Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes

Proteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The COPII coat mediates the anterograde transport of newly synthesised proteins from the endoplasmic reticulum, engaging cargoes with a wide range of size and biophysical properties. The native architecture of the COPII coat and how cargo might influence COPII carrier morphology remain poorly understood. Here, we have reconstituted COPII-coated membrane carriers using purified S. cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated.