Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23971
標題: XpsE蛋白N2區域在XpsE形成多聚體及其與XpsL蛋白相互作用中的重要性
Significance of the XpsE N2 domain in XpsE oligomerization and its interaction with XpsL
作者: 趙怡姍
Chao, Yi-Shan
關鍵字: N2 domain;多聚體
出版社: 生物化學研究所
引用: 1.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(9): 1121. 2.Bitter W., Koster M., Latijnhouwers M., Cock H. de, 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(1): 209. 3.Cascales E. and Christie P.J. (2003) The versatile bacterial type IV secretion systems. Nat. Rev. Microbiol. 1(2): 137. 4.Chen L.Y., Chen D.Y., Miaw J., and Hu N.T. (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J. Biol. Chem. 271: 2703. 5.Chen Y., Shiue S.J., Huang C.W., Chang J.L., Chien Y.L., Hu N.T. and Chan N.L. (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J. Biol. Chem. USA 280: 42356. 6.Christie P.J. and Cascales E. (2005) Structural and dynamic properties of bacterial type IV secretion systems (review). Mol. Membr. Biol. 22(1-2): 51. 7.d'Enfert C., Ryter A., and Pugsley A.P. (1987) Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J. 6: 3531. 8.Dums F., Dow J.M., and Daniels M.J. (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(3): 357. 9.Higgins C.F., Hiles I.D., Salmond G.P.C., Gill D.R., Downie J.A., Evans I.J., Holland I.B., Gray L., Buckel S.D., Bell A.W., and Hermodson M.A. (1986) A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature 323: 448. 10.Hu N.T., Hung M.N., Chiou S.J., Tang F., Chiang D.C., Huang H.Y., and Wu C.Y. (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. 11.Hu N.T., Hung, M.N. Liao C.T. and Lin M.H. (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology 141: 1395. 12.Hu N.T., Hung M.N., Chen D.C. and. Tsai R.T (1998) Insertion mutagenesis of XpsD, an outermembrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris. Microbiology 144: 1479. 13.Hu N.T., Leu W.M., Lee M.S., Chen A., Chen S.C., Song Y.L., and Chen L.Y. (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem. J., 365(Pt 1): 205. 14.Hueck C.J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62(2): 379. 15.Keyzer J. de, Does C. van der, and Driessen A.J. (2003) The bacterial translocase: a dynamic protein channel complex. Cell Mol. Life Sci. 60: 2034. 16.Lee H.M., Wang K.C., Liu Y.L., Yew H.Y., Chen L.Y., Leu W.M., Chen D.C., and Hu N.T. (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. 17.Lee H.M., Tyan S.W., Leu W.M., Chen L.Y., Chen D.C., and 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. 18.Lee M.S., Chen L.Y., Leu W.M., Shiau R.J., and 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. 19.Nouwen N., Ranson N., Saibil H., Wolpensinger B., Engel A., Ghazi A., and Pugsley A.P. (1999) Secretin PulD: Association with pilot PulS, structure, and ion-conducting channel formation. Proc. Natl. Acad. Sci. USA 96: 8173. 20.Planet P.J., Kachlany S.C., DeSalle R., and Figurski D.H. (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. USA 98: 2503. 21.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(3): 659. 22.Robien M.A., Krumm B.E., Sandkvist M. and. Ho W.G.J. (2003) Crystal Structure of the Extracellular Protein Secretion NTPase EpsE of Vibrio cholerae. J. Mol. Biol. 333: 657 23.Sandkvist M., Bagdasarian M., Howard S.P., and 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(8): 1664. 24.Sandkvist M. (2001a) Type II Secretion and Pathogenesis. Infect. Immun. 69: 3523. 25.Sandkvist M. (2001b) Biology of Type II secretion. Mol. Microbiol. 40(2): 271. 26.Savvides S.N., Yeo H.J., Beck M.R., Blaesing F., Lurz R., Lanka E., Buhrdorf R., Fischer W., Haas R. and Waksman G. (2003) VirB11 ATPases are dynamic hexameric assemblies: New insights into bacterial type IV secretion. EMBO J. 22: 1969. 27.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(7): 1426. 28.Shiue S.J., Chien I.L., Chan N.L., Leu W.M., and Hu N.T. (2007) Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol. Microbiol. 65(2): 401. 29.Silverstein R., Lin C.C., and Rawitch A.B. (1980) Evidence for an essential hydrophobic domain in the maintenance of phosphoenolpyruvate carboxykinase activity. Site-specific binding and inactivation by 1-anilinonaphthalene- 8-sulfonate. J. Biol. Chem. 255: 1374. 30.Thomas J.D., Reeves P.J., andSalmond G.P. (1997) The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF. Microbiology 143: 713. 31.Wei C.J. (2006) Analysis of comformationation change at the N domain of XpsE protein by utilizing fluorescence probes. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R.O.C. 32.Yamagata A. and Tainer J.A. (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J. 26: 878. 33.Yo T.T. (2003) Detection of interaction 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 (Xanthomonas campestris pv. campetris) 屬於格蘭氏陰性菌,利用第二型分泌機制 (type II secretion) 分泌水解酵素而感染十字花科植物,第二型分泌機制含有 11 個蛋白,包括位於外膜形成分泌孔道的 XpsD,貫穿內外膜的 pseudopilus (XpsG-H-I-J-K),以及位於內膜的 XpsN(C)、M、L、F,其中 XpsE 是第二型分泌機制的唯一胞內蛋白,也是本研究所要探討的蛋白。它含有 4 個 nucleotide binding motifs,本身具有 ATPase 活性,XpsE 在與 ATP 結合的狀態下會形成六聚體 (hexamer),並與內膜上的 XpsL 蛋白 N 端 (XpsLN) 產生交互作用而附著於內膜,XpsL 蛋白會刺激 XpsE 的 ATPase 活性,促使 ATP 水解以提供能量。根據過去的研究顯示,XpsE 蛋白 N2 區域的 Arg286 突變成Ala後,此突變蛋白會形成六聚體但卻失去與 XpsL 結合的能力,並失去分泌功能。為探討此區域的重要性,本研究將 XpsE 的 N2 區域與其他參與第二型分泌或第四型纖毛組裝的蛋白進行胺基酸序列比對,發現 Ser201、Asp202、His204、Arg217、Asp219 均具有高度保留性,於是將這些胺基酸分別突變成 Ala,並送入 xpsE 缺損株 XC1723 中,發現 XpsED202A、XpsEH204A、XpsER217A 皆無法回復 α-amylase 的分泌功能;只有 XpsED202A 會嚴重干擾野生株 XC1701 的正常分泌。in vitro 實驗發現,在 AMPPNP (ATP analog) 存在下,XpsED202A 和 XpsEH204A與XpsLN結合,而 XpsER217A 則失去和 XpsL 結合的能力;in vivo 的蛋白分佈實驗顯示,XpsED202A、XpsEH204A 可正常附著至內膜,XpsER217A 則不會附著到內膜上。至於各突變蛋白是否因與 AMPPNP 結合而形成六聚體,仍需進一步分析。

Xanthomonas campestris pv. campetris belongs to Gram-negative bacteria and utilizes type II secretion apparatus to secrete hydrolytic enzymes in their infection of crucifer plants. Type II secretion apparatus is a multi-protein system. XpsD is an outer membrane lipoprotein, and forms the pore to allow extracellular protein pass through. Spanning between outer membrane and cytoplasmic membrane is a pilus-like structure composed of one major, GspG protein, and possibly four minor (GspH, -I, -J, -K) pseudopilins. The four integral cytoplasmic membrane proteins are XpsL, XpsM, XpsN(C) and XpsF. XpsE protein is the only cytoplasmic protein component and the focus of this study. It has four conserved nucleotide binding motifs and exhibits ATPase activity. When bound to ATP, XpsE oligomizes. The hexameric XpsE was apparently capable of XpsLN binding, and associating with cytoplasmic membrane. The ATPase activity of XpsE is stimulated by XpsL, presumable providing the energy for extracellular protein secretion from periplam across outer membrane. Previous studies indicated that the XpsE mutated at R286, which is located in the N2 domain, can no longer associate with XpsL despite forming hexamer. It also lost the ability to support type II secretion. In order to determine the significance of the XpsE N2 domain, five more conserved residues were identified by aligning six proteins of TypeII/IV secretion NTPases superfamily. They are Ser201, Asp202, His204, Arg217 and Asp219. Each was mutated to Ala and transformed into the xpsE mutant XC1723 and analyzed for their function. XpsED202A, XpsEH204A and XpsER217A were unable to resume secretion of α-amylase in XC1723. However, only XpsED202A interferes with normal secretion in the wild type strain XC1701. In vitro experiments showed that, in presence of AMPPNP (ATP analog), both XpsED202A and XpsEH204A are able to bind to XpsLN, whereas XpsER217A is not. When expressed in XC1723, while XpsED202A and XpsEH204A remain attached to cytoplasmic membrane, but XpsER217A is not. As to the AMPPNP triggered oligomerization property of each mutant, further studies are required.
URI: http://hdl.handle.net/11455/23971
其他識別: U0005-3007200717463500
Appears in Collections:生物化學研究所

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