Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain

Abstract

In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network

“…Consistent with this, the outer coat on tubules reconstituted with Sec31-ΔCTD appears less ordered than in wild-type conditions. Interestingly, human Sec31 proteins lacking the CTD assemble into cages 29 , indicating that either the vertex is more stable for human proteins, or that the CTD is important in the context of membrane budding but not for cage formation in high salt conditions. Our data paints a picture of the assembled coat where the outer coat C-terminal disordered region reaches down to bind and stabilise the inner coat, and then loops back to lock onto the outer coat lattice.…”

“…Sec31 also contains a putative helical C-terminal domain (CTD), that is separated from the cage-forming elements by a long flexible PRD 27 . No role for the CTD has yet been assigned, but limited proteolysis experiments and secondary structure prediction suggest an ordered helical domain of ~18 kDa 28 , 29 .…”

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network

“…Consistent with this, the outer coat on tubules reconstituted with Sec31-ΔCTD appears less ordered than in wild type conditions. Interestingly, human Sec31 proteins lacking the CTD assemble into cages (Paraan et al, 2018), indicating that either the vertex is more stable for human proteins, or that the CTD is important in the context of membrane budding but not for cage formation in high salt conditions. Our data paints a new picture of the assembled coat, where the outer coat C-terminal disordered region reaches down to bind and stabilise the inner coat, and then loops back to lock onto the outer coat lattice.…”

“…Sec31 also contains a putative helical C-terminal domain (CTD), that is separated from the cage-forming elements by a long flexible proline-rich domain (PRD) (Fath et al, 2007). No role for the CTD has yet been assigned, but limited proteolysis experiments and secondary structure prediction suggest an ordered helical domain of ~ 18 kDa (Dokudovskaya et al, 2006; Paraan et al, 2018).…”

COPII‐mediated trafficking at the ER/ERGIC interface

“…Additionally, the angle between the terminal beta‐propeller domains and alpha‐helical central rod is flexible, creating a hinge that allows the cage to vary in dimension, depending on the size of the transport carrier it must encase . A second potential hinge was identified more recently, which allows the beta‐propeller domains of Sec13 and Sec31 to move relative to one another . Based on cryoEM studies, the underlying inner coat is positioned such that Sec23 protomers appear below each vertex and in the middle of triangular faces of the outer cage, and Sec24 subunits are located in the middle of square faces of cuboctahedrons or pentagons of icosidodecahedrons, providing sufficient space to accommodate the cytosolic domains of integral membrane cargoes .…”