Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36301
標題: 阿拉伯芥泛素受體的功能性分析
Functional analyses of the major ubiquitin receptors in Arabidopsis thaliana
作者: 林雅玲
Lin, Ya-Ling
關鍵字: 26S proteasome
蛋白酶體
ubiquitin receptor
RPN10
Arabidopsis
泛素受體
RPN10
阿拉伯芥
出版社: 生物科技學研究所
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摘要: 選擇性蛋白質分解在生物生長與發育的各個面向都扮演重要角色。生物體內短暫存在而需要嚴密調控的蛋白質,乃至折疊出錯的蛋白質,會先被連接上四個單位以上的泛素鏈,再送到26S蛋白酶體進行分解。負責傳送泛素化受質的泛素受體可分成直接和間接兩種,26S蛋白酶體單元RPN10與RPN13能直接接收泛素化受質,而可以同時和泛素鏈以及蛋白酶體結合的RAD23與DSK2,則歸類為間接接收受體。阿拉伯芥(Arabidopsis thaliana)的RPN10能利用ubiquitin-interacting motif 3 (UIM3)和UIM1,分別與RAD23與DSK2結合,因此同時參與了直接與間接接收受質的途徑。雖然理論上將泛素化受質送到蛋白酶體是蛋白質分解不可或缺的步驟,但是詳細的分子作用機制尚待研究。目前泛素受體在不同物種的重要性不僅有爭議,分析也不夠全面,尤其是泛素受體通常有多個不同的功能區域(例如RPN10利用不同區域接收泛素化受質和影響蛋白酶體的穩定),因此必須研究缺少特定蛋白功能區域的轉殖生物性狀,才能證明剔除泛素受體造成的性狀確實是破壞接收泛素化受質造成的。為了了解不同泛素受體的功能,本研究選取阿拉伯芥做為材料,利用基因缺失或低蛋白質表達突變株研究主要泛素受體的功能,其中包括RPN10、RPN13、RAD23、DSK2與DDI1。我們發現只有剔除RPN10的植物(rpn10-2)受到顯著影響,同時雙帽蛋白酶體的含量也低於野生型。我們將三個UIM都突變的RPN10(命名為u123)放回rpn10-2,發現轉殖植物顯示野生型性狀,雙帽蛋白酶體的含量也大致回復。本研究證實在阿拉伯芥中,植物缺少RPN10所顯現的性狀可能是蛋白酶體結構出現問題所造成,而非無法接收受質。令人驚訝地,當我們移除u123轉殖植物體內的RPN13,植物亦顯現野生型性狀。我們的結果指出,已知的主要泛素受體功能不但互相重疊,也暗示其他泛素受體或傳遞途徑的存在。
Selective protein degradation by ubiquitin/26S proteasome-mediated proteolysis (UPP) has been shown to be involved in numerous cellular processes of eukaryotes. During UPP, substrates are covalently marked with Lys-48-linked ubiquitin chain of at least four units and targeted to the 26S proteasome for degradation. Current model suggests that ubiquitylated substrates are delivered to the 26S proteasome directly through proteasome subunits RPN10 and RPN13 or indirectly through shuttle receptors RAD23 and DSK2 that can interact simultaneously with both the 26S proteasome and ubiquitylated substrates. However, the functional roles and mechanisms of delivering pathways in any given species are not well established. Interestingly, Arabidopsis RPN10 interacts with RAD23 and DSK2 via its ubiquitin-interacting motif 3 (UIM3) and UIM1, respectively, indicating RPN10 also plays a role in indirect targeting pathway. To further characterize the functional roles of individual targeting pathways in Arabidopsis, we established the T-DNA-inserted or RNA silencing lines for genes encoding the major ubiquitin receptors, including RPN10, RPN13, RAD23, DSK2 and DDI1. We found only RPN10 null mutant, rpn10-2, showed pleiotropic growth phenotypes with accumulation of the ubiquitylated proteins and distorted abundance of the proteasome complexes. Surprisingly, all of the characterized RPN10 null phenotypes is complemented by a triple UIM-site-mutated RPN10 variant (designed as u123), incapable of interacting with the ubiquitylated substrates and shuttle receptors. The data suggest that rpn10-2 phenotypes are likely caused by structural defects of proteasome but not substrate receiving functions of RPN10. Notably, when knocking out RPN13 from the u123-complementation line, the plants displayed wild-type phenotypes. Our results suggest RPN10 is required for maintaining proper structure of 26S proteasome and the major ubiquitin receptors are functionally redundant, indicating additional substrate delivering pathways exist.
URI: http://hdl.handle.net/11455/36301
其他識別: U0005-2511201111445700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2511201111445700
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