Three-dimensional structure of a COPII prebudding complex

The coat protein complex II (COPII) catalyzes transport vesicle formation from the endoplasmic reticulum. Crystallographic analysis of a Sec23/24-Sar1 prebudding complex of COPII now provides a molecular view of this GTPase-directed coat assembly mechanism.     

The structure of the Sec13/31 COPII cage bound to Sec23

Structural studies have revealed some of the organizing principles and mechanisms involved in the assembly of the COPII coat including the location of the Sec23/24 adapter layer. Previous studies, however, were unable to unambiguously determine the positions of Sec23 and Sec24 in the coat. Here, we have determined a cryogenic electron microscopic structure of Sec13/31 together with Sec23. Electron tomography revealed that the binding of Sec23 induces Sec13/31 to form a variety of different geometries including a cuboctahedron, as was previously characterized for Sec13/31 alone. Single-particle reconstruction of the Sec13/31-23 cuboctahedra revealed that the binding of Sec23 induces a conformational change in Sec13/31, resulting in a more extended conformation. Docking Sec23 crystal structures into the electron microscopy map suggested that Sec24 projects its cargo binding surface out into the large open faces of the coat. These results have implications for the mechanisms by which COPII transports large cargos, cargos with large intracellular domains, and for tethering complexes that must project out of the coat in order to interact with their binding partners. Furthermore, Sec23 binds Sec13/31 at two unique sites in the coat, which suggests that each site may have unique roles in the mechanisms of COPII vesiculation.     

The osmotic permeability of a tubule wall

The influence of unstirred layers in osmotic experiments designed to measure the osmotic permeability of a tubule wall is considered. Results are given in the form of a set of graphs whose axes are closely related to observed and known experimental parameters. These enable osmotic permeability values to be obtained which are closer to the true values.     

Making COPII coats

Newly synthesized proteins en route to the Golgi apparatus are exported from the endoplasmic reticulum by COPII coated vesicles. Fath et al. (2007) now reveal the structure of a large portion of the yeast Sec13/31 complex, which comprises the coat framework of COPII-coated vesicles. Their findings suggest a mechanism by which the COPII cage assembles and accommodates cargo of different sizes.     

Self-assembly of minimal COPII cages

The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40–60 nm. Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60–120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these stuctures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.     

Developmental changes in Malpighian tubule cell structure.

Structural changes which occur in the Malpighian tubule yellow region primary cells during larval-pupal-adult development of the skipper butterfly Calpodes ethlius are described. The developmental changes in cell structure are correlated with functional changes in fluid transport (Ryerse, 1978a) in a way which supports osmotic gradient models of fluid secretion. Larval tubules are specialized for fluid secretion with deep basal infolds and elongate mitochondria-containing apical microvilli which provide channels in which osmotic gradients could be set up. The Malpighian tubule cells are extensively remodelled at pupation when fluid transport is switched off, but they persist intact through metamorphosis. At this time, the basement membrane doubles in thickness, the mitochondria are retracted from the microvilli and are isolated for degradation in autophagic vacuoles, and both apical and basal plasma membranes are internalized via coated vesicles for degradation in multivesicular bodies, which results in the shortening of the microville and the disappearance of the basal infolds. Mitochondria are re-inserted into the microvilli, and the basal infolds re-form in pharate adult stage Malpighian tubules when fluid secretion resumes. Adult tubules are similar in general structure to larval tubules and contain mitochondria in the microvilli and basal infolds. However, they differ from larval tubules in that they are capable of very rapid fluid transport, have a reduced tubule diameter and tubule wall thickness, a much thicker basement membrane and peripherally associated tracheoles. Mineral concretions of calcium phosphate accumulate in larval tubules, persist through metamorphosis and decline in number in adults, suggesting they serve some anabolic role.     

The genetic basis of a craniofacial disease provides insight into COPII coat assembly

Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.     

Regulation of a COPII component by cytosolic O-glycosylation during mitosis

Endoplasmic reticulum (ER)-to-Golgi transport is blocked in mammalian cells during mitosis; however, the mechanism underlying this blockade remains unknown. Since COPII proteins are involved in this transport pathway, we investigated at the biochemical level post-translational modifications of COPII components during the course of mitosis that could be linked to inhibition of ER-to-Golgi transport. By comparing biochemical properties of cytosolic COPII components during interphase and mitosis, we found that Sec24p isoforms underwent post-translational modifications resulting in an increase in their apparent molecular weight. No such modification was observed for the other COPII components Sec23p, Sec13p, Sec31p or Sar1p. Analyzing in more details Sec24p isoforms in interphase and mitotic conditions, we found that the interphase form of Sec24p was O-N-acetylglucosamine modified, a feature lost upon entering into mitosis. This mitotic deglycosylation was coupled to Sec24p phosphorylation, a feature likely responsible for the increase in apparent molecular weight of these molecules. These modifications correlated with an alteration in the membrane binding properties of Sec24p. These data suggest that when entering into mitosis, the COPII component Sec24p is simultaneously deglycosylated and phosphorylated, a process which may contribute to the observed mitotic ER-to-Golgi traffic block.     

Molecular mechanisms of COPII vesicle formation

The first step in protein secretion from eukaryotic cells is mediated by COPII vesicles, known for the cytoplasmic coat proteins that are the minimal machinery required to generate these small transport carriers. The five COPII coat components coordinate to create a vesicle by locally generating membrane curvature and populating the incipient bud with the appropriate cargo. This review describes the molecular details of how the COPII coat sculpts vesicles from the endoplasmic reticulum and highlights some unresolved questions regarding the regulation of this process in the complex environment of the eukaryotic cell.     

Lysophospholipids Facilitate COPII Vesicle Formation

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The fine structure of the nephronic tubule of the mudskipper Periophthalmus koelreuteri (Pallas)

Ultrastructural examination of the head kidney of Periophthalmus koelreuteri (Pallas) (Teleostei, Gobiidae) revealed that the nephronic tubule cells are bound by tight junctions and desmosomes with little intercellular space. The first proximal segment (PI) consists of low columnar cells with well developed brush borders, indented nuclei, and numerous apical endocytic vesicles and lysosomes. A second cell type possessing clusters of apical cilia and lacking brush border and lysosomes is occasionally found between PI cells. The second proximal segment (PII) is formed of high columnar cells with brush border, regular spherical nuclei and numerous mitochondria located between well developed infoldings of the basal membrane. Single ciliary structures protrude into the lumen from PI and PII cells. The distal segment is lined by low columnar epithelium with few microvilli, regular spherical nuclei, numerous scattered mitochondria, and microbodies. The collecting tubule cells are cuboidal with few euchromatic nuclei, some mitochondria, and secondary lysosomes.     

Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.     

SNARE selectivity of the COPII coat

The COPII coat buds transport vesicles from the endoplasmic reticulum that incorporate cargo and SNARE molecules. Here, we show that recognition of the ER-Golgi SNAREs Bet1, Sed5, and Sec22 occurs through three binding sites on the Sec23/24 subcomplex of yeast COPII. The A site binds to the YNNSNPF motif of Sed5. The B site binds to Lxx-L/M-E sequences present in both the Bet1 and Sed5 molecules, as well as to the DxE cargo-sorting signal. A third, spatially distinct site binds to Sec22. COPII selects the free v-SNARE form of Bet1 because the LxxLE sequence is sequestered in the four-helix bundle of the v-/t-SNARE complex. COPII favors Sed5 within the Sed5/Bos1/Sec22 t-SNARE complex because t-SNARE assembly removes autoinhibitory contacts to expose the YNNSNPF motif. The COPII coat seems to be a specific conductor of the fusogenic forms of these SNAREs, suggesting how vesicle fusion specificity may be programmed during budding.     

The COPII cage: unifying principles of vesicle coat assembly

Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers - vesicles and tubules - that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.     

A new role for a COPII cargo adaptor in autophagy

ABSTRACT

Endoplasmic reticulum (ER) homeostasis is maintained by the removal of misfolded ER proteins via different quality control pathways. Aggregation-prone proteins, including certain disease-linked proteins, are resistant to conventional ER degradation pathways and require other disposal mechanisms. Reticulophagy is a disposal pathway that uses resident autophagy receptors. How these receptors, which are dispersed throughout the ER network, target a specific ER domain for degradation is unknown. We recently showed in budding yeast, that ER stress upregulates the reticulophagy receptor, triggering its association with the COPII cargo adaptor complex, Sfb3/Lst1-Sec23 (SEC24C-SEC23 in mammals), to discrete sites on the ER. These domains are packaged into phagophores for degradation to prevent the accumulation of protein aggregates in the ER. This unconventional role for Sfb3/Lst1 is conserved in mammals and is independent of its role as a cargo adaptor on the secretory pathway. Our findings may have important therapeutic implications in protein-aggregation linked neurodegenerative disorders.     

p38 MAPK regulates COPII recruitment

Here, we investigate regulation of coat protein complex II (COPII) recruitment onto ER export sites in permeabilized cells. In cytosols from nocodazole treated HeLa cells we find COPII loading is inhibited. The stress kinase p38 MAPK is activated in these cytosols and COPII loading can be rescued by depletion of p38 MAPK α or by the p38 MAPK inhibitor (SB203580) but not by inhibition/depletion of cdc2. These observations indicate regulation of the early secretory pathway by p38 MAPK.     

COPII vesicles get supersized by ubiquitin

Some proteins are too big to fit into conventional COPII-coated vesicles, which raises the question of how large cargo, such as procollagen fibrils, are exported from the endoplasmic reticulum. Jin?et?al. (2012) in Nature now report that the creation of oversized vesicles is facilitated by the ubiquitination of the COPII component Sec31p.     

Poliovirus Infection Transiently Increases COPII Vesicle Budding

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication "organelles" of PV (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.     

Proteomic Profiling of Mammalian COPII and COPI Vesicles

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