The Rate of Folding Dictates Substrate Secretion by the Escherichia coli Hemolysin Type 1 Secretion System
The Rate of Folding Dictates Substrate Secretion by the Escherichia coli Hemolysin Type 1 Secretion System
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DOI：
10.1074/jbc.m110.173658
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发表时间：
2010-12-24
影响因子：
4.8
通讯作者：
Schmitt, Lutz
中科院分区：
文献类型：
--
作者：
Bakkes, Patrick J.;Jenewein, Stefan;Schmitt, Lutz
大肠杆菌毒素溶血素A(HlyA)的分泌是由膜蛋白复合体HlyB-HlyD-TolC催化的，需要位于HlyA最后60个氨基酸范围内的分泌序列。当N端与该分泌序列融合时，HLY转位复合体会输出各种乘客蛋白。然而，并不是所有的融合都能有效地分泌。在这里，我们证明了麦芽糖结合蛋白(男性)缺乏其自然输出信号，并与HlyA分泌信号融合在一起，HLY系统分泌不良。我们预计折叠动力学可能会限制分泌，因此我们引入了“折叠”突变Y283D。事实上，这种突变融合蛋白的分泌水平要高得多。第二个男性折叠突变(V8G或A276G)的引入进一步提高了这一水平。分泌物不需要分子伴侣SecB。折叠分析表明，所有突变都降低了底物的重折叠速度，而去折叠速度没有受到影响。因此，HLY系统的分泌效率由底物的折叠率决定。此外，我们证明了在出口中有缺陷的融合蛋白可以在保持功能的同时被改造成分泌。
Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.