Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly
Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly
复制标题
DOI：
10.1074/jbc.m109.047084
复制
发表时间：
2010-03-05
影响因子：
4.8
通讯作者：
Wang, Yanzhuang
中科院分区：
文献类型：
--
作者：
Chen, Xuequn;Simon, Eric S.;Wang, Yanzhuang
在有丝分裂过程中，高尔基体的堆叠结构经历了一个连续的破碎过程。产生的有丝分裂片段均匀地分布到子细胞中，并重新组装成新的高尔基体。这种分解和重组过程对于细胞分裂过程中高尔基体的生物发生至关重要，但其潜在的分子机制却知之甚少。在这项研究中，我们使用体外试验概述了这一过程，并使用蛋白质组学方法分析了与间期和有丝分裂高尔基膜相关的蛋白质。孵育纯化的大鼠肝脏高尔基体膜与有丝分裂的HeLa细胞胞质溶胶导致的膜碎片，这些膜与间期胞质溶胶的后续治疗允许重新组装成新的高尔基体堆栈的高尔基体片段。这些膜，然后用于定量蛋白质组学分析相结合的相对和绝对定量方法与OFFGEL等电聚焦分离和液相色谱基质辅助激光解吸电离串联质谱的同量异位素标签。在三个独立的实验中，共鉴定和定量了1,193种高尔基体相关蛋白。这些包括广泛的功能类别，如高尔基体结构蛋白、高尔基体驻留酶、SNARE、Rab GTP酶、货物和细胞骨架蛋白。更重要的是，定量方法与蛋白质印迹法的结合使我们能够揭示84个蛋白质，与有丝分裂条件下相比，间期条件下的丰度有显着变化。在这些蛋白质中，几种COPI外被体亚基（α、β、γ和δ）是特别感兴趣的。总之，这个系统的定量蛋白质组学研究揭示了候选蛋白的分子机制，控制高尔基体的拆卸和重新组装过程中的细胞周期。
During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma, and delta) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.