Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23976
標題: XpsF位於胞內的區域與XpsLN及與XpsE蛋白之 相互作用關係
Interactive relationship of cytoplasmic domains of XpsF with XpsLN and with XpsE
作者: 范芝瑋
Fan, Chih-Wei
關鍵字: 相互作用;XpsF
出版社: 生物化學研究所
引用: Arts J, de Groot A, Ball G, Durand E, El Khattabi M, Filloux A, Tommassen J, Koster M (2007) Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF). Microbiology 153: 1582-92 Anderson DM, Schneewind O (1999) Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more. Curr Opin Microbiol 2: 18-24 Bleves S, Voulhoux R, Michel G, Lazdunski A, Tommassen J, Filloux A (1998) The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family). Mol Microbiol 27: 31-40 Bally M, Filloux A, Akrim M, Ball G, Lazdunski A, Tommassen J (1992) Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. 6: 1121-31 Bitter W, Koster M, Latijnhouwers M, de Cock H, Tommassen J (1998) Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27: 209-19 Braibant M, Gilot P, Content J (2000) The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis. FEMS Microbiol Rev 24: 449-67 Camberg JL, Sandkvist M (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187: 249-256 Chen LY, Chen DY, Miaw J, Hu NT (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer J Biol Chem 2: 2703-8 Dums F, Dow JM, 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 229: 357-64. Douville K, Price A, Eichler J, Economou A, Wickner W (1995) SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem 25: 20106-11 Fullner KJ, Lara JC, Nester EW (1996) Pilus assembly by Agrobacterium T-DNA transfer genes. Science 23: 1107-9 Filloux A (2004) The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694: 163-179 Hsu Shu Yin (2004) Analysis of Interactions between XpsF and XpsL, or XpsE, in the Type II Secretion Apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 174: 2679-2687 Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song YL, Chen LY (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J 365: 205-211 Lin C C (2002) 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. Lee C T (2002) Affinity-Chromatography and Yeast-two hybrid Analysis of the Complex Formation among XpsE, -F, -L, -M and -N in Xanthomonas campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Lee MS, Chen LY, Leu WM, Shiau RJ, Hu NT (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: 4585-91 Lee HM, Tyan SW, Leu WM, Chen LY, Chen DC, 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 183: 528-535 Linderoth NA, Simon MN, Russel M (1997) The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 28: 1635-8 Michel G, Bleves S, Ball G, Lazdunski A, Filloux A (1998) Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa. Microbiology 144: 3379-86 Nouwen N, Ranson N, Saibil H, Wolpensinger B, Engel A, Ghazi A, Pugsley AP (1999) Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A 96: 8173-7 Nunn DN, Lory S (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 175: 4375-82 Py B, Loiseau L, Barras F (2001) An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep 2: 244-8 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 Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57: 50-108 Pan H W (2002) Characterization of the roles of XpsE and XpsF proteins in the type II secretion pathway of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 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 SJ, Kao KM, Leu WM, Chen LY, Chan NL, Hu NT (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25: 1426-35 Sandkvist M, Bagdasarian M, Howard SP, DiRita VJ (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 Sandkvist M (2001) Biology of type II secretion. Mol Microbiol 40: 271-283 Sandkvist M (2001) Type II secretion and pathogenesis. Infect Immun 69: 3523-35 Sandkvist M, Hough LP, Bagdasarian MM, Bagdasarian M (1999) Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. J Bacteriol 181: 3129-35 Tsai RT, Leu WM, Chen LY, 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 367: 865-871 Wandersman C, Delepelaire P (1990) TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A 87: 4776-80. Walker JE, Saraste M, Runswick MJ, Gay NJ (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: 945-51 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 Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Zhu Z L (2006) Type II Secretion Apparatus of Xanthomonas campestris : Expression and Functional Characterization of the Cytoplasmic Domains of XpsF. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
摘要: 
十字花科黑腐病菌 ( Xanthomonas campestris ) 屬於格蘭氏陰性菌,需仰賴第二型蛋白分泌系統將各種水解酵素分泌至胞外,藉此感染十字花科植物。第二型蛋白分泌機器由XpsD~XpsO等十二個蛋白所組成。XpsE是唯一的胞內蛋白,和內胞膜上的XpsL蛋白有交互作用關係;並且擁有四個nucletide binding motifs,具有微弱的ATPase活性。在與ATP結合情況下,會促進XpsE形成六聚體,進而與XpsL朝向胞內的N端區域 ( XpsLN ) 結合,隨後XpsLN則會促進XpsE蛋白水解ATP。XpsF為穿膜三次的內胞膜蛋白,由穿膜區將其切割成N端(XpsFN)及C端(XpsFC)兩個位於胞內的蛋白區塊,目前對其功能尚有待釐清。過去分析XpsF蛋白功能時,發現xpsF基因缺損會使菌株喪失分泌澱粉酵素的功能,顯示XpsF蛋白在此機器運作中扮演必要角色。先前利用yeast two hybrid system針對位於胞內的Xps蛋白區塊之間相互作用的分析,發現XpsFc分別與XpsFN、全長XpsE以及XpsLN皆有交互作用發生,暗示XpsE、XpsF以及XpsL三者之間有互動關係。為了進一步探討XpsF蛋白與XpsL或XpsE之間的相互作用,本文利用in vitro pull down 實驗進行分析,發現無論是否添加不同的nucleotides,XpsFN或XpsFC與全長XpsE蛋白或與XpsLN的交互作用皆極微弱,即使有MBP-XpsLN或XpsFC蛋白的存在也無顯著促進現象。其次,利用ADP-AlF4模擬ATP transition state偵測XpsE與XpsLN的結合情形以及XpsF在這當中可能扮演的角色,發現XpsE與ADP-AlF4結合不會影響它與XpsLN的交互作用,且XpsE與XpsLN的結合也沒有因為XpsFN或XpsFC的存在而改變。Gel filtration結果指出XpsE結合ADP-AlF4會促進六聚體的形成,因此推測當XpsE水解ATP進入transition state時,可能仍維持在六聚體的狀態 ,並依然結合在XpsL上,而XpsF在此分泌機制中所擔任的任務仍有待進一步探討。

Xanthomonas campestris pv. campestris is a Gram-negative bacterium that relies on type II secretion system for secreting hydrolytic enzymes in their infection of plant leaves. The X. campestris type II secretion system is composed of 12 Xps protein components, of which XpsE is the only cytoplasmic protein consisting of four conserved nucleotide binding motifs. It is known to interact with the inner membrane protein XpsL. Triggered by ATP-binding, XpsE oligomerizes and associates with the N terminal domain of XpsL. Such interaction appears to stimulate ATP hydrolysis by XpsE. XpsF is predicted to be a polytopic inner membrane protein with two large cytoplasmic domains divided by three transmembrane sequences. Its biological function in type II secretion system remains poorly understood. Previous yeast two hybrid analyses revealed that the C-terminal domain of XpsF (XpsFC) interacts with its own N-terminal domain (XpsFN), with the N-terminal domain of XpsL (XpsLN) and with XpsE, implicating interactive relationship among XpsE, XpsF and XpsL. To further characterize physical interaction of XpsF with XpsL and with XpsE, detailed in vitro pull down assays were performed in this study. The results indicated that the interactions between XpsFN or XpsFC and XpsLN or XpsE, with or without preincubating with different nucleotides, are all very weak. Neither could stimulation be observed in presence of MBP-XpsLN or Nus-XpsFch,. Interaction between XpsE and XpsLN were also analyzed in presence of ADP-AlF4, an analogue mimicking ATP transition state, or in presence of XpsFN or XpsFC. Pull down assay revealed that ADP-AlF4 has no effect on the interaction between XpsE and XpsLN. Neither does XpsFN or XpsFC. Gel filtration chromatography revealed that preincubation of XpsE with ADP-AlF4 stimulated its oligomerization, implying XpsE bound to ATP transition state remains oligomeric and associated with XpsLN. As to the role of XpsF in type II secretion system, further studies are required.
URI: http://hdl.handle.net/11455/23976
其他識別: U0005-3107200715374000
Appears in Collections:生物化學研究所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.