Proteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The coat protein complex II (COPII) mediates the anterograde transport of newly synthesized 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 reconstituted COPII-coated membrane carriers using purified Saccharomyces 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.

蛋白质通过细胞器之间的膜运输穿过真核生物分泌途径。外壳蛋白复合物 II （COPII） 介导新合成蛋白质从内质网的顺行转运，吸引具有各种大小和生物物理特性的货物。COPII 外套的原生结构以及货物如何影响 COPII 载体形态仍然知之甚少。在这里，我们使用纯化的酿酒酵母蛋白和细胞衍生的微粒体作为天然膜来源重构了 COPII 包被的膜载体。使用冷冻电子断层扫描和亚断层照片平均，我们证明 COPII 涂层结合货物并从天然膜形成大部分球形囊泡。我们揭示了内涂层和外涂层的结构，并阐明了球形载体是如何形成的。我们的结果提供了对 COPII 外壳的结构和调节的见解，并推进了我们目前对膜曲率如何产生的理解。