Abstract
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.
Highlights
The Golgi complex is the central organelle in the secretory pathway, essential for post-translational modifications, sorting, and trafficking of newly synthesized secretory and membrane proteins and lipids in all eukaryotic cells
It is believed that the cell cycle-regulated Golgi disassembly and reassembly processes involve interactions between cytosolic and membrane proteins, and many studies have been performed in efforts to identify these proteins using biochemical and cell biology approaches
Applying isobaric tags for relative and absolute quantification (iTRAQ)-based Quantitative Proteomics to Cell-free Golgi Disassembly and Reassembly Assay—In this study, large amounts of stacked Golgi membranes were isolated from rat liver as described previously [28]
Summary
The Golgi complex is the central organelle in the secretory pathway, essential for post-translational modifications, sorting, and trafficking of newly synthesized secretory and membrane proteins and lipids in all eukaryotic cells. It is believed that the cell cycle-regulated Golgi disassembly and reassembly processes involve interactions between cytosolic and membrane proteins, and many studies have been performed in efforts to identify these proteins using biochemical and cell biology approaches. Comparative Analysis of Golgi Disassembly and Reassembly the cytosol by centrifugation through a sucrose cushion and processed for biochemical and morphological analyses This approach has contributed to the discovery and examination of many of the currently identified proteins that mediate Golgi membrane tethering [15, 16], fusion [13, 17,18,19,20], and cisternal stacking [21,22,23]. This study revealed candidate proteins involved in the regulation of Golgi morphological changes during the cell cycle
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