在蛋白質合成的路徑中，需要tRNA將胺基酸帶到核醣體進行催化反應。而胺基酸接上對應的tRNA形成胺醯化tRNA是由aminoacyl-tRNA synthetases (aaRS) 催化而成。在酵母菌Saccharomyces cerevisiae的細胞質中，Gln-tRNAGln的合成是由GlnRS直接催化而成的，但在其粒線體中，缺乏GlnRS，Gln-tRNAGln是由GluRSc催化Glutamate接上tRNAGln，再經由Glutamyl-tRNAGln amidotransferase (GluAdT)，將Glutamate轉變成Glutamine，之前我們曾研究在Escherichia coli GlnRS (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。我們進一步研究Thermus thermophilus GlnRS (TtGlnRS)，發現和EcGlnRS不同的是，若我們在胺基端接上Arc1p無法提供GLN4 剔除株生長所必須的酵素活性，但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(MTS)則此融合蛋白質可以取代粒線體內間接合成Gln-tRNAGln的路徑。根據西方墨點法的實驗結果，發現Arc1p可以增加TtGlnRS在S. cerevisiae的表現量，進一步做蛋白質降解實驗，發現TtGlnRS在粒線體較穩定，體外試驗發現Arc1p-TtGlnRS比TtGlnRS對粒線體tRNAmGln有較高的活性。在Schizosaccharomyces pombe中的GluAdT目前只能找到GatAB兩個次單元，尚未發現GluAdT的第三個次單元，因此我們利用pulldown assay，尋找會和GatB有交互作用的蛋白質，再進一步利用LC/MS/MS進行分析，找到S. pombe中系統編號SPCC777.11，可能是GluAdT的第三次單元體。;Aminoacylation of tRNA is catalyzed by a group of enzymes, called aminoacyl-tRNA synthetases. The resultant aa-tRNA is then delivered to ribosomes for protein translation. In Saccharomyces cerevisiae, cytosolic Gln-tRNAGln is generated by the direct pathway, but mitochondrial Gln-tRNAGln is formed by an indirect pathway involving mischarging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNAGln amidotransferase, a heterotrimeric GatFAB. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for both the direct and indirect pathways of Gln-tRNAGln synthesis. We showed herein that fusion of Arc1p to Thermus thermophilus GlnRS enabled the bacterial enzyme to substitute for the indirect, but not the direct, pathway of Gln-tRNAGln synthesis. In otherwise, the fission yeast, Schizosaccharomyces pombe use the same mode to synthesis the Gln-tRNAGln by two different pathway. We predict the amidotransferase GatAB ortholog, but cannot figure out the GatC or GatF ortholog. We use the TAP pulldown and LC/MS/MS to demonstrate the third subunit of amidotransferase in S. pombe, and figure out the systematic ID is SPCC777.11.
