Articoli di riviste: "AP-1 adaptor complex"

1

Yeung, Bonny G., Huan L. Phan e Gregory S. Payne. "Adaptor Complex-independent Clathrin Function in Yeast". Molecular Biology of the Cell 10, n. 11 (novembre 1999): 3643–59. http://dx.doi.org/10.1091/mbc.10.11.3643.

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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, n. 9 (settembre 2006): 4014–26. http://dx.doi.org/10.1091/mbc.e06-02-0103.

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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

Tong, Xiao, Werner Boll, Tomas Kirchhausen e Peter M. Howley. "Interaction of the Bovine Papillomavirus E6 Protein with the Clathrin Adaptor Complex AP-1". Journal of Virology 72, n. 1 (1 gennaio 1998): 476–82. http://dx.doi.org/10.1128/jvi.72.1.476-482.1998.

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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.

4

Camus, Grégory, Carolina Segura-Morales, Dorothee Molle, Sandra Lopez-Vergès, Christina Begon-Pescia, Chantal Cazevieille, Peter Schu, Edouard Bertrand, Clarisse Berlioz-Torrent e Eugenia Basyuk. "The Clathrin Adaptor Complex AP-1 Binds HIV-1 and MLV Gag and Facilitates Their Budding". Molecular Biology of the Cell 18, n. 8 (agosto 2007): 3193–203. http://dx.doi.org/10.1091/mbc.e06-12-1147.

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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.

5

Lefkir, Yaya, Marilyne Malbouyres, Daniel Gotthardt, Adrian Ozinsky, Sophie Cornillon, Franz Bruckert, Alan A. Aderem, Thierry Soldati, Pierre Cosson e François Letourneur. "Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis". Molecular Biology of the Cell 15, n. 2 (febbraio 2004): 861–69. http://dx.doi.org/10.1091/mbc.e03-06-0365.

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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.

6

Zlatic, Stephanie A., Emily J. Grossniklaus, Pearl V. Ryder, Gloria Salazar, Alexa L. Mattheyses, Andrew A. Peden e Victor Faundez. "Chemical-genetic disruption of clathrin function spares adaptor complex 3–dependent endosome vesicle biogenesis". Molecular Biology of the Cell 24, n. 15 (agosto 2013): 2378–88. http://dx.doi.org/10.1091/mbc.e12-12-0860.

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A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.

7

Peden, Andrew A., Viola Oorschot, Boris A. Hesser, Cary D. Austin, Richard H. Scheller e Judith Klumperman. "Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins". Journal of Cell Biology 164, n. 7 (29 marzo 2004): 1065–76. http://dx.doi.org/10.1083/jcb.200311064.

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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.

8

Duncan, Mara Colleen. "Regulation of clathrin adaptor protein complex‐1(AP‐1) by a Laa1 protein containing complex". FASEB Journal 34, S1 (aprile 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.00639.

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9

Bonifacino, Juan S. "Adaptor proteins involved in polarized sorting". Journal of Cell Biology 204, n. 1 (6 gennaio 2014): 7–17. http://dx.doi.org/10.1083/jcb.201310021.

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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.

10

Traub, L. M., J. A. Ostrom e S. Kornfeld. "Biochemical dissection of AP-1 recruitment onto Golgi membranes." Journal of Cell Biology 123, n. 3 (1 novembre 1993): 561–73. http://dx.doi.org/10.1083/jcb.123.3.561.

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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.

11

Fölsch, Heike, Marc Pypaert, Peter Schu e Ira Mellman. "Distribution and Function of Ap-1 Clathrin Adaptor Complexes in Polarized Epithelial Cells". Journal of Cell Biology 152, n. 3 (5 febbraio 2001): 595–606. http://dx.doi.org/10.1083/jcb.152.3.595.

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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.

12

Okamoto, Curtis T., e Young Y. Jeng. "An immunologically distinct β-adaptin on tubulovesicles of gastric oxyntic cells". American Journal of Physiology-Cell Physiology 275, n. 5 (1 novembre 1998): C1323—C1329. http://dx.doi.org/10.1152/ajpcell.1998.275.5.c1323.

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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.

13

Craige, Branch, Gloria Salazar e Victor Faundez. "Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3–sorting Motif and a Kinase Domain That Are Both Required for Endosome Traffic". Molecular Biology of the Cell 19, n. 4 (aprile 2008): 1415–26. http://dx.doi.org/10.1091/mbc.e07-12-1239.

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The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II α (PI4KIIα) is one of several proteins possessing catalytic domains that regulate AP-3–dependent sorting. Here we present evidence that PI4KIIα uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIα form a complex that requires a dileucine-sorting motif present in PI4KIIα. Mutagenesis of either the PI4KIIα-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIα to LAMP-1–positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIα are necessary to rescue endosomal PI4KIIα siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

14

Zhu, Yunxiang, Linton M. Traub e 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, n. 6 (giugno 1998): 1323–37. http://dx.doi.org/10.1091/mbc.9.6.1323.

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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.

15

Ooi, Chean Eng, Esteban C. Dell'Angelica e Juan S. Bonifacino. "ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes". Journal of Cell Biology 142, n. 2 (27 luglio 1998): 391–402. http://dx.doi.org/10.1083/jcb.142.2.391.

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Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

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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, n. 26 (30 giugno 2016): 3382–86. http://dx.doi.org/10.1182/blood-2015-06-650689.

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Čopič, Alenka, Trevor L. Starr e Randy Schekman. "Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast". Molecular Biology of the Cell 18, n. 5 (maggio 2007): 1803–15. http://dx.doi.org/10.1091/mbc.e06-11-1000.

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The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

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Sorkina, T., A. Bild, F. Tebar e A. Sorkin. "Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors". Journal of Cell Science 112, n. 3 (1 febbraio 1999): 317–27. http://dx.doi.org/10.1242/jcs.112.3.317.

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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.

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BAROIS, Nicolas, e Oddmund BAKKE. "The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study". Biochemical Journal 385, n. 2 (7 gennaio 2005): 503–10. http://dx.doi.org/10.1042/bj20041010.

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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.

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Scheele, Urte, Christoph Kalthoff e Ernst Ungewickell. "Multiple Interactions of Auxilin 1 with Clathrin and the AP-2 Adaptor Complex". Journal of Biological Chemistry 276, n. 39 (24 luglio 2001): 36131–38. http://dx.doi.org/10.1074/jbc.m106511200.

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21

Sawasdee, Nunghathai, Mutita Junking, Piengpaga Ngaojanlar, Nattakan Sukomon, Duangporn Ungsupravate, Thawornchai Limjindaporn, Varaporn Akkarapatumwong, Sansanee Noisakran e Pa-thai Yenchitsomanus. "Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)". Biochemical and Biophysical Research Communications 401, n. 1 (ottobre 2010): 85–91. http://dx.doi.org/10.1016/j.bbrc.2010.09.015.

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22

Zhu, Yunxiang, Linton M. Traub e Stuart Kornfeld. "High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors". Molecular Biology of the Cell 10, n. 3 (marzo 1999): 537–49. http://dx.doi.org/10.1091/mbc.10.3.537.

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Abstract (sommario):

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

23

Ghosh, Pradipta, e Stuart Kornfeld. "AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation". Journal of Cell Biology 160, n. 5 (25 febbraio 2003): 699–708. http://dx.doi.org/10.1083/jcb.200211080.

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Abstract (sommario):

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.

24

Fölsch, Heike, Marc Pypaert, Sandra Maday, Laurence Pelletier e Ira Mellman. "The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains". Journal of Cell Biology 163, n. 2 (27 ottobre 2003): 351–62. http://dx.doi.org/10.1083/jcb.200309020.

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Abstract (sommario):

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.

25

Newell-Litwa, Karen, Gloria Salazar, Yoland Smith e Victor Faundez. "Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles". Molecular Biology of the Cell 20, n. 5 (marzo 2009): 1441–53. http://dx.doi.org/10.1091/mbc.e08-05-0456.

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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.

26

Margeta, M. A., G. J. Wang e K. Shen. "Clathrin adaptor AP-1 complex excludes multiple postsynaptic receptors from axons in C. elegans". Proceedings of the National Academy of Sciences 106, n. 5 (21 gennaio 2009): 1632–37. http://dx.doi.org/10.1073/pnas.0812078106.

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27

Bian, Jingwei, Yuzhong Zhu, Panhui Tian, Qiqi Yang e Zijian Li. "Adaptor protein HIP-55 promotes macrophage M1 polarization through promoting AP-1 complex activation". Cellular Signalling 117 (maggio 2024): 111124. http://dx.doi.org/10.1016/j.cellsig.2024.111124.

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28

Foote, Christopher, e Steven F. Nothwehr. "The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast". Journal of Cell Biology 173, n. 4 (15 maggio 2006): 615–26. http://dx.doi.org/10.1083/jcb.200510161.

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Abstract (sommario):

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F→A)–alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F→A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of ∼60 min. Delivery of A(F→A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F→A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F→A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN→EE→PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F→A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.

29

Roeth, Jeremiah F., Maya Williams, Matthew R. Kasper, Tracey M. Filzen e 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, n. 5 (29 novembre 2004): 903–13. http://dx.doi.org/10.1083/jcb.200407031.

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Abstract (sommario):

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.

30

Lefkir, Yaya, Benoît de Chassey, Annick Dubois, Aleksandra Bogdanovic, Rebecca J. Brady, Olivier Destaing, Franz Bruckert, Theresa J. O'Halloran, Pierre Cosson e 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, n. 5 (maggio 2003): 1835–51. http://dx.doi.org/10.1091/mbc.e02-10-0627.

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Abstract (sommario):

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.

31

Di Pietro, Santiago M., Juan M. Falcón-Pérez, Danièle Tenza, Subba R. G. Setty, Michael S. Marks, Graça Raposo e 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, n. 9 (settembre 2006): 4027–38. http://dx.doi.org/10.1091/mbc.e06-05-0379.

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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.

32

Doray, Balraj, e Stuart Kornfeld. "γ Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly". Molecular Biology of the Cell 12, n. 7 (luglio 2001): 1925–35. http://dx.doi.org/10.1091/mbc.12.7.1925.

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Abstract (sommario):

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from thetrans-Golgi network (TGN). The β1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the β chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the γ subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the γ hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the γ subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-γ hinge, but not by GST-γ appendage. However, the hinge and appendage domains of γ function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the γ and β1 subunits of AP-1.

33

Keyel, Peter A., James R. Thieman, Robyn Roth, Elif Erkan, Eric T. Everett, Simon C. Watkins, John E. Heuser e Linton M. Traub. "The AP-2 Adaptor β2 Appendage Scaffolds Alternate Cargo Endocytosis". Molecular Biology of the Cell 19, n. 12 (dicembre 2008): 5309–26. http://dx.doi.org/10.1091/mbc.e08-07-0712.

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Abstract (sommario):

The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites.

34

Morris, Kyle L., Cosmo Buffalo, Xuefeng Ren e James H. Hurley. "High Resolution cryo-EM Structure of a HIV Nef-Inhibited AP-1 Clathrin Adaptor Complex". Biophysical Journal 114, n. 3 (febbraio 2018): 163a. http://dx.doi.org/10.1016/j.bpj.2017.11.913.

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35

Ren, Xuefeng, Ginny G. Farías, Bertram J. Canagarajah, Juan S. Bonifacino e James H. Hurley. "Structural Basis for Recruitment and Activation of the AP-1 Clathrin Adaptor Complex by Arf1". Cell 152, n. 4 (febbraio 2013): 755–67. http://dx.doi.org/10.1016/j.cell.2012.12.042.

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36

Fujita, Hideaki, Masayo Saeki, Kumiko Yasunaga, Tadashi Ueda, Taiji Imoto e Masaru Himeno. "In VitroBinding Study of Adaptor Protein Complex (AP-1) to Lysosomal Targeting Motif (LI-Motif)". Biochemical and Biophysical Research Communications 255, n. 1 (febbraio 1999): 54–58. http://dx.doi.org/10.1006/bbrc.1998.0140.

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37

Schneider, Helga, Margarita Martin, Fernando A. Agarraberes, Li Yin, Iris Rapoport, Tomas Kirchhausen e 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, n. 4 (15 agosto 1999): 1868–79. http://dx.doi.org/10.4049/jimmunol.163.4.1868.

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Abstract (sommario):

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.

38

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, n. 24 (15 dicembre 2011): 4854–67. http://dx.doi.org/10.1091/mbc.e11-07-0592.

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Abstract (sommario):

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.

39

Burgess, Jason, Miluska Jauregui, Julie Tan, Janet Rollins, Sylvie Lallet, Peter A. Leventis, Gabrielle L. Boulianne et al. "AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila". Molecular Biology of the Cell 22, n. 12 (15 giugno 2011): 2094–105. http://dx.doi.org/10.1091/mbc.e11-01-0054.

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Abstract (sommario):

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

40

Pujol, François M., Vibor Laketa, Florian Schmidt, Markus Mukenhirn, Barbara Müller, Steeve Boulant, Dirk Grimm, Oliver T. Keppler e Oliver T. Fackler. "HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites". Journal of Virology 90, n. 15 (11 maggio 2016): 6709–23. http://dx.doi.org/10.1128/jvi.00504-16.

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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.

41

Hirst, Jennifer, Georg H. H. Borner, James Edgar, Marco Y. Hein, Matthias Mann, Frank Buchholz, Robin Antrobus e Margaret S. Robinson. "Interaction between AP-5 and the hereditary spastic paraplegia proteins SPG11 and SPG15". Molecular Biology of the Cell 24, n. 16 (15 agosto 2013): 2558–69. http://dx.doi.org/10.1091/mbc.e13-03-0170.

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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.

42

Chen, Buxin, David P. Siderovski, Richard R. Neubig, Mark A. Lawson e JoAnn Trejo. "Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins". Journal of Biological Chemistry 289, n. 3 (2 dicembre 2013): 1580–91. http://dx.doi.org/10.1074/jbc.m113.528273.

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The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 420AKKAA424 mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

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Martina, José A., Cecilia J. Bonangelino, Rubén C. Aguilar e Juan S. Bonifacino. "Stonin 2". Journal of Cell Biology 153, n. 5 (28 maggio 2001): 1111–20. http://dx.doi.org/10.1083/jcb.153.5.1111.

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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.

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Lubben, Nienke B., Daniela A. Sahlender, Alison M. Motley, Paul J. Lehner, Philippe Benaroch e Margaret S. Robinson. "HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2". Molecular Biology of the Cell 18, n. 9 (settembre 2007): 3351–65. http://dx.doi.org/10.1091/mbc.e07-03-0218.

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Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2–depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment.

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Huizing, Marjan, Rangaprasad Sarangarajan, Erin Strovel, Yang Zhao, William A. Gahl e Raymond E. Boissy. "AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes". Molecular Biology of the Cell 12, n. 7 (luglio 2001): 2075–85. http://dx.doi.org/10.1091/mbc.12.7.2075.

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Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the β3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the β3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3–deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

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Almomani, Ensaf Y., Jennifer C. King, Janjuree Netsawang, Pa-Thai Yenchitsomanus, Prida Malasit, Thawornchai Limjindaporn, R. Todd Alexander e 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, n. 5 (1 settembre 2012): C554—C566. http://dx.doi.org/10.1152/ajpcell.00124.2012.

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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.

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Jakob, Viktor, Alexander Schreiner, Ritva Tikkanen e Anna Starzinski-Powitz. "Targeting of Transmembrane Protein Shrew-1 to Adherens Junctions Is Controlled by Cytoplasmic Sorting Motifs". Molecular Biology of the Cell 17, n. 8 (agosto 2006): 3397–408. http://dx.doi.org/10.1091/mbc.e05-11-1034.

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We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that μ1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms.

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Noviello, Colleen M., Serge Benichou e John C. Guatelli. "Cooperative Binding of the Class I Major Histocompatibility Complex Cytoplasmic Domain and Human Immunodeficiency Virus Type 1 Nef to the Endosomal AP-1 Complex via Its μ Subunit". Journal of Virology 82, n. 3 (5 dicembre 2007): 1249–58. http://dx.doi.org/10.1128/jvi.00660-07.

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ABSTRACT Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the μ but not the γ/σ subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical μ-binding motif. These data identify the μ subunit of AP-1 (μ1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with μ1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based μ-binding signal in the MHC-I CD by AP-1.

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Vince, James E., Dedreia L. Tull, Timothy Spurck, Merran C. Derby, Geoffrey I. McFadden, Paul A. Gleeson, Suzanne Gokool e Malcolm J. McConville. "Leishmania Adaptor Protein-1 Subunits Are Required for Normal Lysosome Traffic, Flagellum Biogenesis, Lipid Homeostasis, and Adaptation to Temperatures Encountered in the Mammalian Host". Eukaryotic Cell 7, n. 8 (30 maggio 2008): 1256–67. http://dx.doi.org/10.1128/ec.00090-08.

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ABSTRACT The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite Leishmania mexicana mexicana, the AP-1 μ1 and σ1 subunits are not required for growth at 27°C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence. The μ1 and σ1 subunits were localized to late Golgi and endosome membranes of the major parasite stages. Parasite mutants lacking either AP-1 subunit lacked obvious defects in Golgi structure, endocytosis, or exocytic transport. However, these mutants displayed reduced rates of endosome-to-lysosome transport and accumulated fragmented, sterol-rich lysosomes. Defects in flagellum biogenesis were also evident in nondividing promastigote stages, and this phenotype was exacerbated by inhibitors of sterol and sphingolipid biosynthesis. Furthermore, both AP-1 mutants were hypersensitive to elevated temperature and perturbations in membrane lipid composition. The pleiotropic requirements for AP-1 in membrane trafficking and temperature stress responses explain the loss of virulence of these mutants in the mammalian host.

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Ishizaki, Ray, Hye-Won Shin, Hiroko Mitsuhashi e 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, n. 6 (giugno 2008): 2650–60. http://dx.doi.org/10.1091/mbc.e07-10-1067.

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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.

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