{"_buckets": {"deposit": "e4e4b540-d350-4ccb-9a36-9b2b8289681b"}, "_deposit": {"created_by": 21, "id": "1447", "owners": [21], "pid": {"revision_id": 0, "type": "depid", "value": "1447"}, "status": "published"}, "_oai": {"id": "oai:ir.soken.ac.jp:00001447", "sets": ["21"]}, "author_link": ["0", "0", "0"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2009-03-24", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "大岡, 杏子"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_creator_3": {"attribute_name": "フリガナ", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "オオオカ, キョウコ"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_date_granted_11": {"attribute_name": "学位授与年月日", "attribute_value_mlt": [{"subitem_dategranted": "2009-03-24"}]}, "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_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "2009045", "subitem_description_type": "Other"}]}, "item_1_description_12": {"attribute_name": "要旨", "attribute_value_mlt": [{"subitem_description": "　　　Autophagy is a major self-degradative process in eukaryotic cells that plays\u003cbr /\u003efundamental roles in cellular and organismal homeostasis, and is involved in many\u003cbr /\u003ephysiological and pathological situations. When autophagy is induced, cytoplasmic\u003cbr /\u003ematerials and organelles are sequestered into newly emerging double-membrane\u003cbr /\u003evesicles called autophagosomes, and delivered to the lysosome or the vacuole for\u003cbr /\u003edegradation. \u003cbr /\u003e　　　In the past decade, many \u003ci\u003eATG\u003c/i\u003e (\u003cu\u003ea\u003c/u\u003eu\u003cu\u003et\u003c/u\u003eopha\u003cu\u003eg\u003c/u\u003ey-related) genes have been identified by\u003cbr /\u003egenetic approaches using the yeast Saccharomyces cerevisiae. Atg8, a ubiquitin-like\u003cbr /\u003eprotein (Ubl), is one of the proteins essential for autophagosome formation. The cysteine protease Atg4 first removes the C-terminal arginine of Atg8 to expose the glycine as the new terminus. This glycine forms a thioester bond with Atg7, an activating enzyme (E1), and transfers to and also forms a thioester bond with Atg3, a conjugation enzyme (E2). Atg8 is eventually conjugated to the amino group in the hydrophilic head of\u003cbr /\u003ephosphatidylethanolamine (PE).  Atg8 is anchored to isolation membrane and\u003cbr /\u003eautophagosomal membranes probably as this lipid-modified form, and thought to directly participate in the formation of these membranes. Atg4 also catalyzes the deconjugation of Atg8-PE after it has fulfilled its role in autophagosome formation, thus Atg8 is reused. Because the details of this sequential reaction of Atg8 lipidation are unclear, I focus on the mechanism of Atg8 lipidation, in this study. \u003cbr /\u003e　　　The Atg8 conjugation system was reconstituted using purified proteins expressed in\u003cbr /\u003e\u003ci\u003eEscherichia coli\u003c/i\u003eand PE-containing liposomes in vitro. First, I successfully capture\u003cbr /\u003eauthentic thioester intermediates, Atg8-Atg7 and Atg8-Atg3, which can not have been\u003cbr /\u003edetected because of their lability. This allows me to analyze the sequential reaction of\u003cbr /\u003eAtg8 lipidation. \u003cbr /\u003e　　　It was shown that Atg8 could be conjugated with phosphatidylserine (PS) as\u003cbr /\u003eefficiently as PE in vitro. However, PE was identified as the sole lipid conjugated to the\u003cbr /\u003eC-terminal glycine of Atg8 in vivo. It suggests that there exists a mechanism that directs\u003cbr /\u003eAtg8 conjugation preferentially to PE in the cell. In this study, I show that, in contrast to\u003cbr /\u003ePE conjugation, the PS conjugation of Atg8 is markedly suppressed at physiological\u003cbr /\u003e(neutral) pH. Then, I show that both of the Atg8-Atg7 and the Atg8-Atg3 intermediates\u003cbr /\u003eare formed in the presence of PS liposomes as rapidly as in the presence of PE\u003cbr /\u003eliposomes, and transfer of Atg8 from Atg3 to PS is specifically retarded at neutral pH. \u003cbr /\u003eFurthermore, the addition of acidic phospholipids to liposomes is also suggested to result in the preferential formation of the Atg8−PE conjugate. I also show that the acidic\u003cbr /\u003ephospholipids specifically promote the recruitment of the Atg8-Atg7 and the Atg8-Atg3\u003cbr /\u003ethioester intermediates to the membrane. Furthermore, .it was reported that the\u003cbr /\u003eAtg12-Atg5 conjugate, which is formed by ubiquitin-like conjugation reaction, is\u003cbr /\u003eindispensable for Atg8-PE production in vivo, and that recombinant Atg12-Atg5 indeed\u003cbr /\u003estimulates Atg8-PE and Atg8-PS production in vitro. \u003cbr /\u003e　　　The preferential formation of Atg8-PE can be achieved by combination of neutral\u003cbr /\u003epH, acidic phospholipids, and the Atg12-Atg5 conjugate. Furthermore, I show that PS is\u003cbr /\u003enot essential for autophagosome formation even if Atg8 is conjugated to PS in vivo, \u003cbr /\u003ebecause the autophagic activity of cells deficient for the PS synthesis enzyme-deficient\u003cbr /\u003e(\u003ci\u003epss1△\u003c/i\u003e) cells was normal. However, in vitro, the less efficient but significant production of Atg8-PS was still observed, suggesting that the exclusive formation of Atg8-PE requires precise in vivo settings for these factors and/or other facto(s). Alternatively, this result may imply the production of Atg8-PS in vivo, although its amount should be much less than the PE oonjugate. Previously, PE was detected as the sole lipid conjugated to Atg8 and its mammalian homolog LC3 (microtubule-associated protein light chain 3) in vivo. However, lipidated Atg8 and LC3 were forced to accumulate by mutation or treatment with lysosomal inhibitors under nutrient-replete conditions. Therefore, there also remains an alternative possibility that Atg8-PS is formed in starved cells undergoing autophagy, so, I try to purify lipidated Atg8 from the cells under starvation conditions for detailed analysis of lipids conjugated to Atg8 by LC-MS/MS. \u003cbr /\u003e　　　Next, I perform gel filtration chromatography to know the interaction of the\u003cbr /\u003ecomponents in the Atg8 conjugation reaction in vitro. Atg3 interacts with Atg7 as it shown, and Atg3 also interacts with the Atg8-Atg7 thioester intermediate. Furthermore, I find the\u003cbr /\u003eAtg8-Atg3 thioester intermediate releases from Atg7. It is reasonable because Atg7 is\u003cbr /\u003ereused rapidly for the next cycle of the reaction. Furthermore, I show that Atg3 and Atg7\u003cbr /\u003eare interacted via the disulfide bond between active center cysteines in Atg3 and Atg7. I\u003cbr /\u003eshow the model of the sequential reaction of Atg8 lipidation.", "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1253号", "subitem_description_type": "Other"}]}, "item_1_select_14": {"attribute_name": "所蔵", "attribute_value_mlt": [{"subitem_select_item": "有"}]}, "item_1_select_16": {"attribute_name": "複写", "attribute_value_mlt": [{"subitem_select_item": "複写不可"}]}, "item_1_select_17": {"attribute_name": "公開状況", "attribute_value_mlt": [{"subitem_select_item": "全文公開可"}]}, "item_1_select_8": {"attribute_name": "研究科", "attribute_value_mlt": [{"subitem_select_item": "生命科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "19 基礎生物学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "2008"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "OH-OKA, Kyoko", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "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", "download_preview_message": "", "file_order": 0, "filename": "甲1253_要旨.pdf", "filesize": [{"value": "310.7 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 310700.0, "url": {"label": "要旨・審査要旨", "url": "https://ir.soken.ac.jp/record/1447/files/甲1253_要旨.pdf"}, "version_id": "ad496fc1-f821-4766-8646-03d0dd2dc9c4"}, {"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 1, "filename": "甲1253_本文.pdf", "filesize": [{"value": "33.3 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 33299999.999999996, "url": {"label": "本文", "url": "https://ir.soken.ac.jp/record/1447/files/甲1253_本文.pdf"}, "version_id": "05c6fe1e-7662-4b18-9f0b-0f19ec554b8c"}]}, "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": "Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation"}, {"subitem_title": "Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "21", "path": ["21"], "permalink_uri": "https://ir.soken.ac.jp/records/1447", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-03-24"}, "publish_date": "2010-03-24", "publish_status": "0", "recid": "1447", "relation": {}, "relation_version_is_last": true, "title": ["Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation"], "weko_shared_id": -1}