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De novo formation of transitional ER sites and Golgi structures in Pichia pastoris

Nature Cell Biology volume 4, pages 750–756 (2002)Cite this article

Abstract

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

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Figure 1: Sec13–GFP labels a subdomain of the ER in P. pastoris.
Figure 2: tER sites form de novo and fuse with one another.
Figure 3: Golgi structures form and fuse in conjunction with associated tER sites.
Figure 4: De novo formation of Golgi structures visualized with a transmembrane protein.
Figure 5: Late Golgi elements are transported toward sites of polarized growth.
Figure 6: Model of tER site organization in P. pastoris.

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Acknowledgements

We thank T. Karr, C. Lassy, Y. Gottlieb, V. Bindokas and R. Blocker for support with confocal imaging; J. Soderholm for the Sec13–GFP construct; D. Strongin for improving the GFP–Gos1 expression plasmid; and S. Mogelsvang, A. Staehelin and the members of the Glick lab for suggestions. This work was supported by grants from the American Cancer Society, the NIH and the Pew Charitable Trusts.

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Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, 60637, Illinois, USA

    Brooke J. Bevis, Adam T. Hammond, Catherine A. Reinke & Benjamin S. Glick

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  1. Brooke J. Bevis
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  2. Adam T. Hammond
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Correspondence to Benjamin S. Glick.

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Supplementary information

Movie 1

P. pastoris cells containing Sec13p-GFP-labeled tER sites were imaged for ~ 33 min. Several de novo formation and fusion events can be seen. Weak nuclear fluorescence is also visible. This movie is accelerated 60-fold relative to real time. (MOV 4192 kb)

Movie 2

P. pastoris cells containing Sec13p-GFP-labeled tER sites were imaged for ~ 30 min. Several de novo formation and fusion events can be seen, particularly in the cell on the lower left. Weak nuclear fluorescence is also visible. This movie is accelerated 60-fold relative to real time. (MOV 3597 kb)

Movie 3

P. pastoris cells containing Sec13p-GFP-labeled tER sites and Sec7p-DsRed-labeled Golgi structures were imaged for ~ 14 min. The formation and growth of new Golgi structures is linked to that of the associated tER sites, as seen most clearly in the pair of cells on the left. This movie is accelerated 60-fold relative to real time. (MOV 1332 kb)

Movie 4

P. pastoris cells containing Sec13p-GFP-labeled tER sites and Sec7p-DsRed-labeled Golgi structures were imaged for ~ 15 min. In the cell on the left, two tER sites fuse, and the associated Golgi structures also fuse. This movie is accelerated 60-fold relative to real time. (MOV 903 kb)

Movie 5

P. pastoris cells containing GFP-Gos1p-labeled Golgi structures were imaged for ~ 21 min. Several de novo formation and fusion events can be seen, particularly in the cell on the lower left. This movie is accelerated 60-fold relative to real time. (MOV 2644 kb)

Movie 6

P. pastoris cells containing containing Sec13p-GFP-labeled tER sites and Sec7p-DsRed-labeled Golgi structures were imaged for ~ 4 min. In the cell on the left, a Golgi structure moves away from its associated tER site toward the bud. This movie is accelerated 12-fold relative to real time. (MOV 244 kb)

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Bevis, B., Hammond, A., Reinke, C. et al. De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nat Cell Biol 4, 750–756 (2002). https://doi.org/10.1038/ncb852

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