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Dynamics of the COPII coat with GTP and stable analogues

Nature Cell Biology volume 3, pages 531–537 (2001)Cite this article

Abstract

We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time. We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds. The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.

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Figure 1: Sequential assembly of the COPII coat onto liposomes as detected by light scattering.
Figure 2: Transient assembly of the COPII coat on liposomes preloaded with Sar1pGTP.
Figure 3: Sec13/31p stimulates the GAP activity of Sec23/24p.
Figure 4: Translocation of the COPII components in the presence of the phosphate analogue BeFx.
Figure 5: Morphology of major–minor mix liposomes incubated with COPII proteins and with no activator (a), GMP-PNP (b) or BeFx (c).
Figure 6: Assembly and disassembly of the COPII coat with Sar1pGTP at low temperature.

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Acknowledgements

We thank B. Lesch and C. F. Chan for the preparation of COPII proteins, and M. Welsh for the use of the fluorimeter. This work was supported by the HHMI (R.S.), the Swiss National Science Foundation (L.O.), CNRS, NATO and HFSP (B.A.).

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  1. Bruno Antonny

    Present address: IPMC–CNRS, 660 route des Lucioles, 06560, Valbonne, France

Authors and Affiliations

  1. Department of Molecular and Cell Biology, Howard Hugues Medical Institute, Stanley Hall, University of California, Berkeley, 94720, California, USA

    Bruno Antonny, David Madden, Susan Hamamoto & Randy Schekman

  2. Département de morphologie, Université de Genève, Centre Médical Universitaire, 1 Rue Michel Servet, Genève, CH12-11 4, Suisse

    Lelio Orci

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Correspondence to Randy Schekman.

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Antonny, B., Madden, D., Hamamoto, S. et al. Dynamics of the COPII coat with GTP and stable analogues. Nat Cell Biol 3, 531–537 (2001). https://doi.org/10.1038/35078500

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