利用異位及異種表現的方式探討衣藻細胞核版粒線體呼吸鏈相關基因在人類細胞中之表現

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研究生：	林于廷 Lin, Yu-Ting
論文名稱：	利用異位及異種表現的方式探討衣藻細胞核版粒線體呼吸鏈相關基因在人類細胞中之表現 Allotopic and xenotopic expression of respiratory subunits in human cells with nuclear genes from Chlamydomonas reinhardtii encoding cognate mitochondrial proteins
指導教授：	高茂傑 Kao, Mou-Chieh
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	66
中文關鍵詞：	粒線體、衣藻、異位表現、異種表現
外文關鍵詞：	mitochondria, Chlamydomonas reinhardtii, Allotopic expression, xenotopic expression
相關次數：	點閱：2 下載：0
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內共生的發生使得粒線體的基因轉移到細胞核內。然而，仍然有一些基因依然保留在粒線體的基因體(genome)內，這些基因表現的蛋白涉及氧化磷酸化的反應。這些蛋白包含了complex I中的ND1~ND6和ND4L次單元蛋白，complex III的cytochrome c，complexIV的Cox1~Cox3次單元蛋白，以及complex V的ATP6和ATP8次單元蛋白。有趣的是，衣藻的粒線體基因體內缺乏nd3、nd4l、cox2、cox3、atp6以及atp8等基因。先前的文獻指出衣藻的nd3、nd4l、cox2、cox3以及atp6等基因已經轉移到細胞核中，其中cox2基因還分離為兩個獨立的基因。
在這個研究當中，異位及異種表現的策略被應用在評估這些方法對於治療粒線體疾病的可能性。當衣藻的ND4L、ND3、ATP6、COX3及COX2a的粒線體標的訊號(mitochondrial targeting signal , MTS)與enhanced green fluorescent protein (EGFP)結合後，可以成功的將結合蛋白運送進入HeLa和HEK293細胞的粒線體內。然而，當衣藻的ND4L及ND3的MTS分別和它們的人類同源蛋白結合後，異位運送結合蛋白進入粒線體的效果卻很有限。為了加強運送蛋白的效率，異種表現衣藻的細胞核版本之全長粒線體相關蛋白被運用來探討粒線體蛋白在人類細胞之運送。藉由這種方式，運送complex I的ND4L次單元至粒線體的效果大幅的改善。儘管如此，當HIV的轉錄活化子( the transactivator of the transcription, TAT)片段結合至衣藻轉殖基因的前方時，這種新的設計對於改善蛋白運送進入粒線體內的幫助並不大。然而，當親水性的EGFP蛋白被結合到衣藻ND4L次單元的C端時，這個方法卻可以有效降低運送蛋白的疏水性並且增加運送蛋白至粒線體的能力。本篇研究證實衣藻的ND4L次單元在人類的細胞中擁有被運送進入粒線體的能力，以及蛋白的疏水性對於運送至粒線體的過程中扮演了一個非常重要的角色。

The endosymbiotic event leads mitochondrial genes to be translocated into nuclei. However, there are still a few genes conserved in the mitochondrial genome which code for proteins involved in oxidative phosphorylation. These proteins include ND1~ND6 and ND4L subunits of complex I, cytochrome c of complex III, Cox1~Cox3 subunits of complex IV, ATP6 and ATP8 subunits of complex V. Interestingly, the mitochondrial genome in Chlamydomonas reinhardtii lacks nd3, nd4l, cox2, cox3, atp6 and atp8 genes. Previous report suggested that nd3, nd4l, cox2, cox3 and atp6 were transfered to nuclei in C. reinhardtii, and cox2 was further divided into two independent nuclear genes.
In this study, allotopic and xenotopic expression strategies were adopted to evaluate the possibilities of applying these methods as treatment concepts for mtDNA diseases. When the mitochondrial targeting signal (MTS) of ND4L, ND3, ATP6, COX3, and COX2a of C. reinhardtii was fused with enhanced green fluorescent proteins (EGFP), the resultant fusion proteins were successfully imported into mitochondria of HeLa and HEK293 cells. However, when the MTSs of the ND4L and ND3 were individually fused with its human homologue, only a limited mitochondrial targeting was observed. To improve the import efficiency, xenotopic expression of several full-length C. reinhardtii nuclear-encoded mitochondrial proteins were directly applied for mitochondrial targeting studies in human cells. By this approach, a significant improvement of mitochondrial import was observed in the targeted complex I ND4L subunit. Nevertheless, when the transactivator of transcription (TAT) region from the HIV was added in front of these constructed C. reinhardtii transgenes, the new designs did not show a significant improvement on mitochondrial targeting. However, when the hydrophilic EGFP protein was added at the C-terminus of the full-length C. reinhardtii ND4L subunit, the process could decrease the hydrophobicity of the importing protein effectively and improve the ability of the mitochondrial targeting. In this report, we proved that the C. reinhardtii ND4L subunit possesses the capability of the mitochondrial targeting in human cells and the hydrophobicity of the importing proteins plays an important role for mitochondrial targeting.

中文摘要    i
Abstracts    ii
Abbreviations    vi
Introduction    1
Materials and methods    13
Results    19
1. Isolation of Chlamydomonas reinhardtii genes and construction of expression vectors    19
2. Expression of algal mitochondrial-targeted signal fused EGFP in human cells    19
3. Allotopic expression of human recoded ND3 and ND4L with the corresponding algal leader peptide    20
4. Xenotopic expression of C. reinhardtii nuclear encoded mitochondrial proteins in human cells    21
5. Expression of TAT- algal nuclear encoded proteins in human cells    22
6. Fusion of hydrophilic EGFP to an entire algal nuclear-encoded ND4L    23
Discussion    25
Table    30
Figure    35
Figure 1. Sequence alignments of ND4L polypeptides from various species.    35
Figure 2. The mitochondrial targeting efficiency of C. reinhardtii ND4LMTS, ND3MTS, ATP6MTS, COX3MTS, and COX2aMTS fused with EGFP in HeLa and HEK293 cells.    39
Figure 3. The cleavage of mitochondrial targeting sequence from various C. reinhardtii nuclear-encoded mitochondrial proteins in HeLa cell.     40
Figure 4. The subcellular location of H. sapiens ND3 and ND4L subunits fused with the corresponding C. reinhardtii mitochondrial targeting sequence in HeLa cells and HEK293 cells.    42
Figure 5. The subcellular location of various C. reinhardtii nuclear-encoded mitochondrial proteins in HeLa cells and HEK293 cells.    46
Figure 6. The subcellular location of various C. reinhardtii nuclear-encoded mitochondrial proteins fused with the HIV TAT peptide in the N-terminus in HeLa and HEK293 cells.    49
Figure 7. Expression of EGFP fused full-length nuclear-encoded C. reinhardtii mitochondrial protein ND4L or ND3 in the HeLa and HEK293 cells.    51
Figure 8. Immunoblotting analyses of C. reinhardtii ND4L-EGFP and C. reinhardtii ND3-EGFP imported into mitochondria in the HeLa and HEK293 cells.    52
Figure 9. Mesohydrophobicity and hydrophobicity plot of mitochondrial proteins from C. reinhardtii and H. sapiens.    53
Reference    54
Appendixes    60

                                

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