酵母菌粒線體Gln-tRNAGln的形成

Formation of Gln-tRNAGln in the yeast mitochondria

廖芝淇(Chih-chi Liao)

指導教授 ： 王健家

國立中央大學/理學院/生命科學系/碩士(2011年)

摘要

在細胞中，有兩種合成Gln-tRNAGln的路徑，一種是和GlnRS有關的直接路徑，另一種是和轉胺酶GatFAB有關的間接路徑。之前的研究發現，在酵母菌Saccharomyces cerevisiae中，部分細胞質的GlnRS會被送到粒線體中，因此他們推測酵母菌粒線體可能是利用直接路徑來合成Gln-tRNAGln，但是最近的研究發現酵母菌粒線體是經由間接路徑來合成Gln-tRNAGln，那麼酵母菌粒線體中GlnRS執行甚麼功能有待進一步的研究。在本篇論文中，我們利用報導基因試驗以及免疫螢光分析來標定酵母菌GlnRS的粒線體標的訊號，實驗結果顯示: 粒線體標的訊號不在其胺基端，而是位於GlnRS中靠近活化區的一段序列。這是一個非常特別的例子，GlnRS的標的訊號在蛋白質進到粒線體後不會被切除。另外，我們發現，在EcGlnRS的胺基端接上Arc1p可以提供GLN4 剔除株生長所必須的酵素活性，若進一步在此融合蛋白質的胺基端加上一段粒線體標的訊號則此融合蛋白質可以取代粒線體內間接合成Gln-tRNAGln的路徑，這些發現突顯了基因平行轉移的可能性，利用直接合成Gln-tRNAGln的路徑取代間接合成路徑。

關鍵字

tRNA合成酶 ； 酵母菌 ； 粒線體

並列摘要

There are two pathways for Gln-tRNAGln formation in the cell, a direct pathway, which involves GlnRS, and an indirect pathway, which involves a GatFAB transamidase. In Saccharomyces cerevisiae, early research indicated that a portion of cytoplasmic GlnRS was transported into the mitochondria. It was thus proposed that the yeast mitochondria may employ a direct pathway to form Gln-tRNAGln. However, a recent study argued that an indirect pathway is actually involved in the synthesis of mitochondrial Gln-tRNAGln. As a result, the true biological function of the imported GlnRS in the mitochondria is still elusive. In this study, we used reporter gene assays, and immunofluorescence analysis to map the mitochondrial targeting signal of yeast GlnRS. Our results showed that the signal is located at an internal segment close to the active site of the enzyme. This might be one of few examples of mitochondrial matrix proteins that use an uncleavable internal sequence as the mitochondrial targeting signal. On the other hand, we found that fusion of Arc1p to EcGlnRS enables the bacterial enzyme to rescue the growth defect of a GLN4 knockout strain. Further fusion of a mitochondrial targeting signal to the fusion enzyme Arc1p-EcGlnRS enabled the enzyme to replace the indirect pathway for Gln-tRNAGln formation in the mitochondria. These findings underscore the possibility of a horizontal transfer event, where an indirect pathway for Gln-tRNAGln formation is substituted for by a direct pathway.

並列關鍵字

tRNA synthetase ； yeast ； mitochondria

參考文獻

葉耀榮 (2008) 探討酵母菌Glutaminyl-tRNA synthetase對於粒線體功能之影響。 中央大學碩士論文

張嘉珮 (2007) 酵母菌使用罕見轉譯起始密碼的可能性探討。 中央大學碩士論文

Arnez, J. G., and Moras, D. (1997) Structural and functional considerations of the aminoacylation reaction. Trends Biochem Sci. 22: 211-216.

Asahara, H. and Uhlenbeck, O.C. (2002) The tRNA specificity of Thermus thermophilus EF-Tu. Proc. Natl. Acad. Sci. 99: 3499-3504.

Biswas T.K., and Getz G.S. 2002 Import of yeast mitochondrial transcription factor (Mtf1p) via a nonconventional pathway. J Biol Chem. 277: 45704-45714.