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研究生:陳冠毓
研究生(外文):Kuan-Yu Chen
論文名稱:葉綠體運輸機組次複合體的分析
論文名稱(外文):Identification of translocon subcomplexes associated with chloroplast protein import intermediates
指導教授:李秀敏李秀敏引用關係
指導教授(外文):Hsou-min Li
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:50
中文關鍵詞:葉綠體複合體運輸機組蔗糖密度梯度離心蛋白質輸入
外文關鍵詞:chloroplastransloconprotein importBN-PAGEsucrose gradient
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大部分葉綠體蛋白質是由細胞核基因所轉錄,於細胞質中合成再送入葉綠體。由細胞核基因轉錄轉譯而來的葉綠體前驅蛋白質(precursor protein)在N端具有訊息胜肽(transit peptide),會經由葉綠體外膜及內膜上的運輸機組(translocon complex)一連串的作用輸入葉綠體中。運輸機組成員中位於葉綠體外膜者稱為Toc (translocon at the outer envelope membrane of chloroplasts)蛋白,位於內膜者稱為Tic (translocon at the inner envelope membrane of chloroplasts)蛋白。過去10年來,科學家對於這些運輸機組成員的研究已有十分大的進展,至今已發現10個以上的成員,但對於這些成員如何協調、組合、及以什麼樣的步驟將前驅蛋白送入葉綠體,則至今仍不清楚。我們利用碗豆葉片的葉綠體做為實驗材料,輸入放射線標定前驅蛋白,希望觀察到葉綠體在運輸過程中負責運送前驅蛋白的運輸機組所形成的各式複合體。利用化學交連結固定這些複合體後,由蔗糖密度梯度離心以及藍色原性電泳進行蛋白質分析,我觀察到四個複合體。在蔗糖密度梯度離心中,前驅蛋白最主要分布在兩個區塊。在蔗糖密度較重的區塊,利用西方點漬法可以鑑定出Tic與Toc成員都會聚集在此處,因此推論此處含有一個Tic/Toc超級複合體,並推測此複合體的大小至少超過2 MDa。接著將蔗糖密度較輕處的區塊樣本由蛋白質原性電泳分析後,發現到兩個不同分子量的複合體C1及C2。C1分子量約為700 kDa,C2分子量約為800 kDa。經由抗體偏移蛋白質原性電泳分析,我發現到C1由兩個複合體所構成,C1-1為一首次發現,只含有Toc34、Toc75不含Toc159的Toc次複合體, C1-2則含有Toc34、Toc75及 Toc159,為完整的Toc複合體。而C2則含有Toc34、Toc75、Toc159、Tic110及Hsp93。經由追蹤輸入的方式可以得知這些複合體都是會運送前驅蛋白進入葉綠體的有功能複合體。另外在葉綠體輸入的一開始,即葉綠體表面黏接前驅蛋白時,這三個複合體也都可以被發現到,因此推斷C1-1、C1-2與C2皆為葉綠體輸入蛋白時位於前期的步驟,它們會轉變為Tic/Toc超級複合體繼續完成將前驅蛋白送入葉綠體的工作。我也試著輸入了其他不同的前驅蛋白,同樣可以發現到前述四種複合體。根據上述資料可以推論,葉綠體在輸入前驅蛋白的過程中,至少會有這四種次複合體形成。
Most chloroplast proteins are encoded by the nucleus, synthesized in the cytosol and post-translationally imported into chloroplasts. These nucleus-encoded proteins are synthesized as precursors with cleavable N-terminal transit peptides, and are transported into chloroplasts by a set of translocon complex located in the chloroplast outer and inner envelope membranes. Translocon components are termed Toc (translocons at the outer envelope membrane of chloroplasts) and Tic (translocons at the inner envelope membrane of chloroplasts) proteins. In the past ten years, researchers have made great progresses in identification of the Tic / Toc components. They have identified more than 10 Tic/Toc components. However, we still do not understand how these components work, interact, and cooperate. We also do not know the sequential steps of import into chloroplasts. Therefore, I tried to import radio-labeled precursor proteins into pea chloroplasts and identify intermediate steps in the import process. I used a chemical crosslinker to fix the intermediate complexes, and then used sucrose density gradient centrifugation and blue native polyacrylamide gel electrophoresis (BN-PAGE) to separate and identify these intermediate complexes. I found 4 groups of complexes in total. First, I found a TicToc supercomplex containing all of the Tic、Toc components. Its molecular mass is at least 2 mDa. Then I found a lighter population of precursor-protein-containing complexes which can be chased to TicToc. Combined with BN-PAGE, I found this lighter population contained a ~700-kDa complex and an ~800-kDa complex. In further experiments, I found that the 700-kDa complex contained two populations of complexes. One population contained Toc75 and Toc34, and the other population contained Toc75, Toc34 and Toc159, which is a complete Toc complex. I also identified a TicToc subcomplex of 800 kDa which contained Toc75, Toc34, Toc159, Tic110 and Hsp93 but not Tic40. My results suggest that these complexes represent early intermediate steps in the import process and are general intermediates for all precursors.
壹、 縮寫表................................................I
貳、 中文摘要............................................III
參、 英文摘要..............................................V
肆、 前言..................................................1
伍、 材料與方法
一、 材料
1. 抗體...................................................6
2. 標記 (Marker) .........................................6
3. 植物材料...............................................7
二、 方法
1. 碗豆葉綠體的製備.....................................7
2. 以硫酸十二脂鈉聚丙烯醯胺膠片進行電泳分析(SDS-PAGE)..8
3. 以藍色原態蛋白質電泳進行分析 (BN-PAGE)..............9
4. 活體外轉錄 (In vitro transcription)...............11
5. 活體外轉譯 (In vitro translation).................12
6. 活體外轉譯蛋白之去鹽...............................13
7. 蛋白質濃度測定.....................................13
8. 抗體偏移雜合 (antibody-shift hybridization).........14
9. 西方點漬法 (western blot) ..........................14
10. 活體外前驅蛋白持續輸入及追蹤輸入葉綠體 (percursor protein import & chase into chloroplasts) .........16
11. 活體外前驅蛋白表面黏接及追蹤輸入葉綠體 (percursor protein binding & chase into chloroplasts).........17
12. 蔗糖密度梯度離心與電泳分析..........................17
陸、 結果
1. Tic/Toc運輸機組會形成一個Tic及Toc超級複合體TicToc ,與前人結果相同,其大小以BN-PAGE估測超過2 MDa....19
2. 兩群新發現的複合體C1、C2是前驅蛋白輸入葉綠體的中間步驟
3. C1含有兩群複合體,一群由Toc75與Toc34 所組成,另外 一群含有Toc75、Toc34及Toc159......................21
4. 複合體 C2是由Toc75、Toc34、Toc159與Tic110 所 組成的TicToc次複合體,並部份含有Hsp93.............22
5. 次複合體C1與C2可在前驅蛋白黏接於葉綠體表面時發現 ,因此與Akita等人所發現的複合體應為相同............22
6. 次複合體C1 & C2是葉綠體蛋白運輸的共同複合.........23
柒、 討論................................................25
捌、 參考文獻............................................30
玖、 圖表
圖一 前驅蛋白輸入葉綠體示意圖.........................33
圖二 蔗糖密度梯度離心分析前驅蛋白prSS輸入到葉綠體的過程...............................................34
圖三 前驅蛋白經由BN-PAGE分析後可在分子量約440 kDa 處發現Rubisco holozyme .........................35
圖四 在第4-9管間可發現前驅蛋白與Tic/Toc成員分部位置重疊................................................36
圖五 位於蔗糖密度梯度離心較重處的前驅蛋白會形成巨大的複合體,推測是TicToc超級複合體.....................37
圖六 位於-5~-2管間的C1 C2複合體......................38
圖七 經由輸入與追蹤輸入前驅蛋白prSS,含有C1與C2的-5~-2管的前驅蛋白量會逐漸減少............................39
圖八 追蹤輸入10分鐘後,-5~-2管內的複合體仍為C1與C2.40
圖九 C1為Toc次複合體,至少含有Toc75與Toc34.........41
圖十 複合體C1經由不同濃度抗體偏移證實內含有兩個複合體42
圖十一 複合體C2為一個TicToc次複合體..................43
圖十二 位於較輕端的前驅蛋白複合體在葉綠體接受前驅蛋白初期即會產生,並會追蹤輸入到葉綠體內..................44
圖十三 追蹤輸入10分鐘後,-5~-2管中的複合體仍為C1與C2.................................................45
圖十四 抗體偏移BN-PAGE證明-5、-4管間的複合體為C1.................................................46
圖十五 抗體偏移BN-PAGE證明-3~-2管間的複合體為C2.................................................47
圖十六 輸入前驅蛋白prPOR B到葉綠體後,其蔗糖密度梯度離心與BN-PAGE結果....................................48
圖十七 C1POR B 的抗體偏移BN-PAGE.....................49
圖十八 推測葉綠體輸入蛋白路徑圖.......................50
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