Abstract
In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.
Highlights
The interphase Golgi apparatus, as seen by light or fluorescence microscopy, is a compact juxta-nuclear reticulum, located most often in the peri-centriolar region of the cell [1]
The discovery of these proteins that are involved in regulation of Golgi membrane dynamics has contributed much to our understanding of the biogenesis of the Golgi apparatus, it is unclear whether these proteins are sufficient to control mitotic Golgi disassembly and reassembly, as all these studies used cytosol, or cell extract, in the reconstitution assays
Our results show that the disassembly process is mediated by two independent but interactive processes: cisternal membrane unstacking mediated by mitotic kinases, and membrane vesiculation mediated by the COPI vesicle budding machinery
Summary
Reagents—All reagents were from Sigma, Roche Applied Sciences, or Calbiochem, unless otherwise stated. To dephosphorylate GRASP65, the membrane pellets were directly resuspended in KHM buffer (20 mM Hepes-KOH, pH7, 0.2 M sucrose, 60 mM KCl, 5 mM Mg(OAc) mM ATP, 1 mM GTP, 1 mM glutathione, protease inhibitors) containing 100 g interphase cytosol, or 0.5 units of purified phosphatases (except for PP2A1, for which 0.125 milliunits was used), and incubated at 30 °C for 60 min. Golgi membranes (200 g) were mixed with 10 mg of mitotic cytosol, or with purified coatomer (100 g), recombinant myristoylated ARF1 (50 g), 1 mM GTP, and an ATP-regenerating system in MEB buffer, in a final volume of 1000 l. Results were quantitated from three independent experiments and the statistical significance was assessed by Student’s t test
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