BIOGENESIS OF THE GOLGI APPARATUS

在高尔基体中发生的处理、分类和信号传递功能被广泛理解 对健康和疾病有重要意义。因此，重要的是要确定赋予 高尔基体的功能组织。高尔基体-一组细长的高尔基体蛋白， 在一系列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， 未知为了鉴定高尔基体结构和功能所需的蛋白质， 靶向候选物，特别是高尔基体，正与基于细胞的高尔基体组装和运输测定一起使用。 因此，我们在概念上定义了高尔基体组装中的离散步骤以及这些步骤的要求。 步此外，通过分析在给定步骤中高尔基体组装被阻断的细胞，我们正在识别 高尔基体组织特征的重要性，如区室化，定位和带状形成。 本提案的重点是高尔基体p115和GM 130，我们认为这是高尔基体的要求 区室化和带状形成。基因替换与 将使用透化细胞测定和活体成像来分析p115和GM 130的结构/功能 关系。到目前为止，我们已经确定了两个必要的p115结构域：SNARE结合cc 1结构域和， 在未发表的工作中，保守的同源区1（HR 1）结构域。值得注意的是，HR 1结合COPI 囊泡外壳和消除这种相互作用的点突变在基因置换时破坏p115活性。 这些结果为COPI囊泡对接中p115/COPI相互作用和p115/SNARE的测试奠定了基础 SNARE配对中的相互作用。对于GM 130，我们已经证明它需要其GRASP 65结合结构域， GRASP 65本身是高尔基带形成所必需的，在没有高尔基带的情况下， 酶不能平衡，导致有缺陷的加工。接下来，我们将测试假设， 通过GM 130募集到高尔基体，相邻微堆上的GRASP 65寡聚化驱动SNARE- 介导的膜融合以形成带。值得注意的是，我们在高尔基带形成复合体方面的工作， 还阐明了一种新的细胞周期控制机制。我们已经证明，GRASP家族成员 GRASP 55通过MEK/ERK途径被有丝分裂磷酸化，在未发表的工作中，GRASP 55 也需要将高尔基体小堆连接成带状。此外，MEK抑制延迟G2/M细胞周期 过渡，这是抑制事先解开高尔基体。因此，我们将测试假设， G2晚期GRASP 55的MEK/ERK磷酸化解除高尔基体促进CDK 1活化和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 To date, we have identified two required p115 domains: the SNARE-binding cc1 domain and, in unpublished work, the conserved homology region 1 (HR1) domain.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.