Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli

Molecular Microbiology - Tập 39 Số 1 - Trang 47-53 - 2001
Joanna Smith1, Richard A. Daniel2, Jeff Errington2, Colin Robinson1
1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
2Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

Tóm tắt

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.

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Molecular Microbiology

Tập 39 Số 1

47-53

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