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標題: 十字花科黑腐病菌第二類型胞外蛋白分泌系統:XpsE 蛋白和 XpsLN 蛋白間交互作用關係之探討
Type II Secretion Apparatus of Xanthomonas campestris:Analysis of Interactive Relationship between XpsLN and XpsE
作者: 薛聖潔
Shiue, Sheng-Jie
關鍵字: Type II secretion pathway;第二類型胞外蛋白分泌系統
出版社: 生物化學研究所
格蘭氏陰性菌的第二類型分泌途徑 (type II secretion pathway) 負責將胞外蛋白由胞質週緣區 (periplasm) 運送至胞外。構成十字花科黑腐病菌第二型分泌機制的必要組成份子至少有 12 個蛋白,其中只有 XpsE 蛋白是胞內蛋白並含有 4 個 nucleotide binding motifs,被預測為 ATPase 或是 kinase。由 limited trypsinolysis 實驗顯示 XpsE 蛋白可能被區分為 17 kDa 的 N 端區域 (XpsEN) 及 42 kDa 的 C 端區域 (XpsEC),而 nucleotide binding motifs 位於 XpsEC domain。 XpsL 蛋白是 N 端朝向胞內穿膜一次的內膜蛋白,文獻顯示在其它細菌中 E 蛋白和 L 蛋白具有交互作用的關係。為了進一步瞭解 XpsE 與 XpsL 間的交互作用,本研究首先比較只具胞內區域的 XpsLN 與全長 XpsL 在胞內的含量,發現在細胞內 XpsL 和 XpsE 蛋白間互相穩定的關係可透過 XpsLN 達成。若在細胞內以質體同時表現 XpsLN 和 XpsE-Strep 蛋白,XpsLN 蛋白可與 XpsE-Strep 蛋白共純化,說明 XpsLN 蛋白和 XpsE 蛋白之間有直接的結合關係。進一步在試管內以 MBP pull-down assay 觀察此交互作用關係,同樣可偵測到全長 XpsE 蛋白和 MBP-XpsLN 蛋白的結合,而且這樣的結合關係會因 ATP 的加入而促進。另一方面,在試管內加入 XpsLN 蛋白會促進 XpsE 蛋白的 ATPase 活性,暗示 XpsE 在胞內可能藉由與 XpsL 的交互作用,水解 ATP 協助分泌進行。此外,利用 MBP pull-down assay 觀察 XpsEN 及 XpsEC 蛋白各自與 MBP-XpsLN 蛋白的結合,發現兩者的結合能力都比全長的 XpsE 蛋白弱;有趣的是 XpsEC 蛋白對於 MBP-XpsLN 蛋白的結合必須要外加 ATP 才觀察得到。推測在胞內 XpsE 的 N 端及 C 端兩個區域互相合作,呈現出全長 XpsE 與 XpsLN 結合的特性。

The type II secretion pathway in Xanthomonas campestris pv. campestris requires at least 12 components for the extracellular proteins to traverse across the outer membrane. XpsE is the only cytosolic protein among the 12 components. It has four conserved nucleotide binding motifs, and is postulated to be an ATPase or a kinase. Limited trypsinolysis of XpsE revealed that it could be divided into an N-terminal domain of 17 kDa (XpsEN) and a C-terminal domain of 45 kDa (XpsEC) where the nucleotide binding motifs reside. XpsL is a monotopic integral cytoplasmic membrane protein with its N-terminal (XpsLN) facing the cytoplasm. In this study, we observed that the XpsE protein level is reduced significantly in the xpsL mutant XC1712. Normal protein level of XpsE was restored by introducing a plasmid-encoded xpsLN, as well as the full-length xpsL. In agreement with the above observation, the XpsLN protein level, when expressed in the absence of other xps genes, was raised significantly by co-expressing with XpsE. Moreover, XpsLN protein co-eluted with Strep-tagged XpsE on affinity chromatography. These results indicate that XpsLN interacts directly with XpsE. We further analyzed this interaction in vitro by performing maltose binding protein (MBP) pull-down assay. The full-length XpsE was pulled down by immobilized MBP-XpsLN. Such interaction was enhanced in the presence of ATP. Through in vitro ATPase assay, adding XpsLN stimulated ATPase activity of XpsE. To further dissect the XpsE for its interaction domains with XpsLN, XpsE was replaced with XpsEN or XpsEC in the MBP pull-down assay. XpsEN or XpsEC bound XpsLN with much lower affinity than the full-length XpsE. Interestingly, the interaction between XpsEC and XpsLN was almost completely ATP-dependent. We hypothesize that the N-terminal domain and the C-terminal domain of XpsE may interact with XpsLN in synergy.
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