变的组成部分的标准。 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我们提出的是DGK0的PKD依赖性激活的时间，这基本上可以防止DAG的过早消耗，这将抑制该提案的第一个实验的第一个目标。 siRNA的果蝇基因组并确定了130个转运成分（＃4）是从该池中识别的，这些库是在PKD依赖的膜裂变中＃5的实验。在导致膜裂变的事件中，蛋白质和修饰的脂质（例如DAG）和磷脂酰肌醇4-磷酸盐（PIP）将揭示出生长因子，激素和神经肽的重要受体，是将这些组成量的不适当送达的。