RESUMO
The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.
Assuntos
Fator de Transcrição AP-1/ultraestrutura , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/ultraestrutura , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/ultraestrutura , Proteínas Adaptadoras de Transporte Vesicular , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Antígeno 2 do Estroma da Médula Óssea/ultraestrutura , Clatrina , Complexo de Golgi , Células HEK293 , HIV-1 , Humanos , Transporte Proteico/fisiologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Secretórias/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/química , Membrana Celular/metabolismo , Microscopia de Fluorescência , Modelos Teóricos , Conformação Proteica , Família de Proteínas da Síndrome de Wiskott-Aldrich/químicaRESUMO
Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de srcRESUMO
Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.
Assuntos
Membrana Celular/metabolismo , Endocitose , Transdução de Sinais , Animais , Evolução Biológica , Membrana Celular/química , Homeostase , HumanosRESUMO
Protein coats are supramolecular complexes that assemble on the cytosolic face of membranes to promote cargo sorting and transport carrier formation in the endomembrane system of eukaryotic cells. Several types of protein coats have been described, including COPI, COPII, AP-1, AP-2, AP-3, AP-4, AP-5, and retromer, which operate at different stages of the endomembrane system. Defects in these coats impair specific transport pathways, compromising the function and viability of the cells. In humans, mutations in subunits of these coats cause various congenital diseases that are collectively referred to as coatopathies. In this article, we review the fundamental properties of protein coats and the diseases that result from mutation of their constituent subunits.
Assuntos
Endossomos/química , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Proteínas de Transporte Vesicular/genética , Animais , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/terapia , Humanos , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismoRESUMO
Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with ß-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.
Assuntos
Membrana Celular/ultraestrutura , Clatrina/química , Complexo I de Proteína do Envoltório/química , Vesículas Revestidas/ultraestrutura , Células Eucarióticas/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/química , Transporte Ativo do Núcleo Celular , Membrana Celular/química , Membrana Celular/metabolismo , Clatrina/genética , Clatrina/metabolismo , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Vesículas Revestidas/química , Vesículas Revestidas/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Evolução Molecular , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestrutura , Expressão Gênica , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios ProteicosRESUMO
DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Endocitose , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Asas de Animais/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Ligantes , Receptores Notch/metabolismoRESUMO
Most single animal cells have an internal vector that determines where recycling membrane is added to the cell's surface. Because of the specific molecular composition of this added membrane, a dynamic asymmetry is formed on the surface of the cell. The consequences of this dynamic asymmetry are discussed, together with what they imply for how cells move. The polarity of a single-celled embryo, such as that of the nematode Caenorhabditis elegans, is explored in a similar framework.
Assuntos
Membrana Celular/química , Endocitose , Animais , Biologia/métodos , Caenorhabditis elegans , Movimento Celular , Citoesqueleto/metabolismo , Dictyostelium , Fibroblastos/metabolismo , Células HeLa , Hemaglutininas/química , Humanos , Proteínas de Membrana/química , Proteínas/químicaRESUMO
Biological membranes are integral cellular structures that can be curved into various geometries. These curved structures are abundant in cells as they are essential for various physiological processes. However, curved membranes are inherently unstable, especially on nanometer length scales. To stabilize curved membranes, cells can utilize proteins that sense and generate membrane curvature. In this review, we summarize recent research that has advanced our understanding of interactions between proteins and curved membrane surfaces, as well as work that has expanded our ability to study curvature sensing and generation. Additionally, we look at specific examples of cellular processes that require membrane curvature, such as neurotransmission, clathrin-mediated endocytosis (CME), and organelle biogenesis.
Assuntos
Membrana Celular , Membrana Celular/metabolismo , Humanos , Endocitose/fisiologia , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Clatrina/metabolismoRESUMO
The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, shuttles the glucose transporter GLUT4 from the ERGIC (endoplasmic-reticulum-to-Golgi intermediate compartment) directly to an intracellular GLUT4 storage compartment (GSC), from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. We show that the C-terminal trimerization domain of CHC22 interacts with SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both N- and C-termini of the heavy chain, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi-bypass route for GLUT4 trafficking. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.
RESUMO
Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Endocitose , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Microscopia Crioeletrônica , Ligação Proteica , Proteólise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico , Endossomos/metabolismo , Corpos Multivesiculares/metabolismo , Transporte BiológicoRESUMO
Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) in an inward/ER-directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis-localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS-CoV-2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions.
Assuntos
Retículo Endoplasmático , Neoplasias , Humanos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Neoplasias/metabolismo , Transporte ProteicoRESUMO
Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.
Assuntos
Vesículas Revestidas por Clatrina , Endocitose , Mamíferos , Animais , Vesículas Revestidas por Clatrina/metabolismo , Mamíferos/metabolismo , Plantas/metabolismo , Plantas/microbiologia , Humanos , Clatrina/metabolismo , Leveduras/metabolismoRESUMO
Clathrin assembles into honeycomb-like lattices at the plasma membrane but also on internal membranes, such as at the Golgi and tubular endosomes. Clathrin assemblies primarily regulate the intracellular trafficking of different cargoes, but clathrin also has non-endocytic functions in cell adhesion through interactions with specific integrins, contributes to intraluminal vesicle formation by forming flat bilayered coats on endosomes and even assembles on kinetochore k-fibers during mitosis. In this Cell Science at a Glance article and the accompanying poster, we review our current knowledge on the different types of canonical and non-canonical membrane-associated clathrin assemblies in mammalian cells, as observed by thin-section or platinum replica electron microscopy in various cell types, and discuss how the structural plasticity of clathrin contributes to its functional diversity.
Assuntos
Clatrina , Animais , Humanos , Membrana Celular/metabolismo , Clatrina/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismoRESUMO
Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Dinaminas , Endocitose , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clatrina/metabolismo , Clatrina/genética , Dinaminas/metabolismo , Dinaminas/genética , Endocitose/genética , Proteínas de Ligação ao GTP , Mutação/genéticaRESUMO
Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.
Assuntos
Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Domínios C2/fisiologia , Células COS , Chlorocebus aethiops , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Clatrina/fisiologia , Endocitose/fisiologia , Células HEK293 , Homeostase , Humanos , Lipídeos/fisiologia , Espectrometria de Massas , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Transdução de SinaisRESUMO
In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide-MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F-actin dependent microclusters as the T cell spreads over the APC. Within 1-5 min, clathrin is recruited by the ESCRT-0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT-mediated ectocytosis (CEME). After 5-10 min, Hrs is displaced by the endocytic clathrin adaptor epsin-1 to induce clathrin-mediated trans-endocytosis (CMTE) of TCR-pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation.
Assuntos
Clatrina , Linfócitos T , Clatrina/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Receptores de Antígenos de Linfócitos T , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , ComunicaçãoRESUMO
Ligation of T cell receptor (TCR) to peptide-MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC-TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.
Assuntos
Clatrina , Sinapses Imunológicas , Clatrina/metabolismo , Receptores de Antígenos de Linfócitos T , Linfócitos T , Ativação LinfocitáriaRESUMO
Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.