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標題: 十字花科黑腐病菌XpsE與XpsF蛋白於第二類型胞外蛋白分泌路徑之角色探討
Characterization of the Roles of XpsE and XpsF Proteins in the Type II Secretion Pathway of Xanthomonas campestris pv. campestris
作者: 潘亨文
Pan, Heng-Wen
關鍵字: GSP
Type II secretion pathway
出版社: 農業生物科技學研究所
摘要: 革蘭氏陰性菌的細胞結構由內而外,依次分為細胞質、內胞膜、胞質週緣區、細胞壁及外胞膜,不同的細菌發展出不同的分泌途徑以將多種蛋白分泌至胞外。第二類型胞外蛋白分泌途徑常見於革蘭氏陰性菌中,又稱為一般分泌途徑 (general secretory pathway; GSP) ,其分泌過程可分成兩個步驟,首先由SEC系統輔助將胞外蛋白送到胞質週緣區,再由另一群12-14個蛋白所組成的GSP系統將之分泌到胞外。十字花科黑腐病菌的胞外蛋白分泌系統是由XpsD~N等十一個蛋白組成,其中XpsE是唯一的細胞質蛋白,推測具有ATP-binding motif,可能扮演能量供應者的角色;XpsF蛋白以電腦分析預測其為一個穿過膜三次的內膜蛋白,主體為N端與C端兩個位向細胞質的大片段,但功能未明,本實驗以遺傳與生化實驗策略探討XpsE及XpsF蛋白的角色。首先利用同源性基因置換的方式,製備XpsE蛋白Walker A box單點突變菌株,破壞其與ATP的結合能力,實驗結果證實此菌株無法將澱粉酵素分泌至胞外。此外,在野生株中大量表現XpsF蛋白,發現可導致菌株的分泌能力降低,暗示XpsF蛋白可能與其它相對微量的蛋白分子具有複合體關係;相反的,過量表達XpsE Walker A box單點突變蛋白則不影響分泌。為了進一步解析這些菌株分泌機器的變化,我們以免疫共沉澱實驗策略偵測已知之XpsL/M/N及XpsL/E 等蛋白複合體是否發生明顯的含量變化,並觀察XpsF是否亦參與形成蛋白複合體。結果顯示XpsL/M/N蛋白複合體的形成不會受到上述條件影響,亦未明顯偵測到XpsF蛋白參與複合體的形成。
There are five subcellular compartments including the cytoplasm, inner membrane, periplasm, cell wall and the outer membrane in the gram-negative bacteria. Several distinct routes have evolved for protein export in various bacteria. The type II secretion pathway, also known as the General Secretory Pathway (GSP), is one of such system that widely distributed among bacteria. In this system, the secreted proteins are firstly translocated across the cytoplasmic membrane via the sec machinery. The second step requires 12-14 Gsp proteins for assisting proteins across the outer membrane. These proteins are designated XpsD-N in Xanthomonas campestris pv. campestris. XpsE, the only cytoplasmic protein, is associated with cytoplasmic membrane through interaction with XpsL and may act as a kinase or ATPase. XpsF is predicted as a polytopic inner membrane protein with a small periplasmic loop and two large cytoplasmic domains connected by three transmembrane regions. Roles of XpsF remain unclear thus far. We investigated the roles of XpsE and XpsF involved in the assembly of the secretion machinery by genetic and biochemical strategies. A chromosomal xpsE mutant with point mutation on the Walker A box was constructed by homologous recombination. We concluded that an intact Walker A box is essential for function, although XpsEw did not result in overproduction inhibition. In contrast, overproducing XpsF hampered the secretion process, indicates that XpsF may interact with proteins in a limited amount in the wild type strain. To further resolve the mechanism of secretion, co-immunoprecipitation was performed to detect if there was any qualitative change in the known complexes such as XpsL/M/N and XpsL/E, etc. Simultaneously, roles of XpsF were explored if it involved in complex formation. We concluded that the XpsL/M/N complex formation was not affected in the strain analyzed. Complex formation between XpsF and other Xps proteins was not detected.
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