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

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Title:	酵母菌粒線體內Gln-tRNAGln的合成機制;Gln-tRNAGln formation in yeast mitochondria
Authors:	簡鈺真;Chien, Yu-Chen
Contributors:	生命科學系
Keywords:	Glu-tRNAGln 轉胺酶;酵母菌;aaRS輔因子;胺酰-tRNA合成酶;生物素化;Glu-tRNAGln amidotransferase;Schizosaccharomyces pombe;Arc1p;aminoacyl-tRNA synthetases;biotinylation
Date:	2019-01-19
Issue Date:	2019-04-02 14:21:51 (UTC+8)
Publisher:	國立中央大學
Abstract:	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。;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.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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