A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Abstract

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.

Sialin ( SLC17A5 ) functions as a nitrate transporter in the plasma membrane

“…2A), suggesting that salivary glands take up and secrete nitrate. The nitrate transporters identified to date belong to the highly conserved major facilitator superfamily (MFS) (11)(12)(13)(14)(15). To determine the molecular component mediating 2NO 3 − /H + cotransport in salivary glands, we analyzed the expression of 127 MFS genes (11,16,17) in human parotid and submandibular glands (n = 3) using Affymetrix U133Plus 2.0 microarrays.…”

Physiological and Biochemical Characterization of AnNitA, the Aspergillus nidulans High-Affinity Nitrite Transporter

“…Mutant strains lacking NrtA and NrtB showed no 13 NO 3 Ϫ tracer uptake yet are capable of growth on nitrite and a net uptake of nitrite (40). Our previous studies included obtaining accurate K m and V max values using 13 N-nitrate influx for the A. nidulans NrtA and NrtB nitrate transporters (36). In this article, we extended this line of study by generating the short-lived tracer 13 N-nitrite for nitrite transport studies.…”

“…Our previous studies included obtaining accurate K m and V max values using 13 N-nitrate influx for the A. nidulans NrtA and NrtB nitrate transporters (36). In this article, we extended this line of study by generating the short-lived tracer 13 N-nitrite for nitrite transport studies. Such tracer experiments permit a more accurate determination of kinetic constants than can be obtained by use of assays of net nitrite uptake.…”

“…Previous 13 NO 3 Ϫ tracer studies showed that wild-type strains of A. nidulans possess two high-affinity nitrate transporters (NrtA and NrtB) that, in common with high-affinity nitrate transporters from other organisms (8), also transport nitrite (40). Mutant strains lacking NrtA and NrtB showed no 13 NO 3 Ϫ tracer uptake yet are capable of growth on nitrite and a net uptake of nitrite (40). Our previous studies included obtaining accurate K m and V max values using 13 N-nitrate influx for the A. nidulans NrtA and NrtB nitrate transporters (36).…”

“…These transporters include Escherichia coli EcNirC (3,12,13), Aspergillus nidulans AnNitA (40), and C. reinhardtii CrNar1, which possesses no fewer than six paralogs (18,23). Such proteins belong to a family quite separate from the MFS, known as the formate/ nitrite transporter (FNT) family (TC 2.A.44), and many FNT members are thought to transport nitrite and/or the structurally related formate.…”