已知在酵母菌及哺乳動物中,copper chaperone for superoxide dismutase (CCS)是負責提供並將銅離子鑲嵌至超氧歧化酵素(superoxide dismutase,SOD)的蛋白質,而阿拉伯芥也有一個可能的CCS基因(At1g12520,AtCCS)。此基因之產物與其他物種的CCS具相似性,也可區分為三個不同的domains,分別是位於N端具有可抓住銅離子的ATX1-like domain (具MXCXXC metal binding motif)、胺基酸序列與CuZnSOD蛋白質有部分相似的central domain以及位於C端屬於CCS蛋白質所特有之序列,此區段亦具有可抓住銅離子的CXC motif。己知阿拉伯芥有三個CuZnSOD基因,分別為CSD1、CSD2及CSD3,其中CSD1及CSD2的活性分別位於細胞質及葉綠體中,由於CSD3蛋白質的C端具有peroxisome targeting signal (AKL),所以被認為會被送入peroxisomes。然而,可能的AtCCS基因卻僅有一個,為瞭解此基因與三種CuZnSOD之間的關係,本研究利用一個同時失去三種CuZnSOD活性之Atccs knockout突變株為材料,將由The Arabidopsis Information Resource (TAIR) database中所預測的AtCCS cDNA編譯區段轉入此Atccs突變株,發現只有細胞質及peroxisome中的CuZnSOD活性會回復。在由Munich Information Centre for Protein Sequences (MIPS)中所預測的AtCCS cDNA編譯區段中,可轉譯出比TAIR database所預測AtCCS蛋白質的N端多了66個胺基酸,當我們將此基因轉入Atccs突變株,發現除上述兩種CuZnSOD外,葉綠體型CuZnSOD的活性也可被回復。經離體葉綠體蛋白質運移實驗證明,此N端多出來的66個胺基酸確實與AtCCS蛋白質進入葉綠體有關。實驗結果顯示單一AtCCS基因的產物可同時活化三種CuZnSOD活性。我們也將僅含有central domain及C-terminal domain的AtCCS蛋白質表現在Atccs突變株,發現轉植株之CuZnSOD活性並未被回復,此結果顯示在植物體中,ATX1-like domain對於AtCCS蛋白質的功能是必要的區段。
此外,我們也將水稻中可能的CCS基因(OsCCS)之cDNA對應至mature peptide的編譯區段,轉殖到Atccs突變株進行功能性互補試驗(functional complementation assay),發現細胞質及peroxisome中的CuZnSOD活性可被回復,顯示此OsCCS基因確實具有CCS的功能,驗證了除了酵母菌的lys7突變株外,Atccs突變株也是一個適合用來探討其他植物CCS homologues功能的系統。The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in Saccharomyces cerevisiae and mammals. In Arabidopsis thaliana, only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains, a central domain flanked by an ATX1-like and a C-terminal domains. The ATX1-like and the C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by the Arabidopsis Information Resource (TAIR) complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences (MIPS), and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic, CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.
The mature peptide of a putative CCS homologue in Oryza sativa (OsCCS) was ectopically expressed in the Atccs mutant and complemented the cytosolic and peroxisomal CuZnSOD activities. The results indicate that the OsCCS protein functions as a copper chaperone donating copper ions to CuZnSOD. The Atccs mutant can serve as another system, in addition to the yeast lys7 mutant, to confirm the function of the other CCS homologues of plants.Abstract in Chinese I
Abstract in English III
Abbreviations V
Introduction 1
Copper Ion in Living Organisms 2
Copper Trafficking Factors 2
Plant Homologues of the Copper Trafficking Factors 5
ATX1-Like Domain Study 7
Aims of the Dissertation 8
Materials and Methods 9
Plants, Growth condition, and CuSO4 Treatment 9
Analysis of the CCS Gene Sequence 9
Southern-Blot Analyses 10
Northern-Blot Analyses 10
RT-PCR and 5'-RACE of the AtCCS Gene 11
Protein Extraction and Quantification 12
Electrophoresis and SOD Activity Analyses 12
Immunoblot Analyses 12
In Vitro Transcription/Translation of AtCCScyt and AtCCScp Proteins, and Protein Import into Isolated Chloroplasts 13
Gene Construction and Plant Transformation 14
Construction and Transient Expression of the Reporter Genes 15
Results 17
AtCCS Gene in A. thaliana 17
Identification and Characterization of Superoxide Dismutase Activities in A. thaliana 18
CuZnSOD Activity and AtCCS Expression Pattern 19
Characterization of the Atccs Knockout Mutant 19
Different Recovery of CuZnSOD Activities in the Atccs Mutant Complemented with AtCCScyt, AtCCScp, and AtCCScp-mut 20
Chloroplastic Localization of AtCCScp 22
Localization of AtCCS Protein In Vivo 22
Importance of the ATX1-like Domain in Conferring CuZnSOD Activity 23
OsCCS Gene in Oryza sativa 24
Discussion 26
Figures 32
References 5