十字花科黑腐病菌中XpsF與XpsL或XpsE蛋白交互作用之分析

Abstract

革蘭氏陰性菌中負責將多種蛋白如水解酵素和毒素分泌至胞外的機制，第二型蛋白分泌途徑屬於其中之一，為一般分泌途徑 ( general secretion pathway，GSP ) 之主要類型。欲分泌至胞外的蛋白首先經位於內胞膜的Sec分泌機器送到細菌的胞質週緣區 ( periplasm )，再藉由12~15個蛋白所組成的胞外蛋白分泌系統輔助通過外胞膜，送至胞外。十字花科黑腐病菌 ( Xanthomonas campestris pv. campestris ) 的胞外蛋白分泌系統是由XpsD~O等十二個蛋白所組成，XpsF預測為穿膜三次的內膜蛋白，主要區域居均朝向細胞質以第一與第二個穿膜區域為界可分為N端與C端兩個區塊；而XpsE是細胞質蛋白，推測具有ATP binding motif，需藉由內膜蛋白XpsL附著在內胞膜上，可能扮演能量供應者的角色。對於XpsF尚未有證據指出其功能，故本實驗利用Strep-tag親和性管柱層析法、酵母菌雙雜交法及Ni-NTA親和性管柱層析之蛋白下拉實驗三種策略，嘗試偵測XpsF與其它位於胞內Xps 蛋白區塊是否具有交互作用關係。以XpsL互補菌株的細胞膜胞囊 ( membrane vesicle ) 懸浮液為材料，發現唯有加入一種蛋白交聯劑 ( crosslinker )，DSP，方可使C端接有Strep-tag的XpsL與少量的XpsF形成穩定複合體。由此推測XpsF與XpsL的交互作用關係可能易被萃取膜蛋白用的清潔劑 ( detergent ) 拆散，或是本來兩者之間即為短暫動態式的複合體關係。進一步以酵母菌雙雜交法分析XpsF與XpsL或XpsE蛋白的交互作用關係及其作用區塊，發現除了文獻上已知具有交互作用關係的XpsE/XpsE、XpsE/XpsLC和XpsLN/XpsLN等組合外，XpsFC/XpsE、XpsFC/XpsFN與XpsFC/XpsLN蛋白組合也具有強烈的反應，其中前二者僅單向組合具強烈反應，最後一組則反向XpsLN/XpsFC蛋白亦具有反應。再將XpsE蛋白切割成XpsEN與XpsEC區域進行酵母菌雙雜交法測試，分析可知XpsLN/XpsEN與XpsFC/XpsEN具有明顯的交互作用關係，其中XpsFC/XpsEN是文獻上未曾報導過的。我們結論Xps蛋白分泌機器在細胞內具有交互作用的蛋白包括了XpsLN或XpsEC蛋白自我形成的蛋白複合體，XpsFC與XpsFN具有交互作用關係，以及XpsFC、XpsLN與XpsEN蛋白至少彼此兩者間具有交互作用。

The type II secretion pathway, also called the general secretion pathway (GSP), is widely used by Gram-negative bacteria for secretion of a diverse group of proteins. Secreted proteins are first translocated via a Sec-dependent system into the periplasm, and subsequently transported across the outer membrane by the Gsp, constituted by 12~15 members. These proteins are designated XpsD-O in Xanthomonas campestris pv. campestris. XpsF is predicted to be a polytopic inner membrane protein with a small periplasmic loop and two large cytoplasmic domains connected by three transmembrane regions. XpsE is a cytoplasmic protein with the ATP-binding motif and is associated with the cytoplasmic membrane through the interaction with an inner membrane protein, XpsL. Because the function of XpsF is largely unknown, we aim to examine its interacting partners by Strep-tag affinity chromatography analysis, yeast two-hybrid system and Ni-NTA resin pull-down assay. The Strep-tagged XpsL can retain a small amount of XpsF only when the membrane vesicles are pretreated with DSP, a reversible cross-linker. This result suggests that the interaction between XpsF and XpsL is either easily disrupted by detergent or it only exists temporarily. To further dissect the interaction domains of secretion machinery located in the cytoplasmic side, Xps proteins were separated into XpsLN (N stands for N-terminal), XpsLC (C stands for C-terminal), XpsFN, XpsFC, XpsEN, and XpsEC. Through yeast two-hybrid system analyses, we found that beside the well-documented stable complexes such as XpsE/XpsE, XpsE/XpsLN, and XpsLN/XpsLN, there are detectable interactions between XpsFC/XpsE, XpsFC/XpsFN, XpsFC/XpsLN, and XpsLN/XpsFC. Dissection of the XpsE protein revealed that XpsEN, but not XpsEC, interacts with XpsLN and XpsFC. We conclude that in the cytoplasmic side of Xps machinery, XpsLN and XpsEC may form self-multimers while XpsFC, XpsEN, and XpsLN may form complexes with each other.