以受體特定剪切達到年齡對葉綠體蛋白運輸的調控

Specific receptor cleavage mediates age-dependent import regulation

陳婷芳(Ting-Fang Chen)

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(2021年)

https://doi.org/10.6342/NTU202101122

摘要

我們實驗室先前的研究發現葉綠體前驅蛋白可分為三種年齡選擇性組別：第一組前驅蛋白傾向運輸至年輕的葉綠體，其中包括核酮糖小亞基前驅蛋白 (prRBCS)；第二組前驅蛋白對於年齡無偏好性；第三組前驅蛋白則傾向運輸至成熟的葉綠體，其中包括葉綠體內膜機組Tic40前驅蛋白(prTic40)和葉綠體熱休克同源前驅蛋白70-1 (prcpHsc70-1)。我們並進一步發現在導向序列上的雙陽性模體（兩個帶正電荷殘基）是調控前驅蛋白優先選擇運輸至成熟葉綠體的重要關鍵，但對於雙陽性模體的受體仍不清楚。為了找出此受體與了解年齡調控前驅蛋白運輸的分子機制，我們利用接上FLAG標籤的prTic40 (prAtTic40-FLAG)作為材料，將其導向序列上雙陽性模體兩側的殘基突變成半胱氨酸，當prAtTic40-FLAG與成熟葉綠體進行結合時，利用BMH交聯劑將接近雙陽性模體附近的半胱氨酸進行交聯，再將prAtTic40-FLAG以免疫沈澱法分離出來，此時被交聯的蛋白可通過串聯式質譜儀識別。根據我的實驗結果，受體最有可能是葉綠體外膜機組蛋白159 （Toc159，其由A、G、M三個結構域組成，以下稱為 Toc159AGM）的A結構域截斷體，也就是Toc159GM。先前許多研究中皆表明A結構域經常受到降解，但原因尚不明瞭，而我在豌豆(Pisum sativum)的葉綠體觀察到A結構域受到剪切的程度會隨著年齡而增加。另外發現prcpHsc70-1及prTic40運輸進入僅表達Toc159GM的葉綠體效率較好，而他們雙陽性模體破壞的變體prcpHsc70-1(KR2324AA)及prTic40(RK2930EE)則是運輸進入含有Toc159AGM的野生型葉綠體效率較佳。此外在體外沉降實驗中亦證實prTic40導向序列會偏好與Toc159GM結合，而prRBCS導向序列則偏好與Toc159AGM結合。綜合以上結果發現Toc159的A結構域剪切可以調控年齡對葉綠體蛋白運輸的調控。

關鍵字

葉綠體 ； 蛋白運輸 ； 雙陽性模體 ； 葉綠體外膜機組蛋白159（Toc159）

並列摘要

Our lab has previously shown that chloroplast preproteins are divided into three age-selective groups: group 1 preproteins, which include prRBCS, prefer to be imported into young chloroplasts; group 2 preproteins have no age preference; group 3 preproteins, which include prTic40 and prcpHsc70-1, prefer to be imported into old chloroplasts. A twin-positive motif (two consecutive positively charged residues) in the transit peptide is necessary for old chloroplast preference (Teng et al., 2012). However, the receptor recognizing this twin-positive motif remains unknown. To identify the receptor and gain molecular understanding of age-dependent import regulation, we used prTic40 from Arabidopsis as a model substrate (prAtTic40-FLAG) with cysteines engineered flanking the twin positive motif, and used the 1,6-bis-maleimidohexane (BMH), a membrane-permeable homobifunctional cysteine-cysteine crosslinker, to crosslink proteins close to the twin-positive motif when prAtTic40-FLAG was bound to old chloroplasts. Proteins crosslinked to prAtTic40-FLAG were identified by tandem mass spectrometry. On the basis of the results, the receptor is most likely Toc159GM, an A domain truncated variant of the major receptor Toc159, which is composed of three domains: A, G and M, and is hereafter referred to as Toc159AGM. Many studies have reported that the A domain is often cleaved for an unknown reason. I first showed that in pea (Pisum sativum), cleavage of the A domain increased with age. I then showed that prcpHsc70-1 and prTic40 imported better into chloroplasts expressing only Toc159GM; prcpHsc70-1(KR2324AA) and prTic40(RK2930EE), in which the twin-positive motif is mutated, imported better into wild-type chloroplasts containing Toc159AGM. Furthermore, I performed in vitro pull-down assays and the data showed that prTic40 transit peptide preferentially bound to Toc159GM and prRBCS transit peptide preferentially bound to Toc159AGM. These results suggest that specific cleavage of the A domain from Toc159 mediates the age-selection import of chloroplast preproteins.

並列關鍵字

Chloroplasts ； Protein import ； Twin-positive motif ； Translocon at the outer envelope membrane of chloroplast 159 (Toc159)

參考文獻

Agne, B., Infanger, S., Wang, F., Hofstetter, V., Rahim, G., Martin, M., Lee, D.W., Hwang, I., Schnell, D., and Kessler, F. (2009). A toc159 import receptor mutant, defective in hydrolysis of GTP, supports preprotein import into chloroplasts. Journal of Biological Chemistry 284, 8670-8679.

Andrès, C., Agne, B., and Kessler, F. (2010). The TOC complex: preprotein gateway to the chloroplast. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1803, 715-723.

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