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Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers.
Nature Cell Biology
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 approximately 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.
COP-Coated Vesicles/metabolism, COP-Coated Vesicles/ultrastructure, Carcinoma, Hepatocellular/metabolism, Carcinoma, Hepatocellular/pathology, Cells, Cultured, Endoplasmic Reticulum/metabolism, Endoplasmic Reticulum/ultrastructure, Golgi Apparatus/metabolism, Golgi Apparatus/ultrastructure, Humans, Intracellular Membranes/metabolism, Intracellular Membranes/ultrastructure, Liver Neoplasms/metabolism, Liver Neoplasms/pathology, Microscopy, Immunoelectron, Models, Molecular, Protein Transport, R-SNARE Proteins/metabolism, Tomography, X-Ray Computed, Tumor Cells, Cultured
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