Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36109
標題: 十字花科黑腐病菌中第二型分泌機器: 藉由表現XpsF膜蛋白之部分胞內區塊探討蛋白功能
Type II Secretion Apparatus of Xanthomonas campestris: Expression and Functional Characterization of the Cytoplasmic Domains of XpsF
作者: 朱志龍
關鍵字: Xanthomonas campestris pv. campestris
十字花科黑腐病菌
type II secretion apparatus
XpsF protein
第二型分泌機器
XpsF蛋白
出版社: 生物科技學研究所
引用: 林主祈, 2000 〝十字花科黑腐病菌中內膜蛋白XpsF與XpsLMN及XpsE間交互作用之探討〞國立中興大學農業生物科技學研究所 (現為生物科技學研究所) 碩士論文 潘亨文, 2002 〝十字花科黑腐病菌XpsE與XpsF蛋白於第二類型胞外蛋白分泌路徑之探討〞國立中興大學農業生物科技學研究所 (現為生物科技學研究所) 碩士論文 李振東, 2002 〝以金屬離子親和性管柱層析及酵母菌二雜交法分析十字花科黑腐細菌XpsE、XpsF、XpsL、XpsM與XpsN蛋白之複合體關係〞國立中興大學農業生物科技學研究所 (現為生物科技學研究所) 碩士論文 簡伊翎, 2002 〝十字花科黑腐病菌XpsE蛋白之純化及特性分析〞國立中興大學生物化學研究所碩士論文 游婷婷, 2003 〝十字花科黑腐病菌中XpsF與XpsL或XpsE蛋白交互作用之分析〞國立中興大學生物科技學研究所碩士論文 薛聖潔, 2004 〝十字花科黑府病菌第二類型胞外蛋白分泌系統:XpsE蛋白和XpsLN蛋白間交互作用關係之探討〞國立中興大學生物化學研究所碩士論文 徐書尹, 2004 〝十字花科黑腐病菌中XpsF與XpsL或XpsE蛋白交互作用之分析〞國立中興大學生物科技學研究所碩士論文 Ball G, Chapon-Herve V, Bleves S, Michel G, and Bally M. 1999. Assembly of XcpR in the cytoplasmic membrane is required for extracellular protein secretion in Pseudomonas aeruginosa. J Bacteriol. Jan;181(2):382-8. Bally M, Filloux A, Akrim M, Ball G, Lazdunski A, and Tommassen J. 1992. Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. May;6(9):1121-31. Bleves S, Lazdunski A, and Filloux A.1996. Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa. J Bacteriol. Jul;178(14):4297-300. Bleves S, Voulhoux R, Michel G, Lazdunski A, Tommassen J, and Filloux A. 1998. The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family). Mol Microbiol. Jan;27(1):31-40. Chen LY, Chen DY, Miaw J, and Hu NT.1996. XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem. Feb 2;271(5):2703-8. Crowther, L.J., Yamagata, A., Craig, L., Tainer, J.A., and Donnenberg, M.S. (2005). The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J Biol Chem 280, 24839-24848. Driessen AJ, Fekkes P, van der Wolk JP. 1998. The Sec system. Curr Opin Microbiol. Apr;1(2):216-22. Dums F, Dow JM, and Daniels MJ.1991. Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet. Oct;229(3):357-64. Hardie KR, Lory S, and Pugsley AP. 1996. Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J. Mar 1;15(5):978-88. Hu NT, Hung MN, Huang AM, Tsai HF, Yang BY, Chow TY, and Tseng YH. 1992a. Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris. J Gen Microbiol. Aug;138 ( Pt 8):1647-55. Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, and Wu CY. 1992b. Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol. Apr;174(8):2679-87. Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song YL, and Chen LY. 2002. XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J. Jul 1;365(Pt 1):205-11. Kazmierczak BI, Mielke DL, Russel M, and Model P. 1994. pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J Mol Biol. Apr 29;238(2):187-98. Lee HM, Wang KC, Liu YL, Yew HY, Chen LY, Leu WM, Chen DC, and Hu NT. 2000. Association of the cytoplasmic membrane protein XpsN with the outer membrane protein XpsD in the type II protein secretion apparatus of Xanthomonas campestris pv. campestris. J Bacteriol. Mar;182(6):1549-57. Lee HM, Tyan SW, Leu WM, Chen LY, Chen DC, and Hu NT. 2001. Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol. Jan;183(2):528-35. Michel G, Bleves S, Ball G, Lazdunski A, and Filloux A. 1998. Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa. Microbiology. Dec;144 ( Pt 12):3379-86. Possot OM, Vignon G, Bomchil N, Ebel F, and Pugsley AP. 2000. Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE. J Bacteriol. Apr;182(8):2142-52. Pugsley AP, d''Enfert C, Reyss I, and Kornacker MG. 1990. Genetics of extracellular protein secretion by gram-negative bacteria. Annu Rev Genet.;24:67-90. Py B, Loiseau L, and 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. 1999 Jun 11;289(3):659-70. Py B, Loiseau L, and Barras F. 2001. An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep. Mar;2(3):244-8. Sandkvist M, Bagdasarian M, Howard SP, and DiRita VJ. 1995. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J. Apr 18;14(8):1664-73. Sandkvist M, Hough LP, Bagdasarian MM, and Bagdasarian M. 1999. Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. J Bacteriol. May;181(10):3129-35. Sandkvist M. 2001a. Type II secretion and pathogenesis. Infect Immun. Jun;69(6):3523-35. Sauvonnet N, Vignon G, Pugsley AP, and Gounon P. 2000. Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J. May 15;19(10):2221-8. Shevchik VE, Robert-Baudouy J, and Condemine G. 1997. Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins. EMBO J. Jun 2;16(11):3007-16. Shiue, S.J., Kao, K.M., Leu, W.M., Chen, L.Y., Chan, N.L., and Hu, N.T. (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. Embo J 25, 1426-1435. Thomas JD, Reeves PJ, and Salmond GP. 1997. The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF. Microbiology. Mar;143 ( Pt 3):713-20. Tsai RT, Leu WM, Chen LY, and Hu NT. 2002. A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J. Nov 1;367(Pt 3):865-71 Turner LR, Lara JC, and Lory S. 1993. Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J Bacteriol. August;175(16):4962–9
摘要: 十字花科黑腐病菌屬於革蘭氏陰性細菌,會侵染白菜、甘藍、花椰菜等十字花科的蔬菜造成黑腐病,其致病性依賴完整的第二型蛋白分泌系統,以將多種蛋白如果膠酵素、蛋白水解酵素、澱粉水解酵素等分泌至胞外,藉此感染植物。十字花科黑腐病菌的第二型蛋白分泌系統是由XpsD~O等十二個蛋白所組成,其中除了XpsF、XpsE 與XpsL蛋白外,絕大多數的蛋白區塊分佈於胞質周緣區或外膜上。XpsE為一個細胞質蛋白,推測具有ATP-binding motif,可藉由內膜蛋白XpsL而附著於內膜上;XpsF為一個穿過內膜三次的蛋白,由穿膜區可分割成兩個大的蛋白區塊XpsFN與XpsFC,均位於細胞質。為了探討XpsF在第二型分泌機器中所扮演的角色,本研究將XpsF蛋白兩個位於細胞質的區塊大量表達以做分析。雖然在pull-down實驗中發現XpsF的兩個區塊與其他Xps蛋白位於細胞質的區塊均具有交互作用關係,但僅XpsFN蛋白區塊在黑腐病菌中大量表現時,會產生干擾分泌的現象。另一方面,XpsEKMRA是一個ATP-binding site突變的XpsE蛋白,僅能形成monomer,當以蛋白下拉實驗測試時,測得XpsFN-(His)6可滯留XpsEKMRA,此結果暗示XpsFN可與monomer狀態的XpsE結合,此點與XpsL僅能與XpsE的oligomer form結合的性質相異,暗示兩者在第二型分泌機器中所扮演的角色不同。進一步的,我們也發現當在XpsE/XpsL蛋白複合體中加入XpsFN蛋白區塊時,會使XpsE/XpsL蛋白複合體部分瓦解,此結果顯示XpsF可能扮演將XpsE與XpsL分開的角色。
Xanthomonas campestris pv. campestris is a Gram-negative bacteria that infect crucifers and cause black-rot. Ability to secrete proteins extracellularly by the type II secretion apparatus is critical for its pathogenicity. Among the 12 members (XpsD-O) in secretion apparatus, most proteins have no major domains located at the cytoplasmic side except for XpsF, XpsE, and XpsL. XpsE is a cytoplasmic protein with an ATP-binding motif and is associated with the inner membrane through interaction with XpsL, a inner membrane protein. XpsF is predicted to be a polytopic inner membrane protein with two large cytoplasmic-located subdomains connected by three trans-membrane fragments. To analyze roles of XpsF in the secretion machinery, the two cytoplasmic parts of XpsF were separately overexpressed for analysis. Although both subdomains of XpsF were found to interact with all the other cytoplamic-located Xps protein domains in vitro, only XpsFN exhibited secretion interference when overproduced in vivo. XpsEKMRA is an ATP-binding mutant and exist only as monomer form when produced in E. coli. Retention of XpsEKMRA by XpsFN-(His)6 suggested that XpsFN, different from XpsL, can bind XpsE in its monomer status. Moreover, disruption of interaction between XpsL/XpsE was demonstrated by adding the XpsF subdomains. This result indicates that XpsF may play roles in dissociating XpsE from XpsL and back to cytosol.
URI: http://hdl.handle.net/11455/36109
其他識別: U0005-1408200611125500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1408200611125500
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