酵母菌 alanyl-tRNA synthetase 的細胞內傳輸機制

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趙恆億(Hen-Yi Chao) 查詢紙本館藏

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論文名稱

酵母菌 alanyl-tRNA synthetase 的細胞內傳輸機制
(The transport mechanism of alanyl-tRNA synthetase in Saccharomyces cerevisiae)

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摘要(中)

真核生物除了細胞質，粒線體也會進行蛋白質的合成，因此在細胞體內必須要有兩套不同的蛋白質合成酵素。ALA1基因是酵母菌(Saccharomyces cerevisiae)中唯一的alanyl-tRNA synthetase (AlaRS)基因，此基因可同時轉譯出兩種AlaRS異構型，分別作用在細胞質及粒線體內。它是利用最靠近5，端的ATG1 轉譯出細胞質的異構型，利用ATG1上游的二個重複ACG (即ACG-25和ACG-24) 作出粒線體異構型。本研究則是著重在進一步探討粒線體AlaRS的細胞內傳輸機制及功能。藉由功能性互補實驗、點突變、及西方式點墨法的測試，我們發現AlaRS的標的訊號不只包括Met1 上游的前序列，尚含括了Met1 下游的18個胺基酸。只有前序列或胺基端18個胺基酸只能將部份AlaRS運送到粒線體內作用，其餘的則殘留在細胞質內。此外，西方點墨法的實驗顯示粒線體AlaRS的基質蛋白質酶切割位似乎不是如PSORTII預測位於Asn16及Thr17之間，而更可能位在上游的序列上。另外在別種低等真核酵母菌: 如C. albicans中我們也發現了其利用唯一的ALA1基因轉譯出細胞質和粒線體的AlaRS蛋白質的例子，同時更確定C. albicans的AlaRS 可以互補於S. cerevisiae AlaRS的生理活性，因此找出其粒線體標的訊號的所在及探討對蛋白質的傳輸影響是接下來重要的課題。總結我們的實驗結果，S. cerevisiae AlaRS的功能除了取決於蛋白質表現量多寡之外，也受到其標的訊號的強弱影響。

摘要(英)

In eukaryotic cells, protein synthesis occurs not only in the cytoplasm but also in the mitochondria, so cells need to have more than one set of this enzyme for function in different organelles. It was recently shown that ALA1 is the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase (AlaRS). ALA1 was found to encode both cytoplasmic and mitochondrial isoforms of AlaRS. The former is translationally initiated at the first ATG codon (designated ATG1) at the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-ATG codons (i. e., ACG-25 and ACG-24). In this thesis, we focused on the transport mechanism and physiological function of mitochondrial AlaRS. The results of functional tests, site directed mutagenesis, and Western blotting all suggest that the mitochondrial targeting signal of ALA1 includes not only the upstream presequence but also the first eighteen amino acids downstream of Met1 (from codons ACG-25 to TTT+18). When only the presequence or first 18 amino acids of the cytoplasmic N-terminus is used to test for mitochondrial targeting activity, only a portion of the protein products were transported into mitochondria, while the rest remained in the cytoplasm. In addition, the results of Western blotting also reveal that the mitochondrial matrix processing peptidase cutting site may not be between Asn16 and Thr17 as previously predicted by PSORTII, but is rather located at sequences further upstream. In addition, we have also demonstrated that the low eukaryote Candida albicans also uses a single ALA1 gene to encode both cytoplasmic and mitochondrial AlaRS isoforms, and the protein product of this gene has complementing activity in Saccharomyces cerevisiae. Thus finding the mitochondrial targeting signal of this protein and elucidating its effects on protein transport is the next major step of this project. In conclusion, the activity of AlaRS is dependent on both the amount of protein synthesized and the strength of the mitochondrial targeting signal.

關鍵字(中)

★ 傳輸機制

關鍵字(英)

★ alanyl-tRNA synthetase

論文目次

目錄1
圖、表目錄3
縮寫檢索表4
中文摘要 5
英文摘要 6
第一章緒論 8
第二章材料與方法 13
? 使用之菌株、載體及培養基 13
? 大腸桿菌勝任細胞的製備與轉型作用 14
? 酵母菌勝任細胞的製備與轉型作用 16
? 質體之建構 18
? 點突變 19
? 功能性互補試驗(Complementation ) ―測試細胞質功能 20
? 功能性互補試驗(Complementation ) ―測試粒線體功能 21
? 蛋白質製備(Protein Preparation) 22
? SDS-PAGE之蛋白質分子量分析 23
? 西方點漬法(Western blotting) 24
第三章結果 27
一、前導序列不具備完整的粒線體傳輸功能
27
二、AlaRS粒線體標的訊號包含讀碼框N端18個胺基酸
27
三、用西方點漬法證實AlaRS存在的不同形式
30
四、Candida albicans的AlaRS同時具有細胞質及粒線體
功能且互補於S. cerevisiae 的ALA1 剔除菌株
31
五、改變ATG1下游讀碼框序列破壞大部分細胞質及粒線體AlaRS活性 33
第四章討論 35
I. GRS1和ALA1的比較 35
II. C. albicans 的alanyl-tRNA synthetase
36
第五章參考文獻 39
圖表 43

參考文獻

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指導教授

王健家(Chien-Chia Wang)

審核日期

2004-7-16

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