XpsE ATPase活性在其與第二型分泌機器結合或解離中的重要性

Abstract

十字花科黑腐病菌中的第二型分泌系統 (T2SS) 除了含有內、外胞膜蛋白，並含有一個胞內蛋白XpsE，可以區分成N端及C端兩個獨立區域。胺基酸序列比對顯示其C端區域含有四個高度保留性之nucleotide binding motifs，與ATPase活性相關。根據本實驗室的研究顯示: XpsE與ATP結合後聚集形成六聚體，進而與內膜蛋白XpsL結合，參與第二型分泌系統之運轉。先前研究發現K331M突變會導致XpsE ATPase活性降低，但仍維持部分與ATP結合的能力，KMRA (K331M, R504A) 雙突變則會使XpsE失去與ATP結合的能力，不再形成六聚體。若將XpsE與其同源性蛋白比對，發現其R385胺基酸可能扮演著Arginine finger的角色，推測可穩定ATP水解過程中的中間產物，E357此胺基酸不但同源性極高，且其同源性蛋白PaPilT結構顯示此胺基酸參與和鎂離子的結合，可能也與ATPase活性相關。D387可能扮演著XpsE兩分子界面相互結合之重要胺基酸。先前研究顯示上述胺基酸突變均會使XpsE失去分泌功能。本研究使用綠色螢光蛋白 (superfolder GFP; sfGFP)，融合於XpsE C端並利用廣寄主性載體送入十字花科黑腐病菌中表現，以觀察XpsE或其突變蛋白在活體細胞之分佈情形。先前研究顯示孔道蛋白XpsD不存在時，螢光蛋白在細胞邊緣聚集成點，反之則分散於細胞質中，進ㄧ步分析顯示XpsE蛋白僅在參與分泌時呈現與分泌系統結合的現象，分泌完成則解散至細胞質中。為探討XpsE的ATPase活性在其聚散中扮演的角色，本研究利用廣寄主性載體攜帶 xpsER385A-sfgfp、xpsED387A-sfgfp及xpsEE357Q-sfgfp，並觀察綠色螢光在胞內的分佈情形，發現當孔道蛋白XpsD不在時XpsER385A-sfGFP及XpsEE357Q-sfGFP呈現明顯的點狀分佈，說明ATPase活性對於XpsE的聚集並非必要，恢復孔道蛋白XpsD使得XpsER385A-sfGFP點狀分佈的數量減少，強度減弱但並非完全解散，而XpsEE357Q-sfGFP的點狀分佈則維持不變，暗示XpsE的解散可能需要ATPase的ATP水解活性。XpsED387A-sfGFP的觀察結果顯示無論孔道蛋白XpsD存在與否，皆無明顯的螢光點存在，暗示D387A突變可能破壞了XpsE與XpsE之間的結合。

The type II secretion system (T2SS) of Xanthomonas campestris pv. campestris contains, in addition to several inner and outer membrane proteins, a cytoplasmic protein XpsE, which can be divided into an N and a C domain. Four conserved nucleotide binding motifs that are related to its ATPase activity are located in the C domain. Based on previous observations made in this laboratory, the XpsE forms hexamer after it binds to ATP and further interacts with the inner membrane protein XpsL, participating the secretion process. Previous studies showed that the K331M mutation caused reduction in the XpsE ATPase activity, but the mutated protein kept part of the ATP-binding activity. The double mutation K331M, R504A (KMRA), however caused the XpsE deficient in ATP-binding, and the mutated protein was unable to form hexamer. Sequence alignment of XpsE with its homologous proteins reveals that the R385 residue may act as the “ariginine finger” stabilizing the intermediate of ATP hydrolysis. The E357 residue is equivalent to a conserved glutamate in a homologous protein PaPilT that was suggested to bind Mg2+ in the crystal structure, presumably being essential in ATP hydrolysis. An equivalent of the D387 residue was predicted to be significant in the XpsE-XpsE interaction. Previous studies indicated mutation of the residues described above caused the XpsE nonfunctional in T2S. In this study, for monitoring cellular distribution of XpsE, I fused the superfolder GFP (sfGFP) to its C-terminus. Using a broad host-range plasmid, I introduced the fused gene into X. campestris pv. campestris in order to observe the distribution of XpsE or its variants in the cell. Previous studies have shown that in the xpsD- strain, the fluorescent protein-labeled XpsE appeared as foci at the cell boundary. In contrast, the XpsE was diffused in the cytoplasm. Further analysis suggested the XpsE is associated with the T2SS only when taking part in the secretion process. When secretion is completed, the XpsE returns to diffused state in the cytoplasm. To examine the significance of ATP hydrolysis by XpsE in its foci formation or disassembly, I used a broad host-range plasmid to carry the xpsER385A-sfgfp、xpsED387A-sfgfp or xpsEE357Q-sfgfp gene and observed distribution of green fluorescence in the cell. In absence of the secretin XpsD, both XpsER385A-sfGFP and XpsEE357Q-sfGFP exhibited distinct foci, implicating ATPase activity is not essential for the assembly of XpsE. Introduction of a plasmid encoding the secretin XpsD made XpsER385A-sfGFP foci reduced in number and fluoresecence intensity. However, the XpsEE357Q-sfGFP foci number or intensity remained unchanged, agreeing with the hypothesis that ATP hydrolysis is required for XpsE dissolution. As contrast, regardless of the presence or absence secretin XpsD, the XpsED387A-sfGFP appeared diffused implicating D387A mutation may have destroyed the interaction between XpsE and XpsE.