Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli.
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.
Thomas
J D
JD
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Daniel
R A
RA
Errington
J
J
Robinson
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase