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 towards the appropriate localization of 30% of eukaryotic proteins. Coat protein complexes COPII and COPI are involved in the 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 organization of transport carriers in mammalian cells is subject to debate. 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 visualize 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 organization of the mammalian early secretory pathway.