In situ cryo-ET defines the ultrastructure of ER exit sites in human cells

Trafficking of secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus comprises the first, essential steps toward the appropriate localisation of 30% of eukaryotic proteins. Coat protein complexes COPII and COPI are involved in forward and retrograde transport of cargo and cargo receptors between the ER and the Golgi, respectively. Although COPII forms coated vesicles in vitro, the biogenesis, morphology and organisation of transport carriers in mammalian cells is debated. Here we use in situ cryo-electron tomography and super-resolution fluorescence microscopy to reveal the molecular architecture of ER exit sites in human cells that were not perturbed with drugs, temperature blocks, or overexpression systems. We visualise ribosome-exclusion zones enriched with COPII and COPI-coated vesicles and thus resolve the debate regarding the existence of COPII coated vesicles. COPII vesicles derive from ER membranes, whereas COPI vesicles originate from vesicular-tubular clusters that constitute the ER-Golgi intermediate compartment (ERGIC). We quantify coated vesicle morphology and positioning with respect to other ER exit site components, providing a molecular description of the organisation of the mammalian early secretory pathway.