{"created":"2023-06-20T13:21:14.875983+00:00","id":1363,"links":{},"metadata":{"_buckets":{"deposit":"bb89a256-f7ee-4b48-b46e-74f6477b9580"},"_deposit":{"created_by":1,"id":"1363","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1363"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001363","sets":["2:430:27"]},"author_link":["9700","9701","9699"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"奈良, 篤樹"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ナラ, アツキ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2002-03-22"}]},"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":"Endosomes act as sorting platforms for multiple membrane traffics in eukaryotic cells.The system is summarized in Figure 1.Endocytic pathway begins with internalization of extracellular or plasma-membrane proteins.The internalized proteins rapidly enter into endosomes and finally deliver to lysosomes via multivesicular body, or to recycle back to the plasma membrane.Although the process from internalization to fuse of endocytic vesicles with early endosomes is well studied and characterized, the late stage of endocytic pathway from early endosomes is still unclear.Newly synthesized lysosomal proteins are delivered from the endoplasmic reticulum via Golgi apparatus and endosomes(biosynthetic pathway). Autophagic pathway is a transport process mediated by a double membrane structure， autophagosomes, cytoplasmic contents being delivered to lysosomes.Convergence of autophagic and endocytic pathway has been known, while its functional meaning has been obscure.The mouse SKD1 is an ATPase associated with cellular activities(AAA） family protein whose yeast homologue, Vps4p, is implicated as a molecule regulating endosomal membrane trafficking.It is, therefore, possible that SKD1 is involved in endosomal trafficking in mammalian cells.<br />To investigate the function of SKD1, we constructed the mutant SKD1 (SKD1E235Q) by exchanging the conserved ATPase hydrolysis site, from glutamic acid to glutamine.While green fluorescent protein(GFP)-tagged SKD1 localized over the cytoplasm, GFP-SKD1E235Q showed not only diffused pattern but also punctate pattern in the perinuclear region.In these GFP-SKD1E235Q-positive compartments(E235Q- compartments), transferrin receptor(TfR), endocytic tracers such as dextran, and various endosomal membrane markers were accumulated.In electron microscopy, the <br />E235Q-compartment was an exaggerated and tubulo-vesicular membrane structure，decorated with SKD1E235Q proteins.These results suggest that overexpression of SKD1E235Q causes a dominant negative effect on endosomes.<br />To attain more understanding of role of endosomes and molecular mechanisms underlying sorting and transport in endosomes, I started the analysis of the mouse SKD1. In cells overexpressing the mutant SKD1, endocytosed epidermal growth factor(EGF) was also accumulated in the E235Q-compartments and its degradation was inhibited.This rose a possibility that overexpression of the mutant SKD1 affects the transport to lysosomes via endosomes.To examine this quantitatively, I constructed high expression system mediated by recombinant adenovirus.Consistent with a result of immunofluorescent microscopic analysis, [125I]-EGF intracellular degradation was severely inhibited in the mutant protein-overexpressing cells. <br />Internalization of[125I]-EGF was normal, suggesting that transport of EGF to lysosomes was inhibited or degradation of[125I]-EGF delivered to lysosomes was inhibited.To determine which is true, I established the endosome-lysosome transport assay system.Compared with control cells, transport of streptavidin, endocytosed cargo marker, from endosomes to lysosomes was clearly inhibited in cells overexpressing the mutant SKD1.<br />These results indicate that SKD1 regulates outgoing transport from endosomes.Once overexpressing the mutant SKD1， it was tightly associated with <br />endosomal membranes.It is likely that the dominant negative effect of the mutant SKD1 is due to formation of oligomers consisting of the mutant and endogenous proteins, since I found that both bound each other in two-hybrid assay.In the cells, the E235Q-compartments were formed and endosomal sorting and transport system including traffics to lysosomes and to plasma membrane were inhibited.<br /> I next discovered inhibition of autophagic degradation in cells overexpressing the GFP-SKD1E235Q.Accumulation of autophagosomes in cells overexpressing GFP- SKD1E235Q was observed by electron microscopy.While autophagosomes cannot observed in nutrient condition, in the cells,the amount of autophagosomes in this condition was increased 2.7 times as many as in starved cells.On the other band, the amount of autolysosomes, which is formed by fusion of autophagosomes with lysosomes, was decreased to a half.These results suggest that fusion of autophagosomes with lysosomes is inhibited in cells overexpressing the mutant SKD1.I hypothesized that the inhibition of the fusion step might result from blockade of provision of endosomal membranes to autophagosomes.To investigate this possibility, I examined the lysobisphosphatidic acid(LBPA) localization in cells overexpressing the mutant SKD1.In control cells, LBPA, which is mainly distributed in late endosomes, became associated with autophagosomes if autophagy was induced.In contrast, it was not colocalized with autophagosomes in cells overexpressing the mutant SKD1.This suggests that LBPA is delivered to autophagosomes depending on SKD1 function.Distribution of the mutant SKD1 was distinct from that of an autophagosome marker, unlikely suggesting that SKD1 directly act on autophagosomes. I also investigate a time course of the effects. Autophagosome accumulation could be detected 1.5 hours slow after defect of TfR recycling, supporting that inhibition of autophagy is due to impairment of endosomal functions.<br /><br />Altogether, I concluded that SKD1 is a novel regulatory protein in endosomal sorting and transport of various cargoes.Moreover, I produced a first functional evidence that that transport from endosomes to autophagosomes is required for the autophagic process．","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611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":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"X2 分子生物機構論専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2001"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"NARA, Atsuki","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611_要旨.pdf","filesize":[{"value":"296.6 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result ","url":"https://ir.soken.ac.jp/record/1363/files/甲611_要旨.pdf"},"version_id":"1618551c-3968-4d78-a930-f970580be0e7"}]},"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":"Regulation of sorting and transport in endosomes : studies on SKD1","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Regulation of sorting and transport in endosomes : studies on SKD1"},{"subitem_title":"Regulation of sorting and transport in endosomes : studies on SKD1","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["27"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1363","relation_version_is_last":true,"title":["Regulation of sorting and transport in endosomes : studies on SKD1"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:44:47.879870+00:00"}