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Journal of Bacteriology, April 2009, p. 2091-2101, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.00949-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Differential Interactions between Tat-Specific Redox Enzyme Peptides and Their Chaperones{triangledown} ,{dagger}

Catherine S. Chan, Limei Chang, Kenton L. Rommens, and Raymond J. Turner*

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

Received 10 July 2008/ Accepted 30 December 2008

The twin-arginine translocase (Tat) system is used by many bacteria to move proteins across the cytoplasmic membrane. Tat substrates are prefolded and contain a conserved SRRxFLK twin-arginine (RR) motif at their N termini. Many Tat substrates in Escherichia coli are cofactor-containing redox enzymes that have specific chaperones called redox enzyme maturation proteins (REMPs). Here we characterized the interactions between 10 REMPs and 15 RR peptides of known and predicted Tat-specific redox enzyme subunits. A combination of in vitro and in vivo experiments demonstrated that some REMPs were specific to a redox enzyme(s) of similar function, whereas others were less specific and bound peptides of unrelated enzymes. Results from Biacore surface plasmon resonance (SPR) and bacterial two-hybrid experiments identified interactions in addition to those found in far-Western experiments, suggesting that conformational freedom and/or other cellular factors may be required. Furthermore, we show that the interaction of the two prevents both from being proteolytically degraded in vivo, and kinetic data from SPR show up to 10-fold-tighter binding to the expected RR substrate when multiple binding partners existed. Investigations using full-length sequences of the RR proteins showed that the mature portion for some redox enzyme subunits is required for detection of the interactions. Sequence alignments among the REMPs and RR peptides indicated that homology between the REMPs and the hydrophobic regions following the RR motifs in the peptides correlates to cross-recognition.

* Corresponding author. Mailing address: Department of Biological Sciences, BI 156 Biological Sciences Bldg., University of Calgary, 2500 University Dr., N.W., Calgary, Alberta, Canada T2N 1N4. Phone: (403) 220-3581. Fax: (403) 220-9311. E-mail: turnerr{at}ucalgary.ca

{triangledown} Published ahead of print on 16 January 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, April 2009, p. 2091-2101, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.00949-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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