1 Structure of the Sec13/31 COPII coat cage. 2 Function of the COP II Mediate cargo export from ER...
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Transcript of 1 Structure of the Sec13/31 COPII coat cage. 2 Function of the COP II Mediate cargo export from ER...
![Page 1: 1 Structure of the Sec13/31 COPII coat cage. 2 Function of the COP II Mediate cargo export from ER to Golgi complex.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649cbe5503460f94983f37/html5/thumbnails/1.jpg)
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Structure of the Sec13/31 COPII coat cage
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Function of the COPII
Mediate cargo export from ER to Golgi complex
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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.
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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.
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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.
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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)
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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.
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Images of the specimen preservedin vitreous ice showeda population of cage-like particles, most of which were symmetricand 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
cryo-EM analysis
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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
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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
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Cuboctahedrons
roughly spherical polyhedronsE=24 V=12 F=14 8 triangles 6 squaresexhibit 4 3 2 or octahedral symmetry
four edges intersect at each vertex( clathrin geometries are defined by vertices
formed from only three edges )
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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
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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
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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
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Reconstruct cage structure
molecular mass 5.4-9.6 MDa
diameter along its longest diagonal
600 Å
the length of an edge
300 Å
the width of an edge 40 Å
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The asymmetric unit (ASU)
• the smallest unit that can be repeated to generate the full structure
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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 )
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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
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The asymmetric unit (ASU)
We propose that the 24 off-centre, dimeric ASUs comprising the Sec13/31 cage correspond to 24 Sec13/31 heterotetramers .
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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
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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.
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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
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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
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• 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
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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
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An alternative model
• Sec23 is expected to bind near the Sec31-Sec31 dimer interface, whereas Sec24 should bind towards the ends of the ASU
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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
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Contrast of clathrin and sec13/31
Clathrin Sec13/31
intersect to form the vertices
three four
cage formed from
four overlapping clathrin heavy chains
a Sec1 3/31 heterotetramer
diameter 100 A 40 A
interdigitated
Not extensively extensively
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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.
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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.
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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.