Twin-arginine translocase may have a role in the chaperone function of NarJ from Escherichia coli

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Abstract

NarJ is a chaperone involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase A from Escherichia coli. It has also been shown that NarJ exhibits sequence homology to a family of chaperones involved in maturation and cofactor insertion of E. coli redox enzymes that are mediated by twin-arginine translocase (Tat) dependent translocation. In this study, we show that NarJ binds the N-terminal region of NarG through Far Western studies and isothermal titration calorimetry, and the binding event occurs towards a short peptide sequence that contains a homologous twin-arginine motif. Fractionation experiments also show that the interaction of NarJ to the cytoplasmic membrane exhibits Tat-dependence. Upon further investigation through Far Western blots, the interactome of NarJ also exhibits Tat-dependence. Together the data suggest that the Tat system may play a role in the maturation pathway of nitrate reductase A.

Section snippets

Materials and methods

Constructs and growth conditions. The gene sequence corresponding to the first 50 amino acids of the N-terminus of NarG, NarG50, was isolated through amplification of E. coli HB101 [20] genomic DNA using the primers TDMS-76 (5′-ATATCCATGGCTAGTAAATTCCTGGACC-3′) and TDMS-77 (5′-ATATGGTACCAATGGATCCGTGGGTAGAGCGGACG-3′) where the underlined sequences correspond to restriction enzymes sites NcoI, KpnI, and BamHI, respectively. The PCR product of narG50 and the vector pBEc-SBP-SET1 (Stratagene) were

NarJ binding towards the N-terminal region of NarG

If NarJ was indeed a chaperone of similar functions with DmsD, then one would expect that NarJ interacts with the N-terminal region of NarG, which contains the vestige twin-arginine motif, in a similar manner that DmsD interacts with the leader peptide of DmsA [18]. A previous study from our laboratory has shown far Western blotting to be a useful assay in demonstrating the binding of DmsD. Purified recombinant DmsA-leader:GST was spotted onto membranes, which was subsequently incubated with

Discussion

It was demonstrated in this study that binding of E. coli NarJ towards the N-terminal region of mature NarG is towards the first 15 amino acids of its sequence. This region contains a homologous sequence towards the twin-arginine motif, and was described by Turner et al. [17] as a ‘vestige’ motif. The findings here support the observations seen here and in [30], where the first 50 or 40 amino acids of NarG, respectively, were found to bind NarJ, and that the minimal binding sequence consists of

Acknowledgments

The research conducted in this paper was funded by the Canadian Institute of Health Research to R.J. Turner. We thank Frank Sargent (University of East Anglia Norwich, Norfolk, UK) for the generous donation of the E. coli DADE strain. We also thank Tara Winstone and Aaron Yamniuk for technical assistance and Andrew Binding for protein production.

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