Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli
Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli
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DOI：
10.1046/j.1365-2958.2001.02253.x
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发表时间：
2001-01-01
影响因子：
3.6
通讯作者：
Robinson, C
中科院分区：
文献类型：
--
作者：
Thomas, JD;Daniel, RA;Robinson, C
双精氨酸易位 (Tat) 系统通过带有双精氨酸基序的不同信号肽，将含有辅因子的蛋白质靶向穿过大肠杆菌细胞质膜。在本研究中，我们利用与 TMAO 还原酶 (TorA) 的双精氨酸信号肽融合的绿色荧光蛋白 (GFP) 分析了大肠杆菌 Tat 系统的机制和功能。分级研究和荧光测量表明 GFP 被输出到周质，在那里它完全活跃。在 tat 缺失突变体中，输出几乎完全被阻断，这表明在野生型细胞中观察到的输出主要（如果不是完全）通过 Tat 途径发生。成像研究揭示了野生型细胞中与输出的周质形式相对应的荧光晕；在 tat 突变体中，GFP 均匀分布在整个细胞质中。因为之前的工作表明 GFP 无法在周质中折叠，所以我们建议 GFP 以完全折叠的活性状态输出。这些数据还首次表明异源蛋白质可以通过 Tat 途径以活性形式输出。
The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.