Ize Bérengère, Gérard Fabien, Zhang Ming, Chanal Angélique, Voulhoux Romé, Palmer Tracy, Filloux Alain, Wu Long-Fei
Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie CNRS, 31 chemin Joseph Aiguier, F-13402, Marseille cedex 20, France.
J Mol Biol. 2002 Mar 29;317(3):327-35. doi: 10.1006/jmbi.2002.5431.
The bacterial Tat pathway is capable of exporting folded proteins carrying a special twin arginine (RR) signal peptide. By using two in vivo reporter proteins, we assessed factors that affect Tat pathway transport. We observed that, like the intact RR signal peptide, those with a KR or RK substitution were still capable of mediating the translocation of the folded green fluorescent protein (GFP). However, the translocation efficiency decreased in the order of RR>KR>RK. The KK motif was unable to mediate GFP translocation. The translocation of the RR-GFP fusion required TatA, TatB and TatC proteins. By exploiting the periplasmic bactericidal property of colicin V (ColV), we constructed a translocation-suicide probe, RR-ColV. The translocation of RR-ColV fully inhibited the growth of wild-type Escherichia coli and those of the DeltatatD and DeltatatE mutants. In contrast, the deletion of the tatC gene blocked RR-ColV in the cytoplasm and this strain exhibited a normal growth phenotype. Interestingly, the growth of DeltatatA and tatB mutants was inhibited partially by RR-ColV. Moreover, KR, RK and KK motifs were capable of mediating the ColV translocation with a decreasing RR=KR>RK>KK efficiency. In addition to TatE and TatC proteins, either TatA or TatB was sufficient for the translocation of RR-ColV or KR-ColV. In contrast, TatA plus the conserved N-terminal domain of TatB were required to mediate the killing effect of ColV fused to the less-efficient RK signal peptide. Taken together, these results suggest that a fully efficient Tat pathway transport is determined by the sequence of the signal peptide, the composition of the Tat apparatus, and the intrinsic characteristics of exported proteins.
细菌的Tat途径能够输出携带特殊双精氨酸（RR）信号肽的折叠蛋白。通过使用两种体内报告蛋白，我们评估了影响Tat途径转运的因素。我们观察到，与完整的RR信号肽一样，那些具有KR或RK替代的信号肽仍然能够介导折叠的绿色荧光蛋白（GFP）的转运。然而，转运效率按RR>KR>RK的顺序降低。KK基序无法介导GFP转运。RR-GFP融合蛋白的转运需要TatA、TatB和TatC蛋白。通过利用大肠杆菌素V（ColV）的周质杀菌特性，我们构建了一种转运自杀探针RR-ColV。RR-ColV的转运完全抑制了野生型大肠杆菌以及DeltatatD和DeltatatE突变体的生长。相比之下，tatC基因的缺失将RR-ColV阻断在细胞质中，并且该菌株表现出正常的生长表型。有趣的是，DeltatatA和tatB突变体的生长被RR-ColV部分抑制。此外，KR、RK和KK基序能够介导ColV的转运，效率依次降低，即RR=KR>RK>KK。除了TatE和TatC蛋白外，TatA或TatB中的任何一种对于RR-ColV或KR-ColV的转运都是足够的。相比之下，需要TatA加上TatB保守的N末端结构域来介导与效率较低的RK信号肽融合的ColV的杀伤作用。综上所述，这些结果表明，高效的Tat途径转运由信号肽的序列、Tat装置的组成以及输出蛋白的内在特性决定。
