In vivo dissection of the Tat translocation pathway in Escherichia coli.
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.
Ize
Bérengère
B
Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie CNRS, 31 chemin Joseph Aiguier, F-13402, Marseille cedex 20, France.
Gérard
Fabien
F
Zhang
Ming
M
Chanal
Angélique
A
Voulhoux
Romé
R
Palmer
Tracy
T
Filloux
Alain
A
Wu
Long-Fei
LF
D020816:Amino Acid Motifs; D003087:Colicins / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003087:Colicins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins