十字花科黑腐病菌第二類型胞外蛋白分泌系統中與蛋白交互作用之探討

Abstract

十字花科黑腐病菌屬於革蘭氏陰性菌，會利用第二類型分泌途徑，將胞外蛋白由胞內送至胞外。這種分泌途徑需經兩個步驟，首先藉由Sec蛋白系統，將胞外蛋白送至胞質週緣區 (periplasm) ，再由另一群至少12個蛋白所組成的分泌系統，將胞外蛋白從胞質週緣區送達胞外。本研究欲探討第二步分泌系統中兩個蛋白XpsE和XpsNL兩者間的關係。XpsE具有與核苷酸結合的氨基酸保留區(Walker A box)，被預測分佈在細胞質內。XpsL是一個以N端朝向胞內、穿過膜一次的內胞膜蛋白，其N端位在胞內的序列被稱為XpsNL。在xpsL基因缺損株中，XpsE蛋白含量明顯下降；分別將表現XpsL或XpsNL蛋白的基因送入此基因缺損株，皆可使XpsE蛋白含量恢復正常，說明XpsL可能會藉由位在胞內的氨基酸序列與XpsE蛋白形成複合體，本研究因而進一步分析經部分純化的XpsNL與XpsE蛋白在試管內的交互作用關係。先構築一利用tac啟動子表現GST-NL融合蛋白的基因，利用glutathione S-transferase與glutathione的親和性，將GST-NL融合蛋白固定在glutathione-Sepharose resin上，與經過純化的C端接有Strep-tag的XpsE-Strep蛋白以等莫耳數混合，經batch binding後，收集flow through與elute fraction，進行西方墨點法分析XpsE與XpsNL的分佈。結果發現約有二分之一的XpsE蛋白會與XpsNL結合，且兩者間的結合不因核苷酸的加入、或XpsE蛋白中Walker A box的突變受到影響。然而，N端缺少36個氨基酸且無功能的突變XpsE(D1-36)蛋白，與GST-NL融合蛋白的結合，卻會受ATP或ATPgS的促進，而上升為幾乎百分之百。

Xanthomonas campestris pv. campestris is a Gram-negative bacterium that could utilize the type II secretion pathway to secrete extracellular proteins. It takes two steps to accomplish the secretion process. In the first step, the secreted proteins are translocated across cytoplasmic membrane via the Sec machinery. In a second step, it takes more than 12 proteins for the proteins to traverse across the outer membrane. In this study, we attempt to study the association between XpsE and XpsNL, both of which are involved in the second step of the secretion pathway. The XpsE protein with a conserved nucleotide binding motif (Walker A box) is predicted to be located in cytoplasm. The XpsL protein is a monotopic integral inner membrane protein with its N terminal facing cytoplasm. We designate the N terminal domain of XpsL as XpsNL. The amount of XpsE protein is reduced significantly in the xpsL mutant. Introduction of plasmid that expresses XpsL or XpsNL into the xpsL mutant makes the amount of XpsE recover to normal level, suggesting that XpsL, via its N domain, may form complex with XpsE. We further analyzed the in vitro interactive relationship between XpsNL and XpsE, both of which were partially purified. First, we constructed the plasmid pCY1 that expresses a GST-NL fusion protein from the tac promoter. By taking advantange of the affinity between glutathione S-transferase (GST) and glutathione, we immobilized GST-NL fusion protein on glutathione-Sepharose resin. We then incubated the resin with purified XpsE-Strep protein that is C-terminally tagged with Strep-tag. By performing Western blotting, we analyzed the distribution of XpsE and XpsNL in the unbound and bound fractions. The results indicate that approximatly half of the XpsE protein could bind with XpsNL. Furthermore, addition of different nucleotides or mutation at Walker A box did not affect such interaction. However, significant enhancement in binding with GST-NL was observed for the nonfunctional XpsE(D1-36) when ATP or ATPgS was included in the incubation.