The transport of group 2 capsular polysaccharides across the peripiasmic space in Escherichia coli -: Roles for the KpsE and KpsD proteins
The transport of group 2 capsular polysaccharides across the peripiasmic space in Escherichia coli -: Roles for the KpsE and KpsD proteins
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DOI：
10.1074/jbc.m008183200
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发表时间：
2001-02-09
影响因子：
4.8
通讯作者：
Roberts, IS
中科院分区：
文献类型：
--
作者：
Arrecubieta, C;Hammarton, TC;Roberts, IS
第2组荚膜多糖的细胞表面表达涉及到多糖从胞质膜内表面的合成位点转移到细胞表面。多糖的转运过程与多糖的重复结构无关，跨周质转运需要细胞质膜锚定蛋白KpsE和周质蛋白KpsD。在本文中，我们建立了KpsE蛋白的拓扑结构，并证明了C端与细胞质膜的周质面相互作用。通过化学交联，我们发现KpsE可能以二聚体的形式存在，并且二聚化不依赖于其他Kps蛋白或荚膜多糖的合成。化学交联无法证明KpsD和KpsE之间的相互作用，尽管在KpsE和Lpp存在的情况下，KpsD可以与身份未知的7-kDa蛋白交联。此外，我们证明了KpsD不仅存在于周质中，还存在于细胞质和外膜中，并且KpsD的正确膜结合依赖于KpsE， Lpp和分泌的多糖分子。在不同的突变背景下，KpsD和KpsE都显示出增加的蛋白酶K敏感性，这反映了由于运输过程的中断，HpsD和KpsE蛋白的构象发生了变化。总的来说，这些数据表明，跨质周输出涉及到KpsD作为细胞质膜转运蛋白和外膜之间的联系，而KpsE则促进这一运输过程。
The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD, In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide, No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the HpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.