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1
Yeung, Bonny G., Huan L. Phan, and Gregory S. Payne. "Adaptor Complex-independent Clathrin Function in Yeast." Molecular Biology of the Cell 10, no. 11 (November 1999): 3643–59. http://dx.doi.org/10.1091/mbc.10.11.3643.
Анотація:
Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes inSaccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.
2
Salazar, G., B. Craige, M. L. Styers, K. A. Newell-Litwa, M. M. Doucette, B. H. Wainer, J. M. Falcon-Perez, et al. "BLOC-1 Complex Deficiency Alters the Targeting of Adaptor Protein Complex-3 Cargoes." Molecular Biology of the Cell 17, no. 9 (September 2006): 4014–26. http://dx.doi.org/10.1091/mbc.e06-02-0103.
Анотація:
Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1–deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.
3
Fölsch, Heike, Marc Pypaert, Sandra Maday, Laurence Pelletier, and Ira Mellman. "The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains." Journal of Cell Biology 163, no. 2 (October 27, 2003): 351–62. http://dx.doi.org/10.1083/jcb.200309020.
Анотація:
Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD μ1A or μ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B–containing membranes that were both distinct from AP-1A–positive TGN elements and more closely apposed to transferrin receptor–positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.
4
Kim, Myung-Hee, and Louis B. Hersh. "The Vesicular Acetylcholine Transporter Interacts with Clathrin-associated Adaptor Complexes AP-1 and AP-2." Journal of Biological Chemistry 279, no. 13 (January 14, 2004): 12580–87. http://dx.doi.org/10.1074/jbc.m310681200.
5
Sorkina, T., A. Bild, F. Tebar, and A. Sorkin. "Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors." Journal of Cell Science 112, no. 3 (February 1, 1999): 317–27. http://dx.doi.org/10.1242/jcs.112.3.317.
Анотація:
Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.
6
Fölsch, Heike, Marc Pypaert, Peter Schu, and Ira Mellman. "Distribution and Function of Ap-1 Clathrin Adaptor Complexes in Polarized Epithelial Cells." Journal of Cell Biology 152, no. 3 (February 5, 2001): 595–606. http://dx.doi.org/10.1083/jcb.152.3.595.
Анотація:
Expression of the epithelial cell–specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD μ subunit, μ1A, being replaced by the closely related μ1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged μ1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.
7
BAROIS, Nicolas, and Oddmund BAKKE. "The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study." Biochemical Journal 385, no. 2 (January 7, 2005): 503–10. http://dx.doi.org/10.1042/bj20041010.
Анотація:
The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin–Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.
8
Lefkir, Yaya, Benoît de Chassey, Annick Dubois, Aleksandra Bogdanovic, Rebecca J. Brady, Olivier Destaing, Franz Bruckert, Theresa J. O'Halloran, Pierre Cosson, and François Letourneur. "The AP-1 Clathrin-adaptor Is Required for Lysosomal Enzymes Sorting and Biogenesis of the Contractile Vacuole Complex in Dictyostelium Cells." Molecular Biology of the Cell 14, no. 5 (May 2003): 1835–51. http://dx.doi.org/10.1091/mbc.e02-10-0627.
Анотація:
Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The γ-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing γ-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1–coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.
9
Nie, Z. "The Arf GAPs AGAP1 and AGAP2 distinguish between the adaptor protein complexes AP-1 and AP-3." Journal of Cell Science 118, no. 15 (August 1, 2005): 3555–66. http://dx.doi.org/10.1242/jcs.02486.
10
LUNDMARK, Richard, та Sven R. CARLSSON. "The β-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2". Biochemical Journal 362, № 3 (8 березня 2002): 597–607. http://dx.doi.org/10.1042/bj3620597.
Анотація:
Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the β-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the β-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the β-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel β-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.
11
Coleman, Scott H., Nanette Van Damme, John R. Day, Colleen M. Noviello, Douglas Hitchin, Ricardo Madrid, Serge Benichou, and John C. Guatelli. "Leucine-Specific, Functional Interactions between Human Immunodeficiency Virus Type 1 Nef and Adaptor Protein Complexes." Journal of Virology 79, no. 4 (February 15, 2005): 2066–78. http://dx.doi.org/10.1128/jvi.79.4.2066-2078.2005.
Анотація:
ABSTRACT The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the μ subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the σ1 plus γ subunits of AP-1 or the σ3 plus δ subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the μ subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The μ and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.
12
Boehm, Markus, and Juan S. Bonifacino. "Adaptins." Molecular Biology of the Cell 12, no. 10 (October 2001): 2907–20. http://dx.doi.org/10.1091/mbc.12.10.2907.
Анотація:
Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. In this article, we report the results of a survey for adaptins from sequenced genomes including those of man, mouse, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the plant Arabidopsis thaliana, and the yeasts, Saccharomyces cerevisiae andSchizosaccharomyces pombe. We find that humans, mice, and Arabidopsis thaliana have four AP complexes (AP-1, AP-2, AP-3, and AP-4), whereas D. melanogaster,C. elegans, S. cerevisiae, and S. pombe have only three (AP-1, AP-2, and AP-3). Additional diversification of AP complexes arises from the existence of adaptin isoforms encoded by distinct genes or resulting from alternative splicing of mRNAs. We complete the assignment of adaptins to AP complexes and provide information on the chromosomal localization, exon-intron structure, and pseudogenes for the different adaptins. In addition, we discuss the structural and evolutionary relationships of the adaptins and the genetic analyses of their function. Finally, we extend our survey to adaptin-related proteins such as the GGAs and stonins, which contain domains homologous to the adaptins.
13
Schneider, Helga, Margarita Martin, Fernando A. Agarraberes, Li Yin, Iris Rapoport, Tomas Kirchhausen та Christopher E. Rudd. "Cytolytic T Lymphocyte-Associated Antigen-4 and the TCRζ/CD3 Complex, But Not CD28, Interact with Clathrin Adaptor Complexes AP-1 and AP-2". Journal of Immunology 163, № 4 (15 серпня 1999): 1868–79. http://dx.doi.org/10.4049/jimmunol.163.4.1868.
Анотація:
Abstract The negative signaling receptor cytolytic T lymphocyte-associated Ag-4 (CTLA-4) resides primarily in intracellular compartments such as the Golgi apparatus of T cells. However, little is known regarding the molecular mechanisms that influence this accumulation. In this study, we demonstrate binding of the clathrin adaptor complex AP-1 with the GVYVKM motif of the cytoplasmic domain of CTLA-4. Binding occurred primarily in the Golgi compartment of T cells, unlike with AP-2 binding that occurs mostly with cell surface CTLA-4. Although evidence was not found to implicate AP-1 binding in the retention of CTLA-4 in the Golgi, AP-1 appears to play a role in shuttling of excess receptor from the Golgi to the lysosomal compartments for degradation. In support of this, increased CTLA-4 synthesis resulted in an increase in CTLA-4/AP-1 binding and a concomitant increase in the appearance of CTLA-4 in the lysosomal compartment. At the same time, the level of intracellular receptor was maintained at a constant level, suggesting that CTLA-4/AP-1 binding represents one mechanism to ensure steady state levels of intracellular CTLA-4 in T cells. Finally, we demonstrate that the TCRζ/CD3 complex (but not CD28) also binds to AP-1 and AP-2 complexes, thus providing a possible link between these two receptors in the regulation of T cell function.
14
Newell-Litwa, Karen, Gloria Salazar, Yoland Smith, and Victor Faundez. "Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles." Molecular Biology of the Cell 20, no. 5 (March 2009): 1441–53. http://dx.doi.org/10.1091/mbc.e08-05-0456.
Анотація:
Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.
15
Tong, Xiao, Werner Boll, Tomas Kirchhausen, and Peter M. Howley. "Interaction of the Bovine Papillomavirus E6 Protein with the Clathrin Adaptor Complex AP-1." Journal of Virology 72, no. 1 (January 1, 1998): 476–82. http://dx.doi.org/10.1128/jvi.72.1.476-482.1998.
Анотація:
ABSTRACT The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the γ subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.
16
Di Pietro, Santiago M., Juan M. Falcón-Pérez, Danièle Tenza, Subba R. G. Setty, Michael S. Marks, Graça Raposo, and Esteban C. Dell’Angelica. "BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes." Molecular Biology of the Cell 17, no. 9 (September 2006): 4027–38. http://dx.doi.org/10.1091/mbc.e06-05-0379.
Анотація:
The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.
17
Camus, Grégory, Carolina Segura-Morales, Dorothee Molle, Sandra Lopez-Vergès, Christina Begon-Pescia, Chantal Cazevieille, Peter Schu, Edouard Bertrand, Clarisse Berlioz-Torrent, and Eugenia Basyuk. "The Clathrin Adaptor Complex AP-1 Binds HIV-1 and MLV Gag and Facilitates Their Budding." Molecular Biology of the Cell 18, no. 8 (August 2007): 3193–203. http://dx.doi.org/10.1091/mbc.e06-12-1147.
Анотація:
Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1μ obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1μ enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1μ. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.
18
Pujol, François M., Vibor Laketa, Florian Schmidt, Markus Mukenhirn, Barbara Müller, Steeve Boulant, Dirk Grimm, Oliver T. Keppler, and Oliver T. Fackler. "HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites." Journal of Virology 90, no. 15 (May 11, 2016): 6709–23. http://dx.doi.org/10.1128/jvi.00504-16.
Анотація:
ABSTRACTThe host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process.IMPORTANCECD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.
19
Chakrabarti, R., M. Joly, and S. Corvera. "Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells." Journal of Cell Biology 123, no. 1 (October 1, 1993): 79–87. http://dx.doi.org/10.1083/jcb.123.1.79.
Анотація:
Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.
20
Traub, L. M., J. A. Ostrom, and S. Kornfeld. "Biochemical dissection of AP-1 recruitment onto Golgi membranes." Journal of Cell Biology 123, no. 3 (November 1, 1993): 561–73. http://dx.doi.org/10.1083/jcb.123.3.561.
Анотація:
Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.
21
Theos, Alexander C., Danièle Tenza, José A. Martina, Ilse Hurbain, Andrew A. Peden, Elena V. Sviderskaya, Abigail Stewart, et al. "Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to Melanosomes." Molecular Biology of the Cell 16, no. 11 (November 2005): 5356–72. http://dx.doi.org/10.1091/mbc.e05-07-0626.
Анотація:
Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.
22
Hirst, Jennifer, Georg H. H. Borner, James Edgar, Marco Y. Hein, Matthias Mann, Frank Buchholz, Robin Antrobus, and Margaret S. Robinson. "Interaction between AP-5 and the hereditary spastic paraplegia proteins SPG11 and SPG15." Molecular Biology of the Cell 24, no. 16 (August 15, 2013): 2558–69. http://dx.doi.org/10.1091/mbc.e13-03-0170.
Анотація:
The AP-5 complex is a recently identified but evolutionarily ancient member of the family of heterotetrameric adaptor proteins (AP complexes). It is associated with two proteins that are mutated in patients with hereditary spastic paraplegia, SPG11 and SPG15. Here we show that the four AP-5 subunits can be coimmunoprecipitated with SPG11 and SPG15, both from cytosol and from detergent-extracted membranes, with a stoichiometry of ∼1:1:1:1:1:1. Knockdowns of SPG11 or SPG15 phenocopy knockdowns of AP-5 subunits: all six knockdowns cause the cation-independent mannose 6-phosphate receptor to become trapped in clusters of early endosomes. In addition, AP-5, SPG11, and SPG15 colocalize on a late endosomal/lysosomal compartment. Both SPG11 and SPG15 have predicted secondary structures containing α-solenoids related to those of clathrin heavy chain and COPI subunits. SPG11 also has an N-terminal, β-propeller–like domain, which interacts in vitro with AP-5. We propose that AP-5, SPG15, and SPG11 form a coat-like complex, with AP-5 involved in protein sorting, SPG15 facilitating the docking of the coat onto membranes by interacting with PI3P via its FYVE domain, and SPG11 (possibly together with SPG15) forming a scaffold.
23
Rodionov, Dmitrii G., and Oddmund Bakke. "Medium Chains of Adaptor Complexes AP-1 and AP-2 Recognize Leucine-based Sorting Signals from the Invariant Chain." Journal of Biological Chemistry 273, no. 11 (March 13, 1998): 6005–8. http://dx.doi.org/10.1074/jbc.273.11.6005.
24
Puffer, Bridget A., S. C. Watkins, and Ronald C. Montelaro. "Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly." Journal of Virology 72, no. 12 (December 1, 1998): 10218–21. http://dx.doi.org/10.1128/jvi.72.12.10218-10221.1998.
Анотація:
ABSTRACT We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.
25
Larimore, Jennifer, Karine Tornieri, Pearl V. Ryder, Avanti Gokhale, Stephanie A. Zlatic, Branch Craige, Joshua D. Lee, et al. "The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse." Molecular Biology of the Cell 22, no. 24 (December 15, 2011): 4854–67. http://dx.doi.org/10.1091/mbc.e11-07-0592.
Анотація:
Dysbindin assembles into the biogenesis of lysosome-related organelles complex 1 (BLOC-1), which interacts with the adaptor protein complex 3 (AP-3), mediating a common endosome-trafficking route. Deficiencies in AP-3 and BLOC-1 affect synaptic vesicle composition. However, whether AP-3-BLOC-1–dependent sorting events that control synapse membrane protein content take place in cell bodies upstream of nerve terminals remains unknown. We tested this hypothesis by analyzing the targeting of phosphatidylinositol-4-kinase type II α (PI4KIIα), a membrane protein present in presynaptic and postsynaptic compartments. PI4KIIα copurified with BLOC-1 and AP-3 in neuronal cells. These interactions translated into a decreased PI4KIIα content in the dentate gyrus of dysbindin-null BLOC-1 deficiency and AP-3–null mice. Reduction of PI4KIIα in the dentate reflects a failure to traffic from the cell body. PI4KIIα was targeted to processes in wild-type primary cultured cortical neurons and PC12 cells but failed to reach neurites in cells lacking either AP-3 or BLOC-1. Similarly, disruption of an AP-3–sorting motif in PI4KIIα impaired its sorting into processes of PC12 and primary cultured cortical neuronal cells. Our findings indicate a novel vesicle transport mechanism requiring BLOC-1 and AP-3 complexes for cargo sorting from neuronal cell bodies to neurites and nerve terminals.
26
Wang, Ying Jie, Jing Wang, Hui Qiao Sun, Manuel Martinez, Yu Xiao Sun, Eric Macia, Tomas Kirchhausen, Joseph P. Albanesi, Michael G. Roth, and Helen L. Yin. "Phosphatidylinositol 4 Phosphate Regulates Targeting of Clathrin Adaptor AP-1 Complexes to the Golgi." Cell 114, no. 3 (August 2003): 299–310. http://dx.doi.org/10.1016/s0092-8674(03)00603-2.
27
Prandini, Alberto, Valentina Salvi, Francesca Colombo, Daniele Moratto, Luisa Lorenzi, William Vermi, Maria Antonia De Francesco, et al. "Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency." Blood 127, no. 26 (June 30, 2016): 3382–86. http://dx.doi.org/10.1182/blood-2015-06-650689.
28
Ishizaki, Ray, Hye-Won Shin, Hiroko Mitsuhashi, and Kazuhisa Nakayama. "Redundant Roles of BIG2 and BIG1, Guanine-Nucleotide Exchange Factors for ADP-Ribosylation Factors in Membrane Traffic between the trans-Golgi Network and Endosomes." Molecular Biology of the Cell 19, no. 6 (June 2008): 2650–60. http://dx.doi.org/10.1091/mbc.e07-10-1067.
Анотація:
BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.
29
Lefkir, Yaya, Marilyne Malbouyres, Daniel Gotthardt, Adrian Ozinsky, Sophie Cornillon, Franz Bruckert, Alan A. Aderem, Thierry Soldati, Pierre Cosson, and François Letourneur. "Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis." Molecular Biology of the Cell 15, no. 2 (February 2004): 861–69. http://dx.doi.org/10.1091/mbc.e03-06-0365.
Анотація:
The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.
30
Mitsunari, Takashi, Fubito Nakatsu, Noriko Shioda, Paul E. Love, Alexander Grinberg, Juan S. Bonifacino, and Hiroshi Ohno. "Clathrin Adaptor AP-2 Is Essential for Early Embryonal Development." Molecular and Cellular Biology 25, no. 21 (November 1, 2005): 9318–23. http://dx.doi.org/10.1128/mcb.25.21.9318-9323.2005.
Анотація:
ABSTRACT The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the μ2 subunit of AP-2 in the mouse. We found that μ2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no μ2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that μ2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.
31
Bonifacino, Juan S. "Adaptor proteins involved in polarized sorting." Journal of Cell Biology 204, no. 1 (January 6, 2014): 7–17. http://dx.doi.org/10.1083/jcb.201310021.
Анотація:
Polarized cells such as epithelial cells and neurons exhibit different plasma membrane domains with distinct protein compositions. Recent studies have shown that sorting of transmembrane proteins to the basolateral domain of epithelial cells and the somatodendritic domain of neurons is mediated by recognition of signals in the cytosolic domains of the proteins by adaptors. These adaptors are components of protein coats associated with the trans-Golgi network and/or recycling endosomes. The clathrin-associated adaptor protein 1 (AP-1) complex plays a preeminent role in this process, although other adaptors and coat proteins, such as AP-4, ARH, Numb, exomer, and retromer, have also been implicated.
32
Peden, Andrew A., Viola Oorschot, Boris A. Hesser, Cary D. Austin, Richard H. Scheller, and Judith Klumperman. "Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins." Journal of Cell Biology 164, no. 7 (March 29, 2004): 1065–76. http://dx.doi.org/10.1083/jcb.200311064.
Анотація:
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.
33
Nadiminti, Sravanthi S. P., Shirley B. Dixit, Neena Ratnakaran, Anushka Deb, Sneha Hegde, Sri Padma Priya Boyanapalli, Sierra Swords, Barth D. Grant та Sandhya P. Koushika. "LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α". PLOS Genetics 20, № 5 (9 травня 2024): e1011253. http://dx.doi.org/10.1371/journal.pgen.1011253.
Анотація:
Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.
34
Braun, Virginie, Chantal Deschamps, Graça Raposo, Philippe Benaroch, Alexandre Benmerah, Philippe Chavrier, and Florence Niedergang. "AP-1 and ARF1 Control Endosomal Dynamics at Sites of FcR–mediated Phagocytosis." Molecular Biology of the Cell 18, no. 12 (December 2007): 4921–31. http://dx.doi.org/10.1091/mbc.e07-04-0392.
Анотація:
Phagocytosis, the mechanism of ingestion of large material and microorganisms, relies on actin polymerization and on the focal delivery of intracellular endocytic compartments. The molecular mechanisms involved in the formation and delivery of the endocytic vesicles that are recruited at sites of phagocytosis are not well characterized. Here we show that adaptor protein (AP)-1 but not AP-2 clathrin adaptor complexes are recruited early below the sites of particle attachment and are required for efficient receptor-mediated phagocytosis in murine macrophages. Clathrin, however, is not recruited with the AP complexes. We further show that the recruitment of AP-1–positive structures at sites of phagocytosis is regulated by the GTP-binding protein ARF1 but is not sensitive to brefeldin A. Furthermore, AP-1 depletion leads to increased surface levels of TNF-α, a cargo known to traffic through the endosomes to the plasma membrane upon stimulation of the macrophages. Together, our results support a clathrin-independent role for AP complexes in endosomal dynamics in macrophages by retaining some cargo proteins, a process important for membrane remodeling during phagocytosis.
35
Martina, José A., Cecilia J. Bonangelino, Rubén C. Aguilar, and Juan S. Bonifacino. "Stonin 2." Journal of Cell Biology 153, no. 5 (May 28, 2001): 1111–20. http://dx.doi.org/10.1083/jcb.153.5.1111.
Анотація:
Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH2-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the μ subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP–stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.
36
Wyss, Stéphanie, Clarisse Berlioz-Torrent, Michael Boge, Guillaume Blot, Stefan Höning, Richard Benarous, and Markus Thali. "The Highly Conserved C-Terminal Dileucine Motif in the Cytosolic Domain of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Is Critical for Its Association with the AP-1 Clathrin Adapter." Journal of Virology 75, no. 6 (March 15, 2001): 2982–92. http://dx.doi.org/10.1128/jvi.75.6.2982-2992.2001.
Анотація:
ABSTRACT Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adapter proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adapter complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD–AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env–AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.
37
Okamoto, Curtis T., та Young Y. Jeng. "An immunologically distinct β-adaptin on tubulovesicles of gastric oxyntic cells". American Journal of Physiology-Cell Physiology 275, № 5 (1 листопада 1998): C1323—C1329. http://dx.doi.org/10.1152/ajpcell.1998.275.5.c1323.
Анотація:
Clathrin and the γ-adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 ( Cell Physiol. 43): C1017–C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the γ-, μ1-, and ς1-adaptin subunits. Although the putative β-adaptin subunit in this fraction is not immunoreactive with the anti-β-adaptin monoclonal antibody (MAb), this β-adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val640, a region conserved between β1- and β2-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti-γ-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the β-adaptin that did not react with the anti-β-adaptin MAb but did react with the anti-β-adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a β-adaptin that was recognized by both the anti-β-adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct β-adaptin. This immunologically distinct β-adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.
38
Mitchell, Richard S., Rittik Chaudhuri, O. Wolf Lindwasser, Kristie A. Tanaka, David Lau, Rudy Murillo, Juan S. Bonifacino, and John C. Guatelli. "Competition Model for Upregulation of the Major Histocompatibility Complex Class II-Associated Invariant Chain by Human Immunodeficiency Virus Type 1 Nef." Journal of Virology 82, no. 16 (June 4, 2008): 7758–67. http://dx.doi.org/10.1128/jvi.02668-07.
Анотація:
ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL165, was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI8 and EQLPML17, were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.
39
Almomani, Ensaf Y., Jennifer C. King, Janjuree Netsawang, Pa-Thai Yenchitsomanus, Prida Malasit, Thawornchai Limjindaporn, R. Todd Alexander, and Emmanuelle Cordat. "Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells." American Journal of Physiology-Cell Physiology 303, no. 5 (September 1, 2012): C554—C566. http://dx.doi.org/10.1152/ajpcell.00124.2012.
Анотація:
Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85–91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.
40
Zhu, Yunxiang, Linton M. Traub, and Stuart Kornfeld. "ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes." Molecular Biology of the Cell 9, no. 6 (June 1998): 1323–37. http://dx.doi.org/10.1091/mbc.9.6.1323.
Анотація:
Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.
41
Ghosh, Pradipta, and Stuart Kornfeld. "AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation." Journal of Cell Biology 160, no. 5 (February 25, 2003): 699–708. http://dx.doi.org/10.1083/jcb.200211080.
Анотація:
The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its β1 and μ1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)–mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated μ1. Once on the membrane, the μ1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of μ1 (and μ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70–mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
42
Lindwasser, O. Wolf, William J. Smith, Rittik Chaudhuri, Peter Yang, James H. Hurley, and Juan S. Bonifacino. "A Diacidic Motif in Human Immunodeficiency Virus Type 1 Nef Is a Novel Determinant of Binding to AP-2." Journal of Virology 82, no. 3 (November 21, 2007): 1166–74. http://dx.doi.org/10.1128/jvi.01874-07.
Анотація:
ABSTRACT A key function of the Nef protein of immunodeficiency viruses is the downregulation of the T-cell and macrophage coreceptor, CD4, from the surfaces of infected cells. CD4 downregulation depends on a conserved (D/E)XXXL(L/I)-type dileucine motif in the C-terminal, flexible loop of Nef, which mediates binding to the clathrin adaptor complexes AP-1, AP-2, and AP-3. We now report the identification of a consensus (D/E)D motif within this loop as a second, conserved determinant of interaction of Nef with AP-2, though not with AP-1 and AP-3. Mutations in this diacidic motif abrogate both AP-2 binding and CD4 downregulation. We also show that a dileucine motif from tyrosinase, both in its native context and in the context of Nef, can bind to AP-2 independently of a diacidic motif. These results thus identify a novel type of AP-2 interaction determinant, support the notion that AP-2 is the key clathrin adaptor for the downregulation of CD4 by Nef, and reveal a previously unrecognized diversity among dileucine sorting signals.
43
Meyer, Daniel M., Pascal Crottet, Bohumil Maco, Elena Degtyar, Dan Cassel, and Martin Spiess. "Oligomerization and Dissociation of AP-1 Adaptors Are Regulated by Cargo Signals and by ArfGAP1-induced GTP Hydrolysis." Molecular Biology of the Cell 16, no. 10 (October 2005): 4745–54. http://dx.doi.org/10.1091/mbc.e05-06-0568.
Анотація:
The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1·GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 “GTPase timer.” In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1·GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.
44
Coleman, Scott H., Ricardo Madrid, Nanette Van Damme, Richard S. Mitchell, Jerome Bouchet, Cecile Servant, Satish Pillai, Serge Benichou, and John C. Guatelli. "Modulation of Cellular Protein Trafficking by Human Immunodeficiency Virus Type 1 Nef: Role of the Acidic Residue in the ExxxLL Motif." Journal of Virology 80, no. 4 (February 15, 2006): 1837–49. http://dx.doi.org/10.1128/jvi.80.4.1837-1849.2006.
Анотація:
ABSTRACT The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E160xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins.
45
Suckow, Arthur T., Branch Craige, Victor Faundez, William J. Cain та Steven D. Chessler. "An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet β-cells". American Journal of Physiology-Endocrinology and Metabolism 299, № 1 (липень 2010): E23—E32. http://dx.doi.org/10.1152/ajpendo.00664.2009.
Анотація:
Pancreatic islet β-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since β-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in β-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates β-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 β-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits β3B and μ3B are expressed in β-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that β-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to β-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in β-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 δ-subunit expression. Our findings suggest that β-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.
46
Janvier, Katy, Yukio Kato, Markus Boehm, Jeremy R. Rose, José A. Martina, Bong-Yoon Kim, Sundararajan Venkatesan та Juan S. Bonifacino. "Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes". Journal of Cell Biology 163, № 6 (22 грудня 2003): 1281–90. http://dx.doi.org/10.1083/jcb.200307157.
Анотація:
The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3.
47
Fessart, Delphine, May Simaan та Stéphane A. Laporte. "c-Src Regulates Clathrin Adapter Protein 2 Interaction with β-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization". Molecular Endocrinology 19, № 2 (1 лютого 2005): 491–503. http://dx.doi.org/10.1210/me.2004-0246.
Анотація:
Abstract β-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to β-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, β-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between β-arrestin2 and the β-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both β-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and β-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and β-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.
48
Crottet, Pascal, Daniel M. Meyer, Jack Rohrer, and Martin Spiess. "ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes." Molecular Biology of the Cell 13, no. 10 (October 2002): 3672–82. http://dx.doi.org/10.1091/mbc.e02-05-0309.
Анотація:
At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation.
49
Janvier, Katy, and Juan S. Bonifacino. "Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins." Molecular Biology of the Cell 16, no. 9 (September 2005): 4231–42. http://dx.doi.org/10.1091/mbc.e05-03-0213.
Анотація:
The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.
50
Roeth, Jeremiah F., Maya Williams, Matthew R. Kasper, Tracey M. Filzen, and Kathleen L. Collins. "HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail." Journal of Cell Biology 167, no. 5 (November 29, 2004): 903–13. http://dx.doi.org/10.1083/jcb.200407031.
Анотація:
To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the μ1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef–MHC-I complex is an important step required for inhibition of antigen presentation by HIV.