葉綠體內膜運輸機組蛋白Tic40之導引序列分析

Functional Characterizations of Sequence Motifs in the Transit Peptide of Pisum sativum Tic40

詹博婷(PO-TING CHAN)

指導教授 ： 李秀敏

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02407

摘要

葉綠體是植物細胞所具有的特殊胞器，其功能主要為進行光合作用以提供養份使植物生長。大部分的葉綠體蛋白質都是藉由細胞核內基因所轉錄，於細胞質中轉譯，再自細胞質送入葉綠體執行功能。在還未送入葉綠體前，細胞質內的葉綠體前驅蛋白質(precursor protein)在N端帶有一段可被葉綠體膜上受器所辨認的導引序列(transit peptide)，之後便藉由葉綠體外膜及內膜上的運輸機組(translocon complex)送入葉綠體。而該段導引序列在前驅蛋白質被送入葉綠體後，即會被葉綠體基質處理酶(stromal processing peptidase)所切除而成為成熟蛋白質(mature protein)。目前的研究成果已經確認葉綠體蛋白質之導引序列，對於葉綠體蛋白質的運輸是扮演著一個充分且必要的角色。但這些導引序列無論是胺基酸組成或是長度，都存在著高度相異性，因此關於葉綠體蛋白質的導引序列，至今我們仍然不甚瞭解。
過去關於葉綠體蛋白質的研究發現：葉綠體蛋白質可依照其喜歡的葉綠體年紀分為三大類，分別是喜歡進年輕葉綠體、喜歡進年老葉綠體、以及在年輕或年老葉綠體皆無差異者；而這種對年齡選擇的能力，是存在於葉綠體的導引序列。在這篇論文中，我主要針對喜歡進年老葉綠體的蛋白質進行導引序列功能性分析。利用丙氨酸置換(alanine scanning)及定點突變(site-directed mutagenesis)等技術將豌豆 Tic40 前驅蛋白質(prPsTic40)之導引序列進行區段分析，發現影響偏好進年老葉綠體的關鍵區域位在皆帶正電的第29個精氨酸(arginine)與第30個賴氨酸 (lysine)。當拿掉這兩個胺基酸時，就會發現Tic40不喜歡進老的葉綠體；而再把這兩個胺基酸放回導引序列後，便可發現Tic40恢復喜歡進年老葉綠體的特性。除此之外，在另一個也具有喜歡進年老葉綠體特性的蛋白質prAtL11，其導引序列也發現兩個帶正電的lysine扮演著同樣會影響年紀選擇的角色。這些研究結果顯示：偏好進年老葉綠體的蛋白質，其位於導引序列的連續兩個攜帶正電氨基酸，是影響年紀選擇的關鍵。

關鍵字

葉綠體 ； 導引序列 ； 年紀選擇

並列摘要

Most chloroplast proteins are encoded in the nucleus, translated in the cytosol, and then transported into chloroplasts through the Toc (Translocon at the outer envelope membrane of chloroplasts) and Tic (Translocon at the inner envelope membrane of chloroplasts) complexes. Nucleus-encoded chloroplast proteins are usually synthesized as higher molecular weight precursors with N-terminal targeting signals called the transit peptides. Even though transit peptides are necessary and sufficient for chloroplast precursor proteins import, what constitute the critical sequence features for receptor recognition in the transit peptides is still poorly understood. Previous research has shown that precursors can be classified into three groups based on their preference to be imported into chloroplasts of different ages and the age-selective signal is located within the transit peptide of each precursor. In this study, I tried to identify the transit peptide features that confer preference for older chloroplasts. I divided the transit peptide of prPsTic40 (precursor of Tic40 from pea Pisum sativum), one of the precursors that prefer to be imported into older chloroplasts, into eight blocks and generated serial alanine scanning mutants. When block 4 (residues 28-36) was mutated, import efficiency into older chloroplasts was significantly decreased, while import efficiency into younger chloroplasts was not affected. Further mutations within block 4 through site-directed mutagenesis revealed that two positive charges, arginine and lysine of residues 29 and 30, are necessary and sufficient to confer preference for older chloroplasts. Moreover, two positive charges in the transit peptide of prAtL11 (precursor of L11 from Arabidopsis thaliana), another precursor that also prefers to be imported into older chloroplasts, are also responsible for prAtL11 preference for older chloroplasts. These results indicate that two consecutive positive charges in the transit peptide are the critical feature constituting the age-selective signal for older chloroplasts preference.

並列關鍵字

chloroplast ； transit peptide ； age selectivity

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