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J. Biol. Chem., Vol. 282, Issue 11, 8309-8316, March 16, 2007

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Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides^*

Danielle Tullman-Ercek¹, Matthew P. DeLisa, Yasuaki Kawarasaki^¶, Pooya Iranpour, Brian Ribnicky, Tracy Palmer^||**2, and George Georgiou^¶3

From the Departments of Chemical and Biomedical Engineering and the ^¶Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, the ^||Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom, and ^**School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a +1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Received for publication, November 10, 2006

^* This work was supported in part by grants from the Foundation for Research and National Institutes of Health (RO-01 GM069872). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S5-S7, Tables S2 and 3, and supplemental data.

¹ Supported by a National Science Foundation graduate fellowship.

² A Medical Research Council Senior Non-clinical Research Fellow.

³ To whom correspondence should be addressed: Dept. of Chemical Engineering, University of Texas at Austin, 1 University Station C04000, Austin, TX 78712. Tel.: 512-471-6975; Fax: 512-471-7963; E-mail: gg{at}che.utexas.edu.

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