STRUCTURE/FUNCTION OF THE TRANS GOLGI NETWORK

我们的假设是，有一套独特的分子和结构 参与将蛋白质和脂类移出肝细胞TGN。我们 已经确定了六个在本构途径中起作用的分子。 这些对于形成不同的囊泡种群是必不可少的。 含有PIGA受体(PIGA-R)：(1)TGN38，跨膜 P62cplx的受体；(2)p62；(3)与之相关的25kD GTP酶 P62(统称为p62cplx)；(4)约100kD的催化PI (5)磷脂酰肌醇转移蛋白(PITP)和 (6)动力素。在这个提案中，我们扩展了对这些分子的研究 以考察它们在退出高尔基体中的作用 途径(S.A.1)。到目前为止，被证明对PIGA至关重要的分子- R萌芽似乎是信号分子。S.A.2的目标将是 寻找与PIGA-R囊泡相关的传统外壳蛋白 采用二维凝胶分析。潜在的外壳蛋白将被测序并 功能化的。2D凝胶分析扩展了 我们的无细胞检测，提供了跟踪多个 蛋白质。两种分拣分析，对出境货物进行预分拣 不同的跨池和分成两个囊泡/小管种群 在退出TGN后，将开发出。一种主要的信号分子 是p62cplx相关的PI3-激酶，它协同作用于 PITP参与了PI(3)的形成。 高尔基体膜上二酰甘油(DAG)平衡的调节。S.A. 3重点了解这些脂质、PI(3)P和 达格，从高尔基的出口开始工作。这包括测试 假设修饰的内质网对细胞表面的作用 高尔基(以前称为Gerl)是接受DAG生产的 高尔基。在S.A.4中，我们将测试小管，而不是小泡， 离开运载构成货物的跨高尔基山脉。专门化 高分辨率、最佳保存EM的设备和专业知识 对不稳定结构(即小管)的形态研究是可用的。 通过我们与博尔德实验室的合作获得3D罚款 结构。此外，信号脂和信号转导之间的相互作用 将对TGN的动力剂进行检查。最后，有可能 Dynamin是控制小管与囊泡产生的分子 将对TGN进行评估。总的来说，使用不同的方法获得的数据 方法将帮助我们实现我们的最终目标：理解 反式高尔基体的结构功能及其分子细节 分泌物的过程。

Our hypothesis is that there are unique sets of molecules and structures involved in moving proteins and lipids out of the hepatocyte TGN. We have identified six molecules functioning in the constitutive pathway. These are essential for the formation of a distinct vesicle population containing the pIgA-receptor (pIgA-R): (1) TGN38, the transmembrane receptor for the p62cplx; (2) p62; (3) the 25 kD GTPase associated with p62 (together called p62cplx); (4) a approximately 100 kD catalytic PI 3-kinase subunit; (5) phosphatidylinositol transfer protein (PITP) and (6) dynamin. In this proposal we extend our studies of these molecules to examine their roles in exit from the Golgi in the clathrin-mediated pathway (S.A.1). To date, the molecules shown to be essential for pIgA- R budding appear to be signaling molecules. A goal of S.A. 2 will be to search for traditional coat-proteins associated with pIgA-R vesicles using 2D gel analysis. Potential coat-proteins will be sequenced and functionally characterized. The 2D gel analysis extends the power of our cell-free assay, providing the capacity to follow sorting of many proteins. Two sorting assays, pre-sorting of exiting cargo between distinct trans-cisternae and sorting into two vesicle/tubule populations upon exit from the TGN will be developed. A major signaling molecule is the p62cplx associated PI 3-kinase which acts synergistically with PITP in the formation of PI(3)P. PITP is predicted to be involved in the regulation of diacylglycerol (DAG) balance in Golgi membranes. S.A. 3 focuses on understanding the mechanism of how these lipids, PI(3)P and DAG, function in exit from the Golgi. This includes testing the hypothesis that the function of the modified ER on the trans-face of the Golgi (formerly known as the GERL) is to accept DAG produced in the Golgi. In S.A. 4 we will test whether tubules, rather than vesicles, leave the trans-Golgi carrying constitutive cargo. The specialized equipment and expertise for high resolution, optimal preservation EM morphological studies of labile structures (i.e. tubules) are available to us through our collaboration with the Boulder Laboratory for 3D fine structure. In addition, the interplay between signaling lipids and dynamin at the TGN will be examined. Finally, the possibility that dynamin is the molecule controlling tubule versus vesicle production at the TGN will be evaluated. Together, the data obtained using diverse approaches will help us reach our ultimate goal of understanding the structure function of the trans-Golgi and the molecular details of the process of secretion.