本計劃是以本實驗室先前的發現為基礎。此發現已於C012-1中描述，證實了啤酒酵母菌之YJR129Cp為一真實具有活性之新穎蛋白質甲基轉移酶。此新發現利用一維電泳分析顯示，其蛋白質受質之分子量為95 kDa, 72 kDa, 55 kDa, 43 kDa，pI值為 6~7。本計劃將進一步利用蛋白質體學方法找出YJR129Cp之蛋白質受質；即進一步利用二維電泳分離及質譜分析來鑑定出蛋白質受質種類。並將利用定點突變、基因剔除、及甲基化反應等方式來證明是否YJR129Cp於體外及體內皆具有專一的活性。最後，計劃將延伸至功能方面的探討，此部分將借助於雙雜合篩選及合成致死基因篩選等技術。 This proposal is based on the previous finding in our lab that YJR129Cp of Saccharomyces cerevisiae is a bona fide, novel protein methyltransferase, as presented in C012-1. This new discovery indicates that the protein substrate has a molecular weight of 95 kDa, 72 kDa, 55 kDa, 43 kDa, and pI 6~7 in one-dimensional electrophoretic analysis. Here, identification of the protein substrate is planned by the proteomics approach; that is, two-dimensional electrophoretic separation followed by mass spectrometry. The specificity of both in vitro and in vivo activity of YJR129Cp is also planned to be verified using site-directed mutagenesis, gene deletion, and both in vitro and in vivo methylation assay. Finally, the study will be extended to the functional aspect by two-hybrid screening and synthetic lethal screening.
