酵母菌粒線體內Gln-tRNAGln的合成機制

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簡鈺真(Yu-Chen Chien) 查詢紙本館藏

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論文名稱

酵母菌粒線體內Gln-tRNAGln的合成機制
(Gln-tRNAGln formation in yeast mitochondria)

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摘要(中)

在蛋白質合成過程中，aminoacyl-tRNA synthetase (aaRS)將胺基酸接到相對應的tRNA形成aa-tRNA，接著aa-tRNA被送到核醣體進行轉譯作用。在酵母菌細胞質中，Gln-tRNAGln是藉由直接路徑合成(Gln + tRNAGln → Gln-tRNAGln)，而在粒線體內則是藉由間接路徑合成Gln-tRNAGln，首先利用glutamyl-tRNA synthetase (GluRSc)催化合成Glu-tRNAGln，接著再用轉胺酶(GluAdT或GatFAB)催化合成Gln-tRNAGln。奇怪的是在酵母菌Schizosaccharomyces pombe粒線體中，我們可以找到GatA及GatB的同源基因，卻找不到GatF的同源基因。藉由GatB-TAP的蛋白質體外結合實驗及LC/MS/MS分析，我們找到一個大小約14 kDa的蛋白質(SPCC777.11) (稱為GatX)，GatX與GatF在序列上差異頗大，且缺少一段N端序列。GatX是一個粒線體蛋白質，且會與GatA、GatB、GluRSc有交互作用，在細胞中是必要基因，因此在Sch. pombe中GatX可能取代S. cerevisiae中GatF的功能，成為轉胺酶複合體中的第三個次單元。論文的第二個部分，我們探討Arc1p的生物素化，Arc1p會與細胞質methionyl-tRNA synthetase (MetRSc)和GluRSc形成三元複合體，Arc1p不只幫助此二酵素進行胺醯化反應，也會調控它們在細胞內的分佈。我們發現：Arc1p的生物素化會受培養基中的生物素濃度調控，而且在Arc1p/GluRSc/MetRSc複合體中，與我們所想的不同，Arc1p生物素化的比例高達85%，從這邊的實驗中，似乎酵母菌不能藉由生物素化來調控Arc1p/GluRSc/MetRSc的複合體形成。

摘要(英)

In protein synthesis, aminoacyl-tRNA synthetases (aaRS) charge amino acids to their cognate tRNAs to become aa-tRNAs. The aa-tRNA then localizes to ribosomes for translation. In the yeast cytoplasm, Gln-tRNAGln synthesis occurs via a direct pathway (Gln + tRNAGln → Gln-tRNAGln). However, Gln-tRNAGln is synthesized by an indirect pathway in mitochondria through mischarging by a non-discriminating glutamyl-tRNA synthetase (GluRSc) followed by transamidation via a specific Glu-tRNAGln amidotransferase (GluAdT or GatFAB). Strangely, while GatA and GatB orthologues were readily identified in Schizosaccharomyces pombe genome, no GatF orthologue was found. Using GatB-TAP pulldown assay by LC/MS/MS assays we identified a GatB-interacting protein with a molecular weight of 14 kD (SPCC777.11) (GatX). GatX and GatF diverge greatly in sequence similarity, with GatX lacking an N-terminal domain. GatX is an essential gene and its protein product is localized in mitochondria. Therefore, GatX may as the third subunit in the multiplex. In the second part of the study, we focused on Arc1p biotinylation in S. cerevisiae. The yeast cytoplasmic methionyl-tRNA synthetase (MetRSc) and GluRSc form a ternary complex with Arc1p, thus regulating both their aminoacylation activities and subcellular localization. We found that biotinylation of Arc1p was modulated by biotin concentration in the medium. Contrary by our belief, the biotinylation level of Arc1p in the complex is as high as 85%. These result suggest that biotinylation is not involved in Arc1p/GluRSc/MetRSc complex formation.

關鍵字(中)

★ Glu-tRNAGln 轉胺酶
★ 酵母菌
★ aaRS輔因子
★ 胺酰-tRNA合成酶
★ 生物素化

關鍵字(英)

★ Glu-tRNAGln amidotransferase
★ Schizosaccharomyces pombe
★ Arc1p
★ aminoacyl-tRNA synthetases
★ biotinylation

論文目次

中文摘要 i
ABSTRACT ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 viii
第一章緒論 1
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 1
1.1.1 aaRS的功能 1
1.1.2. aaRS的分類 1
1.2 Glutamyl-tRNA synthetase (GluRS)的簡介 2
1.2.1 GluRS的生化特性 2
1.2.2 GluRS的演化起源 3
1.3 Arc1p的簡介 3
1.3.1 Arc1p的特性 3
1.3.1 Arc1p的生物素化後轉譯調控 3
1.4 Gln-tRNAGln的形成 4
1.4.1 Gln-tRNAGln的形成途徑：直接或間接路徑 4
1.4.2 Gln-tRNAGln的間接形成途徑 4
1.5 Glu-tRNAGln amidotransferase ( GluAdT；Glu-tRNAGln (轉胺酶)的生化特性 5
Glu-tRNAGln(轉胺酶)的生化特性 5
1.6 不同酵母菌株 5
1.6.1 Saccharomyces cerevisiae 6
1.6.2 Schizosaccharomyces pombe 6
1.7 研究目的 7
1.7.1 S. cerevisiae中Arc1p的生物素化是否會影響EMA複合體構型與功能 7
1.7.2 Sch. pombe的GluAdT 7
第二章材料與方法 8
2.1菌株、載體及培養基 8
2.1.1 菌株、基因型及其來源 8
2.1.2 載體 8
2.1.3 培養基 9
2.2大腸桿菌勝任細胞的製備與轉型作用 11
2.2.1大腸桿菌勝任細胞的製備 11
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) 12
2.3酵母菌勝任細胞的製備與轉型作用 12
2.3.1酵母菌 (S.cerevisiae)勝任細胞的製備 12
2.3.2酵母菌(S.cerevisiae)勝任細胞的轉型作用 13
2.3.3 酵母菌 (Sch.pombe)勝任細胞的製備 13
2.3.4 酵母菌(Sch.pombe)勝任細胞的轉型作用 14
2.4質體之選殖 14
2.5點突變 (Site-directed Mutagenesis) 14
2.6功能性互補試驗 (Complementation)―測試細胞質功能 15
2.7 Sch. pombe 標記 (GFP-tagging) 菌株製備 16
2.8酵母菌菌落PCR 18
2.9酵母菌交配 (mating) 18
2.10蛋白質製備 (Protein preparation) 19
2.10.1 震盪純化法 19
2.10.2 三氯醋酸 (trichloroacetic acid) TCA變性純化法 20
2.11 SDS-PAGE之蛋白質分子量分析 21
2.12西方點墨法 (Western Blotting) 21
2.13酵母菌融合蛋白質的表現與純化 23
2.14醯化作用分析 25
2.15 移動轉移實驗( mobility shift assay) 26
第三章實驗結果 27
3.1 GatXAB的交互作用 27
3.2 GatX、GatA、GatB的細胞分佈分析 27
3.3 GatX是否為必要的基因 28
3.4 GatX (SPCC77.11)之序列分析與演化關係 28
3.5 酵母菌細胞質內Arc1p與生物素化關係 29
3.5.1 Arc1p表現量及生物素化與細胞生長的關係 29
3.5.2壓力環境對Arc1p生物素化的影響 29
3.5.3 不同程度生物素化的Arc1p蛋白質 30
3.5.4 測定K86是否Arc1p上唯一的生物素化位置 31
3.6 生物素化與EMA複合體之間的關係 32
3.7 建構SpBpl1p取代ScBpl1p的酵母菌轉型株 32
3.8 Arc1p生物素化對於EMA複合物的活性影響 33
第四章討論 34
4.1 Sch. pombe GluAdT的第三個次單元 34
4.2 S. cerevisiae中EMA複合體與Arc1p生物素化之間的關聯性 35
參考文獻 39

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指導教授

王健家(Chien-Chia Wang)

審核日期

2019-1-19

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