{"created":"2023-06-20T13:21:16.137821+00:00","id":1389,"links":{},"metadata":{"_buckets":{"deposit":"f2e24480-9571-4575-b9cf-746271be7532"},"_deposit":{"created_by":1,"id":"1389","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1389"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001389","sets":["2:430:27"]},"author_link":["9772","9773","9771"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"小野寺, 純"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"オノデラ, ジュン"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2004-09-30"}]},"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":"　　　Cellular activities require the maintenance of a balance between the synthesis and degradation of proteins. Macroautophagy (hereafter referred to as autophagy) is an intracellular bulk degradation system, which is well conserved in eukaryotes; autophagy transports cytoplasmic components to the lysosome/vacuole for degradation. This degradation is a cellular response to starvation and also plays a role in the recycling of cytoplasmic components, which may be important for cellular remodeling, development and differentiation. A total of 16 genes, which are essential for autophagy, and which are named APG and AUT (current nomenclature is ATG), have been identified by genetic screens in the yeast, Saccharomyces cerevisiae. Much progress has been made in the functional analysis of these genes.<br />　　　Autophagy is initiated by the sequestration of cytoplasmic components in a double-membrane structure termed the autophagosome. Immuno-electron microscopy has shown that ribosomes and typical cytosolic marker enzymes are present in the autophagosome and autophagic bodies at the same densities as in the cytosol. It is indicated that autophagy is a nonselective degradation. If degradation of long-lived proteins is exclusively mediated by autophagy, all proteins might be expected to have similar lifetimes. However, long-lived proteins have a variety of lifetimes; therefore, the autophagic pathway might have some selectivity.<br />　　　To investigate the possibility of selective autophagic degradation, he attempted to compare the amounts of each intracellular protein under growth and starvation conditions in the yeast, S. cerevisiae. He performed a systematic analysis using two-dimensional PAGE and MALDI-TOF mass spectrometry to detect the autophagy dependent degradation of intracellular proteins. During this analysis, he detected that the Mg<SUP>2+</SUP>- and NADPH-dependent cytosolic acetaldehyde dehydrogenase (Ald6p) decreased under nitrogen starvation. This enzyme catalyzes the conversion of acetaldehyde to acetate in the cytosol (acetaldehyde + NADP<SUP>+</SUP> → acetate + NADPH). As assessed by immuno-blot, Ald6p was reduced by greater than 82％ after 24 h of nitrogen starvation. This reduction was dependent on Atg/Apg proteins and vacuolar proteases, but was not dependent on the proteasome degradation, the Cvt pathway or the Vid protein.<br />　　　He hypothesized that the decrease in Ald6p levels was the result of degradation during nitrogen starvation. To examine this possibility, the kinetics of Ald6p degradation was measured by pulse-chase experiments, which suggest that Ald6p is degraded much more rapidly than typical cytosolic proteins. Ald6p was visualized by Ald6p-GFP fusion protein and immuno-electron microscopy analyses. In Δpep4 vacuolar proteinase deficient cells, Ald6p or Ald6p-GFP was localized in autophagic bodies in the vacuole under nitrogen starvation. These results indicate that Ald6p is degraded in the vacuole under nitrogen starvation. Furthermore, using subcellular fractionation and pulse-chase experiments, he also demonstrated that Ald6p was preferentially transported to the vacuole via autophagosome.<br />　　　To address the physiological significance of this preferential degradation, he analyzed cells of Ald6p over-producer and its disruptant. Δatg7 Δald6 double mutant cells were able to maintain higher rates of viability than Δatg7 cells under nitrogen starvation, and ALD6 overexpressing cells were not able to maintain high rates of viability. Furthermore, the Ald6p<SUP>c306s</SUP> mutant, which lacks enzymatic activity, had viability rates similar to Δald6 cells. Ald6p enzymatic activity may be disadvantageous for survival under nitrogen starvation; therefore, yeast cells may preferentially eliminate Ald6p via autophagy.<br />　　　These results show that Ald6p is one example of a preferential substrate for autophagic degradation. Ald6p was the only major protein on the two-dimensional PAGE gel to decrease during starvation; however, it is still possible that other minor proteins behave like Ald6p. If further studies were able to find such proteins,  it would help clarify the molecular mechanisms of selective autophagy and the physiological significance of the preferential degradation.<br />　　　He also found several specific proteins are induced under nitrogen starvation on the above-mentioned screening using two-dimensional PAGE. These proteins included typical proteins of environment stress responses (Eno1p/Hsp48p and Hsp26p), enzyme of amino acid biosynthesis (Arg1p), quenching enzyme of reactive oxygen species (Sod2p) and so on. These proteins did not increase in Δatg7 mutant cells; however, their mRNA levels were high as wild-type cells under nitrogen starvation. Thus, it is possible that these proteins synthesis are inhibited in the translational step.<br />　　　It is generally thought that autophagic protein degradation supplies significant amounts of free amino acids under nitrogen starvation. From this perspective, he quantified the free amino acids in yeast cells. Wild-type cells could maintain the constant level of amino acids pool during nitrogen starvation,  while Δatg7 cells depleted free amino acids after a few hours starvation. This result may indicate that Δatg7 cells cannot keep free amino acids enough to synthesize starvation-induced proteins. To ensure this consequence, he assessed in vivo protein synthesis using [<SUP>14</SUP>C]valine; protein synthesis of Δatg7 cells was even lower level than that of wild-type cells after 6 h starvation. However, when nitrogen starved cells fed free amine acids beforehand protein synthesis of Δatg7 cells was high level as well as that of wild-type cells. These results suggest that the pool size of free amino acids should limit the protein synthesis.<br />　　　It is often presumed that the ubiquitin-proteasome degradation is the most important for protein turnover in all phase. In this study, he showed the direct evidence that autophagy is essential for protein turnover and formation of amino acids pool under nitrogen starvation condition.","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814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":"2004"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ONODERA, Jun","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814_要旨.pdf","filesize":[{"value":"305.5 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/1389/files/甲814_要旨.pdf"},"version_id":"88ea88cf-61e4-4388-b604-c0f811ec6fc4"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲814_本文.pdf","filesize":[{"value":"984.1 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1389/files/甲814_本文.pdf"},"version_id":"11f3611d-34eb-4b22-941c-bf4924c32b5d"}]},"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":"Physiological Significance of Autophagy in Protein Turnover","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Physiological Significance of Autophagy in Protein Turnover"},{"subitem_title":"Physiological Significance of Autophagy in Protein Turnover","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":"1389","relation_version_is_last":true,"title":["Physiological Significance of Autophagy in Protein Turnover"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:44:02.631341+00:00"}