{"created":"2023-06-20T13:20:37.788606+00:00","id":668,"links":{},"metadata":{"_buckets":{"deposit":"042af923-9db6-44be-a94e-79b367c652c3"},"_deposit":{"created_by":1,"id":"668","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"668"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000668","sets":["2:428:15"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"井上, 道雄"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"イノウエ, ミチオ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2005-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　Living organisms are composed of many kinds of protein and metabolize these proteins to maintain their bodies. All eucaryotic cells have membrane-bound organelles, such as nucleus, endoplasmic reticulum, Golgi apparatus, endosomes and lysosomes. Proteins and lipids are mainly transported by membrane traffic between these organelles. The intracellular protein transport is important for cellular function and mistransported cargo proteins can cause serious nervous diseases. In order to elucidate the intracellular transport, I pursued crystallographic study of GGA (Golgi-associated, γ-ear-containing, ARF-binding) protein in the clathrin-mediated membrane traffic <br />　Clathrin-mediated membrane traffic is responsible for protein transport from the plasma membrane or the trans-Golgi network (TGN) to the endosomal/lysosomal system. The recruitment of clathrin is mediated by GGA proteins and adaptor protein (AP) complexes. GGA and AP-1 complex are thought to cooperatively regulate the packaging of cargo receptors such as mannose 6-phosphate receptors (MPR) to transport vesicle at the TGN. GGAs are composed of three domains and one flexible region: the N-terminal VHS (Vps27p/Hrs/STAM) domain which recognizes C-terminal tail of cargo receptors such as MPR, the GAT (GGA and Tom1) domain which interacts with the GTP form of ARF on the TGN membranes, the proline-rich hinge region which interacts with clathrin, and the C-terminal GAE (γ-adaptin ear) domain which interacts with a number of accessory proteins that regulate membrane transport. Accessory proteins localize on TGN membrane, clathrin-coated vesicles, endosomal membrane and so on. Thus, GAE domain may regulate the formation, maintenance and disassembly of clathrin-coated vesicles through the interaction with various accessory proteins.<br />　First, I determined the crystal structure of GAE domain of GGA1. GGA1-GAE forms an immunoglobulin-like β-sandwich fold composed of two β-sheets (strands β1-β2-β3-β5-β6 and β4-β7-β8) and one α-helix. This structure is similar to the γ1-ear structure but GGA1-GAE forms a dimer in the crystal. The dimerization of GAE domain in crystal was the first observation. To elucidate the dimer formation of GGA1-GAE in solution, small angle X-ray scattering (SAXS) of GGA1-GAE was measured. I compared the Kratky plots of SAXS data with the crystal structure. The data shows GGA1-GAE also forms dimer in solution.<br />　To elucidate the interaction mechanism between GGA1-GAE and accessory proteins, I measured the binding affinities of GGA1-GAE to accessory proteins and determined the complex structure of GGA1-GAE with the accessory protein peptide. GGA1-GAE bound to accessory proteins mainly through hydrophobic interaction.<br />　Recently, γ-ear domain of AP-1 which is homologus to GAE domain was shown to interact with the \"WNSF\" motif in the GGA1 hinge region. I found that GGA1-GAE interacts with GGA1 hinge region. I measured the binding affinity of GGA1-GAE to GGA1 hinge in vitro and determined the complex structure of GGA1-GAE with the GGA1 hinge peptide. The complex structure revealed that the accessory proteins and GGA1 hinge interacted with GGA1-GAE at the same binding site. I confirmed that the GGA-GAE and GGA1 hinge interaction competes with accessory protein using fluorescence spectroscopy. These suggest that the autoinhibition of GGA1-GAE by GGA1 hinge controls the clathrin mediated traffic pathway. <br />　In summary, I researched GGA1 to understand clathrin-mediated transport. I analyzed structurally and functionally about the GAE domain of GGA1. I propose that the autoinhibition of GGA1-GAE by GGA1 hinge may regulate the clathrin-mediated transport.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第847号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"高エネルギー加速器科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"13 物質構造科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2004"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"INOUE, Michio","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲847_要旨.pdf","filesize":[{"value":"253.0 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/668/files/甲847_要旨.pdf"},"version_id":"c5bbe270-ab10-41f6-abca-f5d7f7ddb96d"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲847_本文.pdf","filesize":[{"value":"5.0 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/668/files/甲847_本文.pdf"},"version_id":"fa16f808-5b0b-401e-b8ce-c26bb7ea6017"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Structural and functional analysis of intra-cellular transport between Golgi apparatus and endosome/ lysosome system using synchrotron radiation","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Structural and functional analysis of intra-cellular transport between Golgi apparatus and endosome/ lysosome system using synchrotron radiation"},{"subitem_title":"Structural and functional analysis of intra-cellular transport between Golgi apparatus and endosome/ lysosome system using synchrotron radiation","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["15"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"668","relation_version_is_last":true,"title":["Structural and functional analysis of intra-cellular transport between Golgi apparatus and endosome/ lysosome system using synchrotron radiation"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:12:09.553476+00:00"}