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

Abstract

中文摘要 十字花科黑腐細菌在農業上是很重要的致病菌，與其它致病性細菌相同，它的分泌機器之完整與致病能力息息相關，其分泌機制隸屬於第二型的蛋白分泌系統，此分泌系統的最大特色是與SEC蛋白群有依賴關係，亦即欲分泌至胞外的蛋白在細胞質內產生後，需先透過SEC蛋白群送至細菌的細胞周質區（periplasm），再透過第二型分泌途徑送至胞外。黑腐細菌第二型分泌系統相關的蛋白至少有11個，已知其中XpsE為一細胞質蛋白，具有ATP binding motif，可藉由XpsL附著於內胞膜上，一般相信其最主要的角色應為提供分泌機器動作所須的能量；XpsL、XpsM、XpsN三者均為內膜蛋白，以XpsM蛋白居中形成複合體，倘若缺乏其中任何一種蛋白，則另二種蛋白含量均劇降；XpsD為一外膜蛋白，以至少12個左右的單元形成一規律的結構，推測其即為分泌的孔道。至於XpsF則為一穿過膜三次的內胞膜蛋白，推測其有兩種可能的角色，一作為組合分泌機器蛋白群的平台，或者作為輔助蛋白摺疊的chaperone，然而目前均欠缺證據，本篇研究根據XpsF絕大部份的蛋白區塊均面向細胞質的事實，推測XpsF應與XpsL或XpsE蛋白具有交互作用關係。 先前本實驗室曾利用XpsE、XpsF、XpsL、XpsM及XpsN蛋白共同過量表現的菌株，以免疫共沉澱法證實這五個蛋白可形成複合體，惟複合體含量不高。本篇研究則針對蛋白表現並不高但具有分泌能力的菌株，使用金屬離子親和性管柱大量吸附XpsL-(His)6融合蛋白，期望可同時吸附住XpsF蛋白，然而發現仍僅能偵測到XpsL/M/N蛋白複合體。在測試各種xps單一基因缺失的菌株後，我們結論在此些菌株中其XpsL/M/N與XpsL/E等蛋白複合體含量，均不會因為分泌動作無法完成而明顯改變。最後本研究使用酵母菌二雜交系統，分析除去嵌膜區域的各種Xps蛋白區塊是否具有交互作用，並定量分析其作用力的強弱。

Abstact Xanthomonas campestris is an important phytopathogen in which the integrity of secretion machinery is critical for its pathogenicity. The type II secretion pathway (also known as the general secretion pathway) that requires the Sec proteins is known to exist in Xanthomonas. The type II secretion pathway is constituted by a stepwise process: secreted proteins are firstly translocated by sec-dependent mechanism through the inner membrane, released into the periplasm, and transported across the outer membrane by the so-called Gsp proteins. The Gsp proteins in Xanthomonas campestris contain at least 11 members. XpsE, with the ATP-binding motif, is a cytoplasmic protein associated with the cytoplasmic membrane through interaction with XpsL. XpsL, -M and -N are cytoplasmic membrane proteins that assembly as ternary complex. The stabilities of these three proteins are mutually dependent. XpsD is an outer membrane which forms the secretion pore. XpsG probably assemble with XpsH, -I, -J and -K and form pilus-like structure in periplasm. In contract to the above, roles of XpsF, a cytoplasmic membrane protein with two main domains facing inward, remain unknown thus far. It was speculated that XpsF may act, as platform for apparatus assemble or alternatively, as chaperon for protein folding. According to the predicated membrane topology, it is proposed that XpsF may interact with XpsE or XpsL, the other two Xps members with major cytoplasmic domains. Demonstrated by co-immunoprecipitation, XpsF was found to form complex with XpsE and XpsL/M/N, although only to a scarce amount under overexpressed condition. Using metal-affinity chromatography, we aimed to detect the complex formation among the above five proteins in strains with secretion ability. Furthermore, xps chromosomal mutants were examined to evaluate the possibility that the secretion complex may be locked and stabilized while the machinery is partially defective. However, only XpsL/M/N and XpsL/E complex were sucessfully detected by the above two approaches. Using the yeast two-hybrid system, we further dissect the interaction domains by removing the membrane-anchor region of XpsF, XpsL and XpsM.