The Structures of Natively Assembled Clathrin Coated Vesicles

AbstractVesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2-appendages crosslink adjacent CHC β-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

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The structures of natively assembled clathrin-coated vesicles

Science Advances ◽

10.1126/sciadv.aba8397 ◽

2020 ◽

Vol 6 (30) ◽

pp. eaba8397 ◽

Cited By ~ 1

Author(s):

Mohammadreza Paraan ◽

Joshua Mendez ◽

Savanna Sharum ◽

Danielle Kurtin ◽

Huan He ◽

...

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Associated Factors ◽

Structural Model ◽

Protein Complexes ◽

Adaptor Protein ◽

Coated Vesicles ◽

Clathrin Heavy Chain ◽

Assembled Complexes ◽

The Individual

Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

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ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2217 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2217-2229 ◽

Cited By ~ 63

Author(s):

Lisa A. Hannan ◽

Sherri L. Newmyer ◽

Sandra L. Schmid

Keyword(s):

Plasma Membrane ◽

Protein Complexes ◽

Adaptor Protein ◽

Adaptor Proteins ◽

Dual Role ◽

Vesicular Trafficking ◽

Coated Vesicles ◽

Golgi Network ◽

Cytosolic Factor

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

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Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46

Journal of Biological Chemistry ◽

10.1074/jbc.m117.780155 ◽

2017 ◽

Vol 292 (18) ◽

pp. 7462-7473 ◽

Cited By ~ 21

Author(s):

Michael Taschner ◽

André Mourão ◽

Mayanka Awasthi ◽

Jerome Basquin ◽

Esben Lorentzen

Keyword(s):

Protein Complexes ◽

Adaptor Protein ◽

Intraflagellar Transport ◽

Structural Basis ◽

Fallopian Tubes ◽

Large Protein ◽

Terminal Domain ◽

Unicellular Organisms ◽

Motile Cilia ◽

The Individual

Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

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Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent

The Journal of Cell Biology ◽

10.1083/jcb.200601067 ◽

2006 ◽

Vol 173 (4) ◽

pp. 545-557 ◽

Cited By ~ 368

Author(s):

Elizabeth E. Glater ◽

Laura J. Megeath ◽

R. Steven Stowers ◽

Thomas L. Schwarz

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Adaptor Protein ◽

Direct Interaction ◽

Anterograde Transport ◽

Kinesin Light Chain ◽

Energy Demands ◽

Kinesin Heavy Chain ◽

Conventional Kinesin ◽

Dependent Transport

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton–KHC complexes, and mitochondria are present in klc −/− photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH2 terminus–splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain–independent, anterograde transport of mitochondria.

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Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2055 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2055-2062 ◽

Cited By ~ 32

Author(s):

F M Brodsky

Keyword(s):

Monoclonal Antibodies ◽

Heavy Chain ◽

Light Chain ◽

Coated Vesicles ◽

Cell Lysates ◽

Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

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Structural basis for spectrin recognition by ankyrin

Blood ◽

10.1182/blood-2009-11-255604 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4093-4101 ◽

Cited By ~ 62

Author(s):

Jonathan J. Ipsaro ◽

Alfonso Mondragón

Keyword(s):

Membrane Proteins ◽

Structural Model ◽

Membrane Integrity ◽

Adaptor Protein ◽

Principal Component ◽

Structural Basis ◽

Protein Protein Interaction ◽

The Individual ◽

Spectrin Repeats

Maintenance of membrane integrity and organization in the metazoan cell is accomplished through intracellular tethering of membrane proteins to an extensive, flexible protein network. Spectrin, the principal component of this network, is anchored to membrane proteins through the adaptor protein ankyrin. To elucidate the atomic basis for this interaction, we determined a crystal structure of human βI-spectrin repeats 13 to 15 in complex with the ZU5-ANK domain of human ankyrin R. The structure reveals the role of repeats 14 to 15 in binding, the electrostatic and hydrophobic contributions along the interface, and the necessity for a particular orientation of the spectrin repeats. Using structural and biochemical data as a guide, we characterized the individual proteins and their interactions by binding and thermal stability analyses. In addition to validating the structural model, these data provide insight into the nature of some mutations associated with cell morphology defects, including those found in human diseases such as hereditary spherocytosis and elliptocytosis. Finally, analysis of the ZU5 domain suggests it is a versatile protein-protein interaction module with distinct interaction surfaces. The structure represents not only the first of a spectrin fragment in complex with its binding partner, but also that of an intermolecular complex involving a ZU5 domain.

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BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2209 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2209-2219 ◽

Cited By ~ 129

Author(s):

Young-Kwang Lee ◽

Joseph W. Brewer ◽

Rachel Hellman ◽

Linda M. Hendershot

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Protein Complexes ◽

Critical Role ◽

Light Chains ◽

Heavy Chains ◽

Chain Complexes ◽

Chain Synthesis

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

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Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition.

The Journal of Cell Biology ◽

10.1083/jcb.111.3.829 ◽

1990 ◽

Vol 111 (3) ◽

pp. 829-837 ◽

Cited By ~ 116

Author(s):

L M Hendershot

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Secretory Pathway ◽

Protein Transport ◽

Protein Complexes ◽

Binding Protein ◽

Immunoglobulin Heavy Chain ◽

Light Chains ◽

Heavy Chains

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.

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Structure of clathrin cages and coats in ice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014213x ◽

1986 ◽

Vol 44 ◽

pp. 94-97

Author(s):

G.P.A. Vigers ◽

R.A. Crowther ◽

B.M.F. Pearse

Keyword(s):

Electron Microscopy ◽

Heavy Chain ◽

Outer Part ◽

Coated Vesicles ◽

Eukaryotic Cells ◽

Coat Proteins ◽

Terminal Domain ◽

Clathrin Heavy Chain ◽

Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

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