Signal recognition particle-dependent inner membrane targeting of the Pu1G pseudopilin component of a type II secretion system
Signal recognition particle-dependent inner membrane targeting of the Pu1G pseudopilin component of a type II secretion system
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DOI：
10.1128/jb.01230-06
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发表时间：
2007-03-01
影响因子：
3.2
通讯作者：
Pugsley, Anthony P.
中科院分区：
文献类型：
--
作者：
Francetic, Olivera;Buddelmeijer, Nienke;Pugsley, Anthony P.
假脓毒杆菌PulG是产酸克雷伯菌普鲁兰酶特异性11型分泌系统的重要组成部分。PulG是短而细丝的假毛囊的主要亚基，它可能在周质中伸长和收缩，作为一个动态活塞促进普鲁兰酶的分泌。根据对绿色和红色荧光蛋白嵌合体的研究，它有一个类似信号序列的n端片段，将未组装的PulG锚定在内膜上。我们分析了PulG在不同蛋白靶向和输出因子缺陷的大肠杆菌衍生物中的内、膜靶向和插入的早期步骤。在产生PulG-LacZ杂交蛋白的菌株中，当dsbA、dsbB或除secB外的所有see基因被突变破坏时，p -半乳糖苷酶活性显著增加。为了便于分析天然PulG膜插入，在n端跨膜段下游设计了一个先导肽酶切割位点（PrePulG*）。在携带secA、secY、secE和secD基因突变的菌株中检测到未加工的PrePuIG*，包括一些新的secY和secD等位基因。此外，信号识别粒子（SRP）的Ffh组分的耗尽完全消除了PrePulG*处理，而不影响sec依赖性的外质MalE和RbsB蛋白的输出。因此，PulG通过SRP共译靶向内膜Sec转位酶。
The pseudopilin PulG is an essential component of the pullulanase-specific type 11 secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner,membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The P-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all see genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePuIG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.