Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system

Nature Protocols volume 5, pages 758–772 (2010)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Standard Golgi disassembly and reassembly assay in which purified Golgi membranes were treated with mitotic and interphase HeLa cell cytosol (a,b).
Figure 2: Defined Golgi disassembly and reassembly assay using purified proteins (a,b).
Figure 3: Distribution of Golgi proteins in vesicles and Golgi remnants after mitotic Golgi disassembly.

Similar content being viewed by others

References

  1. Acharya, U. et al. The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. Cell 82, 895–904 (1995).

    Article  CAS  Google Scholar 

  2. Jamora, C. et al. Regulation of Golgi structure through heterotrimeric G proteins. Cell 91, 617–626 (1997).

    Article  CAS  Google Scholar 

  3. Kano, F., Nagayama, K. & Murata, M. Reconstitution of the Golgi reassembly process in semi-intact MDCK cells. Biophys. Chem. 84, 261–268 (2000).

    Article  CAS  Google Scholar 

  4. Kano, F. et al. MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts. J. Cell. Biol. 149, 357–368 (2000).

    Article  CAS  Google Scholar 

  5. Misteli, T. & Warren, G. COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system. J. Cell. Biol. 125, 269–282 (1994).

    Article  CAS  Google Scholar 

  6. Rabouille, C., Misteli, T., Watson, R. & Warren, G. Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system. J. Cell. Biol. 129, 605–618 (1995).

    Article  CAS  Google Scholar 

  7. Shorter, J. & Warren, G. Golgi architecture and inheritance. Annu. Rev. Cell. Dev. Biol. 18, 379–420 (2002).

    Article  CAS  Google Scholar 

  8. Wang, Y. Golgi apparatus inheritance. In The Golgi Apparatus. State of the Art 110 Years After Camillo Golgi's Discovery, Vol. Chapter 4.3. (eds. Mironov, A., Pavelka, M. & Luini, A.) 580–607 (Springer-Verlag Gmbh., Wien-New York, 2008).

  9. Barr, F.A., Puype, M., Vandekerckhove, J. & Warren, G. GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 91, 253–262 (1997).

    Article  CAS  Google Scholar 

  10. Wang, Y., Seemann, J., Pypaert, M., Shorter, J. & Warren, G. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor. EMBO J. 22, 3279–3290 (2003).

    Article  CAS  Google Scholar 

  11. Shorter, J. et al. GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system. EMBO J. 18, 4949–4960 (1999).

    Article  CAS  Google Scholar 

  12. Lowe, M. et al. Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis. Cell 94, 783–793 (1998).

    Article  CAS  Google Scholar 

  13. Sonnichsen, B. et al. A role for giantin in docking COPI vesicles to Golgi membranes. J. Cell. Biol. 140, 1013–1021 (1998).

    Article  CAS  Google Scholar 

  14. Satoh, A., Wang, Y., Malsam, J., Beard, M.B. & Warren, G. Golgin-84 is a rab1 binding partner involved in Golgi structure. Traffic 4, 153–161 (2003).

    Article  CAS  Google Scholar 

  15. Shorter, J. & Warren, G. A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling golgi cisternae in a cell-free system. J. Cell. Biol. 146, 57–70 (1999).

    Article  CAS  Google Scholar 

  16. Rabouille, C. et al. Syntaxin 5 is a common component of the NSF- and p97-mediated reassembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro . Cell 92, 603–610 (1998).

    Article  CAS  Google Scholar 

  17. Rabouille, C., Levine, T.P., Peters, J.M. & Warren, G. An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82, 905–914 (1995).

    Article  CAS  Google Scholar 

  18. Kondo, H. et al. p47 is a cofactor for p97-mediated membrane fusion. Nature 388, 75–78 (1997).

    Article  CAS  Google Scholar 

  19. Meyer, H.H., Wang, Y. & Warren, G. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 21, 5645–5652 (2002).

    Article  CAS  Google Scholar 

  20. Wang, Y., Satoh, A., Warren, G. & Meyer, H.H. VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments. J. Cell. Biol. 164, 973–978 (2004).

    Article  CAS  Google Scholar 

  21. Tang, D., Mar, K., Warren, G. & Wang, Y. Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay. J. Biol. Chem. 283, 6085–6094 (2008).

    Article  CAS  Google Scholar 

  22. Lin, C.Y. et al. Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2. Proc. Natl. Acad. Sci. USA 97, 12589–12594 (2000).

    Article  CAS  Google Scholar 

  23. Wang, Y., Satoh, A. & Warren, G. Mapping the functional domains of the Golgi stacking factor GRASP65. J. Biol. Chem. 280, 4921–4928 (2005).

    Article  CAS  Google Scholar 

  24. Xiang, Y., Seemann, J., Bisel, B., Punthambaker, S. & Wang, Y. Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation. J. Biol. Chem. 282, 21829–21837 (2007).

    Article  CAS  Google Scholar 

  25. Otter-Nilsson, M., Hendriks, R., Pecheur-Huet, E.I., Hoekstra, D. & Nilsson, T. Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion. EMBO J. 18, 2074–2083 (1999).

    Article  CAS  Google Scholar 

  26. Muller, J.M. et al. An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion. Nat. Cell. Biol. 1, 335–340 (1999).

    Article  CAS  Google Scholar 

  27. Muller, J.M. et al. Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. J. Cell. Biol. 157, 1161–1173 (2002).

    Article  Google Scholar 

  28. Uchiyama, K. et al. p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis. Dev. Cell. 11, 803–816 (2006).

    Article  CAS  Google Scholar 

  29. Hui, N., Nakamura, N., Slusarewicz, P. & Warren, G. Purification of rat liver Golgi stacks. In Cell Biology: A Laboratory Handbook, Vol. 2 (ed. Celis, J.) 46–55 (Academic Press, San Diego, California, 1998).

  30. Wang, Y., Taguchi, T. & Warren, G. Purification of rat liver Golgi stacks. In Cell Biology: A Laboratory Handbook, 3rd edn. (ed. Celis, J.) 33–39 (Elsevier Science, San Diego, California, 2006).

  31. Pavel, J., Harter, C. & Wieland, F.T. Reversible dissociation of coatomer: functional characterization of a beta/delta-coat protein subcomplex. Proc. Natl. Acad. Sci. USA 95, 2140–2145 (1998).

    Article  CAS  Google Scholar 

  32. Randazzo, P.A., Weiss, O. & Kahn, R.A. Preparation of recombinant ADP-ribosylation factor. Methods Enzymol. 257, 128–135 (1995).

    Article  CAS  Google Scholar 

  33. Ha, V.L., Thomas, G.M., Stauffer, S. & Randazzo, P.A. Preparation of myristoylated Arf1 and Arf6. Methods Enzymol. 404, 164–174 (2005).

    Article  CAS  Google Scholar 

  34. Whiteheart, S.W. et al. N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J. Cell. Biol. 126, 945–954 (1994).

    Article  CAS  Google Scholar 

  35. Levine, T.P., Rabouille, C., Kieckbusch, R.H. & Warren, G. Binding of the vesicle docking protein p115 to Golgi membranes is inhibited under mitotic conditions. J. Biol. Chem. 271, 17304–17311 (1996).

    Article  CAS  Google Scholar 

  36. Satoh, A., Beard, M. & Warren, G. Preparation and characterization of recombinant golgin tethers. Methods Enzymol. 404, 279–296 (2005).

    Article  CAS  Google Scholar 

  37. Beard, M., Satoh, A., Shorter, J. & Warren, G. A cryptic Rab1-binding site in the p115 tethering protein. J. Biol. Chem. 280, 25840–25848 (2005).

    Article  CAS  Google Scholar 

  38. Molloy, S.S., Thomas, L., Kamibayashi, C., Mumby, M.C. & Thomas, G. Regulation of endosome sorting by a specific PP2A isoform. J. Cell. Biol. 142, 1399–1411 (1998).

    Article  CAS  Google Scholar 

  39. Lucocq, J.M., Berger, E.G. & Warren, G. Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J. Cell. Biol. 109, 463–474 (1989).

    Article  CAS  Google Scholar 

  40. Bisel, B. et al. ERK regulates Golgi and centrosome orientation towards the leading edge through GRASP65. J. Cell. Biol. 182, 837–843 (2008).

    Article  CAS  Google Scholar 

  41. Xiang, Y. & Wang, Y. GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking. J. Cell. Biol. 188, 237–251 (2010).

    Article  CAS  Google Scholar 

  42. Shima, D.T., Haldar, K., Pepperkok, R., Watson, R. & Warren, G. Partitioning of the Golgi apparatus during mitosis in living HeLa cells. J. Cell. Biol. 137, 1211–1228 (1997).

    Article  CAS  Google Scholar 

  43. Altan-Bonnet, N. et al. Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities. Mol. Biol. Cell. 17, 990–1005 (2006).

    Article  CAS  Google Scholar 

  44. Malsam, J., Satoh, A., Pelletier, L. & Warren, G. Golgin tethers define subpopulations of COPI vesicles. Science 307, 1095–1098 (2005).

    Article  CAS  Google Scholar 

  45. Nickel, W. et al. Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro . J. Cell. Sci. 111 (Part 20): 3081–3090 (1998).

    CAS  PubMed  Google Scholar 

  46. Serafini, T. & Rothman, J.E. Purification of Golgi cisternae-derived non-clathrin-coated vesicles. Methods Enzymol. 219, 286–299 (1992).

    Article  CAS  Google Scholar 

  47. Vielemeyer, O. et al. Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping. J. Biol. Chem. 284, 20791–20795 (2009).

    Article  CAS  Google Scholar 

  48. Satoh, A., Malsam, J. & Warren, G. Tethering assays for COPI vesicles mediated by golgins. Methods Enzymol. 404, 125–134 (2005).

    Article  CAS  Google Scholar 

  49. Meyer, H.H., Kondo, H. & Warren, G. The p47 co-factor regulates the ATPase activity of the membrane fusion protein, p97. FEBS Lett. 437, 255–257 (1998).

    Article  CAS  Google Scholar 

  50. Meyer, H.H., Shorter, J.G., Seemann, J., Pappin, D. & Warren, G. A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192 (2000).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA

    Danming Tang, Yi Xiang & Yanzhuang Wang

Authors
  1. Danming Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yanzhuang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Yanzhuang Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.38

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing