Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly
| Autor: | Philip C. Andrews, Yi Xiang, Xuequn Chen, Yanzhuang Wang, Maureen Kachman, Eric S. Simon |
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| Rok vydání: | 2010 |
| Předmět: |
Proteomics
Proteome Molecular Sequence Data Quantitative proteomics Golgi Apparatus Biology Biochemistry symbols.namesake Cytosol Animals Cluster Analysis Humans Molecular Biology Mitosis Cell-Free System Cell Cycle Computational Biology Membrane Proteins Intracellular Membranes Cell Biology COPI Golgi apparatus Rats Cell biology Liver Membrane protein Golgi disassembly symbols Rab Peptides HeLa Cells |
| Zdroj: | Journal of Biological Chemistry. 285:7197-7207 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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