有著“能量工廠”之稱的粒線體能為真核細胞運轉提供大部分的能源，而位在其內膜中的氧化磷酸化系統則由電子傳遞鏈和ATP合成裝置這兩部分所構成。Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7)為電子傳遞鏈第一蛋白質複合體中核心蛋白之一次單元，其具備了能與[4Fe-4S]鐵硫中心N2鍵結的保留序列，因此由細胞核基因所編碼的NDUFS7扮演著粒線體第一蛋白質複合體中電子傳遞最後接受者的重要角色。先前的研究顯示，NDUFS7的N端具有一段粒線體引導序列(MTS)，其C端則分別具有細胞核座落訊號(NLS)和細胞核運出訊號(NES)，且也出現粒線體、細胞質和細胞核等位置。NDUFS7的缺損已被發現與萊氏症候群(Leigh syndrome)及一些神經性疾病有所關聯。蛋白的分佈狀態與功能時常受到各種轉譯後修飾的調控，而其中又以磷酸化修飾在這一方面的涉及最為普遍。從我們之前的研究結果中可得知，主要位於細胞質中的c-Src酵素能對NDUFS7進行磷酸化修飾，故本研究首先驗證了無論在in vivo還是in vitro的實驗中NDUFS7皆能被c-Src磷酸化，接著藉由免疫沉澱的方式確認兩者可以互相結合，另外指出NDUFS7上的Tyr160位置為c-Src磷酸化修飾的重要位點且能對此蛋白的穩定性造成影響。在不影響NDUFS7於細胞核與細胞質中分佈的情況下，由c-Src所調控的NDUFS7磷酸化能增加該蛋白在粒線體中成熟型式的數量，意味著c-Src能促進NDUFS7進入粒線體中並轉化以成熟的形式存在。此外，NDUFS7的SUMOylation修飾會拮抗NDUFS7的磷酸化反應。根據測試在不同壓力反應的結果顯示，NDUFS7磷酸化的程度顯著受到由氯化鈷(CoCl2)所誘發的缺氧壓力及分別由過氧化氫(H2O2)和魚藤酮(Rotenone)所誘發的氧化壓力影響。另一方面，NDUFS7磷酸化程度反而在飢餓環境所產生的壓力下、粒線體膜電位受損及細胞凋亡的狀態中皆未受到顯著影響。之後我們的研究將著重於NDUFS7磷酸化修飾對粒線體功能的影響及其與其他修飾關聯性的細部探討。

Mitochondria, the powerhouse of cells, provide most of energy for cellular operation in eukaryotic cells. The oxidative phosphorylation (OXPHOS) system located on mitochondrial inner membrane consists of an electron transport chain and an ATP synthesis machinery. Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of core subunits of mitochondrial complex I in the electron transport chain. NDUFS7 contains a conserved sequence for binding to the [4Fe-4S] cluster N2. This nuclear-encoded protein is involved in electron transport and plays a key role as “the last electron receptor” in mitochondrial complex I. Previously, we have demonstrated that NDUFS7 contains a mitochondrial targeting sequence (MTS) at the N-terminus, a nuclear localization signal (NLS) and a nuclear export signal (NES) at the C-terminus, and is present in mitochondria, the cytosol and the nucleus. Defects of NDUFS7 are found to be associated with Leigh syndrome and some neural diseases. The localization and function of a protein are frequently modulated by post-translational modifications and phoshphorylation is the most common modification involved in this aspect. We also found that NDUFS7 can be phosphorylated by c-Src, which is a kinase in the cytosol. In this study, phosphorylation of NDUFS7 by c-Src was verified in both in vivo and in vitro experiments. The physical interaction between NDUFS7 and c-Src was also confirmed by immunoprecipitation. In addition, Tyr160 of NDUFS7 was found as an important site for c-Src-mediated phosphorylation and could affect the stability of this protein. Furthermore, c-Src-mediated phosphorylation was found to increase the mature form of NDUFS7 in mitochondria without affecting the distribution of NDUFS7 in the nucleus and the cytosol. It implies that c-Src can promote the mitochondrial import of NDUFS7 and thus enhance its maturation. In addition, SUMOylation of NDUFS7 was antagonistic to NDUFS7 phosphorylation under the experimental setting used in this study. Based on the data for stress response analyses, the level of c-Src-mediated phosphorylation of NDUFS7 was significantly decreased in CoCl2-induced hypoxia, H2O2-induced and rotenone-induced oxidative stress. On the other hand, the level of c-Src-mediated phosphorylation of NDUFS7 was not significantly affected by serum-induced starvation, dissipation of mitochondrial membrane potential and induction of apoptosis. Further studies will focus on exploring the functional effects of NDUFS7 phosphorylation and the detailed mechanism of the crosstalk between phosphorylation and other modifications on NDUFS7.

摘要 I
Abstracts II
Abbreviations IV
Introduction 1
1. Mitochondria 1
1.1 Mitochondrial genome 2
1.2 Oxidative phosphorylation (OXPHOS) system 3
1.3 The crosstalk between mitochondria and nuclei 4
1.3.1 The function of NLS and NES 6
1.3.2 Mechanism of nucleo-cytoplasmic transport 7
1.3.3 The function of MTS 8
1.3.4 Mechanism of the cytosol-mitochondria transport 8
1.4 Mitochondrial complex I (NADH: ubiquinone oxidoreductase) 9
1.5 Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) 10
1.6 The localization of complex I NDUFS7 subunit 11
1.7 The association between NDUFS7 and diseases 11
2. Phosphorylation 14
2.1 Mechanism and function of phosphorylation 14
2.2 Phosphorylation sites 15
2.3 Protein phosphatases 16
2.4 Protein kinases 17
2.4.1 Src family tyrosine kinases 17
2.4.2 Structural domains of Src family kinases 18
2.4.3 Activation of Src family kinases 19
2.4.4 c-Src 20
2.4.5 Inhibitor of Src family kinases 21
2.5 Phosphorylation in mitochondria 21
2.5.1 Tyrosine phosphorylation and mitochondria 21
2.5.2 Src tyrosine kinases in mitochondria 22
2.6 The association between protein phosphorylation and stress responses 23
3. SUMOylation 23
3.1 SUMOylation in mitochondria 24
3.2 The crosstalk between SUMOylation and phosphorylation 25
Materials and methods 26
1. Cell culture 26
2. Antibodies and chemicals 26
3. Plasmids 27
4. Transient transfection 27
5. Preparation of whole cell extracts 28
6. Immunoprecipitation 29
7. Cell fractionation 29
8. Immunoblotting analysis 31
8.1 Immunoblotting steps for general studies (analyzed by antibodies related to phosphorylation) 31
8.2 Immunoblotting steps for sepcific studies (analyzed by antibodies related to phosphorylation) 32
9. The procedure for mild stripping of membrane for immunoblotting analysis 32
10. Hypoxia-induced assay 33
11. Oxidative stress-induced assay 33
12. Dissipation of mitochondrial membrane potential assay 34
13. Apoptosis-induced assay 34
14. Starvation-induced assay 35
15. Expression of recombinant NDUFS7-His 35
16. Purification of recombinant NDUFS7-His 36
17. Refolding of the purified recombinant NDUFS7-His 37
18. In vitro kinase assay 37
19. Phosphatase treatment 38
20. Statistical analysis 38
Results 39
1. Sequence analysis of NDUFS7 39
2. NDUFS7 is tyrosine-phosphorylated by c-Src in vivo 39
3. c-Src interacts directly with NDUFS7 in vivo 40
4. NDUFS7 is phoshorylated by c-Src in vitro 41
5. c-Src-mediated phosphorylation increases the mature form of NDUFS7 in mitochondria 42
6. Tyr160 of NDUFS7 is the major site for c-Src-mediated phosphorylation and affects protein stability of NDUFS7 43
7. SUMOylation of NDUFS7 has an antagonistic relationship to NDUFS7 phoshphorylation 43
8. The change of c-Src-mediated phosphorylation level of NDUFS7 in response to various stresses 44
Discussion 46
1. The relationship between the mechanism of mitochondrial import of NDUFS7 and c-Src-mediated phosphorylation 46
2. The relationship between phosphorylation and SUMOylation of NDUFS7 47
3. The mechanism of how c-Src transports into the mitochondria. 47
Tables 49
Figures 51
References 76
Appendixes 87

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