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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  第二型分泌機器主要類纖毛蛋白在ATPase聚散中的重要性

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152324

標題: 第二型分泌機器主要類纖毛蛋白在ATPase聚散中的重要性
Significance of the major pseudopilin in the assembly and dispersion of the type II secretion system ATPase
作者: 劉庭如
Liu, Ting-Ru
Contributors: 胡念台
Nien-Tai Hu)
生物化學研究所
關鍵字: 第二型分泌機器;主要類纖毛蛋白
Type II secretion system;major pseudopilin
日期: 2012
Issue Date: 2013-11-07 13:20:44 (UTC+8)
Publisher: 生物化學研究所
摘要: 十字花科黑腐病菌 (Xanthomonas campestris pv. campestris) 之第二型分泌系統由十二個蛋白質所構成,橫跨內外膜。本研究欲探討主要類纖毛蛋白XpsG對於胞內具有ATPase活性的XpsE蛋白在細胞內聚散之重要性,我利用遺傳方式先將ECFP螢光蛋白與XpsE形成融合蛋白,接著以同源重組的方式將xpsE-ecfp基因置換原來染色體表現之xpsE基因,再藉由螢光顯微鏡觀察XpsE-ECFP在胞內分佈情形。無論xpsG基因缺損或正常時,均可觀察到XpsE-ECFP以點狀方式聚集於細胞邊緣,說明主要類纖毛蛋白XpsG可能不是XpsE-ECFP聚集成點之必要條件,利用大量表現XpsG之質體送入xpsG基因缺損株中,觀察到XpsE-ECFP聚集點不像未送入質體的xpsG基因缺損株中那麼細小且集中,猜測XpsG的存在可能有促進XpsE-ECFP聚集點解散的作用。當通道蛋白缺失時,XpsE-ECFP會明顯以點狀方式聚集在細胞邊緣,若同時主要類纖毛蛋白xpsG基因也缺損時,XpsE-ECFP聚集成點的現象明顯較通道基因或xpsG基因單獨缺損時顯著,聚集點的數目增加,螢光訊號增強;而在內膜蛋白XpsF大量表現、通道蛋白及主要類纖毛蛋白同時缺失時,均勻散布於細胞質中的XpsE-ECFP螢光訊號明顯減弱,點狀聚集更為集中;若在此菌株中,送入大量表現XpsG之質體時,分佈在細胞質內的XpsE-ECFP螢光訊號增強,且聚集點由集中轉為粗大形式,進一步支持主要類纖毛蛋白XpsG可能會促使聚集成點的XpsE-ECFP解散的假說。上述結果說明主要類纖毛蛋白XpsG組裝成類纖毛可能會促使聚集在細胞邊緣的XpsE-ECFP解散至細胞質中,假設XpsE-ECFP聚集的目的是藉由類纖毛的組裝將被分泌蛋白送出位於外膜中的分泌通道,先前結果指出當分泌完成時聚集的ATPase會解散至細胞質中,推測類纖毛組裝可能代表分泌完成,而使得ATPase解散,回復到細胞質中均勻分佈的狀態。
The type II secretion system (T2SS) of Xanthomonas campestris pv. campestris is constituted of twelve components spanning the inner and outer membrane. This study is intended to determine significance of the major pseudopilin XpsG in foci formation of the T2SS ATPase XpsE-ECFP during secretion. By taking homologous recombination approach, I replaced the chromosomal xpsE gene with the xpsE-ecfp gene in the genetic background with or without the xpsG gene. Fluorescence microscopic observations were then made for comparing cellular distribution of the XpsE-ECFP. Foci formation of XpsE-ECFP at cell boundary was observed in strains with or without the xpsG gene, suggesting the XpsG protein is not essential for XpsE-ECFP foci formation. Introduction of a plasmid-encoded xpsG gene into the xpsG-null strain resulted in the XpsG protein expressed at elevated level. Meanwhile, the XpsE-ECFP foci appeared more diffused than those in the xpsG-null strain, implicating that presence of abundant XpsG may have a dispersing effect on the XpsE-ECFP foci. As observed before, in absence of the secretin gene encoding component of the secretion channel, the XpsE-ECFP foci were detected at cell boundary. Simultaneous disruption of the xpsG gene enhanced XpsE-ECFP foci formation, as suggested by forming XpsE-ECFP foci with increased fluorescence intensity and abundance, relative to those observed in the xpsG-null or the secretin-null strain. Further enhancement of the XpsE-ECFP foci formation was observed when the inner membrane protein XpsF was overproduced in the xpsG-, secretin-doubly deleted strain. The fluorescence appearing diffused in cytoplasm diminished while the fluorescent foci became even sharper. Introduction of the plasmid-encoded xpsG gene reversed the effect, revealed as increased fluorescence intensity in cytoplasm and less sharp focal-appearing XpsE-ECFP. This observation agrees with the hypothesis that assembly of pseudopilus from its major constituent XpsG may cause XpsE-ECFP foci disperse. Presumably, the foci-appearing XpsE-ECFP is recruited to provide energy for pseudopilus assembly, which in turn generates mechanical force pushing the secreted protein through the secretion channel. As suggested previously, the XpsE-ECFP becomes dispersed when secretion completes. It is plausible that in type II secretion process, successful passage of secreted protein through secretion channel may be signaled to the assembled ATPase to disperse through pseudopilus formation from its building block the major pseudopilin.
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