Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly

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 (␣, ␤, ␥, and ␦) 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.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. The unique feature of the Golgi in almost all eukaryotic cells is the densely packed stacks of the flattened cisternal membranes. Processing enzymes in the Golgi complex, including those involved in modifying bound oligosaccharides, are arranged across the stack in the cis to trans order in which they function. In animal cells, stacks are often interconnected to form a ribbon-like structure that is localized adjacent to the nucleus. Despite its complicated morphology and function,

Synaptotagmin 11 interacts with components of the RNA‐induced silencing complex RISC in clonal pancreatic β‐cells

“…2). As to their localization, NCL, NPM1, RS10, RS18, RS3, RS3A, TBB2C, SET and SND1 had been found on ER-Golgi vesicles or Golgi membranes by proteomic analysis [31,32]. Strikingly, also FMRP, Ago2 and the Q-SNAREs Vti1a/Vti1b, which we subsequently identified as interacting proteins by immunoreactivity, reside on ER and on Golgi structures [32,[34][35][36].…”

“…Similarly, we used KCl instead of NaCl for washes. Moreover, a number of differential interactions were observed and most importantly, co-localization of binding partners including most of the ribosomal proteins identified here could be deduced from published proteomic data and expression of epitope-tagged proteins [31][32][33].…”

Neuronal porosome proteome: Molecular dynamics and architecture

“…The resulting protein pellets (25 µg) were solubilized in 8M urea, 0.4% SDS and 250 mM TEAB, a potent buffer system used in our previous studies for membrane and membrane-associated proteins [29–31]. After reduction and cysteine-blocking with 55 mM iodoacetamide, trypsin was added to the diluted sample solution (1:10 w/w) for overnight digestion at 37 °C.…”

Golgi Isolation

“…These include the intra-Golgi transport assay (including vesicle budding and fusion processes) (Balch et al 1984), the mitotic disassembly and reassembly assays Rabouille et al 1995;Tang et al 2010), and assays examining the binding to and motility of the Golgi apparatus on the cytoskeleton including microtubules and actin filaments, as well as the cytoskeletal dynamics on Golgi membranes (Fullerton et al 1998;Chen et al 2005). Purified Golgi membranes have also been used in several quantitative proteomics analyses that identified thousands of candidate proteins whose roles in Golgi organization and functions are yet to be determined (Bell et al 2001;Gilchrist et al 2006;Chen et al 2010Chen et al , 2012. …”