跳到主要內容

臺灣博碩士論文加值系統

(3.235.174.99) 您好!臺灣時間:2021/07/24 20:16
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:翁宥閎
研究生(外文):You-Hong Wong
論文名稱:以共純化方式分析內膜蛋白XpsF與XpsEATPase或與另一內膜蛋白XpsL間的相互作用關係
論文名稱(外文):Analyzing interaction between the membrane protein XpsF with the XpsE ATPase or with another membrane protein XpsL by examining their co-purification
指導教授:胡念台
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:29
中文關鍵詞:十字花科黑腐病菌第二型分泌共純化
外文關鍵詞:Xanthomonas campestrisType II secretionXpsFco-purification
相關次數:
  • 被引用被引用:0
  • 點閱點閱:99
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
十字花科黑腐病菌 ( Xanthomonas campestris ) 屬於格蘭氏陰性菌,為農作物上常見的病原菌,藉由其第二型分泌系統可將各種水解酵素分泌至胞外,進而破壞並感染宿主細胞。在第二型分泌系統中,XpsE 為一具有 ATPase 活性的可溶性蛋白,推測 XpsE 可藉由與內胞膜蛋白 XpsL 相互作用將水解 ATP 的能量轉換為分泌所需的動能。XpsF 也是一個內胞膜蛋白,但具有三段穿膜序列,以 N-端起始於胞內,C-端結束於胞質週緣區的形式,在胞內形成兩個較大的區域。過去發現當菌株缺少 XpsF 蛋白即無法行使分泌功能,顯示 XpsF 為第二型分泌系統必要的組成蛋白。先前利用免疫共沉澱的分析方式,發現在同時大量表現 XpsF、XpsE、XpsL、XpsM、XpsC 的 xps 缺損株中,可以觀察到 XpsF、XpsE、XpsL 三者的相互作用關係。然而在具有分泌能力的互補菌株 (將 xpsL-strep 送入 xpsL 缺損株中) 所進行的共純化實驗中, XpsF、XpsE、XpsL 三者的相互作用關係則減弱許多,需添加 DSP (crosslinker) 才可被觀察到,推測具備正常分泌功能的菌株中,XpsF、XpsE、XpsL 三者為動態的短暫性結合。為了進一步探討 XpsF 與 XpsE 或 XpsL之間的相互作用,本文利用共純化實驗進行分析,將 C-端具有 His-tag 的全長 XpsFh 蛋白送入 XC1722 (xpsF 基因刪除株),或是將共表現 XpsE 突變蛋白與 XpsFh 的質體送入 XC1736 (xpsE, xpsF 基因刪除株),發現在野生型 XpsE 或是 XpsE¬K331M、XpsEK331M, R504A、XpsER286A、XpsEV11A, L25A, L39A 參與下皆無法觀察到 XpsF 與 XpsL 或 XpsE 及其突變蛋白的相互作用關係,暗示這些由 XpsE 突變蛋白所模擬的 XpsE 不同運作步驟,並非 XpsF 與 XpsL 或 XpsE 相互作用的最佳狀態。本研究採用的 XpsE 與 XpsFh 共表現質體,有助於未來測試其它 XpsE 突變蛋白是否促進 XpsF 與第二型分泌系統中其它組成蛋白的相互作用關係。
Xanthomonas campestris is a common agricultural pathogen. Being a Gram-negative bacterium, X. campestris utilizes the type II secretion system to secret a number of hydrolytic enzymes outside the cell in its attack and infection of plant cells. Of all components in the X. campestris type II secretion system, XpsE is the only soluble protein exhibiting ATPase activity. It was hypothesized that the energy generated from ATP hydrolysis by XpsE is transmitted for secretion by its interaction with a cytoplasmic membrane protein XpsL. In addition to XpsL, XpsF is also a cytoplasmic membrane protein , however with three transmembrane sequences. It is constituted of two main cytoplasmic domains with its N-terminus facing cytoplasm and C-terminus facing periplasm. It was previously shown that the xpsF gene deficiency causes protein secretion defective, suggesting that the XpsF protein is an essential component of the type II secretion system. As shown in previous coimmune precipitation experiment, interaction between XpsF with XpsE and XpsL was observed by simultaneous overexpression of XpsF with XpsE, XpsL, XpsM and XpsC in a mutant stain XC17433 that is deleted in the xps gene cluster. However, when assayed in a secretion-proficient strain, association between XpsL with XpsF or XpsE appeared very weak, as suggested by the observation that a crosslinker DSP is required for copurification of XpsF or XpsE with XpsL-Strep. It was presumed that the association among XpsF, XpsE and XpsL may be transient in a secretion-proficient strain. In order to improve our understanding of interactive relationship among XpsF, XpsE and XpsL, copurification of XpsE or XpsL with the xpsFh gene was introduced into the xpsF-deficient strain XC1722. Alternatively, a plasmid co-expressing XpsFh and XpsE or its variants was introduced into the mutant strain XC1736 that is doubly deficient in xpsE and xpsF. The results indicated that none of the XpsE mutants included here, i.e. XpsE¬K331M, XpsEK331M, R504A, XpsER286A and XpsEV11A, L25A, L39A, enhanced copurification of XpsE or XpsL with XpsFh. The implication is that none of the secretion steps blocked by these mutations in the xpsE gene is favorable for detecting stable association of XpsE or XpsL with XpsF. However, the coexpression plasmid employed in this study may be useful for future test of other xpsE gene mutations in enhancing of XpsF with other protein components of the type II secretion system.
前言 1
材料與方法 8
一、材料 8
二、實驗方法 8
結果 14
一、利用 nickel-affinity column 及 Western blotting 分析第二型分泌系統蛋白與全長 XpsFh 共純化的情形 14
二、利用 XC1736 (xpsE、F 基因刪除株) 共表現 XpsFh 與 XpsE-Strep 或其突變蛋白分析具不同特性的 XpsE 突變蛋白是否促進全長 XpsFh 與 XpsE 或 XpsL 的相互作用關係 18
討論 22
一、XpsF 與 XpsL 的相互作用關係 22
二、XpsF 與 XpsE 的相互作用關係 23
三、XpsF與其它蛋白的相互作用關係 24
參考文獻 25
圖 30
表 42
附錄 51
附錄一: 利用兩次同源性基因交換獲得 XC1736 (△xpsEF)之流程 51
附嫌二: 培養基、抗生素、試劑與緩衝溶液 53
Arts J, de Groot A, Ball G, Durand E, El Khattabi M, Filloux A, Tommassen J and Koster M (2007) Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF). Microbiology 153: 1582-1592.

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 6: 1121-1131.

Bitter W, Koster M, Latijnhouwers M, de Cock H and 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: 209-219.

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 27: 31-40.

Chao Mei-Li (1998) Significance of XpsF protein in Type II secretion pathway of Xanthomonas campestris. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.

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 271: 2703-2708.

Chen Y, Shiue SJ, Huang CW, Chang JL, Chien YL, Hu NT and Chan NL (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem 280: 42356-42363.

Collins RF, Saleem M and Derrick JP (2007) Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV pilus biogenesis protein PilG. J Bacteriol 189: 6389-6396.

Crowther LJ, Yamagata A, Craig L, Tainer JA and Donnenberg MS (2005) The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J Biol Chem 280: 24839-24848.

Durand E, Michel G, Voulhoux R, Kürner J, Bernadac A and Filloux A (2005) XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J Biol Chem 280: 31378-31389.

Forest KT (2008) The type II secretion arrowhead: the structure of GspI-GspJ-GspK. Nat Struct Mol Biol 15: 428-430.

Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY and 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: 2679-87.
Hu NT, Hung MN, Liao CT and Lin MH (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology 141: 1395-406.

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 365: 205-211.

Korotkov KV and Hol WG (2008) Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat Struct Mol Biol 15: 462-468.

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 183: 528-535.

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 182: 1549-1557.
Lee MS, Chen LY, Leu WM, Shiau RJ and 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: 4585-4591.
Lin CC (2000) Interaction of cytoplasmic membrane protein XpsF with XpsL/M/N and XpsE in Xanthomonas campestris. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.

Linderoth NA, Simon MN and Russel M (1997) The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 278: 1635-1638.

Login FH and Shevchik VE (2006) The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi. J Biol Chem 281: 33152-33162.

Nouwen N, Ranson N, Saibil H, Wolpensinger B, Engel A, Ghazi A and Pugsley AP (1999) Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A 96: 8173-8177.

Nunn DN and Lory S (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 175: 4375-4382.

Planet PJ, Kachlany SC, DeSalle R and Figurski DH (2001) Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci U S A 98: 2503-2508.

Pugsley AP ( 1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57: 50-108. Review.
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 289: 659-670.

Py B, Loiseau L and Barras F (2001) An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep 2: 244-248.

Robien MA, Krumm BE, Sandkvist M and Hol WG (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333: 657-674.
Sandkvist M (2001 a) Type II secretion and pathogenesis. Infect Immun 69: 3523-35.

Sandkvist M (2001 b) Biology of type II secretion. Mol Microbiol 40: 271-283.

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 14: 1664-1673.

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 181: 3129-3135.

Savvides SN, Yeo HJ, Beck MR, Blaesing F, Lurz R, Lanka E, Buhrdorf R, Fischer
Voulhoux R, Ball G, Ize B, Vasil ML, Lazdunski A, Wu LF and Filloux A (2001) Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. EMBO J 20: 6735-6741.

W, Haas R and Waksman G (2003) VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. EMBO J 22: 1969-1980.

Shiue SJ, Chien IL, Chan NL, Leu WM and Hu NT (2007) Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol Microbiol 65: 401-412.

Shiue SJ, Kao KM, Leu WM, Chen LY, Chan NL and Hu NT (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25: 1426-1435.

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 367: 865-871.

Yo TT (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.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top