Secretion of the Intimin Passenger Domain Is Driven by Protein Folding
Secretion of the Intimin Passenger Domain Is Driven by Protein Folding
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DOI：
10.1074/jbc.m116.731497
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发表时间：
2016-09-16
影响因子：
4.8
通讯作者：
Linke, Dirk
中科院分区：
文献类型：
--
作者：
Leo, Jack C.;Oberhettinger, Philipp;Linke, Dirk
内膜素是一种必需的粘附素，用于粘附和清除诸如致病性大肠杆菌等生物体。它也是Ve型分泌或逆自动转运的原型，其中细胞外c端区域或乘客在n端跨膜桶结构域的帮助下输出。我们最近报道了一种内膜素分泌阻滞的中间体，其中乘客位于外周质，但-桶已经插入膜。这种突变体的停滞是由于在乘客的N端插入了一个表位标签。在这里，我们研究了这种插入如何破坏自动运输，并发现它导致n端免疫球蛋白（Ig）样结构域D00的错误折叠。我们也可以通过在D00中进行内部缺失来阻止分泌，并将表位标签引入第二个Ig-like结构域D0中，也导致了乘客分泌减少。在许多经典的自体转运蛋白中，客运蛋白的近端折叠核心是分泌所必需的，而D00结构域则是多余的，因为完全缺乏D00的内膜蛋白突变的客运蛋白可以有效地输出。此外，D00结构域的稳定性略低于D0和D1结构域，通过原子力显微镜测量，D00结构域的展开速度约为200皮牛顿（pN），而D0和D1结构域的展开速度约为250皮牛顿。我们的结果支持一个模型，其中乘客的分泌是由细胞外ig样结构域的顺序折叠驱动的，导致乘客结构域沿N到C方向穿过外膜的矢量运输。
Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli. It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane -barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the -barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at approximate to 200 piconewtons (pN) compared with approximate to 250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction.