RESUMEN
γ2 adaptin is homologous to γ1, but is only expressed in vertebrates while γ1 is found in all eukaryotes. We know little about γ2 functions and their relation to γ1. γ1 is an adaptin of the heterotetrameric AP-1 complexes, which sort proteins in and do form clathrin-coated transport vesicles and they also regulate maturation of early endosomes. γ1 knockout mice develop only to blastocysts and thus γ2 does not compensate γ1-deficiency in development. γ2 has not been classified as a clathrin-coated vesicle adaptor protein in proteome analyses and functions for monomeric γ2 in endosomal protein sorting have been proposed, but adaptin interaction studies suggested formation of heterotetrameric AP-1/γ2 complexes. We detected γ2 at the trans-Golgi network, on peripheral vesicles and identified γ2 clathrin-coated vesicles in mice. Ubiquitous σ1A and tissue-specific σ1B adaptins bind γ2 and γ1. σ1B knockout in mice does not effect γ1/σ1A AP-1 levels, but γ2/σ1A AP-1 levels are increased in brain and adipocytes. Also γ2 is essential in development. In zebrafish AP-1/γ2 and AP-1/γ1 fulfill different, essential functions in brain and the vascular system.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Encéfalo/metabolismo , Clatrina/metabolismo , Vesículas Transportadoras/metabolismo , Red trans-Golgi/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/química , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Adipocitos/citología , Adipocitos/metabolismo , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/metabolismo , Encéfalo/crecimiento & desarrollo , Línea Celular , Clatrina/genética , Embrión de Mamíferos , Embrión no Mamífero , Endosomas/metabolismo , Endosomas/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas/genética , Transducción de Señal , Vesículas Transportadoras/ultraestructura , Pez Cebra , Red trans-Golgi/ultraestructuraRESUMEN
Hepatitis B virus (HBV) is an infectious, potentially lethal human pathogen. However, there are no effective therapies for chronic HBV infections. Antiviral development is hampered by the lack of high-resolution structures for essential HBV protein-protein interactions. The interaction between preS1, an HBV surface-protein domain, and its human binding partner, γ2-adaptin, subverts the membrane-trafficking apparatus to mediate virion export. This interaction is a putative drug target. We report here atomic-resolution descriptions of the binding thermodynamics and structural biology of the interaction between preS1 and the EAR domain of γ2-adaptin. NMR, protein engineering, X-ray crystallography and MS showed that preS1 contains multiple γ2-EAR-binding motifs that mimic the membrane-trafficking motifs (and binding modes) of host proteins. These motifs localize together to a relatively rigid, functionally important region of preS1, an intrinsically disordered protein. The preS1-γ2-EAR interaction was relatively weak and efficiently outcompeted by a synthetic peptide. Our data provide the structural road map for developing peptidomimetic antivirals targeting the γ2-EAR-preS1 interaction.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Imitación Molecular , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Secuencias de Aminoácidos , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Receptores Notch/metabolismo , Factor de Transcripción AP-1/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/genética , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Compartimento Celular , Línea Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endosomas/metabolismo , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Humanos , Lisosomas/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , Receptores Notch/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Factor de Transcripción AP-1/química , Factor de Transcripción AP-2/química , Factor de Transcripción AP-2/metabolismo , Red trans-Golgi/metabolismoRESUMEN
BACKGROUND: Native immunoprecipitation followed by protein A-mediated recovery of the immuno-complex is a powerful tool to study protein-protein interactions. A limitation of this technique is the concomitant recovery of large amounts of immunoglobulin, which interferes with down-stream applications such as mass spectrometric analysis and Western blotting. Here we report a detergent-based "soft" elution protocol that allows effective recovery of immunoprecipitated antigen and binding partners, yet avoids elution of the bulk of the immunoglobulin. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the performance of the soft elution protocol using immunoprecipitation of Adaptor protein complex 1 (AP-1) and associated proteins as a test case. Relative to conventional elution conditions, the novel protocol substantially improved the sensitivity of mass spectrometric identification of immunoprecipitated proteins from unfractionated solution digests. Averaging over three independent experiments, Mascot scores of identified AP-1 binding partners were increased by 39%. Conversely, the estimated amount of recovered immunoglobulin was reduced by 44%. We tested the protocol with five further antibodies derived from rabbit, mouse and goat. In each case we observed a significant reduction of co-eluting immunoglobulin. CONCLUSIONS/SIGNIFICANCE: The soft elution protocol presented here shows superior performance compared to standard elution conditions for subsequent protein identification by mass spectrometry from solution digests. The method was developed for rabbit polyclonal antibodies, but also performed well with the tested goat and mouse antibodies. Hence we expect the soft elution protocol to be widely applicable.
Asunto(s)
Inmunoprecipitación/métodos , Inmunoprecipitación/normas , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/aislamiento & purificación , Animales , Electroforesis en Gel de Poliacrilamida , Cabras , Células HeLa , Humanos , Inmunoglobulinas/aislamiento & purificación , Espectrometría de Masas , Ratones , Conejos , Estándares de ReferenciaRESUMEN
Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/química , Virus de la Hepatitis B/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Catálisis , ADN/química , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Lisina/química , Datos de Secuencia Molecular , Ubiquitina-Proteína Ligasas Nedd4 , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Conformación Proteica , Homología de Secuencia de Aminoácido , Tetraspanina 30 , TransfecciónRESUMEN
In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.
Asunto(s)
Complejo 1 de Proteína Adaptadora/química , Complejo 2 de Proteína Adaptadora/química , Proteínas Portadoras/química , Clatrina/química , Proteínas de Transporte de Membrana , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Electroporación , Eliminación de Gen , Glutatión Transferasa/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Células PC12 , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismo , Sacarosa/química , Sacarosa/farmacología , Tirosina/química , Tirosina/metabolismo , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/química , Proteínas Adaptadoras del Transporte Vesicular , Complejo 1 de Proteína Adaptadora , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotinilación , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Clatrina/metabolismo , Secuencia de Consenso , Glutatión Transferasa/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae , Relación Estructura-Actividad , Técnicas del Sistema de Dos HíbridosRESUMEN
Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The gamma-adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 A. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.
Asunto(s)
Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Factores de Ribosilacion-ADP/genética , Complejo 1 de Proteína Adaptadora/química , Complejo 1 de Proteína Adaptadora/genética , Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/genética , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Vesículas Cubiertas por Clatrina/metabolismo , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , Red trans-Golgi/metabolismoRESUMEN
Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab4/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/química , Ratones , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción AP-1/química , Factor de Transcripción AP-1/metabolismo , Proteínas de Unión al GTP rab4/química , Proteínas de Unión al GTP rab4/genéticaRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/química , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Animales , Ataxina-3 , Secuencia Conservada , Bases de Datos de Proteínas , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares , Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras , Alineación de Secuencia , Homología de Secuencia , Relación Estructura-ActividadRESUMEN
Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes. Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes. Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi. Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin. A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined. Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes. The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes. On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Endosomas/fisiología , Aparato de Golgi/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Subunidades gamma de Complejo de Proteína Adaptadora/química , Secuencia de Aminoácidos , Sitios de Unión , Clatrina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endosomas/ultraestructura , Aparato de Golgi/ultraestructura , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, gamma and beta1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the gamma appendage is presented. The binding site for the known gamma appendage ligand gamma-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of gamma appendage and binds to the same site as gamma-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.