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Results for “Adaptor Protein Complex 3”

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PubMed1999

Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

Adaptor Protein Complex 1↗

PubMed2000

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation.

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.

Adaptor Protein Complex 1↗

PubMed2001

PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic.

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.

Adaptor Protein Complex 3↗

PubMed2006

Hermansky-Pudlak syndrome: a disease of protein trafficking and organelle function.

The Hermansky-Pudlak syndrome (HPS) is a collection of related autosomal recessive disorders which are genetically heterogeneous. There are eight human HPS subtypes, characterized by oculocutaneous albinism and platelet storage disease; prolonged bleeding, congenital neutropenia, pulmonary fibrosis, and granulomatous colitis can also occur. HPS is caused primarily by defects in intracellular protein trafficking that result in the dysfunction of intracellular organelles known as lysosome-related organelles. HPS gene products are all ubiquitously expressed and all associate in various multi-protein complexes, yet HPS has cell type-specific disease expression. Impairment of specialized secretory cells such as melanocytes, platelets, lung alveolar type II epithelial cells and cytotoxic T cells are observed in HPS. This review summarizes recent molecular, biochemical and cell biological analyses together with clinical studies that have led to the correlation of molecular pathology with clinical manifestations and led to insights into such diverse disease processes such as albinism, fibrosis, hemorrhage, and congenital neutropenia.

Adaptor Protein Complex 3↗

PubMed2005

Intermediate filaments and vesicular membrane traffic: the odd couple's first dance?

During the last two decades, much attention has been focused on the regulation of membrane traffic by the actin and microtubule cytoskeletal networks. Their dynamic and polarized behavior and associated motors provide a logical framework from which architectural and movement cues can be communicated to organelles. The study of these cytoskeletal systems has been greatly aided by pharmacological agents. In contrast, intermediate filaments (IFs) have largely been neglected as a potential player in membrane traffic, both because a comprehensive pharmacology to perturb them does not exist and because they lack the intrinsic polarity and specific motors that make the other cytoskeletal systems attractive. In this review, we will discuss evidence suggesting that IFs may play roles in controlling organelle positioning and in membrane protein targeting. Furthermore, we will discuss potential mechanisms by which IFs may regulate the localization and function of organelles.

Adaptor Protein Complex 3↗

PubMed2006

A role for adaptor protein-3 complex in the organization of the endocytic pathway in Dictyostelium.

Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium.

Adaptor Protein Complex 3↗

PubMed1998

A structural explanation for the recognition of tyrosine-based endocytotic signals.

Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the mu2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of mu2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

Adaptor Protein Complex 1↗

PubMed2002

Enhancer of garnet/deltaAP-3 is a cryptic allele of the white gene and identifies the intracellular transport system for the white protein.

The white gene encodes an ABC-type transmembrane transporter that has a role in normal eye pigment deposition. In addition, overexpression in Drosophila leads to homosexual male courtship. Its human homologue has been implicated in cholesterol transport in macrophages and in mood disorders in human males. The garnet gene is a member of a group of other Drosophila eye colour genes that have been shown, or proposed, to function in intracellular protein transport. Recent molecular analysis indicates that it encodes the delta subunit of the AP-3 adaptin complex involved in vesicle transport from the trans-Golgi network to lysosomes and related organelles, such as pigment granules. This identification revealed a novel role for intracellular vesicular transport in Drosophila pigmentation. To further analyze this intracellular transport system, we examined the genetic interactions between garnet and a second site enhancer mutation, enhancer of garnet (e(g)). We show here that e(g) is a cryptic allele of the white gene. The white-garnet interaction is highly sensitive to the levels of both gene products but also shows some allele specificity for the white gene. The additive effect on pigmentation and the predicted protein products of these genes suggest that the garnet/AP-3 transport system ensures the correct intracellular localization of the white gene product. This model is further supported by the observation of homosexual male courtship behavior in garnet mutants, similar to that seen in flies overexpressing, and presumably mis-sorting, the white gene product. The w(e(g)) allele also enhances mutations in the subset of other eye-color genes with phenotypes similar to garnet. This observation supports a role for these genes in intracellular transport and leads to a model whereby incorrect sorting of the white gene product can explain the pigmentation phenotypes of an entire group of eye-color genes.

ATP-Binding Cassette Transporters↗

PubMed2006

Loss of genomic imprinting in Drosophila clones.

Genomic imprinting is a process that genetically distinguishes maternal and paternal genomes, and can result in parent-of-origin-dependent monoallelic expression of a gene that is dependent on the parent of origin. As such, an otherwise functional maternally inherited allele may be silenced so that the gene is expressed exclusively from the paternal allele, or vice versa. Once thought to be restricted to mammals, genomic imprinting has been documented in angiosperm plants (J.L. Kermicle. 1970. Genetics, 66: 69-85), zebrafish (C.C. Martin and R. McGowan. 1995. Genet. Res. 65: 21-28), insects, and C. elegans (C.J. Bean, C.E. Schaner, and W.G. Kelly. 2004. Nat. Genet. 36: 100-105.). In each case, it appears to rely on differential chromatin structure. Aberrant imprinting has been implicated in various human cancers and has been detected in a number of cloned mammals, potentially limiting the usefulness of somatic nuclear transfer. Here we show that genomic imprinting associated with a mini-X chromosome is lost in Drosophila melanogaster clones.

Adaptor Protein Complex 3↗

PubMed2003

Aberrant lung structure, composition, and function in a murine model of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS.

Adaptor Protein Complex 3↗

PubMed1999

Genomic structure and chromosome mapping of the genes encoding clathrin-associated adaptor medium chains mu1A (Ap1m1) and mu1B (Ap1m2).

The protein mu1B is a member of the medium chain family of the clathrin-associated adaptor complex and is expressed exclusively in epithelial cells. We determined the genomic structure of previously cloned murine genes for mu1B (Ap1m2) and its closely related homolog, mu1A (Ap1m1). Comparison of their genomic structures revealed that the positions of introns are identical between these two genes, except for the insertion of an additional intron in Ap1m1 (intron 4). By contrast, these structures are different from that of the more distantly related Ap2m1 gene encoding mu2. Taken together with the similarity of amino acid sequences among these genes, the data presented in this study suggest that Ap1m1/2 and Ap2m1 diverged long before the separation of Ap1m1 and Ap1m2, which most likely resulted from a relatively recent gene duplication. We also mapped AP1M2 to human chromosome 19p13.2 and Ap1m2 to the proximal region of mouse chromosome 9. The results are consistent with the fact that these regions are syntenic.

Adaptor Protein Complex 1↗

PubMed2004

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Adaptor Protein Complex 3↗

PubMed2006

Identification of a homozygous deletion in the AP3B1 gene causing Hermansky-Pudlak syndrome, type 2.

We report on the molecular etiology of an unusual clinical phenotype associating congenital neutropenia, thrombocytopenia, developmental delay, and hypopigmentation. Using genetic linkage analysis and targeted gene sequencing, we defined a homozygous genomic deletion in AP3B1, the gene encoding the beta chain of the adaptor protein-3 (AP-3) complex. The mutation leads to in-frame skipping of exon 15 and thus perturbs proper assembly of the heterotetrameric AP-3 complex. Consequently, trafficking of transmembrane lysosomal proteins is aberrant, as shown for CD63. In basal keratinocytes, the incorporated immature melanosomes were rapidly degraded in large phagolysosomes. Despite distinct ultramorphologic changes suggestive of aberrant vesicular maturation, no functional aberrations were detected in neutrophil granulocytes. However, a comprehensive immunologic assessment revealed that natural killer (NK) and NKT-cell numbers were reduced in AP-3-deficient patients. Our findings extend the clinical and molecular phenotype of human AP-3 deficiency (also known as Hermansky-Pudlak syndrome, type 2) and provide further insights into the role of the AP-3 complex for the innate immune system.

Adaptor Protein Complex 3↗

PubMed2006

Innate immunity defects in Hermansky-Pudlak type 2 syndrome.

Adaptor protein-3 (AP-3) is an ubiquitous cytoplasmic complex that shuttles cargo proteins from the trans-Golgi and a tubular-endosomal compartment to endosome-lysosome-related organelles. Lack of the beta3A subunit of this complex causes Hermansky-Pudlak syndrome type 2, an autosomal recessive disease characterized by partial albinism, prolonged bleeding tendency, and immunodeficiency. To investigate the pathogenesis of immunodeficiency, we studied natural killer (NK) cells and neutrophil functions in 2 previously unreported siblings affected by Hermansky-Pudlak type 2 syndrome. In both patients we observed a dramatic reduction of cytolytic activity of freshly isolated and of IL-2-activated NK cells. Levels of perforin were reduced in unstimulated NK cells, thereby accounting for the impairment of NK cytolitic activity. In addition, analysis of neutrophils in these patients demonstrated that intracellular elastase content was largely reduced while CD63 expression on plasma membrane was substantially increased. Taken together, these observations suggest that type 2 Hermansky-Pudlak syndrome is characterized by defects of innate immunity.

Adaptor Protein Complex 3↗

PubMed2002

Nonsense mutations in ADTB3A cause complete deficiency of the beta3A subunit of adaptor complex-3 and severe Hermansky-Pudlak syndrome type 2.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disease consisting of oculocutaneous albinism and a storage pool deficiency resulting from absent platelet dense bodies. The disorder is genetically heterogeneous. The majority of patients, including members of a large genetic isolate in northwest Puerto Rico, have mutations in HPS1. Another gene, ADTB3A, was shown to cause HPS-2 in two brothers having compound heterozygous mutations that allowed for residual production of the gene product, the beta3A subunit of adaptor complex-3 (AP-3). This heterotetrameric complex serves as a coat protein-mediating formation of intracellular vesicles, e.g. the melanosome and platelet dense body, from membranes of the trans-Golgi network. We determined the genomic organization of the human ADTB3A gene, with intron/exon boundaries, and describe a third patient with beta3A deficiency. This 5-y-old boy has two nonsense mutations, C1578T (R-->X) and G2028T (E-->X), which produce no ADTB3A mRNA and no beta3A protein. The associated mu3 subunit of AP-3 is also entirely absent. In fibroblasts, the cell biologic concomitant of this deficiency is robust and aberrant trafficking through the plasma membrane of LAMP-3, an integral lysosomal membrane protein normally carried directly to the lysosome. The clinical concomitant is a severe, G-CSF-responsive neutropenia in addition to oculocutaneous albinism and platelet storage pool deficiency. Our findings expand the molecular, cellular, and clinical spectrum of HPS-2 and call for an increased index of suspicion for this diagnosis among patients with features of albinism, bleeding, and neutropenia.

Adaptor Protein Complex 3↗

PubMed2000

RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis.

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

ATP-Binding Cassette Transporters↗

PubMed1999

A genetic and molecular characterization of the garnet gene of Drosophila melanogaster.

The garnet gene was one of the first genes to be identified in Drosophila melanogaster. Mutations in the garnet gene affect both of the biochemically distinct types of pigments in the eye and disrupt pigmentation of other organs. As an initial step in the analysis of this gene, we have analyzed the pigmentation defects in several of the garnet alleles. We have also cloned the gene and examined its expression in various tissues and at different stages of development. The garnet gene is expressed throughout development and in all tissues examined. Structurally related sequences can be detected in a variety of other eukaryotes. The predicted protein sequence of the garnet product resembles clathrin and nonclathrin adaptin proteins and is highly similar to the delta subunit of the newly isolated mammalian AP-3 adaptin complex, which is associated with the trans-Golgi network and endosomes. This suggests that garnet encodes a protein that acts in the intracellular sorting and trafficking of vesicles from the trans-Golgi network to endosomes, and related specialized organelles such as the pigment granule. This finding provides an explanation for the phenotype of garnet mutations and predicts that other Drosophila eye-colour genes will be a rich resource for the genetic dissection of intracellular vesicle transport.

Adaptor Protein Complex 3↗

PubMed2004

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins.

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.

Adaptor Protein Complex 3↗