RESUMO
Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras , ATPases Transportadoras de Cobre , Endossomos , Transporte Proteico , Receptor IGF Tipo 2 , Rede trans-Golgi , Humanos , Endossomos/metabolismo , Células HeLa , Transporte Proteico/genética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismoRESUMO
BACKGROUND: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue. MATERIALS AND METHODS: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS. RESULTS: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor. CONCLUSION: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/metabolismo , Endossomos/metabolismo , Antígeno Ki-67/metabolismo , Recidiva Local de Neoplasia/metabolismo , Fator de Transcrição AP-1/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismoRESUMO
Adaptor protein 4 (AP-4) is a heterotetrameric complex composed of ε, ß4, µ4, and σ4 subunits that mediates export of a subset of transmembrane cargos, including autophagy protein 9A (ATG9A), from the trans-Golgi network (TGN). AP-4 has received particular attention in recent years because mutations in any of its subunits cause a complicated form of hereditary spastic paraplegia referred to as "AP-4-deficiency syndrome." The identification of proteins that interact with AP-4 has shed light on the mechanisms of AP-4-dependent cargo sorting and distribution within the cell. However, the mechanisms by which the AP-4 complex itself is assembled have remained unknown. Here, we report that the alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34) binds to and stabilizes the AP-4 ε and σ4 subunits, thus promoting complex assembly. The physiological importance of these interactions is underscored by the observation that AAGAB-knockout cells exhibit reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN like those in cells with mutations in AP-4-subunit genes. These findings demonstrate that AP-4 assembly is not spontaneous but AAGAB-assisted, further contributing to the understanding of an adaptor protein complex that is critically involved in development of the central nervous system.
Assuntos
Subunidades do Complexo de Proteínas Adaptadoras , Proteínas de Membrana , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Rede trans-Golgi/metabolismoRESUMO
The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.
Assuntos
Regulação para Baixo , Regulação da Expressão Gênica , Antígeno HLA-A2/metabolismo , Fator de Transcrição AP-1/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia Imunoeletrônica , Transporte Proteico , Linfócitos T/imunologia , Linfócitos T/virologia , Rede trans-Golgi/metabolismoRESUMO
In this study, we expanded our recent work showing that ASCT2, a Na+-dependent neutral amino acid transporter that plays a major role in glutamine uptake in cancer cells, is physically associated with EGFR in human head and neck squamous cell carcinoma cells and in several other types of cancer cells. We found in our current study that ASCT2 can be downregulated by cetuximab, an approved anti-EGFR therapeutic antibody, via cetuximab-induced EGFR endocytosis independently of cetuximab-mediated inhibition of EGFR tyrosine kinase. We further found that ASCT2-EGFR association involves the adaptor-related protein complex 1 gamma 1 subunit (AP1G1), a subunit of clathrin-associated adaptor protein complex 1, which plays a role in membrane protein sorting in endosomes after receptor-mediated endocytosis. We found that AP1G1 is physically associated with both ASCT2 and EGFR and, together with those molecules, forms a heterotrimeric molecular complex. Knockdown of AP1G1 lowered the level of ASCT2-EGFR association, inhibited cetuximab-mediated internalization of ASCT2-EGFR complex, and decreased intracellular glutamine uptake and glutathione biosynthesis. These findings suggest a new therapeutic strategy to overcome cetuximab resistance in cancer cells through combination of cetuximab, which co-targets ASCT2 along with EGFR, with an ROS-inducing agent.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/patologia , Espécies Reativas de Oxigênio/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/metabolismo , Encéfalo/metabolismo , Clatrina/metabolismo , Vesículas Transportadoras/metabolismo , Rede trans-Golgi/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/química , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Subunidades sigma do Complexo de Proteínas Adaptadoras/química , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Encéfalo/crescimento & desenvolvimento , Linhagem Celular , Clatrina/genética , Embrião de Mamíferos , Embrião não Mamífero , Endossomos/metabolismo , Endossomos/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Transporte Proteico/genética , Transdução de Sinais , Vesículas Transportadoras/ultraestrutura , Peixe-Zebra , Rede trans-Golgi/ultraestruturaRESUMO
The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or µ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Antígenos CD4/metabolismo , Regulação para Baixo , HIV-1/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Técnicas do Sistema de Duplo-HíbridoRESUMO
Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oligopeptídeos/metabolismo , Vacúolos/metabolismo , Rede trans-Golgi/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Retículo Endoplasmático/metabolismo , Immunoblotting , Membranas Intracelulares/metabolismo , Leucina/genética , Leucina/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Oligopeptídeos/genética , Peptídeos/genética , Peptídeos/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Protoplastos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Endossomos/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Transporte Biológico , Membrana Celular/enzimologia , Membrana Celular/genética , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Endossomos/enzimologia , Endossomos/genética , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico , Transferrina/genética , Transferrina/metabolismo , Proteínas rab4 de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Mimetismo Molecular , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/química , Motivos de Aminoácidos , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, µ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their µ subunits in plants. Because of uncertain homology, the µ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678-693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057-2068, Zwiewka et al. (2011) Cell Res 21(12):1711-1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215-232]. In contrast, the µ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 µ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Transporte Proteico/fisiologia , Complexo 1 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Citocinese/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Interfase/fisiologia , Microscopia Eletrônica de Transmissão , Mutagênese Insercional , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the µ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Polaridade Celular , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Fatores de Ribosilação do ADP/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Receptores Frizzled/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Transporte Proteico , Interferência de RNA , Receptores Proteína Tirosina Quinases/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Receptores Notch/metabolismo , Fator de Transcrição AP-1/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/química , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Compartimento Celular , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endossomos/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Humanos , Lisossomos/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Receptores Notch/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator de Transcrição AP-1/química , Fator de Transcrição AP-2/química , Fator de Transcrição AP-2/metabolismo , Rede trans-Golgi/metabolismoRESUMO
γ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of γ2-adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of γ2-adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon γ2-adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of γ2-adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify γ2-adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Transdução de Sinais/fisiologia , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Humanos , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Microscopia de Fluorescência , Corpos Multivesiculares/metabolismo , Ligação Proteica , Transporte Proteico , Interferência de RNA , Retroviridae/genética , Retroviridae/crescimento & desenvolvimento , TransfecçãoRESUMO
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Endossomos/metabolismo , Glicoproteínas de Membrana/fisiologia , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Adenosina Trifosfatases/genética , Androstenos/farmacologia , Anticolesterolemiantes/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Ceruloplasmina/metabolismo , ATPases Transportadoras de Cobre , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/metabolismo , Glicoproteínas de Membrana/genética , Mutação , Proteína C1 de Niemann-Pick , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-adaptin. By mapping regions of Nedd4 involved in binding to gamma2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-adaptin. In addition, we show that gamma2-adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-adaptin, a closely related homolog of gamma2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Endossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Fator de Crescimento Epidérmico/metabolismo , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Tropomyosin-related kinase A (TrkA) receptor mediates the effects exerted by nerve growth factor on several subpopulations of neuronal cells. Ligand binding to TrkA induces receptor autophosphorylation on several tyrosine residues and the activation of signaling cascades. In this study, we describe a new site relevant for TrkA regulation, the tyrosine 701 (Y701), which is important for receptor trafficking and activation. Y701 replacement by aspartate or phenylalanine reduces receptor internalization rate and decreases the colocalization and association of TrkA with clathrin heavy chain, demonstrating that Y701 constitutes a YxxPhi (YRKF701-704) trafficking motif relevant for the regulation of receptor endocytosis. In accordance with this hypothesis, the colocalization of Y701 mutant receptors with a lysosomal marker is also reduced giving support to the involvement of the YRKF701-704 motif in the lysosomal targeting of TrkA receptors. Contrary to what was expected, substitution of Y701 for an Asp in order to mimic phosphorylation, impairs TrkA ability to mediate nerve growth factor-induced differentiation, although the mutant receptor retains its in vitro kinase activity. This is the first evidence that a Tyr residue can simultaneously regulate TrkA receptor trafficking and activity.
Assuntos
Receptor trkA/metabolismo , Tirosina/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cadeias Pesadas de Clatrina/metabolismo , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/metabolismo , Mutação/fisiologia , Células PC12 , Transporte Proteico/fisiologia , Ratos , Transfecção/métodosRESUMO
Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Clatrina/química , Fator de Transcrição AP-1/química , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Transporte Biológico , Cristalografia por Raios X , Dimerização , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
Genetic polymorphisms in xenobiotic transporters have recently been clarified to be associated with change in drug distribution and disposition. To expand on recent identification of direct interaction and functional regulation of several transporters by a PDZ (PSD95, Dlg and ZO1) domain containing protein PDZK1, the effect of mutation in PDZK1 on transport activity and subcellular localization of organic cation/carnitine transporters OCTN1 and OCTN2, and oligopeptide transporter PEPT2 was examined in the present study. HEK293 cells stably expressing a mutant transcript PDZK1-E195K (HEK293/PDZK1-E195K) were constructed, followed by transient transfection of cDNA for each transporter. Uptake of tetraethylammonium by OCTN1 was much higher in HEK293/PDZK1 cells, compared with that in the parent HEK293 cells, the uptake in HEK293/PDZK1-E195K cells showing middle range between the two values. Such difference in transport activity was accounted for the difference in transport capacity, with minimal change in affinity of OCTN1 to the substrate or other compounds. The similar difference among HEK293/PDZK1, HEK293/PDZK1-E195K and HEK293 cells was also observed in transport property of OCTN2 and PEPT2, whereas the difference was not so remarkable in each transporter with the last four amino acids deleted, that has much lower interaction potential with PDZK1. Immunohistochemical analysis indicated that OCTN1 was colocalized with PDZK1 on cell-surface, whereas colocalization with PDZK1-E195K was partially observed in cytoplasmic region. These results suggest a novel hypothesis that mutation in PDZK1 potentially changes transport property of various types of xenobiotic transporters by affecting their subcellular localization, possibly leading to change in disposition of various types of substrate drugs.
Assuntos
Proteínas de Transporte/fisiologia , Mutação/genética , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Transportador 1 de Cátions Orgânicos/fisiologia , Simportadores/fisiologia , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Radioisótopos de Carbono , Carnitina/farmacocinética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Dipeptídeos/farmacocinética , Humanos , Proteínas de Membrana , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Ligação Proteica , Pirilamina/farmacocinética , Membro 5 da Família 22 de Carreadores de Soluto , Simportadores/genética , Simportadores/metabolismo , Tetraetilamônio/farmacocinética , Transfecção , Trítio , Técnicas do Sistema de Duplo-Híbrido , Verapamil/farmacocinéticaRESUMO
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.