Oligomeric Properties and Signal Peptide Binding by Escherichia coli Tat Protein Transport Complexes

• Published in: Journal of molecular biology Vol. 322; no. 5; pp. 1135 - 1146
• Main Authors: de Leeuw, Erik, Granjon, Thierry, Porcelli, Ida, Alami, Meriem, Carr, Stephen B., Müller, Matthias, Sargent, Frank, Palmer, Tracy, Berks, Ben C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 04.10.2002
• Subjects: affinity chromatography; Arginine - metabolism; Biological Transport - physiology; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Macromolecular Substances; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - isolation & purification; Membrane Transport Proteins - metabolism; Protein Binding; Protein Sorting Signals; Protein Subunits; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Tat protein transport; tryptophan fluorescence; twin-arginine signal peptide; affinity chromatography; Chaps, 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulphonate; REES, red edge excitation shift; Tat protein transport; IMV, inner membrane vesicle; Escherichia coli; C 12E 9, nona-polyoxyethylene dodecyl ether; tryptophan fluorescence; twin-arginine signal peptide
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(02)00820-3

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.