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

nature structural & molecular biology Article https://doi.org/10.1038/s41594-024-01413-4 Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes Received: 16 January 2024 Euan Pyle , Elizabeth A. Miller4,6 & Giulia Zanetti 1,2,3,5 1,2,3 Accepted: 1 October 2024 Published online: xx xx xxxx Check for updates 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. Eukaryotic cells use the secretory pathway to transport proteins and lipids to their required locations within and outside the cell. Approximately one in three proteins is translocated in the endoplasmic reticulum (ER) upon synthesis and is trafficked to the Golgi apparatus as the first step of the secretory pathway1. Anterograde transport of proteins from the ER to the Golgi is facilitated by coat protein complex II (COPII)-coated membrane carriers. The COPII coat assembles on the cytosolic side of the ER membrane, generating membrane curvature to form coated carriers while specifically recruiting and enveloping newly synthesized cargo proteins2,3. COPII comprises five proteins (Sar1, Sec23, Sec24, Sec13 and Sec31) that are essential and highly conserved from yeast to humans3. COPII assembly is initiated by the small guanosine triphosphate (GTP) hydrolase Sar1, which inserts its N-terminal amphipathic helix into the outer leaflet of the ER upon nucleotide exchange, an event catalyzed by the ER-resident GTP exchange factor (GEF) Sec12 (refs. 4,5). Membrane-bound Sar1 recruits heterodimeric Sec23–Sec24 to form the inner layer of the COPII coat, with Sec24 acting as the main cargobinding subunit6,7. The outer coat layer is formed when heterotetrametric rod-shaped Sec13–Sec31 complexes are recruited to budding sites through the interaction of Sec31 with Sec23–Sar1 and assemble in a cage-like arrangement8–10. Symmetric polyhedral cages assemble in vitro when purified Sec13–Sec31 heterotetramers are incubated in the absence of any membrane10,11. The detachment of Sar1 from the membrane is triggered by GTP hydrolysis, stimulated by its cognate GTPase-activating protein (GAP) Sec23 and further accelerated by binding of Sec31 (ref. 12). Sar1 GTP hydrolysis is thought to destabilize the coat; however, the dynamics and regulation of coat disassembly are poorly understood. We previously determined the structure of the Saccharomyces cerevisiae COPII coat reconstituted in vitro from giant unilamellar vesicles (GUVs) using cryo-electron tomography (cryo-ET) with subtomogram averaging (STA)13–16. We showed that COPII forms coated tubes on GUVs and that the inner and outer coat layers both arrange Institute of Structural and Molecular Biology, Birkbeck College, London, UK. 2Institute of Structural and Molecular Biology, UCL, London, UK. The Francis Crick Institute, London, UK. 4MRC Laboratory of Molecular Biology, Cambridge, UK. 5Present address: EMBL, Heidelberg, Germany. 6 Present address: School of Life Sciences, University of Dundee, Dundee, UK. e-mail: 1 3 Nature Structural & Molecular Biology Article into pseudohelical lattices that wrap around the tubular membrane. High-resolution STA yielded atomic models describing coat interactions that allowed us to design coat mutants where assembly interfaces are disrupted15. We found that the two interfaces that form the outer coat cage, formed by the N-terminal and C-terminal domains of Sec31, are dispensable for membrane budding in vitro and in yeast cells lacking the glycosylphosphatidylinositol-anchored protein cargo adaptor Emp24 (refs. 15,17). Moreover, when the interface between inner coat lattice subunits was weakened by amino acid substitutions, budded membranes switched from a tubular to a spherical profile, indicating that membrane curvature is generated by a complex network of interactions spanning both coat layers15. COPII-coated membrane carriers are known to adopt a range of sizes and shapes, which may be important to adapt to the wide range of cargoes that need to be accommodated. However, it remains unclear how coat assembly is regulated to achieve a variety of membrane carrier sizes3,18,19. Whilst our previous studies found that purified S. cerevisiae COPII forms extended tubules on GUVs, electron microscopy (EM) studies of cell sections suggested that membrane carriers in vivo are spherical vesicles 50–100 nm wide20–22, raising the question of which components of native membrane composition affect coat assembly and budding morphology. It also remains unclear how the tightly packed inner coat assembly is compatible with cargo binding by the Sec24 subunits. To answer these questions, we carried out in vitro reconstitution of COPII budding using native ER membranes derived from yeast, referred to as microsomes. In striking comparison to the tubules formed by COPII on GUVs, cryo-ET revealed that the majority of coated membranes are pseudospherical. We used STA16,23,24 to obtain the structures of the inner and outer coat assembled on native membranes. We found that the inner coat layer can assemble as in its tubular arrangement but forms limited patches of coat that are randomly oriented around a pseudospherical membrane. Cargo density could be detected within the inner coat array, in the space between inner coat subunits, indicating that the lateral assembly of Sar1–Sec23–Sec24 heterotrimers can occur while small or flexible cytosolic domains of cargo molecules are accommodated in between. Lastly, STA analysis of the outer coat layer revealed nonsymmetric cages with a variety of architectures. We characterize multiple points of flexibility, increasing the complexity of the outer coat network and challenging the current model where assembly of the outer coat into a polyhedral cage is the main driver of membrane curvature. Results COPII forms coated pseudospherical vesicles on microsomes To reconstitute COPII budding in vitro from native membrane sources, we incubated purified S. cerevisiae COPII proteins with S. cerevisiae ER-enriched microsomes and a nonhydrolyzable (...truncated)