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標題: 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
Interaction of cytoplasmic membrane protein XpsF with XpsL/M/N and XpsE in Xanthomonas campestris
作者: 林主祈
Lin, Chu-Chi
關鍵字: TypeⅡ secrection pathway;第二型分泌機制;Xanthomonas campestris;General secrection pathway;十字花科黑腐病菌;一般分泌途徑
出版社: 農業生物科技學研究所
革蘭氏陰性細菌分泌酵素或致病因子至胞外環境須要通過二層胞膜,目前已知至少有四種不同的系統單獨或共同存在於單一菌種中,其中第二型系統又稱為一般分泌途徑(General Secretion Pathway,GSP),其特點為使用兩個步驟來完成運送過程,被分泌的蛋白先由Sec蛋白群輔助將之從胞內送至胞質週緣區,再由橫跨內、外膜的Gsp蛋白群輔助抵達胞外。十字花科黑腐病菌的外膜蛋白分泌系統乃由xpsD~N十一個基因的蛋白產物所組成,其中有三個成員蛋白具有位向細胞質的大片段蛋白區域,XpsE是唯一的細胞質蛋白,具有ATP-binding motif,可能扮演能量供應者的角色;XpsL為內膜蛋白,於膜內外側均具有蛋白質功能區,可能扮演橋樑的角色,聯繫細胞質中的XpsE與胞質週緣區的其它Xps成員蛋白;XpsF則功能未明,其絕大部分的蛋白區域均朝向細胞質,本實驗以遺傳及生化策略,探討XpsF是否與位向相同的XpsE及XpsL蛋白具有交互作用關係。首先製備了xpsF或xpsE單一基因缺損菌株,我們證實此二基因均為分泌功能所必需,然而菌體缺乏XpsF蛋白並未造成其它Xps成員蛋白的含量改變,亦不影響XpsE與內胞膜附著的能力;相反的,於野生株中大量製造XpsF蛋白,則發現導致菌體的分泌能力降低,且此現象可由同時大量製造XpsE蛋白得到緩解,暗示XpsF與XpsE之間具有直接或間接的蛋白交互作用關係。進而利用免疫共沉澱策略,証實XpsL/M/N與XpsE或XpsF可分別形成蛋白複合體,並不需要其它Xps成員蛋白的參與,然而XpsE與XpsF之間則無直接的結合關係。我們據此提出XpsE、XpsF與XpsL/M/N蛋白間複合體互動關係的假說,並討論XpsF於分泌機制中可能扮演的角色。

In Gram-negative bacteria, the export of enzymes or virulence factors needs to overcome two successive hydrophobic barriers, the cytoplasmic and outer membranes. To date, four distinct secretion apparatus are known to be used, either alone or simultaneously, in one bacterium. In type II secretion pathway, also known as the general secretory pathway, the sorted proteins were first exported by Sec systems from cytoplasm into periplasm, and then were assisted by the GSP multiprotein complexes formed between two membrane bilayers to reach the extracellular medium. In Xanthomonas, GSP was constituted by at least 11 protein members, the XspD~N. Among them, three proteins are known to contain large cytoplasmic domains. XpsE is located on the cytoplasmic side of inner membrane and might act as a kinase or ATPase. XpsL is an integral inner membrane protein likely to play a bridging role by interacting with both the cytoplasmic protein XpsE and the others, mainly periplasmically located Xps proteins such as XpsM and XpsN. XpsF is also an inner membrane protein, however, contains two domains facing mainly toward the cytoplasmic side. Although essential for the secretory pathway, the roles of XpsF is thus far unknown yet. We aim to elucidate the protein-protein interaction relationship, if any, between XpsF and the physically co localized XpsE and XpsL proteins. The chromosomal xpsF in-frame deletion mutant was generated by homologous recombination. Neither the amount of Xps proteins nor the ratio of membrane-bound XpsE was changed in the absence of XpsF. On the opposite, overproduction of XpsF hampered the secretion process while concomitant overproduction of the XpsE with XpsF alleviated such inhibition, indicated that there are direct or indirect interactions between XpsE and XpsF. Coimmunoprecipitation experiments had shown that XpsF form complex directly with XpsL/M/N, but not with XpsE, in the absence of other Xps components. The interaction between XpsE and XpsF was therefore indirect and might act through XpsL/M/N complex. Together with the XpsE/L/M/N complex detected in the same study, a dynamic relationship of XpsF、L/M/N and E proteins in complex formation was proposed and the roles of XpsF in assembly of the secretory machinery was discussed.
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