Function of the COPII Mediate cargo export from ER to Golgi complex
Componets of COPII Sar1 GTPase Sec 23/24 Sec 13/31 Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles. Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24.
Structure of the “cage” We present a three-dimensional reconstruction of these Sec13/31 cages at 30 A resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers.
Total introduction Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.
Methods 1、Recombinant production and purification 2、Dynamic light scattering（ DLS） 3、cryo-electron microscopy (cryo-EM) 4、Single particle reconstruction. 5、 gel electrophoresis (PAGE) 6、gel filtration chromatography (GFC)
cryo-EM analysis Purified Sec13/31 forms a relatively homogeneous population of assemblies as judged by GFC, analytical ultracentrifugation, DLS, GFC-MALS and electron microscopy analyses of the negatively stained samples We characterized the same samples using cryo-electron microscopy (cryo-EM) and single particle analysis.
cryo-EM analysis Images of the specimen preserved in vitreous ice showed a population of cage- like particles, most of which were symmetric and with an average diameter of ,600 A. These dimensions are in good agreement with the size of COPII cages/vesicles observed in vitro4,12 and in vivo
single particle analysis a total of 9,777 individual cage particles were selected from a set of 516 defocus pairs of micrographs. Particles were first subjected to a reference-free alignment algorithm as implemented in the EMAN package to generate averages with an improved signal-to-noise ratio.
single particle analysis Of the 104 resulting class averages, ten that showed the best signal-to-noise ratio and symmetry were used as reference images in a multi-reference alignment procedure. The resulting class averages exhibited two-fold, three-fold and four-fold symmetry and geometry consistent with that of a cuboctahedron
Cuboctahedrons roughly spherical polyhedrons E=24 V=12 F=14 8 triangles 6 squares exhibit 4 3 2 or octahedral symmetry four edges intersect at each vertex （clathrin geometries are defined by vertices formed from only three edges）
Cuboctahedrons the four-fold rotational axes of symmetry run down the middle of the square faces, the three-fold rotational axes run through the middle of the triangular faces the two-fold rotational axes run through the vertices
Reconstruct cage structure Use a simple cuboctahedron constructed with continuous density for the edges as an initial model The cage structure was refined to a resolution of 30 Å
Reconstruct cage structure • There is excellent agreement between projections of the final model and the individual raw particle images as well as the class averages
The asymmetric unit (ASU) • the smallest unit that can be repeated to generate the full structure
The asymmetric unit (ASU) Two roughly spherical lobes of density at either end（1、2、5、6） connected by a continuous curving stretch of density with a diameter of 40 Å（3、4）
The asymmetric unit (ASU) The lobes at either end are not identical, they appear to be related to each other by a 180°rotation around the centre of the density connecting the two ends. ASU is a dimer The centre of symmetry of the ASU is not in the centre of the edge
The asymmetric unit (ASU) We propose that the 24 off-centre, dimeric ASUs comprising the Sec13/31 cage correspond to 24 Sec13/31 heterotetramers .
Positions of Sec13/31 The positions occupied by Sec13 and Sec31 in the cage remain to be determined. From a structural perspective, Sec13 contains WD40 repeat motifs that are implicated in protein-protein interactions. Biochemical and computational analyses indicate that the Sec13 structure may comprise a single domain, b-propeller fold with six blades
Positions of Sec13/31 heterotetramer model two Sec13 proteins would form part of the continuous density in the centre of the ASU (3 and 4 ) but cannot be resolved as distinct entities at the present resolution. such a model would suggest that Sec13 dimerization is critical for cross-bridging the two halves of the edge.
An alternative model Sec13 forms the vertices of the cuboctahedron • Sec13/31 heterotetramer is arranged as Sec13/Sec31-Sec31/ Sec13 and corresponds to the ASU that constitutes the edges of the cuboctahedron
An alternative model Regions 1 and 6 contain Sec13. The Sec1 3 subunits would interact with each other at the vertices of the cage in two unique ways 1、edge-vertex contacts 2、vertex-vertex contacts
An alternative model • larger globular domains (2 and 5） would correspond to the predicted b -propeller fold comprising the seven WD40 motif repeats/blades of the Sec31 N-terminal domain • smaller globular domains (1 and 6 ）would correspond to the predicted b-propeller fold comprising the six WD40 motif repeats/blades of the entire Sec13 subunit
An alternative model Considering that Sec13 interacts with the N-terminal WD40 repeat domain of Sec31 ，this new model would place the N-terminal domain of Sec31 near the vertex of the cuboctahedral cage. It follows that Sec31 dimerization at the centre of the ASU would be critical for cross-bridging the two halves of the cuboctahedral edge.
An alternative model • Sec23 is expected to bind near the Sec31-Sec31 dimer interface, whereas Sec24 should bind towards the ends of the ASU
Clathrin can also self-assemble in vitro to form empty cages lacking the adaptor components and cargo , all of which comprise the clathrin coat. These are strikingly different from the Sec13/31 cage
overview 1、The function of Sec13/31 analogous to that of clathrin, which self-assembles to form a cage independent of its adaptor proteins. This is in contrast with a recent study that suggested that the Sec23/24 adaptor is required for the self-assembly of a minimal COPII cage.
overview 2、A model for COPII coat formation where Sec23/24, like the clathrin adaptor proteins, coordinates cargo selection with the self-assembly of the Sec13/31 cage to promote budding from the ER.
overview 3、The discovery of the self-assembling properties of Sec13/31 to generate a cage structure provides a new focus for elucidating the biological mechanisms of cargo selection, concentration and budding for transport of nearly one-third of all proteins encoded by the eukaryotic genome.