葉綠體運輸機組次複合體的分析

陳冠毓

doi:10.6342/NTU.2005.00083

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葉綠體運輸機組次複合體的分析

Identification of translocon subcomplexes associated with chloroplast protein import intermediates

陳冠毓 , Masters　　Advisor：李秀敏　　

繁體中文 DOI： 10.6342/NTU.2005.00083

葉綠體；複合體；運輸機組；蔗糖密度梯度離心；蛋白質輸入； chloroplast ； ranslocon ； protein import ； BN-PAGE ； sucrose gradient

Abstract │ Reference (23) │ Altmetrics

Abstract 〈TOP〉

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。

Parallel Abstract 〈TOP〉

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.

Reference ( 23 ) 〈TOP〉

Akita, M., Nielsen, E., and Keegstra, K. (1997). Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. Cell Biol. 136, 983-994.
連結：
Bauer, J., Chen, K., Hiltbunner, A., Wehrli, E., Eugster, M., Schnell, D., and Kessler, F. (2000). The major protein import receptor of plastids is essential for chloroplast biogenesis. Nature 403, 203-207.
連結：
Chen, X., Smith, M.D., Fitzpatrick, L., and Schnell, D.J. (2002). In vivo analysis of the role of atTic20 in protein import into chloroplasts. Plant Cell 14, 641-654.
連結：
Chou, M., Fitzpatrick, L., Tu, S., Budziszewski, G., Potter-Lewis, S., Akita, M., Levin, J., Keegstra, K., and Li, H.-m. (2003). Tic40, a membrane-anchored co-chaperone homologue in the chloroplast protein translocon. EMBO J. 22, 2970-2980.
連結：
Hinnah, S.C., Wagner, R., Sveshnikova, N., Harrer, R., and Soll, J. (2002). The chloroplast protein import channel Toc75:pore properties and interaction with transit peptides. Biophysical J. 83, 899-911.
連結：

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