Biochemical and Biophysical Research Communications
Twin-arginine translocase may have a role in the chaperone function of NarJ from Escherichia coli
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.
References (40)
- et al.
Purification and properties of nitrate reductase from Escherichia coli K12
J. Biol. Chem.
(1974) - et al.
The iron–sulfur cluster composition of Escherichia coli nitrate reductase
J. Biol. Chem.
(1985) - et al.
Characterization of NarJ, a system-specific chaperone required for nitrate reductase biogenesis in Escherichia coli
J. Biol. Chem.
(1997) - et al.
A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins
Cell
(1998) - et al.
Protein targeting by the bacterial twin-arginine translocation (Tat) pathway
Curr. Opin. Microbiol.
(2005) - et al.
Export of complex cofactor-containing proteins by the bacterial Tat pathway
Trends Microbiol.
(2005) - et al.
Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial Tat pathway
J. Biol. Chem.
(1999) - et al.
TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli
J. Biol. Chem.
(1998) Studies on transformation of Escherichia coli with plasmids
J. Mol. Biol.
(1983)- et al.
Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels
J. Mol. Biol.
(1996)
Investigation of Escherichia coli dimethyl sulfoxide reductase assembly and processing in strains defective for the Sec-independent protein translocation system membrane targeting and translocation
J. Biol. Chem.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples
Anal. Biochem.
Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein
Biochem. Biophys. Res. Commun.
TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export
J. Biol. Chem.
The twin-arginine leader-binding protein, DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase
J. Biol. Chem.
Involvement of the molybdenum cofactor biosynthetic machinery in the maturation of the Escherichia coli nitrate reductase A
J. Biol. Chem.
NarI region of the Escherichia coli nitrate reductase (nar) operon contains two genes
J. Bacteriol.
Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer
Mol. Gen. Genet.
A common co-factor for nitrate reductase and xanthine dehydrogenase which also regulates the synthesis of nitrate reductase
Nature
Membrane cytochromes of Escherichia coli chl mutants
J. Bacteriol.
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