Abstract
The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defined Golgi disassembly and reassembly assay that reconstitutes this process using purified proteins instead of cytosol. Treatment of Golgi membranes with mitotic kinases and COPI coat proteins efficiently disassembles the membranes into mitotic Golgi fragments, whereas further incubation with p97 or N-ethylmaleimide-sensitive factor (two AAA ATPases involved in membrane fusion) and their cofactors, in combination with protein phosphatase PP2A, leads to reassembly of the membranes into new Golgi stacks. The whole process takes 3–4 d and is applicable for identification and determination of novel cytosolic and membrane proteins that regulate Golgi membrane dynamics in the cell cycle.
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Acknowledgements
The current Golgi disassembly and reassembly assay was modified from a protocol developed by T. Misteli and C. Rabouille who used purified Golgi membranes, mitotic HeLa cell cytosol and rat liver cytosol5,6. We thank J. Shorter and G. Warren for help and suggestions during our work, M. Jackman, K. Mar, J. Malsam, H. Meyer and T. Taguchi for protein preparations and J. Rothman and G. Thomas for reagents. This work was supported by the Pardee Cancer Research Foundation, the National Institute of Health (GM087364), the American Cancer Society (RGS-09-278-01-CSM), a University of Michigan Rackham Faculty Research Grant, the NIH-funded Michigan Alzheimer's Disease Research Center (P50 AG08761) and an anonymous donation to Y.W.
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Y.W. and Y.X. prepared Golgi membranes, Y.W. performed the Golgi disassembly and reassembly assay and Y.W. and D.T. analyzed the results. Y.W., D.T. and Y.X. wrote the paper.
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Tang, D., Xiang, Y. & Wang, Y. Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system. Nat Protoc 5, 758–772 (2010). https://doi.org/10.1038/nprot.2010.38
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DOI: https://doi.org/10.1038/nprot.2010.38
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