https://scholars.lib.ntu.edu.tw/handle/123456789/160143
標題: | 探討酵母菌核醣核酸蛋白Rbp1p面對環境壓力之後轉譯修飾 Characterization of Posttranslational modification of RNA binding protein, Rbp1p, in Saccharomyces cerevisiae under stress conditions |
作者: | 蔡松蒲 Tsai, Sung-Pu |
關鍵字: | 酵母菌核醣核酸蛋白;Rbp1p | 公開日期: | 2009 | 摘要: | 在真核細胞中,核醣核酸結合蛋白調控著信使核醣核酸(mRNA)的轉錄和轉錄後事件(post-transcriptional events),例如剪接(splicing)、編輯(editing)、運輸出核(export)、穩定性(stability)、位置(localization)和轉譯(translation)。之前實驗室發現酵母菌核醣核酸結合蛋白Rbp1p,分布在細胞質中,為具有抑制酵母菌生長的負調控因子。此外,在葡萄糖剝奪(glucose depletion)、熱壓力(heat shock)等環境壓力下,Rbp1p分別會座落到細胞質中的細胞質處理小體(P-bodies)及壓力體(stress granules; SGs)。利用二維電泳分析,發現在這兩種環境壓力下Rbp1p等電點圖譜皆有往酸性端移動之現象。推測在處在環境壓力下Rbp1p含有不同的轉譯後修飾。藉由質譜儀分析Rbp1p轉譯後修飾,發現了八個磷酸化的位置,並且利用定點突變(site-directed mutagenesis)的技術將它們突變為丙氨酸,觀察到這些突變型的Rbp1p不會影響抑制酵母菌生長的能力。另外,也發現在環境壓力下,這些突變型的Rbp1p依然可以座落到細胞質處理小體。同時在酵母菌雙雜交實驗發現S646A和S649A突變型的Rbp1p和野生型的Rbp1p結合的能力會減弱。藉著共同免疫沉澱實驗,我們發現了Ssa2p為Rbp1p的結合蛋白,並且在熱壓力的環境下結合的能力會下降。此外,在ssa2突變株中大量表現Rbp1p,Rbp1p抑制酵母菌生長的能力增強了。 In eukaryotic cells, RNA binding proteins regulate mRNA transcription and post-transcriptional events, such as splicing, editing, export, stability, localization, and translation. Our laboratory has demonstrated that RNA binding protein1, Rbp1p, appears punctate in cytoplasm and as a negative growth regulator. In addition, Rbp1p localizes to P-bodies and stress granules in cytoplasm upon glucose deprivation and heat shock, respectively. Using two-dimensional electrophoresis analysis, the iso-electric point pattern of Rbp1p shows spots toward acidic pore after glucose deprivation and heat shock treating. The results imply that Rbp1p contains different post-translational modifications in stress conditions. By mass spectrometry analysis, we identify eight phosphorylation sites of Rbp1p (Ser459, Ser462, Ser463, Ser524, Ser526, Thr637, Ser646, and Ser649), and using site-directed mutagenesis approach to substitute alanine for serine/threonine. S646A and S649A mutants of Rbp1p decrease the ability of interaction with Rbp1p by yeast two-hybrid assay. Under glucose deprivation, however, phosphorylation mutants of Rbp1p can still localize to P-bodies, and these mutants cannot affect ability of growth inhibition. In co-immunoprecipitation experiments, I identify that Ssa2p is one of associated proteins with Rbp1p, and the interaction ability diminishes at heat shock. Besides, overexpression of Rbp1p in ssa2Δ strain enhances the growth-inhibition ability. It implies that Ssa2p can inhibit Rbp1p negative regulator growth. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/178734 |
顯示於: | 分子醫學研究所 |
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ntu-98-R94448007-1.pdf | 23.32 kB | Adobe PDF | 檢視/開啟 |
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