阿拉伯芥伴護蛋白CPN20具鐵離子鑲嵌能力活化葉綠體之鐵超氧歧化酶

CPN20 as an iron chaperone mediates chloroplastic FeSODs activation in Arabidopsis

郭文鈺(Wen-Yu Kuo)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00936

摘要

超氧歧化酶是阿拉伯芥葉綠體中主要的抗氧化酵素。其中唯一可以測得活性的鐵超氧歧化酶，是由位於基質中的FE SUPEROXIDE DISMUTASE 1 (FSD1)所賦予，然而位於類囊膜及擬核中之FSD2及FSD3的活性則無法測得。阿拉伯芥FSD2及FSD3基因為葉綠體早期發育所必須，當同時剔除兩基因後，會中止葉綠體的發育。鐵超氧歧化酶需鐵離子輔酶的嵌合才具有活性，然而其如何獲取得鐵離子，至今依舊未知。我們利用逆向色層分析法、膠體過濾法並配合液態層析質譜儀篩選出CHAPERONIN 20 (CPN20)，其可能參與鐵超氧歧化酶的活化。利用酵母菌雙雜合系統及螢光共振能量轉移系統，證實了CPN20與FSD1具蛋白質交互作用。在體外實驗中，CPN20可提升FSD1、FSD2及FSD3超氧歧化酶的活性，確定CPN20對於鐵超氧歧化酶之活化具普遍性的輔助效果。經由感應耦合電漿質譜分析，發現CPN20擁有鐵離子並且可以提升FSD1之鐵離子輔酶的嵌合，推論CPN20能為FSD1鐵鑲嵌之輔助蛋白。我們的體內實驗結果也顯示，無論野生型CPN20或是喪失其伴護蛋白功能之突變型CPN20基因，皆可以提升FSD1的活性，顯示CPN20此一新功能與其所詳知的蛋白質伴護系統之功能不同。此外，在大量表現CPN20之轉植株中，FSD1蛋白質之表現量及活性皆提升，而CPN60蛋白質表現量並無變化；利用病毒誘導基因靜默法降低番茄CPN20基因表現量後，鐵超氧歧化酶活性亦隨之下降。綜合本研究之結果，我們確立了伴護蛋白CPN20具鐵離子鑲嵌能力活化葉綠體中之鐵超氧歧化酶，此功能與其參與蛋白質伴護系統之功能截然不同，為一獨立之新功能。CPN20之研究成果對於鐵超氧歧化酶的活化機制、葉綠體中蛋白質伴護系統，以及葉綠體發育系統之間，提供了一個重要的連結。

關鍵字

超氧歧化酶鐵超氧歧化酶伴護蛋白20 ； 鐵離子鑲嵌蛋白 ； 葉綠體 ； 阿拉伯芥

並列摘要

Iron superoxide dismutases (FeSODs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FE SUPEROXIDE DISMUTASE1 (FSD1) conferred the only detectable FeSOD activity, while the thylakoid membranes and nucleoids co-localized FSD2 and FSD3 double mutant showed chloroplast development arrested. FeSOD requires Fe cofactor for its activity, but how it is activated is unclear. In this study, we characterized the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. Reverse-phase high performance liquid chromatography, gel filtration chromatography and LC-MS/MS analyses were performed to identify CPN20 as a candidate to functionally interact with FSD1. Their physical interaction is demonstrated by yeast two-hybrid and fluorescence resonance energy transfer experiments. The relation between CPN20 and FeSOD was confirmed by in vitro experiments, which showed that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity, suggesting a common role for CPN20 in FeSOD activation. CPN20 could bind Fe, and Fe binding to FeSOD was increased with CPN20 incubation analyzed by inductively coupled plasma mass spectrometry, suggesting CPN20 might act as an Fe chaperone for FeSOD. The in vivo results showing overexpressing CPN20 and mutants with defective co-chaperonin activity increased FSD1 activity, without changing chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also corresponding decreased FeSOD activity. Conclusively, our results implicate CPN20 as an Fe chaperone for FeSOD in chloroplasts, a role which is independent of its well-known function in the chaperonin system. The findings provide a link between the mechanisms of FeSOD activation, the chloroplast chaperonin system, and chloroplast development.

並列關鍵字

SOD ； FeSOD ； CPN20 ； iron chaperone ； chloroplast ； Arabidopsis

參考文獻

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