Ultrastructure of COPII vesicle formation characterised by correlative light and electron microscopy

Abstract

Traffic of proteins out of the endoplasmic reticulum (ER) is driven by the COPII coat, a layered protein scaffold that mediates the capture of cargo proteins and the remodelling of the ER membrane into spherical vesicular carriers. Although the components of this machinery have been genetically defined, and the mechanisms of coat assembly extensively explored in vitro, understanding the physical mechanisms of membrane remodelling in cells remains a challenge. Here we use correlative light and electron microscopy (CLEM) to visualize the nanoscale ultrastructure of membrane remodelling at ER exit sites (ERES) in yeast cells. Using various COPII mutants, we have determined the broad contribution that each layer of the coat makes in membrane remodelling. Our data suggest that inner coat components define the radius of curvature whereas outer coat components facilitate membrane fission. The organization of the coat in conjunction with membrane biophysical properties determine the ultrastructure of vesicles and thus the efficiency of protein transport.

Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat

“…The polar character of the dimer contacts helps ensure these contacts are weak and can break readily when a rearrangement is needed. The idea that a dense cylindrical AP-1:Arf1 inner coat could exist as a normal intermediate in CCV formation is consistent with new thinking about the role of the COPII inner coat at ER exit sites (Melero et al, 2022). In this model, the inner coat sets the radius of membrane curvature, and initiates the subsequent recruitment of clathrin.…”

“…Indeed, the asymmetric unit of the closed trimer docks into the lattice density with no need for conformational adjustments, and only slight shift in the Nef core. The major interactions between the β1-bound Arf1 (hereafter, Arf1 β1 ) γ-bound Arf1 (hereafter, Arf1 γ ) with the GTP-dependent switch regions of the Arf1 molecules is identical to the seen in the BST2 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) -bound closed AP-1 trimer (Morris et al, 2018), and therefore not presented in detail here. The density of the lattice is fully accounted for by AP-1 core , Arf1 myr and Nef myr .…”

“…Subtomograms were pooled based on tube diameter and analyzed separately to maximize structural homogeneity. Aligned and averaged subtomograms from narrow tubes (20-30 nm diameter) resulted in a 20 Å resolution map which could be fit unambiguously with atomic models of the hyper-unlocked AP-1 core (Jia et al, 2014;Morris et al, 2018) (Fig. 1D, Fig.…”

“…Globular densities adjacent to the fitted AP-1 heterotetramers showed density extending downward toward the membrane surface suggesting the densities belonged to the N-termini and globular domains of Arf1 and/or Nef. Placement of AP-1 core models (PDB: 6cm9) (Morris et al, 2018) within the lattice revealed two alternating tracks of AP-1 dimers propagating along the tube axis; one dimer pair created by γ : γ, the other by β1 : β1 contacts.…”

“…The stoichiometry and interfaces between AP-1, Arf1 and Nef within the asymmetric unit match those observed in the asymmetric unit of the soluble (e.g. nonmembrane associated) closed trimeric AP-1 complex consisting of the AP-1 core , Nterminally truncated Arf1 and a genetic fusion of BST2 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and NL4-3 Nef (PDB: 6cm9) (Morris et al, 2018). Indeed, the asymmetric unit of the closed trimer docks into the lattice density with no need for conformational adjustments, and only slight shift in the Nef core.…”