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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 >  第二型分泌機制次要類纖毛蛋白XpsHIJK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/309270000/152321

標題: 第二型分泌機制次要類纖毛蛋白XpsHIJK在ATPase聚散中的重要性
Significance of the minor pseudopilins XpsHIJK in the assembly and dispersion of the type II secretion system ATPase
作者: 黃明分
Huang, Ming-Fen
Contributors: 胡念台
生物化學研究所
關鍵字: 第二型分泌系統;次要類纖毛蛋白;ATP水解酶
T2SS;minor pseudopilins;ATPase
日期: 2013
Issue Date: 2013-11-07 13:20:37 (UTC+8)
Publisher: 生物化學研究所
摘要: 十字花科黑腐病菌利用第二型分泌機制將位在periplasm的分泌性蛋白分泌到胞外以感染宿主細胞。先前研究發現第二型分泌機制中唯一的胞內蛋白XpsE會與ATP結合形成多聚體,經由與內膜蛋白XpsL的交互作用結合至內膜,推測藉此提供能量使位於內膜的類纖毛蛋白(pseudopilin)組裝成類纖毛(pseudopilus),推動分泌性蛋白經由外膜蛋白XpsD形成的通道分泌到胞外。
Pseudopilus的主要成分為XpsG又稱為major pseudopilin,次要成分為XpsHIJK 又稱為minor pseudopilin,根據文獻推測XpsHIJK位在pseudopilus的頂端,可能先進行自我組裝作為pseudopilus組裝的起始構造,隨後XpsG逐一以右旋的方式加入,使pseudopilus得以向外膜方向延長。已知在xpsD-遺傳背景下,XpsE-ECFP會在細胞邊緣呈現點狀分布,在庭如學姊的研究中,發現在xpsD- 情況下大量表現XpsG會使得XpsE-ECFP分散至細胞質中,推測主要類纖毛蛋白組裝類纖毛可能導致聚集的ATPase解散。為進一步檢驗此推論正確與否,且此現象是否需要次要類纖毛蛋白,本研究首先在xpsHIJK基因缺失情況下觀察XpsE-ECFP蛋白在胞內的分佈情形。在xpsHIJK-基因或xpsG-基因缺損株中均可觀察到自染色體基因表現的XpsE-ECFP在胞內聚集成點,暗示主要或次要類纖毛蛋白缺失均會使聚集的XpsE-ECFP數量增加。進一步在xpsD-菌株中以質體表現的XpsE-sfGFP,額外表現主要或次要類纖毛蛋白或是兩者同時表現,觀察XpsE-sfGFP在胞內的分佈。研究結果顯示,額外表現次要類纖毛蛋白對於xpsD-菌株中XpsE-sfGFP在胞內分布沒有顯著影響,而在xpsD-菌株中額外表現主要類纖毛蛋白,可觀察到XpsE-sfGFP分散至細胞質中亮度減少,且點狀聚集的螢光強度明顯加強,同時,在xpsD+菌株中也觀察到相同現象,暗示在細胞中提高主要類纖毛蛋白的表現量對於ATPase聚集的現象有促進作用。
Type II secretion system is utilized by Xanthomonas campestris pv. campestris for translocating proteins across the outer membrane to attack the host plant cells. Previous studies showed that the only cytoplasmic protein XpsE forms oligomer when it binds to ATP and associates with inner membrane through its interaction with the inner membrane protein XpsL. It has been postulated that ATP hydrolysis by XpsE provides the energy for the assembly of pseudopilus from pseudopilin. The growing pseudopilus may push the exoprotein through the secretion channel constituted of secretin XpsD in the outer membrane. Pseudopilus comprises of one major component XpsG named major pseudopilin and 4 minor components XpsHIJK named minor pseudopilins. As suggested by studies in the literature, minor pseudopilins may self-assemble to form so-called tip complex and prime the pseudopilus elongation approaching the outer membrane by incorporating XpsG one by one following the right handed helix rule. Previous studies in this laboratory showed that XpsE-ECFP in the xpsD- strain gathered as focused spots at the cell boundary. It was observed by the former student Ting-Ru Liu that the XpsE-ECFP foci in the xpsD- strain became diffused in cytoplasm when XpsG was overproduced. It is hypothesized that perhaps pseudopilus assembly from the major pseudopilin may cause diffusion of the gathered ATPase. To examine this hypothesis and the requirement of minor pseudopilins in XpsE-ECFP foci formation, I performed experiments to analyze the cellular distribution of XpsE-ECFP in xpsHIJK- strain. The number of foci formed by the chromosome-encoded XpsE-ECFP appeared to increase in xpsHIJK-, as well as in the xpsG- strain. The results implicate that depletion of the major or all minor pseudopilins favors XpsE-ECFP foci formation. I further examined if additionally expressed major, minor or both pseudopilins influences the cellular distribution of plasmid-encoded XpsE-sfGFP in the xpsD- strain. In presence of additionally expressed minor psedopilins in xpsD- strain, there was no significant difference in the cellular distribution of XpsE-sfGFP. However, when the major pseudopilin alone was additionally expressed in the xpsD- strain, the brightness of the XpsE-sfGFP fluorescence in the cytoplasm decreased and the intensity of the XpsE-sfGFP foci gathered at the cell boundary increased. Similar results were observed in the xpsD+ strain. These observations implicate that increasing protein abundance of the major pseudopilin alone in the cell enhances gathering of the ATPase.
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