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Randy Schekman - One of the best experts on this subject based on the ideXlab platform.
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small sequence variations between two mammalian paralogs of the small gtpase sar1 underlie functional differences in coat protein complex ii assembly
Journal of Biological Chemistry, 2020Co-Authors: David B Melville, Sean Studer, Randy SchekmanAbstract:Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.
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unique copii component atsar1a atsec23a pair is required for the distinct function of protein er export in arabidopsis thaliana
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Yonglun Zeng, Kin Pan Chung, Ching Man Lai, Sheung Kwan Lam, Xiangfeng Wang, Yong Cui, Caiji Gao, Ming Luo, Kambo Wong, Randy SchekmanAbstract:Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and SEC13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.
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ALG-2 attenuates COPII budding in vitro and stabilizes the Sec23/Sec31A complex.
PloS one, 2013Co-Authors: Jonas M. La Cour, Adam J. Schindler, Martin W. Berchtold, Randy SchekmanAbstract:Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat SEC13/Sec31A. The Ca2+-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca2+ regulation of COPII vesicle budding. Here we show that ALG-2/Ca2+ is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca2+-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and SEC13/31A to artificial liposomes and was capable of mediating binding of SEC13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca2+ in COPII tethering and vesicle budding.
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Erratum: COPII and the regulation of protein sorting in mammals
Nature Cell Biology, 2012Co-Authors: Giulia Zanetti, Kanika Bajaj Pahuja, Sean Studer, Soomin Shim, Randy SchekmanAbstract:Nat. Cell Biol. 14, 20–28 (2012); published online 22 December 2011; corrected after print 6 January 2012 In the version of this review initially published online and in print, the key in figure 2 was incorrect. The grey dots should represent Sec23–24, the blue lines should represent SEC13–31 and the green dots should represent TRAPPI.
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The genetic basis of a craniofacial disease provides insight into COPII coat assembly.
Developmental cell, 2007Co-Authors: J. Christopher Fromme, Randy Schekman, Susan Hamamoto, Mariella Ravazzola, Mohammed Al-balwi, Wafaa Eyaid, Simeon A. Boyadjiev, Pierre Cosson, Lelio OrciAbstract: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.
Chris A Kaiser - One of the best experts on this subject based on the ideXlab platform.
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Isolation of Pichia pastoris genes involved in ER-to-Golgi transport
Yeast (Chichester England), 2000Co-Authors: William E. Payne, Chris A Kaiser, Brooke Bevis, Jon Soderholm, Irina B. Sears, Benjamin S. GlickAbstract:Pichia pastoris has discrete transitional ER sites and coherent Golgi stacks, making this yeast an ideal system for studying the organization of the early secretory pathway. To provide molecular tools for this endeavour, we isolated P. pastoris homologues of the SEC12, SEC13, SEC17, SEC18 and SAR1 genes. The P. pastorisSEC12, SEC13, SEC17 and SEC18 genes were shown to complement the corresponding S. cerevisiae mutants. The SEC17 and SAR1 genes contain introns at the same relative positions in both P. pastoris and S. cerevisiae, whereas the SEC13 gene contains an intron in P. pastoris but not in S. cerevisiae. Intron structure is similar in the two yeasts, although the favoured 5′ splice sequence appears to be GTAAGT in P. pastoris vs. GTATGT in S. cerevisiae. The predicted amino acid sequences of SEC13p, Sec17p, Sec18p and Sar1p show strong conservation in the two yeasts. By contrast, the predicted lumenal domain of Sec12p is much larger in P. pastoris, suggesting that this domain may help localize Sec12p to transitional ER sites. A comparison of the SEC12 loci in various budding yeasts indicates that the SEC12-related gene SED4 is probably unique to the Saccharomyces lineage. GenBank Accession Nos are: SEC12, AF216960; SEC13, AF242186; SEC17, AF216957; SEC18, AF216958; SAR1, AF216959; ACT1, AF216956. Copyright © 2000 John Wiley & Sons, Ltd.
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physiological regulation of membrane protein sorting late in the secretory pathway of saccharomyces cerevisiae
Journal of Cell Biology, 1997Co-Authors: Kevin J Roberg, Neil Rowley, Chris A KaiserAbstract:In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In SEC13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the SEC13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and SEC13p control Gap1p transport from the Golgi to the plasma membrane.
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Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum.
The Journal of cell biology, 1995Co-Authors: David A. Shaywitz, Mariella Ravazzola, Lelio Orci, Anand Swaroop, Chris A KaiserAbstract:In the yeast Saccharomyces cerevisiae, SEC13p is required for intracellular protein transport from the ER to the Golgi apparatus, and has also been identified as a component of the COPII vesicle coat structure. Recently, a human cDNA encoding a protein 53% identical to yeast SEC13p has been isolated. In this report, we apply the genetic assays of complementation and synthetic lethality to demonstrate the conservation of function between this human protein, designated SEC13Rp, and yeast SEC13p. We show that two reciprocal human/yeast fusion constructs, encoding the NH2-terminal half of one protein and the COOH-terminal half of the other, can each complement the secretion defect of a SEC13-1 mutant at 36 degrees C. The chimera encoding the NH2-terminal half of the yeast protein and the COOH-terminal half of the human protein is also able to complement a SEC13 deletion. Overexpression of either the entire human SEC13Rp protein or the chimera encoding the NH2-terminal half of the human protein and the COOH-terminal half of the yeast protein inhibits the growth of a SEC13-1 mutant at 24 degrees C; this growth inhibition is not seen in a wild-type strain nor in other sec mutants, suggesting that the NH2-terminal half of SEC13Rp may compete with SEC13-1p for a common target. We show by immunoelectronmicroscopy of mammalian cells that SEC13Rp (like the putative mammalian homologues of the COPII subunits Sar1p and Sec23p) resides in the region of the transitional ER. We also show that the distribution of SEC13Rp is not affected by brefeldin A treatment. This report presents the first demonstration of a putative mammalian COPII component functioning in yeast, and highlights a potentially useful approach for the study of conserved mammalian proteins in a genetically tractable system.
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Cytosolic SEC13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.
The Journal of cell biology, 1993Co-Authors: Nancy K. Pryer, Randy Schekman, Nina R Salama, Chris A KaiserAbstract:The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, SEC13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free SEC13p-dependent vesicle formation reaction. SEC13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a SEC13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing SEC13p was added. Cytosolic SEC13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the SEC13p complex restored activity to the SEC13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of SEC13p, suggesting that another member of the complex becomes limiting when SEC13p is overproduced. Overproduced, monomeric SEC13p was inactive in the SEC13p-dependent vesicle formation assay.
Scott M. Stagg - One of the best experts on this subject based on the ideXlab platform.
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flexibility of the SEC13 31 cage is influenced by the sec31 c terminal disordered domain
Journal of Structural Biology, 2018Co-Authors: Mohammadreza Paraan, Nilakshee Bhattacharya, Vladimir N. Uversky, Scott M. StaggAbstract: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.
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Flexibility of the SEC13/31 cage is influenced by the Sec31 C-terminal disordered domain.
Journal of structural biology, 2018Co-Authors: Mohammadreza Paraan, Nilakshee Bhattacharya, Vladimir N. Uversky, Scott M. StaggAbstract: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.
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A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry
Nature structural & molecular biology, 2012Co-Authors: Alex J. Noble, Nilakshee Bhattacharya, Jason O’donnell, Qian Zhang, Hanaa Hariri, Alan G. Marshall, Scott M. StaggAbstract:COPII vesicles transport proteins from the ER to the Golgi apparatus. Previous cryoEM structures of the COPII cage lacked the resolution necessary to determine the residues of SEC13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12A-resolution structure of the COPII cage, where the tertiary structure of SEC13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the SEC13-Sec31 complex to create a reliable pseudo-atomic model of the COPII cage. We combined this model with hydrogen/deuterium exchange mass spectrometry analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the 2-fold symmetry of the Sec31 dimeric region of SEC13-31 is broken on cage formation, and that the resulting hinge is essential to form the proper edge geometry in COPII cages.
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the structure of the SEC13 31 copii cage bound to sec23
Journal of Molecular Biology, 2012Co-Authors: Nilakshee Bhattacharya, Jason O Donnell, Scott M. StaggAbstract: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.
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The structure of the SEC13/31 COPII cage bound to Sec23.
Journal of molecular biology, 2012Co-Authors: Nilakshee Bhattacharya, Jason O′donnell, Scott M. StaggAbstract: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.
Lelio Orci - One of the best experts on this subject based on the ideXlab platform.
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The genetic basis of a craniofacial disease provides insight into COPII coat assembly.
Developmental cell, 2007Co-Authors: J. Christopher Fromme, Randy Schekman, Susan Hamamoto, Mariella Ravazzola, Mohammed Al-balwi, Wafaa Eyaid, Simeon A. Boyadjiev, Pierre Cosson, Lelio OrciAbstract: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.
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sar1p n terminal helix initiates membrane curvature and completes the fission of a copii vesicle
Cell, 2005Co-Authors: Marcus C S Lee, Lelio Orci, Susan Hamamoto, Mariella Ravazzola, Eugene Futai, Randy SchekmanAbstract:Secretory proteins traffic from the ER to the Golgi via COPII-coated transport vesicles. The five core COPII proteins (Sar1p, Sec23/24p, and SEC13/31p) act in concert to capture cargo proteins and sculpt the ER membrane into vesicles of defined geometry. The molecular details of how the coat proteins deform the lipid bilayer into vesicles are not known. Here we show that the small GTPase Sar1p directly initiates membrane curvature during vesicle biogenesis. Upon GTP binding by Sar1p, membrane insertion of the N-terminal amphipathic alpha helix deforms synthetic liposomes into narrow tubules. Replacement of bulky hydrophobic residues in the alpha helix with alanine yields Sar1p mutants that are unable to generate highly curved membranes and are defective in vesicle formation from native ER membranes despite normal recruitment of coat and cargo proteins. Thus, the initiation of vesicle budding by Sar1p couples the generation of membrane curvature with coat-protein assembly and cargo capture.
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Self‐assembly of minimal COPII cages
EMBO reports, 2003Co-Authors: Bruno Antonny, Randy Schekman, Pierre Gounon, Lelio OrciAbstract: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.
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surface structure of the copii coated vesicle
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: K Matsuoka, Randy Schekman, Lelio Orci, John E. HeuserAbstract:The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p ≫ SEC13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles. Sec23/24 resembles a bow tie, and SEC13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of SEC13/31. Partially uncoated profiles revealed strands of SEC13/31p stripped from the vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.
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Dynamics of the COPII coat with GTP and stable analogues
Nature cell biology, 2001Co-Authors: Bruno Antonny, Susan Hamamoto, Lelio Orci, David T. Madden, Randy SchekmanAbstract:We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time. We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds. The two large COPII complexes Sec23/24p and SEC13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the SEC13/31p complex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.
William E. Balch - One of the best experts on this subject based on the ideXlab platform.
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structure of the SEC13 31 sec23 copii coat cage
Biophysical Journal, 2011Co-Authors: Nilakshee Bhattacharya, Abbas Razvi, William E. Balch, Jason Odonnell, Scott M. StaggAbstract:COPII coated vesicles are responsible for packaging and transporting newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII coat consists of SEC13/31, Sec23/24, and Sar1. Mutation in these coat protein cause medical conditions like Anderson disease, chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which highlights the biological relevance of the coat proteins. Previously we solved two different COPII structures (Stagg et. Al., Nature 2006 and Stagg et. Al., Cell 2008) that suggest that the hinge region formed by the four heterotetramer can direct cage expansion to accommodate cargo of various sizes. Recently a tubular structure of Sec 13/31 solved where the tubules were formed by the concatenation of individual SEC13/31 cage (O’Donell et. Al, J. STruc. Biol.). Earlier, we hypothesized that the distribution of Sec23/24 dictates the geometry of the COPII coat. We now show that Sec23 by itself influences the outer geometry of the cage. We have reconstructed a structure of a COPII coat cage assembled from SEC13/31 and Sec23. The assemblies form at least two geometries, and the most common size is 600 A, similar to what has been observed for SEC13/31. We will discuss how the orientation of Sec23 may dictate cage geometry and orient Sar1 to participate in the fission of COPII coated vesicles in the cell.
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Structure of the SEC13/31 & Sec23 COPII coat cage
Biophysical Journal, 2011Co-Authors: Nilakshee Bhattacharya, Jason O’donnell, Abbas Razvi, William E. Balch, Scott M. StaggAbstract:COPII coated vesicles are responsible for packaging and transporting newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII coat consists of SEC13/31, Sec23/24, and Sar1. Mutation in these coat protein cause medical conditions like Anderson disease, chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which highlights the biological relevance of the coat proteins. Previously we solved two different COPII structures (Stagg et. Al., Nature 2006 and Stagg et. Al., Cell 2008) that suggest that the hinge region formed by the four heterotetramer can direct cage expansion to accommodate cargo of various sizes. Recently a tubular structure of Sec 13/31 solved where the tubules were formed by the concatenation of individual SEC13/31 cage (O’Donell et. Al, J. STruc. Biol.). Earlier, we hypothesized that the distribution of Sec23/24 dictates the geometry of the COPII coat. We now show that Sec23 by itself influences the outer geometry of the cage. We have reconstructed a structure of a COPII coat cage assembled from SEC13/31 and Sec23. The assemblies form at least two geometries, and the most common size is 600 A, similar to what has been observed for SEC13/31. We will discuss how the orientation of Sec23 may dictate cage geometry and orient Sar1 to participate in the fission of COPII coated vesicles in the cell.
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Structural Basis for Cargo Regulation of COPII Coat Assembly
Cell, 2008Co-Authors: Scott M. Stagg, Abbas Razvi, Cemal Gurkan, Paul Lapointe, Clinton S. Potter, Bridget Carragher, William E. BalchAbstract:Summary Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and SEC13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the SEC13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 β-propeller domains present in SEC13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.
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the lysophospholipid acyltransferase antagonist ci 976 inhibits a late step in copii vesicle budding
Traffic, 2008Co-Authors: William J Brown, Dan Drecktrah, Helen Plutner, Bret L Judson, William E. BalchAbstract: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.
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Structure of the SEC13/31 COPII coat cage
Nature, 2006Co-Authors: Scott M. Stagg, Cemal Gurkan, Douglas M. Fowler, Paul Lapointe, Ted R. Foss, Clinton S. Potter, Bridget Carragher, William E. BalchAbstract:Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and SEC13 and Sec31 (SEC13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and SEC13/31 can self-assemble to form COPII cage-like particles, we now show that SEC13/31 can self-assemble to form minimal cages in the absence of Sec23/24. 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. 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.