葉綠體具有三個膜系—外膜(outer membrane)、內膜(inner membrane)及類囊膜(thylakoid membrane)，並隔出三個空間，內外膜間隙(intermembrane space)、內膜內部的基質(stroma)與類囊體腔(thylakoid lumen)。蛋白質依據功能不同，會被運送到不同的區域，已知有兩種途徑可將蛋白質送至內膜上，分別是stop-transfer，及post-import。先前的研究僅指出，途徑的選擇是由蛋白質的穿膜區域(transmembrane domain, TMD)所決定，但具體上，兩種TMD的差異為何尚不明確。遂此，我們選取了14個會穿越內膜的蛋白，5個屬於stop-transfer，9屬於個post-import，分析其TMD的胺基酸組成，統計後發現在stop-transfer TMD中具有較多大的胺基酸，post-import TMD中則有較多小的胺基酸，故推測胺基酸大小，是一會影響選擇插入內膜途徑的因素。為測試此一假說，選取了以stop-transfer插入內膜的蛋白質TGD2，實驗結果發現將TGD2 TMD上的tryptophan(W)換成alanine(A) 或是glycine(G) 後，TGD2無法完全停在內膜上，將W 突變成phenylalanine (F)，則無影響。這些結果支持了我們的假說，TMD的胺基酸大小，是決定蛋白質送到葉綠體路徑的因素之一。

已知要進入葉綠體內部的蛋白質是透過內外膜上的運輸機組TOC 與TIC complexes，而外膜蛋白就只需要TOC complex，而位在膜間隙的蛋白質Tic22是如何進入到葉綠體，是否需要TOC complex，則至今仍有爭議。在加強了Tic22前驅蛋白 (prTic22) 進入到葉綠體的效率後，首先我們透過time course及能量的控制，來了解prTic22 進入葉綠體的最佳條件，再利用Tic22與RBCS進入葉綠體的競爭，及prTic22 進入到translocon complex 缺失的突變株所分離的葉綠體中，來研究prTic22 進入葉綠體的路徑。在與RBCS競爭的實驗中發現prTic22會與prRBCS競爭，顯示兩者路徑有所重疊。而進行突變株之進入葉綠體實驗時，發現只有在TOC complex的突變株(toc33、toc75)中，觀察到prTic22 進入葉綠體之效率下降；在TIC complex的tic20突變株中，Tic22進葉綠體的效率不受影響，甚至tic236突變株中有上升的情況。這些結果顯示prTic22應是透過TOC complex穿越外膜，到達膜間隙，無需使用到TIC complex。

Chloroplasts are composed of three independent membrane systems, including the outer membranes (OM), inner membranes (IM) and thylakoid membranes. These three membranes enclose three soluble areas, the intermembrane space, the stroma and the thylakoid lumen. Proteins need to be delivered to the correct compartment in order to be functional. For membrane proteins insertion into IM, two import pathways have been reported, the ‘‘stop-transfer’’ and the ‘‘post-import’’ pathways. It has been shown that the transmembrane domain (TMD) of each IM protein plays a critical role as the pathway determinant. However what features within TMD endow pathway selection is not known. Analysis of TMDs and surrounding amino acids from nine proteins of the post-import pathway and five proteins of the stop-transfer pathway, we found that there are more large amino acids in TMD of protein using the stop-transfer pathway while smaller amino acids are enriched in the post-import group. Thus, we hypothesize that one of determinants for IM insertion pathway selection is the amino acid size in TMD. We tested our hypothesis using TGD2, a protein using the stop-transfer pathway. After site-directed mutagenesis in TMDs and import assays using isolated pea chloroplasts, we found that TGD2 partly lost its ability to stop at IM after mutating a tryptophan (W) at the N terminus of its TMD into alanine (A) or glycine (G) in TMD, while mutating the W to phenylalanine (F) has no effect. These data suggest that N terminal amino acid sizes are important for TMD of chloroplast inner membrane proteins to function as a stop-transfer signal.

Protein import into internal compartments of chloroplasts requires the TOC and TIC translocon complexes on the outer and inner membranes. Protein insertion into the OM only needs the TOC complex. Much less is known about how proteins are imported into the intermembrane space. For example, whether the import of Tic22, the best known intermembrane space protein, needs the TOC complex is still in debates. After enhancing the chloroplast import efficiency of Tic22 perprotein (prTic22), I performed import time course and ATP concentration experiments to characterize the import requirement of prTic22. I further performed import competition experiments using prRBCS and prTic22. My result showed that prTic22 import was competed by prRBCS, indicating that their import pathways at least partially overlap. Finally using chloroplasts isolated from translocon complex mutants, I showed that import of prTic22 was decreased in toc33 and toc75 mutant chloroplasts, was no changed in tic20 mutant chloroplasts and was increased in tic236 mutant chloroplasts. We concluded that prTic22 uses the TOC complex for crossing the OM to arrive at the intermembrance space, and its import does not require the TIC complex.

口試委員審定書 i
致謝 ii
中文摘要 iii
Abstract iv
目錄 vi
前言 1
材料與方法 4
1.植物材料 4
2.植物的種植與生長情況 4
3.引子之序列： 5
4.小量質體DNA抽取 5
5.阿拉伯芥葉綠體抽取 6
6.豌豆葉綠體抽取： 7
7.中量質體DNA抽取 7
8.活體外轉錄合併轉譯(Coupled in vitro transcription and translation ) 8
9.純化包涵體 (inclusion body)中的重組蛋白質 9
10.活體外前驅蛋白質輸入葉綠體 (in vitro protein import assay) 10
11.嗜熱菌酶 (Thermolysin) 處理 11
12.強鹼萃取法(alkaline extraction) 11
13.胰蛋白酶(trypsin)處理 12
14.蛋白質濃度測定 13
15.硫酸十二脂聚丙烯醯胺凝膠電泳分析(SDS-PAGE: Sodium Dodecylsulfate-Polyacryamide Gel Electrophoresis) 13
16.西方墨點法 (Western blotting) 14
17.凝膠過濾活體外轉錄合併轉譯產物 16
結果 17
Stop-transfer及post-import 路徑蛋白質之TMD胺基酸組成 17
Stop-transfer蛋白質TGD2之TMD點突變蛋白 17
TGD2及突變蛋白累積於葉綠體位置的探討 18
prTic22進入葉綠體所需的時間 19
prTic22進入葉綠體所需的ATP濃度 20
prTic22是否與prRBCS 使用相似途徑進入葉綠體 21
在translocon complex突變株中，prTic22進入葉綠體之效率 21
結論 23
圖表 26
圖一、葉綠體內膜蛋白插入之路徑示意圖 26
圖二、TMD胺基酸組成 27
圖三、TGD2之胺基酸序列及TMD點突變 28
圖四、將prTGD2及其TMD突變之蛋白質輸送至分離葉綠體中 29
圖五、TGD2及突變蛋白累積於葉綠體可溶區之確認 30
圖六、prTic22 進入葉綠體之時間進程 (Time course) 31
圖七、prTic22 進入葉綠體對於ATP之需求 32
圖八、prRBCS與prTic22 進入葉綠體的競爭 33
圖九、Tic22 進入到葉綠體膜間隙需要TOC蛋白 34
圖十、prTic22 進入至膜間隙之示意圖 35
表一、TMD胺基酸序列 36
表二、使用post-import pathway插入內膜及穿越內膜的蛋白質TMD與其前後之胺基酸序列 37
表三、使用stop-transfer pathway插入內膜之TMD與其前後之胺基酸序列 39
參考資料 40

Awai, K., Xu, C., Tamot, B., and Benning, C. (2006). A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proceedings of the National Academy of Sciences 103, 10817-10822.
Chen, W., Provart, N.J., Glazebrook, J., Katagiri, F., Chang, H.S., Eulgem, T., Mauch, F., Luan, S., Zou, G., Whitham, S.A., Budworth, P.R., Tao, Y., Xie, Z., Chen, X., Lam, S., Kreps, J.A., Harper, J.F., Si-Ammour, A., Mauch-Mani, B., Heinlein, M., Kobayashi, K., Hohn, T., Dangl, J.L., Wang, X., and Zhu, T. (2002). Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 14, 559-574.
Chen, Y.L., Chen, L.J., Chu, C.C., Huang, P.K., Wen, J.R., and Li, H.M. (2018). TIC236 links the outer and inner membrane translocons of the chloroplast. Nature 564, 125-129.
Froehlich, J. (2011). Studying Arabidopsis envelope protein localization and topology using thermolysin and trypsin proteases. Methods Mol Biol 774, 351-367.
Froehlich, J.E., and Keegstra, K. (2011). The role of the transmembrane domain in determining the targeting of membrane proteins to either the inner envelope or thylakoid membrane. The Plant Journal 68, 844-856.
Gutensohn, M., Schulz, B., Nicolay, P., and Flugge, U.I. (2000). Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus. Plant J 23, 771-783.
Keegstra, K., Lubben, T.H., and Theg, S.M. (1988). Transport of proteins into chloroplasts. Photosynth Res 17, 173-194.
Keegstra, K., Olsen, L.J., and Theg, S.M. (1989). Chloroplastic Precursors and their Transport Across the Envelope Membranes. Annual Review of Plant Physiology and Plant Molecular Biology 40, 471-501.
Kouranov, A., Wang, H., and Schnell, D.J. (1999). Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway. J Biol Chem 274, 25181-25186.
Kouranov, A., Chen, X., Fuks, B., and Schnell, D.J. (1998). Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane. The Journal of cell biology 143, 991-1002.
Li, H.-m.M., and Chiu, C.-C.C. (2010). Protein transport into chloroplasts. Annual review of plant biology 61, 157-180.
Paila, Y.D., Richardson, L.G.L., and Schnell, D.J. (2015). New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development. J Mol Biol 427, 1038-1060.
Richards, F.M. (1974). The interpretation of protein structures: Total volume, group volume distributions and packing density. Journal of Molecular Biology 82, 1-14.
Schnell, D.J., Blobel, G., Keegstra, K., Kessler, F., Ko, K., and Soll, J. (1997). A consensus nomenclature for the protein-import components of the chloroplast envelope. Trends in Cell Biology 7, 303-304.
Shi, L.X., and Theg, S.M. (2013). The chloroplast protein import system: from algae to trees. Biochim Biophys Acta 1833, 314-331.
Stanga, J.P., Boonsirichai, K., Sedbrook, J.C., Otegui, M.S., and Masson, P.H. (2009). A role for the TOC complex in Arabidopsis root gravitropism. Plant Physiol 149, 1896-1905.
Theg, S.M., Bauerle, C., Olsen, L.J., Selman, B.R., and Keegstra, K. (1989). Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J Biol Chem 264, 6730-6736.
Viana, A.A.B., Li, M., and Schnell, D.J. (2010). Determinants for Stop-transfer and Post-import Pathways for Protein Targeting to the Chloroplast Inner Envelope Membrane. Journal of Biological Chemistry 285, 12948-12960.
Vojta, L., Soll, J., and Bolter, B. (2007). Protein transport in chloroplasts - targeting to the intermembrane space. Febs j 274, 5043-5054.
Windsor, B., Roux, S.J., and Lloyd, A. (2003). Multiherbicide tolerance conferred by AtPgp1 and apyrase overexpression in Arabidopsis thaliana. Nature Biotechnology 21, 428-433.