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Volume 152, Issue 11

The NapF protein of the periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA Free

Abstract

The periplasmic nitrate reductase of is important during anaerobic growth in low-nitrate environments. The operon encoding this nitrate reductase comprises seven genes including a gene, , 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 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-tagged protein, exhibited spectral properties characteristic of an iron–sulphur protein. This protein was able to pull down NapA from soluble extracts of . 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.

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Keyword(s): MK, menaquinone , TAT, twin-arginine translocase , UQ, ubiquinone and X-P, 5-bromo-4-chloro-3-indolyl phosphate
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2006-11-01
2024-04-16
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The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA
Microbiology 152, 3227 (2006); https://doi.org/10.1099/mic.0.29157-0
/content/journal/micro/10.1099/mic.0.29157-0
/content/journal/micro/10.1099/mic.0.29157-0
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