Ubiquitin and Cell Cycle Regulation of Golgi Membrane Dynamics

高尔基复合体是一种膜结合的细胞器，它是处理 所有真核细胞中的分泌蛋白。高尔基体结构和功能的改变与 与各种人类疾病，包括自身免疫性疾病，亨廷顿病和阿尔茨海默氏症，病毒 感染和癌症。更好地理解正常高尔基体结构形成之间的关系 在了解它在人类疾病中的作用之前，它的重要细胞功能是必需的。高尔基生物发生 在细胞分裂过程中是由分解和重组过程调节的。它会分解成小管泡 有丝分裂过程中的结构，这些结构被分割成子细胞，在那里它们重新组装 变成一台新的高尔基仪器。重组是由两个ATPase(Nsf和p97)介导的，它们融合了 我们最近发现泛素在p97介导的高尔基体膜融合中发挥作用，这打开了一种 为揭示潜在机制的新方法打开了大门。有丝分裂高尔基体中的泛素化 拆卸，并且是后续重新组装所必需的。重新组装需要 P97/p47和Monoubiquitin以及p97/p47的辅因子VCIP135脱泛素酶的活性 很复杂。我们假设泛素化是高尔基体调节的一种普遍机制。 细胞周期中的膜动力学。我们将使用生化和形态学的组合 泛素化在有丝分裂高尔基体解离过程中如何发生以及它如何调节有丝分裂后 重新组装。其具体目的是：1)鉴定泛素连接酶(E3)并阐明其功能。 体外通过抑制酶，在体内通过击倒蛋白质。2)识别泛素化的 在高尔基体上底物(S)，并使用现有的泛素连接酶和去泛素酶进行确认。3)澄清 有丝分裂高尔基体膜动力学中泛素化的机制。我们将确定相互作用 酶与底物(S)之间的关系与p97介导的膜融合。我们会控制 通过操纵细胞中的泛素连接酶和脱泛素化酶实现泛素化 ShRNA和过表达技术，从而确定在高尔基体膜重组的影响 有丝分裂结束。这些研究将为细胞周期调控的分子机制提供新的见解 高尔基膜动力学。

The Golgi complex is a membrane-bound organelle that serves as a central conduit for the processing of secretory proteins in all eukaryotic cells. Alterations in the Golgi structure and function have been associated with a variety of human diseases, including autoimmune disease, Huntington's and Alzheimer's diseases, viral infections and cancer. A better understanding of the relationship between the normal Golgi structure formation and its vital cellular function is required before its role in human disease can be understood. Golgi biogenesis during cell division is mediated by a disassembly and reassembly process. It disassembles into tubularvesicular structures during mitosis, which are partitioned into the daughter cells where they are reassembled into a new Golgi apparatus. Reassembly is mediated by two ATPases (NSF and p97) that fuse the membranes.Our recent discovery that ubiquitin plays a role in p97-mediated Golgi membrane fusion opens a door for a new approach to uncover the underlying mechanism. Ubiquitination occurs during mitotic Golgi disassembly and is required for subsequent reassembly. Reassembly requires the interaction between the p97/p47 and monoubiquitin and the activity of the deubiquitinating enzyme, VCIP135, a cofactor of the p97/p47 complex. We hypothesize that ubiquitination operates as a general mechanism in regulation of Golgi membrane dynamics during the cell cycle. We will use a combination of biochemical and morphological approaches to elucidate how ubiquitination occurs during mitotic Golgi disassembly and how it regulates postmitotic reassembly. The specific aims are: 1) To identify the ubiquitin ligase (E3) and elucidate its function in vitro by inhibition of the enzyme and in vivo by knocking down the protein. 2) To identify the ubiquitinated substrate(s) on the Golgi and confirm it using the available ubiquitin ligase and deubiquitinase. 3) To elucidate the mechanism of ubiquitination in mitotic Golgi membrane dynamics. We will determine the interactions between the enzymes and the substrate(s) in relation to p97-mediated membrane fusion. We will control ubiquitination by manipulation of both the ubiquitin ligase and the deubiquitinating enzyme in cells using shRNA and overexpression techniques, and thus determine the effects on Golgi membrane reassembly at the end of mitosis. These studies will provide new insights into the molecular mechanisms of cell cycle regulation of Golgi membrane dynamics.