Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER

The COPII complex mediates the selective incorporation of secretory cargo and relevant machinery into budding vesicles at specialised sites on the endoplasmic reticulum membrane called transitional ER (tER). Here, we show using confocal microscopy, immunogold labelling of ultrathin cryosections and...

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Bibliographic Details
Published in:	Journal of cell science Vol. 122; no. 16; pp. 2924 - 2934
Main Authors:	Hughes, Helen, Budnik, Annika, Schmidt, Katy, Palmer, Krysten J, Mantell, Judith, Noakes, Chris, Johnson, Andrew, Carter, Deborah A, Verkade, Paul, Watson, Peter, Stephens, David J
Format:	Journal Article
Language:	English
Published:	England The Company of Biologists Limited 15-08-2009 Company of Biologists
Subjects:	COP-Coated Vesicles - metabolism COP-Coated Vesicles - ultrastructure Endocytosis Endoplasmic Reticulum - metabolism Endoplasmic Reticulum - ultrastructure Fluorescence Recovery After Photobleaching Green Fluorescent Proteins - metabolism HeLa Cells Humans Kinetics Models, Biological Protein Binding Protein Subunits - metabolism Protein Transport Recombinant Fusion Proteins - metabolism Vesicular Transport Proteins - metabolism
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Summary:	The COPII complex mediates the selective incorporation of secretory cargo and relevant machinery into budding vesicles at specialised sites on the endoplasmic reticulum membrane called transitional ER (tER). Here, we show using confocal microscopy, immunogold labelling of ultrathin cryosections and electron tomography that in human cells at steady state, Sec16 localises to cup-like structures of tER that are spatially distinct from the localisation of other COPII coat components. We show that Sec16 defines the tER, whereas Sec23-Sec24 and Sec13-Sec31 define later structures that precede but are distinct from the intermediate compartment. Steady-state localisation of Sec16 is independent of the localisation of downstream COPII components Sec23-Sec24 and Sec13-Sec31. Sec16 cycles on and off the membrane at a slower rate than other COPII components with a greater immobile fraction. We define the region of Sec16A that dictates its robust localisation of tER membranes and find that this requires both a highly charged region as well as a central domain that shows high sequence identity between species. The central conserved domain of Sec16 binds to Sec13 linking tER membrane localisation with COPII vesicle formation. These data are consistent with a model where Sec16 acts as a platform for COPII assembly at ERES.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author for correspondence (david.stephens@bristol.ac.uk) Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/16/2924/DC1 Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE Present address: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PL These authors contributed equally to this work Present address: School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK We would like to thank Harry Mellor, Jon Lane, Nigel Savery, Mark Dillingham, Mark Szczelkun, Pete Cullen, and members of the Stephens laboratory for helpful advice and discussions, and Jeremy Rees of FEI Company for assistance with tomographic reconstructions. We thank the MRC for funding this work through a Senior Non-Clinical Fellowship (to D.J.S., G117/553), and the BBSRC for a research grant (BB/E019633) and doctoral training account studentships. We are also grateful to the University of Bristol, MRC and Wolfson Foundation for providing generous support to establish and develop the Wolfson Bioimaging Facility. Deposited in PMC for release after 6 months.
ISSN:	0021-9533 1477-9137
DOI:	10.1242/jcs.044032