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Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers

Nature Cell Biology volume 8, pages 377–383 (2006)Cite this article

Abstract

Transport from the endoplasmic reticulum (ER) to the Golgi complex requires assembly of the COPII coat complex at ER exit sites. Recent studies have raised the question as to whether in mammalian cells COPII coats give rise to COPII-coated transport vesicles or instead form ER sub-domains that collect proteins for transport via non-coated carriers. To establish whether COPII-coated vesicles do exist in vivo, we developed approaches to combine quantitative immunogold labelling (to identify COPII) and three-dimensional electron tomography (to reconstruct entire membrane structures). In tomograms of both chemically fixed and high-pressure-frozen HepG2 cells, immuno-labelled COPII was found on ER-associated buds as well as on free ∼50-nm diameter vesicles. In addition, we identified a novel type of COPII-coated structure that consists of partially COPII-coated, 150–200-nm long, dumb-bell-shaped tubules. Both COPII-coated carriers also contain the SNARE protein Sec22b, which is necessary for downstream fusion events. Our studies unambiguously establish the existence of free, bona fide COPII-coated transport carriers at the ER–Golgi interface, suggesting that assembly of COPII coats in vivo can result in vesicle formation.

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Figure 1: ERES of high-pressure-frozen HepG2 cells.
Figure 2: COPII labelling of ERES in 2D- and 3D-electron microscopy.
Figure 3: 3D-electron microscopy of high-pressure frozen, rehydrated, COPII-labelled ERES.
Figure 4: Free COPII-coated carriers contain the v-SNARE protein Sec22b.

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Acknowledgements

We thank M. v. Peski and R. Scriwanek for assistance with figure and movie preparation. We also thank A. Verkleij for his continued support and gratefully acknowledge H. Geuze for critical reading of the manuscript. This work was supported by the Dutch Society for Scientific Research (NWO:FOM/ALW 805.47.051) awarded to A.J.K. and J.K.

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Author notes

  1. Dagmar Zeuschner and Willie J.C. Geerts: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Institute of Biomembranes, UMC Utrecht, Heidelberglaan 100, Utrecht, 3584 CX, The Netherlands

    Dagmar Zeuschner, Elly van Donselaar, Jan W. Slot & Judith Klumperman

  2. Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands

    Willie J.C. Geerts, Bruno M. Humbel & Abraham J. Koster

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Correspondence to Judith Klumperman.

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Zeuschner, D., Geerts, W., van Donselaar, E. et al. Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers. Nat Cell Biol 8, 377–383 (2006). https://doi.org/10.1038/ncb1371

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