BIOGENESIS OF THE GOLGI APPARATUS

在高尔基装置上进行的处理、分类和信令功能被广泛理解 在健康和疾病方面有重大意义。因此，重要的是要确定赋予 高尔基仪器的功能组织。高尔基体--一组细长的高尔基体蛋白质 广泛的自身免疫性疾病中的自身抗原是强有力的候选者，但它们的实际作用仍然存在。 未知。基于定向siRNA筛选鉴定高尔基体结构和功能所需的蛋白质 以候选人为目标，特别是高尔基体，正被用于基于细胞的高尔基体组装和贩运分析。 因此，我们在概念上定义了高尔基装配中的离散步骤以及这些步骤的要求 台阶。此外，通过分析高尔基体组装在给定步骤受阻的细胞，我们正在识别 高尔基组织特征的重要性，如划分、定位和带状形成。 这项提案的重点是高尔夫P115和GM130，我们确定这是高尔基的要求 分隔化和条带形成。基因置换与联合 渗透性细胞分析和活体成像将用于分析p115和GM130的结构/功能 两性关系。到目前为止，我们已经确定了两个必需的p115结构域：SNARE结合的cc1结构域和 在未发表的工作中，保守的同源区域1(HR1)结构域。值得注意的是，HR1结合了COPI 囊泡外壳和取消这种相互作用的点突变扰乱了p115在基因替换时的活性。 这些结果为测试p115/COPI在COPI囊泡对接中的相互作用和p115/SNARE奠定了基础 诱捕配对中的相互作用。对于GM130，我们已经证明它需要它的GRASP65结合结构域， GRASP65本身是形成高尔基带所必需的，如果没有带子，则高尔基体 酶不能平衡，导致加工有缺陷。接下来，我们将检验以下假设： GM130招募高尔基人，GRASP65在邻近部门的寡聚推动SNARE- 介导膜融合形成条带。值得注意的是，我们在高尔基带形成络合物方面的工作是 还阐明了一种新的细胞周期调控机制。我们已经证明了GRAPH家族成员 GRASP55通过MEK/ERK通路有丝分裂地磷酸化，在未发表的工作中，GRASP55 也是将高尔基小包连接成丝带所必需的。此外，抑制MEK可延缓G2/M细胞周期 转变，这被高尔基人之前的解链所抑制。因此，我们将检验这一假设 GRASP55在G2晚期的MEK/ERK磷酸化解除了高尔基体促进CDK1激活和G2/M的联系。

The processing, sorting and signaling functions that take place at the Golgi apparatus are widely appreciated to be significant in health and disease. Therefore, it is important to identify the components that confer the functional organization of the Golgi apparatus. Golgins- a set of elongated Golgi proteins that are prominent autoantigens in a wide-array of autoimmune disorders - are strong candidates, but their actual roles remain unknown. To identify proteins required for Golgi structure and function a directed siRNA-based screen targeting candidates, especially golgins, is being used with cell-based Golgi assembly and trafficking assays. As a result, we are defining conceptually discrete steps in Golgi assembly and the requirements at these steps. Further, by analyzing cells with Golgi assembly blocked at a given step, we are identifying the importance of Golgi organizational features such as compartmenalization, positioning, and ribbon formation. The focus of this proposal is on the golgins p115 and GM130 which we identified as requirements for Golgi compartmentalization and ribbon formation, respectively. Gene replacement in conjunction with permeabilized cell assays and live imaging will be used to analyze p115 and GM130 structure/function relationships. To date, we have identified two required p115 domains: the SNARE-binding cc1 domain and, in unpublished work, the conserved homology region 1 (HR1) domain. Significantly, HR1 binds the COPI vesicle coat and a point mutation abolishing this interaction disrupts p115 activity upon gene replacement. These results set the stage for tests of p115/COPI interactions in COPI vesicle docking and of p115/SNARE interactions in SNARE pairing. For GM130, we have shown that it requires its GRASP65-binding domain, that GRASP65 itself is required for Golgi ribbon formation, and that in the absence of the ribbon, Golgi enzymes fail to equilibrate causing defective processing. Next, we will test the hypothesis that after recruitment to the Golgi by GM130, GRASP65 oligomerization on adjacent ministacks drives SNARE- mediated membrane fusion to form the ribbon. Remarkably, our work on Golgi ribbon forming complexes is also elucidating a novel cell cycle control mechanism. We have shown that the GRASP family member GRASP55 is mitotically phosphorylated by the MEK/ERK pathway and, in unpublished work, that GRASP55 is also required for linking Golgi ministacks into ribbons. Further, MEK inhibition delays the G2/M cell cycle transition and this is suppressed by prior unlinking of the Golgi. Thus, we will test the hypothesis that MEK/ERK phosphorylation of GRASP55 in late G2 unlinks the Golgi promoting CDK1 activation and G2/M.