The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA.
The periplasmic nitrate reductase of Escherichia coli is important during anaerobic growth in low-nitrate environments. The nap operon encoding this nitrate reductase comprises seven genes including a gene, napF, that encodes a putative cytoplasmic iron-sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the E. coli cytoplasm. A bacterial two-hybrid protein-protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His(6)-tagged protein, exhibited spectral properties characteristic of an iron-sulphur protein. This protein was able to pull down NapA from soluble extracts of E. coli. A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm.
Nilavongse
Arjaree
A
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Brondijk
T Harma C
THC
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Overton
Tim W
TW
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Richardson
David J
DJ
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Leach
Emily R
ER
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Cole
Jeffrey A
JA
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
D001665:Binding Sites; D020134:Catalytic Domain / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism* / Q000502:physiology; D007506:Iron-Sulfur Proteins / Q000378:metabolism / Q000502:physiology*; D050901:Nitrate Reductase / Q000378:metabolism*; D009876:Operon; D033903:Periplasmic Proteins / Q000378:metabolism; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C515986:NapF protein, E coli; D033903:Periplasmic Proteins; D050901:Nitrate Reductase; C494404:napA protein, E coli