臺灣博碩士論文加值系統

尾端膜蛋白 (Tail-anchored membrane protein，TA protein) 是透過TRC40 / GET3結合因子在哺乳動物和酵母中安全地運送至內質網（ER）膜。但是尾端膜蛋白在植物中遞送的機制相對先前兩者仍然不清楚，這是一個很關鍵且重要的問題。這種獨特的真核細胞膜運輸系統是如何正確地區分出用於各種細胞器的不同尾端膜蛋白，包括粒線體，葉綠體和內質網的尾端膜蛋白。在本篇實驗我們欲表現藻類 (萊茵衣藻) ArsA1（GET3同系物）的蛋白，在改變結構上的重要位點,進而了解ArsA1如何準確的辨認尾端膜蛋白，運送且插入至葉綠體膜上，最後我們希望在往後的實驗找出提升萊茵衣藻光合作用效率的方法。
在本篇文章我們透過 先前實 驗純化 的蛋白進行 結晶，從而得到 綠藻 ArsA1蛋白的結構，另外我們想要了解在綠藻中ArsA1是辨認哪些胞器的尾端膜蛋白並且正確的運送至特定胞器，因此我們在兩種質體中接上ArsA1的序列，在另一質體中接上尾端膜蛋白及His-tag之序列，再進入大腸桿菌中一同表現，當兩種蛋白皆被表現出來時，在純化的過程中只有接有His-tag的尾端膜蛋白會與Ni-column作結合，因此如果在管柱中發現ArsA1則表示兩者蛋白之間是具有交互作用，反之若沒有交互作用時，ArsA1蛋白便不會在管柱中被純化到，我們便是藉由此實驗方法來判斷綠藻中ArsA1是與何種胞器之尾端膜蛋白辨認及運送。

Tail-anchored (TA) membrane protein is safely transported to the endoplasmic reticulum (ER) membrane in mammals and yeast via the TRC40/GET3 binding factor. However, the mechanism by which the TA protein is delivered remains unclear in plants. This unique eukaryotic cell membrane transport system correctly distinguishes between different TA proteins for various organelles, including the mitochondria, chloroplast and ER TA proteins. In this experiment, we want to express the protein of green algae (C. reinhardtii) ArsA1 (GET3 homolog), and to understand how ArsA1 can accurately recognize, transport, and insert the TA proteins into the chloroplast membrane. We also want to find ways to improve ArsA1 activities during the photosynthesis pathway in C. reinhardtii.
In this article, we use the purified ArsA1 overexpressed form E. coli for crystallization, which is similar to our previous experiment, and also found out the structure of ArsA1 protein. Then we want to realize what kind of TA protein that ArsA1 would bind, and then TA protein can be sent to the right place on the specific membrane of the organelles. For this purpose, we constructed two different kind of plasmids. One sequence is the ArsA1, and the other is TA proteins with his-tag on them. We did co-tranformation into the E. coli to express these two protein complex. During the purification step, his-tag in the TA protein complex is the only protein motif specifically binding to the Ni-NTA column rather than ArsA1. We wash out the protein by the imidazole and analysis the solution flowing out. If we can find the ArsA1, than we can say that there is interaction between the ArsA1 and this TA protein.

國立中山大學 研究生學位論文審定書 …………………………………………...…ⅰ
誌謝 …………………………………………………….…………………………………………….….ⅱ
中文 摘要 ………..……………………………………………………………………………..……ⅲ
英文摘要 ……………………………………………………………………………………….…....ⅳ
第一章 序論 …………………………………………………………….………………………………………..1
1-1 前言..…………………………………………………………………………………………..….1
第二章 實驗步驟及材料方法 …………………………………………………………………………...9
2-1 藥品與儀器 ………………………………………………………………………………....9
2-2 Buffer A 與 Buffer B 的製備 ……………………………………………………….10
2-3 SDS-PAGE 電泳膠片的製備(12% Acrylamide) ……..…………..…….10
2-4 Tris-Tricine gel電泳膠片的製備 (12% Acrylamide) .…………..…....11
2-5 質體的來源及製備 ……….……….…………………….…………………...…..…..13
2-6 Ni-column的製備 ……….……….…………………….………………………….…..13
2-7 蛋白濃度測定 ……….……….…………………….…………………….………….…..14
2-8 核酸 濃度 測定 ……….……….………………………….……………….….…….…....14
2-9 實驗 一.基因轉殖 ……………………………………………………….……………...15
2-10 實驗 二.聚合酶連鎖反應 （PCR）………………………………………….....16
2-11 實驗 三.Ligation及 Transformation …………………………………………….17
2-12 實驗 四.質體的抽取 …………………………………………………………………..18
2-13 實驗 五. Transformation ……………………………………………………………...19
2-14 實驗 六.養菌 …………………………………………………………………………..….20
2-15 實驗 七.蛋白純化 ……………………………………………………………………...21
第三章 實驗 結果 ………………………………………….………………………………………..………..23
3-1 Cr-tArsA1蛋白用 Pull down assay的方式觀察與 的方式觀察與 尾端膜蛋白的交互作 用 …………………………………....…..………………..23
3-2 Cr-tArsA1-9N蛋白用 Pull down assay的方式觀察與 的方式觀察與 尾端膜蛋白的交 互作用 ………….……….…......……….…………......23
3-3 Cr-tArsA1-3K蛋白用 Pull down assay的方式觀察與 的方式觀察與 尾端膜蛋白的交 互作用 .….………...……………........………………..24
第四章 討論 …………………………………………………………………………….……………………….25
第五章 結論 ………………………………………….…………………….......................................27
第六章 參考文獻 ………………………………………….……………………...............................50
圖目錄
圖一. GET system Pathway圖 ..……………………….…………………………………….……..4
圖二. Cr-ArsA1序列比對 序列比對 圖 ..……………………….………………………………………….…..6
圖三. TOC34、TOC75、TOC159膜蛋白功能 圖 ..……………………….…………..…7
圖四. 酵母菌 GET3結構 圖 ..……………………….……………………………………….….….8
圖五. 綠藻 ArsA1結構 圖 ..……………………….……………………………………………..….8
圖六. Cr- tArsA1+TOC34-NTC Pull down assay電泳圖 ..………………………..…28
圖七. Cr- tArsA1+TOC159-NTC Pull down assay電泳圖 電泳圖 ..………………………..29
圖八. Cr- tArsA1+TOC159-TC Pull down assay電泳圖 ..……………………….....30
圖九. Cr- tArsA1+TOM7 Pull down assay電泳圖 電泳圖 ..…………………………………….31
圖十. Cr- tArsA1+TOM5 Pull down assay電泳圖 電泳圖 ..…………………………….…..….32
圖十一 . Cr- tArsA1+ sec61β Pull down assay電泳圖 電泳圖 ..……………………………….33
圖十二 . Cr- tArsA1+ VAMP Pull down assay電泳圖 電泳圖 ..…………………………....…34
圖十三 . Cr- tArsA1-9N +TOC34-NTC Pull down assay電泳圖 ..……………....35
圖十四 . Cr- tArsA1-9N + TOC159-NTC Pull down assay電泳圖 ..…………….36
圖十五. Cr- tArsA1-9N +TOC159-TC Pull down assay電泳圖 ..………………..37
圖十六. Cr- tArsA1-9N + TOM5 Pull down assay電泳圖 電泳圖 ..…………………………38
圖十七. Cr- tArsA1-9N +TOM7 Pull down assay電泳圖 電泳圖 ..………………………….39
圖十八. Cr- tArsA1-9N + VAMP Pull down assay電泳圖 電泳圖 ..………………………….40
圖十九. Cr- tArsA1-9N + PEP12 Pull down assay電泳圖 電泳圖 ..…………………………41
圖二十. Cr- tArsA1-9N + Cyt b5 Pull down assay電泳圖 電泳圖 ..…………………………42
圖二十一 . Cr- tArsA1-3K +TOC34-NTC Pull down assay電泳圖 ..………..….43
圖二 十. Cr- tArsA1-3K + TOC159-NTC Pull down assay電泳圖 ..………...44
圖二十三 . Cr- tArsA1-3K + TOM7 Pull down assay電泳圖 ..……………………..45
圖二十四. Cr- tArsA1-3K +VAMP Pull down assay 電泳圖 ..……………………..46
圖二十五. Cr- tArsA1-3K + Pep12 Pull down assay 電泳圖 ..……………..……...47
表目錄
Table.1 綠藻ArsA1 及ArsA2 與酵母菌GET3 及大腸桿菌ArsA 活性測試 ...6
Table.2 實驗中所使用之各類胞器尾端膜蛋白的種類 ……………………………..…..48
Table.3 三種ArsA1 蛋白與尾端膜蛋白的交互作用 ………………………………..……49

1 Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D: Biological Crystallography 66, 213-221 (2010).
2 Beilharz, T., Egan, B., Silver, P. A., Hofmann, K. & Lithgow, T. Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae. Journal of Biological Chemistry 278, 8219-8223 (2003).
3 Kalbfleisch, T., Cambon, A. & Wattenberg, B. W. A bioinformatics approach to identifying tail-anchored proteins in the human genome. Traffic 8, 1687-1694 (2007).
4 Kriechbaumer, V. et al. Subcellular distribution of tail-anchored proteins in Arabidopsis. Traffic 10, 1753-1764 (2009).
5 Rapaport, D. Finding the right organelle: Targeting signals in mitochondrial outer-membrane proteins. EMBO reports 4, 948-952 (2003).
6 Mateja, A. et al. Structure of the Get3 targeting factor in complex with its membrane protein cargo. Science 347, 1152-1155 (2015).
7 Mariappan, M. et al. The mechanism of membrane-associated steps in tail-anchored protein insertion. Nature 477, 61 (2011).
8 Yamagata, A. et al. Structural insight into the membrane insertion of tail-anchored proteins by Get3. Genes to Cells 15, 29-41 (2010).
9 Formighieri, C., Cazzaniga, S., Kuras, R. & Bassi, R. Biogenesis of photosynthetic complexes in the chloroplast of C hlamydomonas reinhardtii requires ARSA 1, a
homolog of prokaryotic arsenite transporter and eukaryotic TRC 40 for guided entry of tail-anchored proteins. The Plant Journal 73, 850-861 (2013).
10 Lee, J., Kim, D. H. & Hwang, I. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria. Frontiers in plant science 5, 173 (2014).
11 Auld, K. L. et al. The conserved ATPase Get3/Arr4 modulates the activity of membrane-associated proteins in Saccharomyces cerevisiae. Genetics 174, 215-227 (2006).
12 Stefanovic, S. & Hegde, R. S. Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell 128, 1147-1159 (2007).
13 Favaloro, V., Spasic, M., Schwappach, B. & Dobberstein, B. Distinct targeting pathways for the membrane insertion of tail-anchored (TA) proteins. Journal of cell science 121, 1832-1840 (2008).
14 Schuldiner, M. et al. The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134, 634-645 (2008).
15 Mateja, A. et al. The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461, 361 (2009).
16 Chartron, J. W., Suloway, C. J., Zaslaver, M. a. & Clemons, W. M. Structural characterization of the Get4/Get5 complex and its interaction with Get3. Proceedings of the National Academy of Sciences 107, 12127-12132 (2010).
17 Chio, U. S., Chung, S., Weiss, S. & Shan, S.-o. A protean clamp guides membrane targeting of tail-anchored proteins. Proceedings of the National Academy of Sciences 114, E8585-E8594 (2017).
18 Yamamoto, Y. & Sakisaka, T. The emerging role of calcium-modulating cyclophilin ligand in posttranslational insertion of tail-anchored proteins into
the endoplasmic reticulum membrane. The Journal of Biochemistry 157, 419-429 (2015).
19 Hsu, C.-M. & Rosen, B. Characterization of the catalytic subunit of an anion pump. Journal of Biological Chemistry 264, 17349-17354 (1989).
20 Zhou, T., Radaev, S., Rosen, B. P. & Gatti, D. L. Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. The EMBO journal 19, 4838-4845 (2000).
21 Maestre-Reyna, M. et al. In search of tail-anchored protein machinery in plants: reevaluating the role of arsenite transporters. Scientific reports 7, 46022 (2017).
22 Lin, T. W. et al. Structural analysis of chloroplast tail-anchored membrane protein recognition by ArsA1. The Plant Journal (2019).
23 Richardson, L. G. et al. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane. Frontiers in plant science 5, 269 (2014).
24 Falkowski, P. G., Barber, R. T. & Smetacek, V. Biogeochemical controls and feedbacks on ocean primary production. Science 281, 200-206 (1998).