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Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport

Abstract

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway1,2. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus2. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.

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Figure 1: Incorporation of εCOP–GFP into functional coatomer complexes and their distribution.
Figure 2: Kinetics of COPI binding to and dissociation from Golgi and ER-to-Golgi transport intermediates in ldlF cells stabily expressing εCOP–GFP.
Figure 3: Golgi association–dissociation kinetics of Arf1–GFP/CFP and εCOP–GFP/YFP in AlF-treated or BFA-treated cells.
Figure 4: Kinetic modelling of coatomer and Arf1 activity and the effects of coatomer depletion on the rate of Arf1 dissociation from membranes.
Figure 5: Testing different models of coatomer function on membranes.

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Acknowledgements

We thank D. Hailey for help in the analysis of Arf1–GFP in yeast cells; M. Krieger for his gift of ldlF cells; F. Wieland and J. E. Rothman for εCOP cDNA; J. Donaldson for Arf1[Q71L] cDNA; G. Romero for the Arf1–GFP plasmid; EG&G Wallac for the use of their Ultravision spinning disc confocal system; and J. Donaldson, J. Bonifacino, C. Jackson, K. Hirschberg, B. Nichols, A. Kenworthy, N. Cole and H. Radhakrishna for valuable discussion. J.F.P. was supported in part by a grant from the Canadian Institutes of Health Research.

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Correspondence to Jennifer Lippincott-Schwartz.

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Presley, J., Ward, T., Pfeifer, A. et al. Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport. Nature 417, 187–193 (2002). https://doi.org/10.1038/417187a

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