title: Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER.
abstract: 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.
authors.affiliation: Cell Biology Laboratories, Department of Biochemistry, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Hughes
authors.forename: Annika
authors.initials: A
authors.lastname: Budnik
authors.forename: Katy
authors.initials: K
authors.lastname: Schmidt
authors.forename: Krysten J
authors.initials: KJ
authors.lastname: Palmer
authors.forename: Judith
authors.initials: J
authors.lastname: Mantell
authors.forename: Chris
authors.initials: C
authors.lastname: Noakes
authors.forename: Andrew
authors.initials: A
authors.lastname: Johnson
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Carter
authors.forename: Paul
authors.initials: P
authors.lastname: Verkade
authors.forename: Peter
authors.initials: P
authors.lastname: Watson
authors.forename: David J
authors.initials: DJ
authors.lastname: Stephens
mesh_terms: D022181:COP-Coated Vesicles / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C517555:SEC16A protein, human; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins