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.     

The fine structure of the terminal segment of the bovine seminiferous tubule

Summary The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.Supported by a grant from the Deutsche Forschungsgemeinschaft     

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.     

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.     

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.     

COPII under the microscope

Transport through the secretory pathway begins with COPII regulation of ER export. Driven by the Sar1 GTPase cycle, cytosolic COPII proteins exchange on and off the membrane at specific sites on the ER to regulate cargo exit. Here recent developments in COPII research are discussed, particularly the use of live-cell imaging, which has revealed surprising insights into the coat's role. The seemingly static ER exit sites are in fact highly dynamic, and the ability to visualise trafficking processes in intact living cells has highlighted the adaptable nature of COPII in cargo transport and the emerging roles of auxiliary factors.     

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.     

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.     

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.     

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.     

The lysophospholipid acyltransferase antagonist CI-976 inhibits a late step in COPII vesicle budding

The mechanism of coat protein (COP)II vesicle fission from the endoplasmic reticulum (ER) remains unclear. Lysophospholipid acyltransferases (LPATs) catalyze the conversion of various lysophospholipids to phospholipids, a process that can promote spontaneous changes in membrane curvature. Here, we show that 2,2-methyl- N -(2,4,6,-trimethoxyphenyl)dodecanamide (CI-976), a potent LPAT inhibitor, reversibly inhibited export from the ER in vivo and the formation of COPII vesicles in vitro . Moreover, CI-976 caused the rapid and reversible accumulation of cargo at ER exit sites (ERESs) containing the COPII coat components Sec23/24 and Sec13/31 and a marked enhancement of Sar1p-mediated tubule formation from ERESs, suggesting that CI-976 inhibits the fission of assembled COPII budding elements. These results identify a small molecule inhibitor of a very late step in COPII vesicle formation, consistent with fission inhibition, and demonstrate that this step is likely facilitated by an ER-associated LPAT.     

The unstirred layer during osmotic flow into a tubule

The interaction between osmotic inflow through the wall of a narrow tubule and bulk flow in the tubule is described. Solution are found by a finite difference method, and two approximate analytic solutions are given. Results given here enable more accurate estimates of osmotic permeability to be obtained for the tubule wall. The theory predicts the behaviour of unstirred layers as experimental parameters are varied and enables tubule experiments to be designed so as to reduce unwanted unstirred layer effects.     

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.     

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.     

Fine structure of the transitional zone of the rat seminiferous tubule

An electron microscopic study was made on the structure of the testicular transitional zone (TZ) in the adult rat. The TZ proper consists of modified Sertoli cellss, with only a few spermatogonia and macrophages, surrounding distally a very narrow lumen. The TZ Sertoli cells have nuclei with a somewhat coarser matrix and more peripheral heterochromatin than Sertoli cell nuclei of the nearby seminiferous tubules, and the electron density of the cytoplasm varies from cell to cell. Smooth endoplasmic reticulum is abundant, but usually there are also scattered ribosomal rosettes and an occasional profile of rough endoplasmic reticulum. Microtubules are very numerous in the columnar portion of the cell, and laminar structures seemingly joining the cell surfaces are sometimes seen. Lipid droplets and lysosmal structures are frequent cellular components in proximal TZ Sertoli cells. Empty intracellular vacuoles are abundant, sometimes arranged around areas of smooth endoplasmic reticulum. Occasionally, membrane-limited fine granules and vacuoles are seen within Sertoli cells and also in the TZ lumen, suggesting a possible secretory activity by these cells. The apical processes of the Sertoli cells form large vacuolar structures, and in the basal parts of the epithelium vacuoles with capillary-like appearance are frequently seen. Phagocytosis of germinal cells by the Sertoli cells occurs in the proximal region of the TZ. Round waste bodies in contact with the Sertoli cell apices protruding into the tubulus rectus, are also common. The tunica propria of the TZ is thickened and somewhat wrinkled, and in the proximal region the myoid cell layer loses its continuity and is replaced by fibroblasts. The epithelium of the tubulus rectus adjacent to the TZ consists of several overlapping epithelial cells. The typical junctional complexes between TZ Sertoli cells appear to be impermeable to the lanthanum tracer.