DOCSLIB.ORG

Multiple and Stepwise Interactions Between Coatomer and ADP-Ribosylation Factor-1 (Arf1)-GTP

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Multiple and Stepwise Interactions Between Coatomer and ADP-Ribosylation Factor-1 (Arf1)-GTP # 2007 The Authors Journal compilation # 2007 Blackwell Publishing Ltd Traffic 2007; 8: 582–593 Blackwell Munksgaard doi: 10.1111/j.1600-0854.2007.00554.x Multiple and Stepwise Interactions Between Coatomer and ADP-Ribosylation Factor-1 (Arf1)-GTP Zhe Sun1,†, Frank Anderl1,†, Kathrin Fro¨ hlich1, suggesting that these complexes are comprised of Liyun Zhao1, Stefan Hanke2, Britta Bru¨ gger1, conserved structural elements (13). The structure of the Felix Wieland1,*, Julien Be´ thune1,* core complexes of adaptor protein (AP) complex 1 and 2 (AP-1 and AP-2) were solved by X-ray crystallography 1Biochemie-Zentrum der Universita¨t Heidelberg (BZH), (14,15). Two 100-kDa subunits are built of a trunk domain Im Neuenheimer Feld 328, D-69120 Heidelberg, connected via a flexible linker to a smaller appendage Germany domain. The trunk domains together with the smaller sub- 2 Max-Planck-Institute for Biochemistry, Am Klopferspitz units m and s form a densely packed core that contains the 18, D-82152 Martinsried, Germany sites for specific interactions with a donor membrane. The †These authors contributed equally to this work coatomer subunits b and g together with d and z are likely to *Corresponding author: Julien Be´thune, [email protected] or Felix Wieland, represent a similar core of coatomer. Structural homology [email protected] was corroborated for the appendage domains of a2and b-adaptin and g-COP by X-ray crystallography (16,17). The small GTPase ADP-ribosylation factor-1 (Arf1) plays Formation of a vesicle includes binding to the donor mem- a key role in the formation of coat protein I (COP I)-coated brane of coat proteins and subsequent membrane deforma- vesicles. Upon recruitment to the donor Golgi membrane tion to sculpt out a bud. In the clathrin system, this is by interaction with dimeric p24 proteins, Arf1’s GDP is achieved by an Arf-GTP-dependent two-step process: bind- exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it ing of an adaptor complex to the GTPase followed by recruits coatomer, the heptameric coat protein complex recruitment of clathrin heavy and light chains. In contrast, of COP I vesicles, from the cytosol. In this process, Arf1 COP I vesicles are formed by an Arf-GTP-dependent one- was shown to specifically interact with the coatomer b step recruitment of coatomer, implying that both adaptor and and g-COP subunits through its switch I region, and with clathrin functions are unified in a single coatomer complex. e-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of b and g-COP. Site- We previously used an in vitro translation technique to directed photolabeling at position 167 in the C-terminal introduce photolabile amino acids at defined positions in helix of Arf1 revealed a novel interaction with coatomer Arf1. Through the use of these photolabile Arf1 derivatives, via a putative longin domain of d-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with we identified the coatomer subunits b-COP and g-COP as membrane-bound Arf1-GTP. These interactions are binding partners for the switch I region of Arf1-GTP located within the core, adaptor-like domain of coatomer, (18,19). In these functional binding assays with myristoy- indicating an organizational similarity between the COP I lated full-length Arf1 derivatives, Golgi enriched mem- coat and clathrin adaptor complexes. branes and coatomer, these GTP-dependent interactions were found after recruitment of coatomer to the mem- Key words: coat protein I, Golgi, membrane traffic, small branes, as well as in isolated COP I vesicles, indicating GTPase, vesicular transport their functional relevance. Another study described binding Received 19 September 2006, revised and accepted for of Arf1-GTP to b-COP and e-COP in a two-hybrid assay publication 8 February 2007, published online 26 March (20). In the clathrin system, GTP-dependent interaction of 2007 Arf1 through its switch I region with the adaptor com- plexes AP-1, 3 and 4, was attributed to the trunk domains of the larger adaptins. Coat protein I (COP I) vesicles mediate lipid and protein transport in the early secretory pathway of eukaryotic cells. In this study, we have extended our Arf1 photo-cross- Coatomer (1–3), the protein coat of these vesicles, is linking studies to map its interactions with coatomer on the recruited to Golgi membranes by the small, 20-kDa, Ras- Golgi membrane. We report that activated Arf1 binds the like GTPase Arf1 in its GTP-bound form (4,5). Inactivation trunk domains of b-COP and g-COP. In addition, we of Arf1 through GTP hydrolysis is sufficient to release describe two novel interactions between Arf1 and coat- coatomer from membranes and thus promotes vesicle omer: one involves a putative longin domain of d-COP and uncoating (6–8). Similarly, Arf1 plays a critical role in the the other the b0-COP subunit. These results underline recruitment of the clathrin adaptor complexes AP-1 (9), AP- structural and functional similarities between clathrin 3 (10) and AP-4 (11). Subunits of coatomer and clathrin adaptor complexes and coatomer and predict a potential adaptor complexes share weak sequence homology (12), interaction of adaptor m-chains with Arf1-GTP. In addition, 582 www.traffic.dk Arf1 and Coatomer Interactions they show differences that might relate to the mechanistic differences in vesicle formation that exist between the clathrin and the COP I systems. Results Expression of Arf1 proteins with site-directed photolabile amino acids Photolabile amino acid derivatives within polypeptides facilitate the identification of specific protein–protein inter- actions. Upon UV irradiation, highly reactive species are generated that covalently cross-link to binding partners within 3-A˚ distance (21). Until recently, generation of site- directed photolabile proteins was dependent on chemical synthesis or in vitro translation systems. Hence, photo- labile proteins were available in very limited quantities, precluding proteomic analysis and restricting identification of cross-link partners to Western blotting for known candidates. Novel binding partners could not be identified using these methods. Recently, an in vivo expression system was described that allows the production of large amounts of photolabile proteins in Escherichia coli using Figure 1: Expression of site-directed photolabile human Arf1 a benzophenone (Bp) amino acid derivative (22). We have proteins in E. coli. A) Sites within Arf1 where photolabile amino adapted this system to produce 16 site-directed photo- acid derivatives were introduced. SW1: switch I region, ISW: labile derivatives of full-length, myristoylated Arf1. Three of interswitch region, SW2: switch II region. The three labeled them could be cross-linked to putative binding partners and positions indicate the derivatives that yielded cross-linked inter- were characterized. In these derivatives, Bp residues action partners and are characterized in the present study. B) replaced amino acid positions I46 and I49 in the switch I Expression of wild-type Arf1 (WT) and photolabile derivatives region, and Y167 in the C-terminus (Figure 1A); each was (lane 2, 3) or was not (lane 1) induced at 278C by arabinose (Ara.) in the presence (lane 3) or absence (lanes 1 and 2) of derivative was obtained in up to microgram quantities p-benzoyl-L-phenylalanine (Bp). Expression products were ana- (Figure 1B). lyzed by Western blotting with an anti-Arf1 (C-terminus) antibody. Note that when produced in E. coli, Arf1 has a mixture of The photoactivable derivatives behave like myristoylated (myr.) and non-myristoylated (nonmyr.) forms (47). wild-type Arf1 For COP I-vesicles formation, Arf1 in its GDP bound form is transiently recruited by dimeric p23 protein to the Golgi Golgi membranes, photolabile Arf1-I46Bp, I49Bp and membrane (23–25). Thereafter, exchange of GDP for GTP Y167Bp were incubated with Golgi membranes and is catalyzed by a guanine nucleotide exchange factor coatomer in the presence of GDPbS or GTPgS, a non- (26,27), and the activated GTP-bound Arf1 is stably asso- hydrolyzable GTP analog. Membranes were then separated ciated with the membrane. As a result, about threefold from unbound material by centrifugation and irradiated at more Arf1-GTP is bound to Golgi membranes than Arf1- 366 nm to drive photo-cross-linking of Bp residues. Analysis GDP (28). by SDS–PAGE and Western blotting with antibodies against Arf1 and coatomer subunits revealed the known interac- To test their functionality, we assessed nucleotide- tions of Arf1 with b-COP and g-COP (Figure 3A and B, lane dependent binding of the Arf1 photolabile proteins to Golgi 4, as indicated). The lower band (Figure 3B, lane 4) repre- membranes. For all Arf1 species, replacement of GDP with sents Arf1 covalently linked to g2-COP, an isotype of g-COP GTP caused a threefold enhancement of Golgi membrane (29,30). Three additional Arf1 complexes were observed binding (Figure 2). This ratio is in very good agreement (Figure 3A and C, indicated as Arf1-I46 þ x, Arf1-I46 þ y, with the previous quantification of nucleotide-dependent Arf1-Y167 þ z) with apparent molecular masses of about binding of Arf1 to Golgi membranes using radiolabeled 150 kDa (Arf1 þ partner y), 130 kDa (Arf1 þ partner x) and Arf1 (28). Thus these photolabile Arf1 species were then 80 kDa (Arf1 þ partner z). used to map the binding sites on b-COP and g-COP and to search for additional binding partners. Arf1-GTP interacts with the trunk domains of b-COP and g-COP GTP-dependent interactions of Arf1 with coatomer Cross-linking and two-hybrid experiments have revealed To probe the interfaces of Arf1 with coatomer under that Arf1-GTP binds clathrin adaptor complexes through functional conditions, i.e.
Load more

Recommended publications
  • Coatomer-Rich Endoplasmic Reticulum LELIO ORCI*, ALAIN PERRELET*, MARIELLA RAVAZZOLA*, Myltne AMHERDT*, JAMES E
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11924-11928, December 1994 Cell Biology Coatomer-rich endoplasmic reticulum LELIO ORCI*, ALAIN PERRELET*, MARIELLA RAVAZZOLA*, MYLtNE AMHERDT*, JAMES E. ROTHMANt, AND RANDY SCHEKMAN* *Department of Morphology, University of Geneva Medical School, 1211 Geneva 4, Switzerland; tDivision of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and tCellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 Contributed by Randy Schekman, August 30, 1994 ABSTRACT We identify in normal cells the existence of digestion and incubated at normal (370C) or low (220C, 15'C, two distinct sites of the transitional endoplasmic reticulum 40C) temperatures under continuous shaking and gassing with (ER), one housing the Sec23p protein complex (the classical 95% 02/5% CO2. Monolayer cultures ofpancreatic endocrine transitional element), the other the coatomer protein complex cells (24) were exposed to low temperatures as for isolated (the coatomer-rich ER). Experimental conditions that reduce islets. ATP depletion in both preparations was induced at transport from the ER to the Golgi complex lead to the 370C with 10 ImM antimycin or 1 mM dinitrophenol or by overexpression of this newly defined coatomer-rich ER. gassing the cells with N2 instead of the 02/CO2 mixture. At the end of the incubations, the samples were fixed with Progress in the identification and characterization of the 1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) carriers involved in membrane traffic from the endoplasmic for 1 hr at the final temperature of the incubation protocol, reticulum (ER) to and through the Golgi complex was pos- washed with buffer, and processed for Epon embedding sible by combining morphological and biochemical methods, (conventional thin sections) or for cryoultramicrotomy ac- cell-free assay systems, and yeast genetics (for review see refs.
    [Show full text]
  • Formation of COPI-Coated Vesicles at a Glance Eric C
    © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs209890. doi:10.1242/jcs.209890 CELL SCIENCE AT A GLANCE Formation of COPI-coated vesicles at a glance Eric C. Arakel1 and Blanche Schwappach1,2,* ABSTRACT unresolved, this review attempts to refocus the perspectives of The coat protein complex I (COPI) allows the precise sorting of lipids the field. and proteins between Golgi cisternae and retrieval from the Golgi KEY WORDS: Arf1, ArfGAP, COPI, Coatomer, Golgi, Endoplasmic to the ER. This essential role maintains the identity of the early reticulum, Vesicle coat secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI- Introduction coated vesicle formation and revisit decades of research in the Vesicle coat proteins, such as the archetypal clathrin and the coat context of recent advances in the elucidation of COPI coat structure. protein complexes II and I (COPII and COPI, respectively) are By calling attention to an array of questions that have remained molecular machines with two central roles: enabling vesicle formation, and selecting protein and lipid cargo to be packaged within them. Thus, coat proteins fulfil a central role in the 1Department of Molecular Biology, Universitätsmedizin Göttingen, Humboldtallee homeostasis of the cell’s endomembrane system and are the basis 23, 37073 Göttingen, Germany. 2Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. of functionally segregated compartments. COPI operates in retrieval from the Golgi to the endoplasmic reticulum (ER) and in intra-Golgi *Author for correspondence ([email protected]) transport (Beck et al., 2009; Duden, 2003; Lee et al., 2004a; Spang, E.C.A., 0000-0001-7716-7149; B.S., 0000-0003-0225-6432 2009), and maintains ER- and Golgi-resident chaperones and enzymes where they belong.
    [Show full text]
  • ADP-Ribosylation Factor, a Small GTP-Binding Protein, Is Required for Binding of the Coatomer Protein Fl-COP to Golgi Membranes JULIE G
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6408-6412, July 1992 Biochemistry ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein fl-COP to Golgi membranes JULIE G. DONALDSON*, DAN CASSEL*t, RICHARD A. KAHN*, AND RICHARD D. KLAUSNER* *Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, and tLaboratory of Biological Chemistry, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Marc Kirschner, April 20, 1992 (receivedfor review February 11, 1992) ABSTRACT The coatomer is a cytosolic protein complex localized to the Golgi complex, although their functions have that reversibly associates with Golgi membranes and is Impli- not been defined. Distinct among these proteins is the ADP- cated in modulating Golgi membrane transport. The associa- ribosylation factor (ARF), originally identified as a cofactor tion of 13-COP, a component of coatomer, with Golgi mem- required for in vitro cholera toxin-catalyzed ADP- branes is enhanced by guanosine 5'-[v-thioltriphosphate ribosylation of the a subunit of the trimeric GTP-binding (GTP[yS]), a nonhydrolyzable analogue of GTP, and by a protein G, (G,.) (19). ARF is an abundant cytosolic protein mixture of aluminum and fluoride ions (Al/F). Here we show that reversibly associates with Golgi membranes (20, 21). that the ADP-ribosylation factor (ARF) is required for the ARF has been shown to be present on Golgi coated vesicles binding of (-COP. Thus, 13-COP contained in a coatomer generated in the presence of GTP[yS], but it is not a com- fraction that has been resolved from ARF does not bind to Golgi ponent of the cytosolic coatomer (22).
    [Show full text]
  • ADP-Ribosylation Factor and Coatomer Couple Fusion to Vesicle Budding Zvulun Elazar,* Lelio Orci,T Joachim Ostermann,* Myl~Nc Amherdt,* Gary Tanigawa,* and James E
    ADP-Ribosylation Factor and Coatomer Couple Fusion to Vesicle Budding Zvulun Elazar,* Lelio Orci,t Joachim Ostermann,* Myl~nc Amherdt,* Gary Tanigawa,* and James E. Rothman* * Program in Cellular Biochemistry and Biophysics, Memorial Sloan Kettering Cancer Center, New York 10021; and ¢Institute of Histology and Embryology, University of Geneva Medical School, 1211 Geneva 4, Switzerland Abstract. The coat proteins required for budding pair directly without an intervening vesicle. Coupling COP-coated vesicles from Golgi membranes, coatomer may therefore result from the sequestration of fuso- and ADP-ribosylation factor (ARF) protein, are shown genic membrane proteins into assembling coated vesi- to be required to reconstitute the orderly process of cles that are only exposed when the coat is removed transport between Golgi cisternae in which fusion of after budding is complete. This mechanism of cou- transport vesicles begins only after budding ends. pling explains the phenomenon of "retrograde transport" When either coat protein is omitted, fusion is uncou- triggered by uncouplers such as the drug brefeldin A. pled from budding-donor and acceptor compartments ow is membrane fusion coupled to vesicle budding? Orci et al., 1989). Purification of COP-coated vesicles (Mal- A transport vesicle must fuse with its target only hotra et al., 1989; Serafini et al., 1991a,b) revealed that their H after its budding from the parental membrane is coats consist of a small GTP-binding protein (ADP-ribosyla- completed. Otherwise, the various membrane-bound com- tion factor, ARF) ~ and a complex of seven distinct proteins partments connected by vesicle shuttles would fuse and the termed coatomer (Waters et al., 1992a; Stenbeck et al., topological organization of the endomembrane system in 1993), whose subunits are or, B, B', 3', ~, e, and ~'-COPs.
    [Show full text]
  • The Structure of the COPI Coat Determined Within the Cell
    RESEARCH ADVANCE The structure of the COPI coat determined within the cell Yury S Bykov1,2, Miroslava Schaffer3, Svetlana O Dodonova1†, Sahradha Albert3, Ju¨ rgen M Plitzko3, Wolfgang Baumeister3*, Benjamin D Engel3*, John AG Briggs1,2* 1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany; 2Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom; 3Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Abstract COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath b’–COP. We find that all coat components *For correspondence: disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, [email protected] (WB); maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo [email protected] (BDE); content as they progress from cis to trans, but the structure of the coat machinery remains [email protected] constant. (JAGB) DOI: https://doi.org/10.7554/eLife.32493.001 Present address: †Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Go¨ ttingen, Germany Introduction Vesicular transport between cellular compartments is a fundamental mechanism of eukaryotic cells.
    [Show full text]
  • Cryo-Tomography of Coat Complexes ISSN 2059-7983
    research papers Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes ISSN 2059-7983 Evgenia A. Markova and Giulia Zanetti* Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, England. Received 8 March 2019 *Correspondence e-mail: [email protected] Accepted 12 April 2019 Coat proteins mediate vesicular transport between intracellular compartments, Keywords: cryo-electron tomography; which is essential for the distribution of molecules within the eukaryotic cell. three-dimensional reconstruction; coat proteins; The global arrangement of coat proteins on the membrane is key to their vesicular transport; COPII; subtomogram function, and cryo-electron tomography and subtomogram averaging have been averaging. used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer performed on in vitro reconstitutions or in situ is summarized. These studies have answered long-standing questions regarding the mechanisms of membrane binding, polymerization and assembly regulation of coat proteins. 1. Introduction Recent advances in cryo-electron microscopy (cryo-EM) have enabled numerous high-resolution studies which have addressed long-standing structural biology questions. The large body of cryo-EM work carried out for protein structure characterization has utilized single-particle electron micro- scopy (Cheng, 2018). Recently, developments in hardware, data collection and data processing have placed cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) at the forefront of structural studies of repetitive assemblies, reaching resolutions comparable to those of single-particle EM (Schur et al., 2016; Wan et al., 2017; Hutchings et al., 2018; Dodonova et al., 2017; Himes & Zhang, 2018).
    [Show full text]
  • Mechanisms of Membrane Fusion: Disparate Players and Common Principles
    REVIEWS Mechanisms of membrane fusion: disparate players and common principles Sascha Martens and Harvey T. McMahon Abstract | Membrane fusion can occur between cells, between different intracellular compartments, between intracellular compartments and the plasma membrane and between lipid-bound structures such as viral particles and cellular membranes. In order for membranes to fuse they must first be brought together. The more highly curved a membrane is, the more fusogenic it becomes. We discuss how proteins, including SNAREs, synaptotagmins and viral fusion proteins, might mediate close membrane apposition and induction of membrane curvature to drive diverse fusion processes. We also highlight common principles that can be derived from the analysis of the role of these proteins. Syncytium Membrane fusion is the process whereby two separate membrane–membrane proximity, by bending and A cell that contains multiple lipid bilayers merge to become one. It is essential for remodelling membranes, or by acting upstream to regu- nuclei and that is formed either communication between membrane-delineated comp- late the lipid or protein composition of the respective by cell–cell fusion or by artments in all eukaryotic cells (FIG. 1). The best-studied lipid bilayers. In the hemifusion model the fusion pore is incomplete cell division. process involving membrane fusion is exocytosis, lipidic, but according to an alternative hypothesis — the Hemifusion whereby vesicles fuse with the limiting membrane of protein-pore model — the initial fusion pore is generated An intermediate stage during a cell in order to release their contents (for example, and lined by transmembrane proteins rather than lipids2. membrane fusion that is hormones or neurotransmitters) into the extracellular There is good evidence that transmembrane domains characterized by the merger of SNARE only the contacting monolayers milieu, or to deposit receptors, transporters, channels of proteins are essential for efficient -dependent and not the two distal or adhesion molecules into the limiting membrane.
    [Show full text]
  • Differential Localization of Coatomer Complex Isoforms Within the Golgi Apparatus
    Differential localization of coatomer complex isoforms within the Golgi apparatus Jo¨ rg Moelleken*, Jo¨ rg Malsam*, Matthew J. Betts†, Ali Movafeghi‡, Ingeborg Reckmann*, Ingrid Meissner*, Andrea Hellwig§, Robert B. Russell†, Thomas So¨ llner*, Britta Bru¨ gger*, and Felix T. Wieland*¶ *Biochemistry Center and §Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany; †Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; and ‡Department of Plant Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran Communicated by James E. Rothman, Columbia University Medical Center, New York, NY, December 20, 2006 (received for review December 8, 2006) Coatomer, the coat protein of coat protein complex (COP)I-vesicles, Genome analyses identified a second isoform for each of the is a soluble protein complex made up of seven subunits, ␣-, ␤-, ␤؅-, two coatomer subunits, ␥- and ␨-COP (14), the paralogs ␥2- and ␥-, ␦-, ␧-, and ␨-COP. Higher eukaryotes have two paralogous ␨2-COP. Previously, the corresponding cDNAs were expressed versions of the ␥- and ␨- subunits, termed ␥1- and ␥2-COP and ␨1- as tagged constructs (15), and the endogenous gene products and ␨2-COP. Different combinations of these subunits are known to were shown to be incorporated into coatomer complexes in a exist within coatomer complexes, and ␥1/␨1-, ␥1/␨2-, and ␥2/␨1- stoichiometric manner, which resulted in three major isoforms of COP represent the major coatomer populations in mammals. The the complex with the compositions ␥1␨1-, ␥1␨2-, and ␥2␨1-COP role of COPI vesicles in the early secretory pathway is the subject (16). To extend this work, we have prepared polyclonal antibod- ␥ ␥ ␨ of considerable debate.
    [Show full text]
  • University of London Thesis
    REFERENCE ONLY UNIVERSITY OF LONDON THESIS Degree fVvO Year""^© *" Name of Author iA vAa ^ V COPYRIGHT This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting the thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the Senate House Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The Senate House Library will provide addresses where possible). B. 1962- 1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975 - 1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied. This thesis comes within category D.
    [Show full text]
  • Coatomer, Arf1p, and Nucleotide Are Required to Bud Coat Protein Complex I-Coated Vesicles from Large Synthetic Liposomes
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11199–11204, September 1998 Cell Biology This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 28, 1998. Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes ANNE SPANG*, KEN MATSUOKA*, SUSAN HAMAMOTO*, RANDY SCHEKMAN*, AND LELIO ORCI† *Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and †Department of Morphology, University of Geneva, CH-1211 Geneva, Switzerland Contributed by Lelio Orci, July 30, 1998 ABSTRACT Synthetic coat protein complex I (COPI)- favorable binding environment for coatomer (14). According coated vesicles form spontaneously from large ('300 nm in to this view, Arf1p-GTP may act indirectly to promote COPI- diameter), chemically defined liposomes incubated with coated vesicle formation. coatomer, Arf1p, and guanosine 5*-[g-thio]triphosphate. Coatomer recruitment to membranes also may be driven by Coated vesicles are 40–70 nm in diameter, approximately the the recognition of protein cargo molecules. The C-terminal size of COPI vesicles formed from native membranes. The KKXX-motif of membrane proteins is known to interact with formation of COPI-coated buds and vesicles and the binding coatomer and to engage COPI vesicles in retrograde transport of Arf1p to donor liposomes depends on guanosine 5*-[g- from the Golgi to the ER. However, although subunits of the thio]triphosphate. In contrast to the behavior of the COPII anterograde carrier, COPII, also interact directly with protein coat, coatomer binds to liposomes containing a variety of cargo leaving the ER membrane (15), the COPII coat is charged or neutral phospholipids.
    [Show full text]
  • June N&V Final
    NEWS AND VIEWS studies is not fully functional, although this is disturbed, whereas binding of the calcium- our knowledge of the various steps in the not proven. An alternative explanation is that activated sytI domain would be sufficient to fusion process and their regulatory mecha- at synapses and in cells, full-length sytI (an promote SNARE zippering and fusion (Fig. 2). nisms. This might ultimately result in the integral membrane protein) is needed not only In conclusion, the results from Tucker et identification of other factors that regulate the for exocytic fusion, but also to initiate vesicle al.1 are an important milestone in the recon- system, in particular those allowing mem- docking to the plasma membrane through stitution of presynaptic events that result in brane fusion to operate in the sub-millisecond binding to SNAP25. In this case, inhibition by neurotransmitter release. On the basis of these timescale as it does in synaptic physiology. the cytosolic domain could be explained by an results, the need for proteins and lipids other 1. Tucker, W. C., Weber, T. & Chapman, E. R. Science unproductive interaction with the t-SNARE than sytI in the calcium regulation of synaptic 304, 435–438 (2004). 5,6 that prevents binding of full-length sytI (refs 9, vesicle fusion can finally be excluded. 2. Weber, T. et al. Cell 92, 759–72 (1998). 10). The reconstituted bilayers, however, could Exocytic fusion, however, is a complex process 3. Geppert, M. et al. Cell 79, 717–727 (1994). 4. Ubach, J. et al. EMBO J. 17, 3921–3930 (1998).
    [Show full text]
  • Vesicle Coats: Structure, Function, And
    Review Vesicle coats: structure, function, and general principles of assembly 1 2 2 1,3 Marco Faini , Rainer Beck , Felix T. Wieland , and John A.G. Briggs 1 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany 2 Heidelberg University Biochemistry Center, Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany 3 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany The transport of proteins and lipids between distinct The three best-characterized types of vesicular carrier cellular compartments is conducted by coated vesicles. involved in intracellular trafficking are distinguished by These vesicles are formed by the self-assembly of coat their different coat proteins and their different trafficking proteins on a membrane, leading to collection of the routes. Clathrin-coated vesicles (CCVs) act in the late vesicle cargo and membrane bending to form a bud. secretory pathway and in the endocytic pathway, Scission at the bud neck releases the vesicle. X-ray COPII-coated vesicles export proteins from the endoplas- crystallography and electron microscopy (EM) have re- mic reticulum (ER), and COPI-coated vesicles shuttle cently generated models of isolated coat components within the Golgi organelle and from the Golgi back to and assembled coats. Here, we review these data to the ER. Despite having different compartment specifici- present a structural overview of the three main coats: ties and different structural components, the mechanisms clathrin, COPII, and COPI. The three coats have similar of their formation follow similar rules. The time and place function, common ancestry, and structural similarities, at which vesicle formation occurs are most often regulated but exhibit fundamental differences in structure and by small GTP-binding proteins.
    [Show full text]