Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23944
標題: 利用點突變探討XpsE蛋白N端區域在其與XpsL交互作用中的重要性
Significance of the N-terminal domain of XpsE in its interaction with XpsL analyzed by site-directed mutation
作者: 曾于娜
Tseng, Yu-Na
關鍵字: Xanthomonas campestris pv. campestris;十字花科黑腐病菌;Type II secretion apparatus;第二型分泌機制
出版社: 生物化學研究所
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S., Robertson, G. T., Farris, M. A., Roop, R. M.,2nd, & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176. Lee, H. M., Tyan, S. W., Leu, W. M., Chen, L. Y., Chen, D. C., & Hu, N. T. (2001). Involvement of the XpsN protein in formation of the XpsL-xpsM complex in xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol 183, 528-535. Lee, M. S., Chen, L. Y., Leu, W. M., Shiau, R. J., & Hu, N. T. (2005). Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J Biol Chem 280, 4585-4591. Lin. C.C. (2000). Interaction of cytoplasmic membrane protein XpsF with XpsL/M/N and XpsE in Xanthomonas campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Nunn, D. N., & Lory, S. (1993). Cleavage, methylation, and localization of the pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 175, 4375-4382. Py, B., Loiseau, L., & Barras, F. (2001). An inner membrane platform in the type II secretion machinery of gram-negative bacteria. EMBO Rep 2, 244-248. Py, B., Loiseau, L., & Barras, F. (1999). Assembly of the type II secretion machinery of erwinia chrysanthemi: Direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL. J Mol Biol 289, 659-670. Robien, M. A., Krumm, B. E., Sandkvist, M., & Hol, W. G. (2003). Crystal structure of the extracellular protein secretion NTPase EpsE of vibrio cholerae. J Mol Biol 333, 657-674. Sandkvist, M. (2001a). Biology of type II secretion. Mol Microbiol 40, 271-283. Sandkvist, M. (2001b). Type II secretion and pathogenesis. Infect Immun 69, 3523-3535. Sandkvist, M., Bagdasarian, M., Howard, S. P., & DiRita, V. J. (1995). Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in vibrio cholerae. EMBO J 14, 1664-1673. Sauvonnet, N., Vignon, G., Pugsley, A. P., & Gounon, P. (2000). Pilus formation and protein secretion by the same machinery in escherichia coli. EMBO J 19, 2221-2228. Shiue, S. J. (2004). Type II secretion apparatus of Xanthomonas campestris: Analysis of interactive relationship between XpsLN and XpsE. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R.O.C. Shiue, S. J., Kao, K. M., Leu, W. M., Chen, L. Y., Chan, N. L., & Hu, N. T. (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25, 1426-1435. Thomas, J. D., Reeves, P. J., & Salmond, G. P. (1997). The general secretion pathway of erwinia carotovora subsp. carotovora: Analysis of the membrane topology of OutC and OutF. Microbiology 143 ( Pt 3), 713-720. Tsai, R. T., Leu, W. M., Chen, L. Y., & Hu, N. T. (2002). A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J 367, 865-871. Walker, J. E., Saraste, M., Runswick, M. J., & Gay, N. J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1, 945-951. Yo, T. T. (2003). Detection of interactions between XpsF and XpsL, or XpsE, in the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
摘要: 
十字花科黑腐病菌利用第二型分泌機制分泌水解酵素,以感染植物葉片。XpsE 蛋白是第二型分泌機制組成蛋白中唯一的非膜蛋白,且含有 4 個高度保留性的 nucleotide binding motifs,被認為可能在分泌過程中扮演提供能量的角色。XpsE 蛋白 N-端 36 個胺基酸缺失嚴重影響其與內膜上的 XpsL 蛋白間的交互作用。為進一步了解 XpsE 蛋白 N-端區域在其與 XpsL 交互作用中的重要性,本研究針對 XpsE 蛋白可能與 XpsL 蛋白交互作用的 N-端 a-helix domain 進行 alanine mutation,以期找出影響分泌功能的突變位置。結果顯示 V11A、L15A 及 L25A 三個單點突變均不會影響 XpsE 的正常分泌能力。兩兩組合的雙點突變蛋白,只有 V11A, L25A 使得突變蛋白的分泌能力降為野生型蛋白的 54%,其餘突變均不影響分泌。三點突變 V11A, L15A, L25A 對分泌影響的程度與 V11A, L25A 相似;另一個三點突變 V11A, L25A, L39A 則嚴重影響分泌功能,它的分泌能力只有野生型蛋白的 13%,暗示 Val11、Leu25 及 Leu39 三個疏水性胺基酸可能為 XpsE 行使正常功能所必需。這個三點突變 V11A, L25A, L39A 並不影響突變蛋白在胞內的含量;且由圓形偏極光光譜 (Circular Dichroism) 及 tryptophan 螢光光譜分析結果推測此三點突變對 XpsE 蛋白的二級及三級結構未造成嚴重影響。試管內 pull down 分析,顯示 V11A, L25A, L39A 三點突變亦造成突變蛋白喪失其與 XpsL 的結合能力。說明 XpsE 蛋白可能藉由 N-端 Val11、Leu25 和 Leu39 包圍形成的疏水性表面 (hydrophobic surface) 和 XpsL 蛋白交互作用,以行使其分泌胞外蛋白的功能。

Type II secretion apparatus is utilized by Xanthomonas campestris pv. campestris for secreting hydrolytic enzymes to infect plants on their leaves. XpsE, without any membrane spanning sequence, is the only cytoplasmic protein in type II secretion apparatus. It consists of four conserved nucleotide binding motifs, and is postulated to provide energy for the secretion process. A truncated XpsE mutant devoid of the N-terminal 36 residues (XpsE∆36) greatly affects its association with the inner membrane protein XpsL. To further understand significance of the N-terminal region of XpsE in its interaction with XpsL, I performed alanine mutation of chosen hydrophobic residues at the N-terminal region of XpsE and looked for mutations that cause XpsE nonfunctional in secretion. In this study, I observed that single mutations V11A, L15A or L25A in XpsE did not affect its normal function in secretion. Of all double mutants constructed, only the mutations at Val11 and Leu25 affect secretion slightly, with 54% secretion ability remaining. Similar level of secretion was also observed for one of the triple mutations V11A, L15A, L25A. In contrast, the other triple mutations V11A, L25A, L39A significantly affect the secretion function of XpsE, reduced to a level of 13% that of the wild-type XpsE, suggesting that intactness of the three hydrophobic residues Val11, Leu25 and Leu39 all together are required for normal function of XpsE. Triple alanine mutations at these three residues of XpsE did not affect its protein abundance. Analysis of circular dichroism and tryptophan fluorescence spectroscopy indicated that the secondary and the tertiary structure of XpsE were probably not significantly changed by the triple alanine mutations. In vitro pull down assay revealed that triple alanine mutations at Val11, Leu25 and Leu39 caused XpsE lose the ability to bind XpsL. This result implies that the hydrophobic patch constituted of the residues Val11, Leu25 and Leu39 are necessary for the association of XpsE with XpsL, which in turn is required for XpsE to be functional.
URI: http://hdl.handle.net/11455/23944
其他識別: U0005-2306200613463000
Appears in Collections:生物化學研究所

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