%0 Journal Article %A Nilavongse, Arjaree %A Brondijk, T. Harma C. %A Overton, Tim W. %A Richardson, David J. %A Leach, Emily R. %A Cole, Jeffrey A. %T The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA %D 2006 %J Microbiology, %V 152 %N 11 %P 3227-3237 %@ 1465-2080 %R https://doi.org/10.1099/mic.0.29157-0 %K UQ, ubiquinone %K X-P, 5-bromo-4-chloro-3-indolyl phosphate %K TAT, twin-arginine translocase %K MK, menaquinone %I Microbiology Society, %X 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 His6-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. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.29157-0