The Experts below are selected from a list of 342 Experts worldwide ranked by ideXlab platform
Kazuhisa Nakayama - One of the best experts on this subject based on the ideXlab platform.
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Molecular Basis for Autoregulatory Interaction Between GAE Domain and Hinge Region of GGA1
Traffic, 2007Co-Authors: Michio Inoue, Tomoo Shiba, Kentaro Ihara, Yusuke Yamada, Satoshi Hirano, Hironari Kamikubo, Mikio Kataoka, Masato Kawasaki, Ryuichi Kato, Kazuhisa NakayamaAbstract:Golgi-localizing, Gamma-Adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The Gamma-Adaptin ear (GAE) domain of GGAs and the Gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that Gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.
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The structure and function of GGAs, the traffic controllers at the TGN sorting crossroads
Cell structure and function, 2003Co-Authors: Kazuhisa Nakayama, Soichi WakatsukiAbstract:GGAs (Golgi-localizing, Gamma-Adaptin ear homology domain, ARF-binding proteins) are a family of monomeric clathrin adaptor proteins that are conserved from yeasts to humans. Data published during the past four years have provided detailed pictures of the localization, domain organization and structure-function relationships of GGAs. GGAs possess four conserved functional domains, each of which interacts with cargo proteins including mannose 6-phosphate receptors, the small GTPase ARF, clathrin, or accessory proteins including Rabaptin-5 and Gamma-synergin. Together with or independent of the adaptor protein complex AP-1, GGAs regulate selective transport of cargo proteins, such as mannose 6-phosphate receptors, from the trans-Golgi network to endosomes mediated by clathrin-coated vesicles.
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GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors
Biochemical Journal, 2002Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Kyoko Toda, Kazuhisa NakayamaAbstract:ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1-ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1-ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1-GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, Gamma-Adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.
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Similar Subunit Interactions Contribute to Assembly of Clathrin Adaptor Complexes and COPI Complex: Analysis Using Yeast Three-Hybrid System
Biochemical and biophysical research communications, 2001Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Mutsumi Futatsumori, Yusaku Yoshida, Hye-won Shin, Kazuhisa NakayamaAbstract:Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, Gamma-Adaptin and beta1-Adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-Adaptin and beta4-Adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, Gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions.
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Adaptor γ Ear Homology Domain Conserved in γ-Adaptin and GGA Proteins That Interact with γ-Synergin
Biochemical and biophysical research communications, 2000Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Kazuhisa NakayamaAbstract:We identified a novel family of proteins that have a VHS domain and an AGEH (adaptor Gamma ear homology) domain that is homologous to the ear domain of the Gamma-Adaptin subunit of the AP-1 clathrin adaptor. When overexpressed, the proteins, called GGA1, GGA2, and GGA3, localized to the trans-Golgi network (TGN) and often caused fragmentation and vacuolation of the compartment. Yeast two-hybrid analysis showed that the AGEH domains of the GGA proteins as well as those of Gamma-Adaptins are able to interact with Gamma-synergin, which was previously shown to localized in the TGN region and interact with Gamma-Adaptin. Furthermore, Gamma-synergin and either of the GGA proteins coexpressed were colocalized in the TGN region. These results suggest that the GGA proteins regulate the function of the TGN or membrane trafficking from this compartment and that the AGEH domains of GGAs and Gamma-Adaptins, like the ear domain of alpha-Adaptin, are involved in interaction with molecules that modulate their functions.
F. Letourneur - One of the best experts on this subject based on the ideXlab platform.
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Acidic clusters target transmembrane proteins to the contractile vacuole in Dictyostelium cells.
Journal of cell science, 2006Co-Authors: V. Mercanti, C. Blanc, Y. Lefkir, P. Cosson, F. LetourneurAbstract:The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the Gamma-Adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.
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Acidic clusters target transmembrane proteins to the contractile vacuole in Dictyostelium cells.
Journal of Cell Science, 2006Co-Authors: V. Mercanti, C. Blanc, Y. Lefkir, P. Cosson, F. LetourneurAbstract:The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the Gamma-Adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the Gamma-Adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.
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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, 2003Co-Authors: Y. Lefkir, P. Cosson, B. Dechassey, André Dubois, A. Bogdanovic, Rj Brady, O. Destaing, F. Bruckert, Tj O'halloran, F. LetourneurAbstract: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 Gamma-Adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing Gamma-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.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 Gamma-Adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing Gamma-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.
Juan S. Bonifacino - One of the best experts on this subject based on the ideXlab platform.
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Definition of the Consensus Motif Recognized by γ-Adaptin Ear Domains
The Journal of biological chemistry, 2003Co-Authors: Rafael Mattera, Brigitte Ritter, Sachdev S. Sidhu, Peter S. Mcpherson, Juan S. BonifacinoAbstract:The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, Gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or Gamma-Adaptin ear (GAE) domains) of two Gamma-Adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.
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RECOGNITION OF ACCESSORY PROTEIN MOTIFS BY THE Gamma-Adaptin EAR DOMAIN OF GGA3
Nature structural biology, 2003Co-Authors: Gregory J. Miller, Juan S. Bonifacino, Rafael Mattera, James H. HurleyAbstract:Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The Gamma-Adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 A. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.
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Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin
Molecular and cellular biology, 2001Co-Authors: Chris Mullins, Juan S. BonifacinoAbstract:The GGAs (Golgi-localized, Gamma-ear-containing, ARF-binding proteins) are ubiquitous adaptor-like proteins that associate with the cytoplasmic face of the trans-Golgi network (TGN) (4, 16, 22, 37, 47). Three GGAs exist in humans (GGA1, GGA2, and GGA3), one each exists in Drosophila melanogaster and Caenorhabditis elegans, and two exist in the budding yeast Saccharomyces cerevisiae (Gga1p and Gga2p). The GGAs are monomeric but have a multidomain structure consisting of VHS (Vps27, Hrs, and STAM), GAT (GGA and TOM), hinge, and GAE (Gamma-Adaptin ear) domains. Biochemical and immunocytochemical analyses have revealed that each of the four GGA domains serves a specific function. The VHS domain of the human GGAs functions as a recognition module for acidic cluster-dileucine sorting signals contained within the cytosolic tails of sortilin (31) and the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) that sort lysosomal hydrolases to lysosomes (38, 56). The GAT domain of human and yeast GGAs mediates interactions with the GTP-bound form of members of the ARF (ADP-ribosylation factor) family of proteins (4, 16, 55). GAT-ARF interactions are responsible for the regulated recruitment of the GGAs from the cytosol to the Golgi complex (4, 16, 39). The hinge domain of all the GGAs contains putative clathrin-binding motifs composed of acidic and bulky hydrophobic amino acids (16, 39). For the human GGAs, these motifs have been shown to mediate interactions with clathrin in vitro and to promote recruitment of clathrin to the TGN in vivo (39). Finally, the GAE domain of the human GGAs binds proteins such as γ-synergin and rabaptin-5, which may function to regulate assembly of GGA-containing coats or formation of coated vesicular carriers (22, 47). These properties of the human GGA domains indicate that they may mediate ARF-dependent recruitment of clathrin to the TGN in order to sort intracellular cargo receptors from the TGN to the endosomal system. Despite the detailed characterization of the properties of GGA domains, the significance of these properties for the function of the GGAs in vivo remains to be assessed. The existence of two GGAs in yeast provides an opportunity to perform analyses of the physiological roles of the GGAs in an organism easily amenable to genetic manipulation. In yeast, biosynthetic protein sorting from the Golgi complex to the vacuole, the equivalent of the mammalian lysosome, is mediated by two principal routes (for reviews, see references 7, 12, 26, and 29). The alkaline phosphatase (ALP) pathway sorts vacuolar proteins such as ALP and the t-SNARE Vam3p from the Golgi complex to the vacuole directly. Formation of Golgi complex-derived carrier vesicles and transport through this pathway require the adaptor protein (AP) complex AP-3 (14, 46) and the VPS (vacuolar protein sorting) gene products Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (32, 49), a member of the dynamin family of proteins (49). In contrast, the carboxypeptidase Y (CPY) pathway sorts vacuolar proteins including CPY and proteinase A (PrA) and subunits of the vacuolar ATPase from the Golgi complex to the vacuole via a prevacuolar endosomal compartment (PVC). Sorting of both CPY and PrA through this pathway is mediated by interactions with the transmembrane receptor Vps10p, which delivers these proteins to the PVC prior to recycling back to the Golgi complex (13, 27). Vesicle formation and transport in the CPY pathway involve the coat-scaffolding protein clathrin (10, 45), the putative aminophospholipid translocase Drs2p (11), and the synaptojanin-like proteins Inp52p and Inp53p (1, 33), as well as numerous VPS gene products, including Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (1, 49). Mutations in factors operating in these respective pathways result, to differing degrees, in impaired vacuolar sorting and defects in vacuole biogenesis. The yeast GGAs appear to play important, redundant roles in biosynthetic sorting to the vacuole based on studies of a mutant gga1Δ gga2Δ strain containing disruptions of both yeast GGA genes. This mutant is defective for transport of pro-CPY to the vacuole and missorts pro-CPY to the periplasmic space (16, 22, 55). The gga1Δ gga2Δ mutant was also found to be defective in sorting of the syntaxin Pep12p from the Golgi complex to late endosomes (2). The presence of a vacuolar morphology defect in gga1Δ gga2Δ cells has, however, been debated, with one study reporting abnormal morphology (22) and another showing no morphological differences between wild-type and gga1Δ gga2Δ strains (55). As is evident from the well-defined phenotypes arising from mutations in the yeast GGA genes, these proteins appear to play an important role in biosynthetic protein sorting. To expand our understanding of the GGAs' function and to assess the relative importance of the different GGA domains in vivo, we have performed a structure-function analysis of yeast Gga1p and Gga2p. First, we elaborate on the gga1Δ gga2Δ mutant phenotype by reporting defects in sorting of additional vacuolar proteins and abnormal vacuolar morphology and a strong defect in maturation of the mating pheromone α-factor. We then analyze the functional requirement of individual GGA domains and find that the VHS, GAT, and hinge domains are important for GGA-mediated pro-CPY sorting and pro-α-factor processing, while the GAE domain appears less important. In addition, we show that, like their human counterparts, the yeast GGAs are capable of binding clathrin via acidic-bulky-hydrophobic motifs in their hinge domains. We also present evidence that these clathrin-binding motifs contribute to GGA-mediated sorting in vivo. Finally, mutational analysis of the Gga2p VHS domain identifies a highly conserved sequence important for this protein's function.
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Altered expression of a novel Adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet
The EMBO journal, 1997Co-Authors: Chean Eng Ooi, Esteban C. Dell'angelica, Jorge E. Moreira, George Poy, David A. Wassarman, Juan S. BonifacinoAbstract:Drosophila eye pigmentation defects have thus far been attributed to mutations in genes encoding enzymes required for biosynthesis of pigments and to ABC-type membrane transporters for pigments or their precursors. We report here that a defect in a gene encoding a putative coat adaptor protein leads to the eye color defect of garnet mutants. We first identified a human cDNA encoding delta-Adaptin, a structural homolog of the alpha- and Gamma-Adaptin subunits of the clathrin coat adaptors AP-1 and AP-2, respectively. Biochemical analyses demonstrated that delta-Adaptin is a component of the adaptor-like complex AP-3 in human cells. We then isolated a full-length cDNA encoding the Drosophila ortholog of delta-Adaptin and found that transcripts specified by this cDNA are altered in garnet mutant flies. Examination by light and electron microscopy indicated that these mutant flies have reduced numbers of eye pigment granules, which correlates with decreased levels of both pteridine (red) and ommachrome (brown) pigments. Thus, the eye pigmentation defect in the Drosophila garnet mutant may be attributed to compromised function of a coat protein involved in intracellular transport processes required for biogenesis or function of pigment granules.
Hugh R B Pelham - One of the best experts on this subject based on the ideXlab platform.
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a selective transport route from golgi to late endosomes that requires the yeast gga proteins
Journal of Cell Biology, 2000Co-Authors: Michael W Black, Hugh R B PelhamAbstract:Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes. By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to Gamma Adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.
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A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins
2000Co-Authors: Michael W Black, Hugh R B PelhamAbstract:Abstract. Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes. By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to Gamma Adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes. Key words: clathrin • endosomes • GGA proteins
Hiroyuki Takatsu - One of the best experts on this subject based on the ideXlab platform.
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GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors
Biochemical Journal, 2002Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Kyoko Toda, Kazuhisa NakayamaAbstract:ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1-ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1-ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1-GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, Gamma-Adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.
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Similar Subunit Interactions Contribute to Assembly of Clathrin Adaptor Complexes and COPI Complex: Analysis Using Yeast Three-Hybrid System
Biochemical and biophysical research communications, 2001Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Mutsumi Futatsumori, Yusaku Yoshida, Hye-won Shin, Kazuhisa NakayamaAbstract:Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, Gamma-Adaptin and beta1-Adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-Adaptin and beta4-Adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, Gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions.
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Adaptor γ Ear Homology Domain Conserved in γ-Adaptin and GGA Proteins That Interact with γ-Synergin
Biochemical and biophysical research communications, 2000Co-Authors: Hiroyuki Takatsu, Kaori Yoshino, Kazuhisa NakayamaAbstract:We identified a novel family of proteins that have a VHS domain and an AGEH (adaptor Gamma ear homology) domain that is homologous to the ear domain of the Gamma-Adaptin subunit of the AP-1 clathrin adaptor. When overexpressed, the proteins, called GGA1, GGA2, and GGA3, localized to the trans-Golgi network (TGN) and often caused fragmentation and vacuolation of the compartment. Yeast two-hybrid analysis showed that the AGEH domains of the GGA proteins as well as those of Gamma-Adaptins are able to interact with Gamma-synergin, which was previously shown to localized in the TGN region and interact with Gamma-Adaptin. Furthermore, Gamma-synergin and either of the GGA proteins coexpressed were colocalized in the TGN region. These results suggest that the GGA proteins regulate the function of the TGN or membrane trafficking from this compartment and that the AGEH domains of GGAs and Gamma-Adaptins, like the ear domain of alpha-Adaptin, are involved in interaction with molecules that modulate their functions.
