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標題: 第二型分泌系統之ATPase 在分泌過程中的聚散現象
Function-related Positioning of the Type II Secretion ATPase
作者: 陳奕霖
Chen, Yih-Lin
Contributors: 胡念台
Nien-Tai Hu
生物化學研究所
關鍵字: 第二型分泌系統;分泌孔道蛋白;蛋白質分泌機器
GspE;T2SS;Secretin;Protein translocation;Secretion machine
日期: 2013
Issue Date: 2013-11-07 13:20:54 (UTC+8)
Publisher: 生物化學研究所
摘要: 格蘭氏陰性菌利用第二型分泌(type II secretion, T2S)系統將細胞間質內摺疊好的被分泌蛋白分泌到胞外,用於攻擊動物或植物細胞,或是從環境中獲取養份。T2S機器是橫跨內、外膜的多蛋白聚合裝置,唯一的ATPase位於細胞質。由於T2S ATPase與內膜蛋白曝露於細胞質內作為ATPase交互作用的蛋白片段共表現後純化的蛋白複合體,可受酸性磷脂的刺激而活化,推測分泌進行中位於膜上且與T2S機器結合的ATPase處於活化的狀態。然而,分泌完成後T2S ATPase如何終止水解ATP的活性並不清楚。本研究利用ECFP螢光蛋白標定感染植物的十字花科黑腐病菌(Xanthomonas campestris pv. campestris) T2S ATPase,發現在分泌功能正常的細胞內,ATPase主要分散在細胞質,只在少數細胞週邊觀察到聚集的螢光點。ATPase螢光點會因為分泌孔道缺失以及被分泌蛋白大量累積於胞內,使螢光點的亮度增強和數量增加。細胞週邊的ATPase螢光點數量與分泌孔道的蛋白分泌功能呈負相關,若逐步恢復分泌孔道分泌功能可使聚集的螢光點數量逐漸減少。且聚集在細胞週邊的ATPase螢光點與分佈於細胞表面的分泌孔道重疊,說明分泌過程中,ATPase以動態的方式短暫的與細胞膜上的T2S機器結合,當分泌完成後解散到細胞質裡,推測T2S ATPase藉由離開T2S機器終止其活化狀態。
Gram-negative bacteria use the type II secretion (T2S) system to secrete exoproteins for attacking animal or plant cells or to obtain nutrients from the environment. The system is unique in helping folded proteins traverse the outer membrane. The secretion machine comprises multiple proteins spanning the cell envelope and a cytoplasmic ATPase. Activity of the ATPase, when copurified with the cytoplasmic domain of an interactive ATPase partner, is stimulated by an acidic phospholipid, suggesting the membrane-associated ATPase is actively engaged in secretion. How the stimulated ATPase activity is terminated when secretion is complete is unclear. We fused the T2S ATPase of Xanthomonas campestris pv. campestris, the causal agent of black rot in the crucifers, with enhanced cyan fluorescent protein (ECFP) and found that the ATPase in secretion-proficient cells was mainly diffused in cytoplasm. Focal spots at the cell periphery were detectable only in a few cells. The discrete foci were augmented in abundance and intensity when the secretion channel was depleted and the exoprotein overproduced. The foci abundance was inversely related to secretion efficiency of the secretion channel. Restored function of the secretion channel paralleled reduced ATPase foci abundance. The ATPase foci colocalized with the secretion channel. The ATPase may be transiently associated with the T2S machine by alternating between a cytoplasmic and a machine-associated state in a secretion-dependent manner. This provides a logical means for terminating the ATPase activity when secretion is completed.
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