Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides

Danielle Tullman-Ercek,George Georgiou,Yasuaki Kawarasaki,Pooya Iranpour,Tracy Palmer,Brian Ribnicky,Matthew P. DeLisa

doi:10.1074/jbc.m610507200

Abstract

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 >or=+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.

Highlights

Binding proteins, and enzymes involved in cell envelope biogenesis [5]
Bioinformatic Identification of Putative Tat Signal Peptides— Putative Tat signal peptides were identified by first using BLAST to search for E. coli open reading frames encoding proteins that contained a twin arginine dipeptide in the first 50 aa and, in addition, any two of the additional four aa that comprise the conserved Tat motif
secretory pathway (Sec) a Shown in bold is the signal peptide c-region and in italics the predicted N-terminal region of the mature protein. b Average hydrophobicity using Kyte Doolittle values is calculated for the h-region, defined as described by Cristóbal et al [9]. c Signal peptide-GFP-SsrA fusions were expressed both in wild type and tatC strains, and whole cell fluorescence values were measured by flow cytometry

Summary

EXPERIMENTAL PROCEDURES

Bioinformatics—Putative Tat signal peptides were identified by conducting a BLAST search using the strings: SRRRFLK, SRRXFLX, TRRXFLX, SRRXXLK, SRRXXLA, TRRXXLK, TRRXXLA, SRRXXLT, SRRXXIK, SRRXXIA, SRRXFIX, SRRXFMK, SRRXFVK, SRRXFVA, SRRQFLK, RRXFLA, and RRXFLK within the first 50 residues. Signal peptides were amplified from E. coli XL1-Blue genomic DNA by PCR using the primers 1–56 shown in supplemental Table S3 and cloned into pKKGS [32], a derivative of plasmid pBAD33 [33]. To test growth on maltose, E. coli HS3018 or its derivatives containing plasmids encoding maltose-binding protein (MBP) fusions were grown overnight, diluted, plated on M9 minimal medium containing 0.4% maltose, and incubated at 37 °C for 2–3 days. E. coli MC4100-P and B1LK0-P containing plasmids encoding GFP-SsrA fusions were grown overnight in Luria Bertani medium as described above, and 500 ␮l of overnight culture were used to inoculate 10 ml of fresh medium. After 1 h of shaking at 37 °C, gene expression was induced with arabinose to a final concentration of 0.01%, the cells were incubated for an additional 4 h, and 1-ml samples were harvested by centrifugation, diluted in phosphate-buffered saline containing 5 ␮g/ml propidium iodide, and analyzed with a BD Biosciences FACSort flow cytometer

RESULTS

Sec ϩ

DISCUSSION