Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway
Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway
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DOI：
10.1073/pnas.0937838100
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发表时间：
2003-05-13
影响因子：
11.1
通讯作者：
Georgiou, G
中科院分区：
文献类型：
--
作者：
DeLisa, MP;Tullman, D;Georgiou, G
为了通过双精氨酸易位（Tat）途径研究折叠和输出能力之间的关系，我们分析了等基因大肠杆菌菌株中八组假定的Tat先导肽和碱性磷酸酶融合物的亚细胞定位，这些融合物允许或不支持细胞质中蛋白质二硫键的形成。我们发现Tat转运子的输出只在细胞质中氧化蛋白折叠的菌株中被观察到。此外，我们发现其他含二硫化物的蛋白质，即单链Fv和异二聚体FAB抗体片段，仅在具有氧化细胞质的菌株中具有出口能力。功能性异二聚体FAB蛋白通过Tat先导肽与重链融合从细胞质中输出，表明在输出之前形成了二硫键二聚体。这些结果表明，在体内只有达到天然构象的蛋白质才会被Tat转运子输出，这表明折叠质量控制机制是输出过程中固有的。输出具有二硫键的蛋白质的能力和Tat途径的折叠特性是生物技术应用的兴趣。
To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric FAB antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric FAB protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.