BIOGENESIS OF THE GOLGI APPARATUS

描述(由申请人提供)：高尔基仪器的处理、分类和信号传递功能被广泛认识到对健康和疾病具有重要意义。因此，重要的是确定赋予高尔基体功能组织的组件。高尔基体是一组细长的高尔基体蛋白，是一系列自身免疫性疾病中的重要自身抗原，是强有力的候选者，但它们的实际作用尚不清楚。为了确定高尔基体结构和功能所需的蛋白质，基于定向siRNA的筛选针对候选蛋白，特别是高尔基体蛋白，正在与基于细胞的高尔基体组装和运输分析一起使用。因此，我们在概念上定义了高尔基体装配中的离散步骤以及这些步骤的要求。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体组织特征的重要性，如区隔、定位和带状形成。这项提案的重点是高尔夫球P115和GM130，我们分别确定这两种球杆为高尔基体分隔和色带形成所需。基因替换结合渗透性细胞分析和实时成像将用于分析p115和GM130的结构/功能关系。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结构域和保守的同源区域1(HR1)结构域。值得注意的是，HR1与COPI囊泡外壳结合，取消这种相互作用的点突变破坏了基因替换时的p115活性。这些结果为在COPI囊泡对接中测试p115/COPI相互作用以及在SNARE配对中测试p115/SNARE相互作用奠定了基础。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，在没有带的情况下，高尔基酶不能平衡，导致加工缺陷。接下来，我们将检验这一假设，即在GM130招募到高尔基体后，相邻小节上的GRASP65寡聚驱动SNARE介导的膜融合形成条带。值得注意的是，我们对高尔基体形成复合体的研究也阐明了一种新的细胞周期控制机制。我们已经证明GRASP55家族成员GRASP55被MEK/ERK通路有丝分裂磷酸化，在未发表的工作中，GRASP55也是连接高尔基体成带所必需的。此外，抑制MEK会延迟G2/M细胞周期的转变，而这会被高尔基体先前去连接所抑制。因此，我们将检验这样的假设，即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。

DESCRIPTION (provided by applicant): 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 compartmentalization, 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 To date, we have identified two required p115 domains: the SNARE-binding cc1 domain and, in unpublished work, the conserved homology region 1 (HR1) domain.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.