STRUCTURE/FUNCTION OF THE TRANS GOLGI NETWORK

我们的假设是有一组独特的分子和结构 参与将蛋白质和脂质移出肝细胞TGN。 我们 已经确定了六种在组成性途径中起作用的分子。 这些对于形成独特的囊泡群体是必不可少的 含有pIgA-受体（pIgA-R）：（1）TGN 38，跨膜 p62 cplx的受体;（2）p62;（3）与p62相关的25 kD GT3 p62（统称为p62 cplx）;（4）约100 kD的催化PI 3-激酶亚基;（5）磷脂酰肌醇转移蛋白（PITP）和 (6)发动蛋白。 在这个建议中，我们扩展了我们对这些分子的研究 研究它们在网格蛋白介导的细胞凋亡中从高尔基体退出的作用。 途径（S.A.1）。 到目前为止，被证明对pIgA至关重要的分子- R出芽似乎是信号分子。 S. A的目标2将 寻找与pIgA-R囊泡相关的传统外壳蛋白 使用2D凝胶分析。 将对潜在的外壳蛋白进行测序， 功能特点。 2D凝胶分析扩展了 我们的无细胞检测，提供了跟踪许多 proteins. 两次分拣试验，对出口货物进行预分拣， 不同的跨池和分类成两个囊泡/小管群体 将在TGN出口处开发。 一种主要的信号分子 是p62 cplx相关的PI 3-激酶，其与 PITP参与PI（3）P的形成。 高尔基体膜甘油二酯（DAG）平衡的调节。 S.A. 3的重点是了解这些脂质，PI（3）P和 DAG，在高尔基体的出口处起作用。 这包括测试 假设修饰的ER在膜的反面上的功能 高尔基（以前称为GERL）是接受DAG生产的， 高尔基 在S.A.我们将测试小管，而不是囊泡， 让trans-Golgi携带组成性货物。 各专门 高分辨率、最佳保存EM的设备和专业知识 可对不稳定结构（即小管）进行形态学研究 通过与博尔德实验室合作， 结构此外，信号脂质和 将检查TGN的发动蛋白。 最后， 发动蛋白是一种控制小管与囊泡产生的分子， 将对TGN进行评估。 总之，使用不同的方法获得的数据 方法将帮助我们达到理解的最终目标 反式高尔基体的结构和功能的分子细节， 分泌的过程。

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.