Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation
Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation
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DOI：
10.1128/jb.187.9.2983-2991.2005
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发表时间：
2005-05-01
影响因子：
3.2
通讯作者：
Beckwith, J
中科院分区：
文献类型：
--
作者：
Huber, D;Boyd, D;Beckwith, J
我们之前已经报道过DsbA信号序列通过细菌信号识别颗粒(SRP)途径促进细胞质蛋白硫氧还蛋白-1的高效共翻译易位。然而，两个常用的信号序列，PhoA和Male的序列，通过翻译后机制促进输出，不输出硫氧还蛋白。我们认为这种输出效率的差异是由于硫氧还蛋白在细胞质中的快速折叠；DsbA信号序列的共翻译输出避免了细胞质折叠的问题(C.F.Schierle，M.Berkmen，D.Huber，C.Kumamoto，D.Boyd和J.Beckwith，J.Baciol)。185：5706-5713,2003)。在这里，我们使用硫氧还蛋白作为报告来区分SRP依赖和非SRP依赖的可切割信号序列。我们基于一种估计疏水性的方法筛选出表现出一系列疏水性值的信号序列。筛选和提纯方法的连续迭代定义了识别SRP所需的阈值疏水性。虽然所有已鉴定的依赖于SRP的信号序列都高于这个阈值，但也有一些高于阈值的信号序列不利用SRP途径。这些结果表明，信号序列疏水性的简单测量是识别SRP的重要指标，但不是充分指标。此外，通过将这两类信号序列融合到DsbA中，我们发现DsbA利用依赖于SRP的信号序列来实现向周质的有效输出。我们的结果表明，那些被发现通过SRP依赖的信号序列输出的蛋白质可能需要这种输出模式，因为它们倾向于在细胞质中快速折叠。
We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.